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Thrombin-activatable fibrinolysis inhibitor and bacterial infectionsValls Serón, M.
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Mercedes Valls Serón
Thrombin-Activatable Fibrinolysis Inhibitor and
Bacterial Infections
Thrombin-Activatable Fibrinolysis Inhibitor and Bacterial Infections
Mercedes Valls Serón
Thrombin-Activatable Fibrinolysis Inhibitor and Bacterial Infections Dissertation, University of Amsterdam, Amsterdam, The Netherlands Copyright © 2011, Mercedes Valls Serón All rights reserved. No part of this thesis may be reproduced or transmitted in nay form by any means, without permission of the author. Author Mercedes Valls Serón Cover Streptococci by www.fotolia.com Printed by Wöhrmann Print Service ISBN 9789085705758 Financial support for the printing of this thesis was kindly provided by The University of Amsterdam and by AMC Medical Research.
Thrombin-Activatable Fibrinolysis Inhibitor and Bacterial Infections
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof.dr. D.C. van den Boom
ten overstaan van een door het college voor
promoties ingestelde commissie, in het openbaar
te verdedigen in de Agnietenkapel
op woensdag 16 november 2011, te 12.00 uur
door
Mercedes Valls Serón
geboren te Zaragoza, Spanje
Promotiecommissie
Promotores: Prof.dr. J. C. M. Meijers
Prof.dr. Ph. G. de Groot
Overige leden: Prof.dr. C. E. Hack
Prof.dr. F. Leebeek
Prof.dr. C. J. F van Noorden
Prof.dr. T. van der Poll
Prof.dr. A. J. Verhoeven
Faculteit der Geneeskunde
Financial support by The Netherlands Heart Fundation for the publication of this thesis is gratefully acknowledged.
A mis padres
Table of contents
CHAPTER 1: General introduction and outline of the thesis 9
CHAPTER 2: Recent developments in thrombin-activatable fibrinolysis 23
inhibitor research
CHAPTER 3: Thrombin-activatable fibrinolysis inhibitor is degraded by 45
Salmonella enterica and Yersinia pestis
CHAPTER 4: Binding characteristics of thrombin-activatable fibrinolysis 65
inhibitor to streptococcal surface collagen-like proteins A
and B
CHAPTER 5: Susceptibility of human TAFI-transgenic mice to 81
Streptococcus pyogenes
CHAPTER 6: Murine TAFI improves survival against Streptococcus pyogenes 93
CHAPTER 7: Summary and general discussion 109
CHAPTER 8: Nederlandse Samenvatting 116
Acknowledgements
List of publications
Introduction
Chapter 1
10
Introduction
Bacterial infections are initiated when bacteria and host come into contact. During the
infection process, the host responds to invading microbes with a number of different defense
mechanisms. In addition to physical barriers, the immune and hemostatic systems are
involved in destruction or contention of the infectious agent. Some bacteria or bacterial
products, however, can employ the hemostatic host response to their own benefit and cause
serious complications. Although different pathogenic bacteria seem to target different stages
of the coagulation and fibrinolytic systems, both systems often have a common consequence
which consists of disease propagation that lead to similar clinical pictures.
The hemostatic system
Hemostasis is a process which involves first blood clotting (coagulation) and subsequent brake
down of existing clots (fibrinolysis). Upon vessel injury, platelets adhere to the sub-
endothelial tissues and aggregate to form a haemostatic plug. At the same time, clotting is
initiated via the extrinsic or intrinsic pathway of coagulation, both of which lead to fibrin
formation through a common pathway, which forms the clot. The primary function of the
coagulation system is to stop bleeding of an injury until repair occurs.
The coagulation system consists of a number coagulation factors that circulate in plasma in
their inactive precursor forms. The extrinsic pathway (Figure 1) is triggered after an event of
injury of the blood vessel wall, when tissue factor (TF) is exposed to plasma and forms a
catalytic complex with coagulation factor VII, leading to the activation of factor X. Activated
factor X (FXa) will initiate the common pathway by assembly in the prothrombinase complex
with activated factor V. The prothrombinase complex (FVa-FXa) then cleaves prothrombin to
thrombin, which then cleaves fibrinogen to fibrin.
The intrinsic pathway (Figure 1) is initiated after activation of the contact system, a process
involving factor XI, factor XII, plasma kallikrein and high molecular weight kininogen. The
Figure 1. The coagulation cascade. The model is explained in detail in the text. The intrinsic and extrinsic pathways result in fibrin formation. Active (a) and inactive factors are represented by their roman numerals. TF: tissue factor.
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contact system assembles on negatively charged surfaces, leading to the reciprocal activation
of FXII and prekallikrein. Activated FXII triggers the sequential activation of FXI, IX, and X, and
subsequent induction of the common pathway.
Overactive coagulation can result wide-spread thrombosis. Therefore, it has to be controlled
by anticoagulation systems. This is achieved through the anticoagulation and fibrinolytic
systems. Together, coagulation, anticoagulation, and fibrinolysis maintain a delicate
physiological balance.
The major anticoagulants include antithrombin (AT), tissue pathway inhibitor (TFPI), and
activated protein C (APC). AT is produced by the liver and inhibits several coagulation factors
such as thrombin, FVIIa, FIXa, and FXa [1]. TFPI is a serine protease that inhibits FXa. In the
presence of FXa, TFPI also inhibits the TF/VIIa complex [2]. Protein C is an inactive plasma
serine protease. When thrombin is produced, it can bind to thrombomodulin present on the
vascular endothelial surfaces. The thrombin/thrombomodulin complex can then cleave
protein C into APC. APC generation is enhanced by the endothelial cell protein C receptor
(EPCR) on the endothelial surface. APC, with cofactor protein S, can cleave and inactivate FVa
and FVIIIa to negatively regulate coagulation [3,4].
At the site of tissue injury, fibrinolysis is initiated when plasminogen is converted to plasmin
by tissue-type plasminogen activator (t-PA) (Figure 2).
Plasmin then degrades the fibrin clot into soluble fibrin degradation products. The C-terminal
lysine residues of fibrin, generated after limited plasmin cleavage, act as a template onto
which both t-PA and plasminogen bind. As a result of t-PA and plasminogen interaction with
fibrin, the catalytic efficiency is 100-1000 fold enhanced. Plasmin formation is regulated by a
thrombin-dependent activation of the plasma protein thrombin-activatable fibrinolysis
Figure 2. The fibrinolytic system. Fibrinolysis is initiated when plasminogen is converted to plasmin by
tissue-type plasminogen activator (t-PA). Fibrin then degrades the fibrin clot into soluble fibrin
degradation products. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) by thrombin (IIa)
together with thrombomodulin (TM) inhibits the formation of plasmin and the degradation of the
fibrin clot.
Chapter 1
12
inhibitor (TAFI). Activated TAFI (TAFIa) cleaves off the C-terminal lysine residues of the
partially degraded fibrin and thereby abrogates the fibrin cofactor function in the t-PA-
mediated plasmin formation. More detailed information about TAFI is described in Chapter 2.
Gram-positive: Streptococcus pyogenes
Like other members of the family Streptococcacae, streptococci are Gram-positive facultative
anaerobic organisms which occur in chains or in pairs. S. pyogenes display a β–hemolytic
pattern of growth on blood agar meaning that bacteria produce a complete hemolysis around
the colonies. S. pyogenes also contain the Lancefield serogroup A carbohydrate on their cell
surface, and are often referred to as group A streptococci (GAS).
Strain characterization of (GAS) has traditionally been based on serological identification of M
protein [5], T-protein and production of streptococcal serum opacity factor (SOF) [6].
Advances in DNA-sequencing technology in the last decade resulted in the development of a
method for determining the M type of GAS from the sequence of the gene encoding M
protein, the emm gene. More than 200 emm types are currently listed in the online C.D.C.
database (http://www.cdc.gov/ncidod/biotech/strep/strepindex.htm).
S. pyogenes is one of the most common and important human bacterial pathogens. Although
it causes relatively mild infections such as pharyngitis (strep throat) and impetigo they may
evolve to life-threatening invasive infections of deeper tissues, the blood stream, and multiple
organs like septicemia and toxic-shock syndrome [7].
S. pyogenes are responsible for an estimated 616 million cases of throat infection
(pharyngitis, tonsillitis) worldwide per year, and 111 million cases of skin infection (primarily
non-bullous impetigo) in children of less developed countries [8]. Based on these numbers,
the bacterium is among the 10 most mortality-causing human pathogens.
S. pyogenes produces several surface-bound and secreted virulence factors that give rise to
these complications. Virulence factors from S. pyogenes include: surface attached virulence
factors such as M proteins [9-11] , streptococcal collagen-like surface protein A and B [12,13]
and fibronectin-binding protein (Protein F1/Sfb1) [14,15]; capsule and cell wall (lipoteichoic
acid and hyaluronic acid [16,17]) and secreted virulence factors such as superantigens [18],
streptokinase [19,20], DNases [21], and streptococcal inhibitor of complement (protein
SIC)[22].
The probably best characterized surface attached virulence factors are the M proteins. M
proteins are composed of two polypeptide chains that form an alpha-helical coiled-coil
configuration. The emm gene encodes for M proteins and is regulated by the Mga regulon,
multiple gene regulator of GAS, which is maximally expressed during the logarithmic growth
phase and in vivo during the acute phase of infection [23,24].
A number of studies have shown that M proteins allow adherence to various host tissues and
extracellular matrix components, trigger internalization into host cells, can provide anti-
phagocytic properties and induce autoimmune reactions in rheumatic fever.
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In addition to the M protein, streptococcal collagen-like surface protein A and B (SclA and
SclB), also contribute to cell adhesion and internalization. It has also been reported that SclA
from M type 41 activates the collagen receptor α2β1 integrin on fibroblasts and interacts with
the low density lipoprotein [25,26], high density lipoprotein [27], fibronectin and laminin [28].
In addition, SclA has been implicated in the inhibition of the alternative pathway of
complement [29,30]. More detailed information about SclA and SclB is described in Chapter 4.
In order to activate the clotting cascade, GAS have developed two mechanisms involving the
intrinsic and the extrinsic pathway of coagulation. M protein expressing bacteria can
assemble factors of the intrinsic pathway on their surface that will lead to fibrin formation. In
addition, soluble M1 and M3, and bacteria from the M1 and M3 serotype can activate the
extrinsic pathway by triggering tissue factor synthesis on isolated human monocytes [31,32]
and induce procoagulant activity on these cells.
Fibrin(ogen) plays multiple roles in the GAS/host interaction. The ability of S. pyogenes
surface to bind fibrinogen via fibrinogen-binding proteins (FgBPs) is believed to be important
in promoting bacterial adherence to host tissues during an infection and seem to have anti-
phagocytic function owing to their ability to impair deposition of complement. During
infection, the host generates fibrin at the local site of infection that can be used to wall off
the site of infection and limit pathogen invasion and spread. However, bacteria within a fibrin
network could be protected from the host defense machinery. To be able to circumvent the
thrombotic host defense, S. pyogenes expresses a number of molecules which confer the
bacterium ability to dissolve formed fibrin clots to facilitate bacterial spread. Streptococci are
proposed to gain fibrinolytic activity through direct binding of plasmin to specific surface
proteins or indirectly by sequential binding of fibrinogen and plasminogen [33].
Specific GAS surface proteins involved in plasminogen-binding are glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) [19,34], streptococcal surface enolase (SEN) [35],
plasminogen-binding group A streptococcal M protein (PAM) [36] and PAM related protein
(Prp) [37]. Moreover, the GAS secreted streptokinase enables GAS to cleave plasminogen to
plasmin without proteolysis [20,38], which degrades connective tissue, extracellular matrix
(ECM), and fibrin clots [39,40].
Of importance for this thesis, SclA and SclB bind to TAFI and is subsequently activated at the
bacterial surface by plasmin and thrombin-thrombomodulin [41]. Furthermore, activation of
TAFI on the surface of S. pyogenes evoked inflammatory reactions by modulating the
kallikrein/kinin systems [42].
Thus, at different stages of the infectious process, S. pyogenes may recruit either thrombotic
or thrombolytic factors to meet the demand for bacterial survival and proliferation.
Gram-negative: Yersinia pestis and Salmonella enterica
Yersinia pestis and Salmonella enterica belong to the Gram-negative family of
Enterobacteriaceae. Both are invasive pathogens.
Chapter 1
14
Yersinia pestis
Yersinia species are anaerobic, non-spore-forming bacilli or coccobacilli. There are multiple
Yersinia species, including the three human pathogens Y. pestis, Yersinia pseudotuberculosis,
and Yersinia enterocolitica.
Y. pestis is the causative agent of plague, an illness that may manifests in bubonic,
pneumonic, or septicemic form. Plague is a zoonotic disease that affects rodents and is
transmitted to humans mainly through the bite of infected fleas. The reservoir of Y. pestis in
nature is wild rodents. Humans are accidental hosts and have no role in its long-term survival
in endemic regions [43].
Upon feeding on blood of infected animals, fleas acquire Y. pestis, which multiply and block
the flea’s foregut. The blocked fleas starve and frenetically bite other rodents and,
incidentally, also humans. [44]. The bacteria are injected into the subcutaneous tissue, where
they promote local proteolysis at the infection site and migrate through the subcutaneous
tissue to the lymph nodes [45,46] where it proliferates, causing bubonic plague.
Bacterial proliferation causes swollen lymph nodes, called buboes. Another route of infection
is via respiratory droplets from an infected mammal to another. The bacteria spread to lungs
within the droplets and multiply causing primary pneumonic plague. The third form of plague
is primary septicaemic plague, where a flea injects the bacteria directly into a blood vessel
[45]. Secondary pneumonic or septicaemic plague occurs if the bacteria spread from buboes
to lungs or to the blood stream, respectively.
Y. pestis has killed millions of humans in three pandemics. According to World Health
Organization (WHO), there are about 2.000 cases and 200 deaths per year, mostly in Africa
and Asia. Because the occurrence of human cases and local epidemics has increased during
the last decades, plague has been classified as a re-emerging disease (WHO).
The genome of Y. pestis consists of a chromosome and three virulence plasmids, a 70-kb pCD
(or pYV), a 96-kb pMT1, and a 9.5-kb pPCP1 [47]. Y. pestis has gathered only a few virulence
factors and they are mostly encoded in plasmids [48].
Y. pestis pathogenesis is mainly caused by the plasminogen activator (Pla). Pla is a
multifunctional virulence factor that belongs to the omptin family of outer membrane
proteases of the Gram-negative bacteria. The gene coding for Pla is located in the pPCP1
plasmid. It has been shown that bacteria that express Pla are highly virulent yielding an LD50
increase of 106 fold compared to the absence of Pla [45,49]. In addition, expression of Pla is
needed for establishment of pneumonic plague [50] and is necessary to initiate bubonic
plague [45].
Lipopolysaccharides (LPS) are found in the cell envelope of Gram-negative bacteria and are
the major components of the outer leaflet of their outer membrane. Typically, an LPS
molecule consists of three structural domains: the lipid A, which holds the endotoxic
biological activity; a non-repeating oligosaccharide core; and a polysaccharide, the O-specific
chain, also known as O-antigen [51]. LPS molecules containing the O-specific chain are termed
smooth LPS while the ones lacking O-antigen are known as rough LPS. Pla and other omptins
Introduction
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require rough LPS to be active [52,53]. Thus, the LPS composition plays an important role in
the proteolysis efficiency of omptins. Y. pestis is naturally rough, which enables the activity of
Pla [52,54,55]. Besides activating plasminogen, Pla also interferes with the regulation of the
fibrinolytic system by inactivating α2-antiplasmin. These two features of Pla, in addition with
its adhesive characteristics, promote uncontrolled proteolysis as well as damage of tissue
barriers at the site of infection [56]. Another proteolytic target of Pla is the serum
complement protein C3 [49]. In addition, Pla proteolytically degrades the main inhibitor of the
initiation phase of blood clotting TFPI, suggesting that inactivation of TFPI may accelerate
blood clotting [57]. Together these interactions facilitate bacterial dissemination.
Salmonella enterica
S. enterica is related to human disease. S. enterica ssp. enterica causes 99% of human
infections: serovars Typhimurium, Enteritidis, Typhi, and Paratyphi are the most common and
most studied serovars. S. enterica serovars Typhimurium and Enteritidis cause gastroenteritis,
and serovars Typhi and Paratyphi cause the life-threatening disease typhoid and paratyphoid
fever, which are severe systemic infections. Gastroenteritis is usually mild and self-limiting,
but the bacteria can spread to distant organs and cause systemic infection [58]. In more than
95% of the cases, the infection initiates as the host ingests food contaminated with
Salmonella cells, which pass the gastrointestinal tract and reach the small intestine [59].
Following the adhesion and colonization of the intestinal tract, bacteria invade the intestinal
mucosa. Upon crossing the intestinal barrier, S. enterica invades macrophages and multiplies
in specific vacuoles known as Salmonella containing vacuoles (SCV)[60] where the bacterium
survive and multiply. Thereafter, S. enterica spreads inside circulating macrophages via blood
to cause systemic disease [61,62]. The ability to survive in immune cells is a major
determinant of pathogenesis and a variety of virulence factors involved in this process have
been identified in S. enterica [63].
According to WHO, Salmonella-gastroenteritis affects millions of people annually, especially in
developing countries, and causes thousands of deaths, and Typhi affects 16-33 million people
with 216.000 deaths per year.
In addition to Pla, PgtE also require rough LPS to be active and is sterically inhibited by the O-
antigen [52,64]. Clinical isolates and cells grown in laboratory medium of S. enterica have LPS
O-antigen oligosaccharide chains and therefore PgtE is apparently inactive. In contrast,
Salmonella isolates from murine macrophages have LPS with altered structure where the LPS
O-antigen is strongly reduced [64,65]. Indicating that expression of PgtE is upregulated during
growth of Salmonella inside macrophages [64,65] since bacteria released from macrophages
exhibit a strong PgtE-mediated proteolytic activity [65].
PgtE from S. enterica Typhimurium cells isolated from SCV of mouse macrophages
proteolytically activates plasminogen to plasmin [49], inactivates the main physiological
inhibitor of plasmin, α2-antiplasmin [65], and mediates bacterial adhesion to extracellular
matrices of human cells [52]. In this way, PgtE mediates degradation of extracellular matrix
Chapter 1
16
components and generates localized proteolytic activity, which may promote migration of
Salmonella through extracellular matrices. PgtE also degrades alpha-helical antimicrobial
peptides [66], which may be important during intracellular growth of the bacterium.
Introduction
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Outline of the thesis
The major objective of the studies described in this thesis is to study the interactions between
TAFI and pathogenic bacteria.
The membrane proteases Pla Y. pestis and PgtE of S. enterica interact with the human
fibrinolytic system by activating plasminogen and inactivating α2-antiplasmin, and PgtE in
addition by activating proMMP-9. TAFI is a regulatory, anti-fibrinolytic protein linking the
coagulation and fibrinolytic systems. In chapter 2, an introduction is given to TAFI and its role
in fibrinolysis and inflammation is explained. In chapter 3, we investigated the effects of the
Gram-negative proteases Pla and PgtE on TAFI.
In chapters 4 to 6, several studies are summarized that investigated the interaction of the
Gram-positive bacteria, S. pyogenes with TAFI and the role of TAFI in the course of
experimental streptococcal infection in vivo.
The binding of TAFI to S. pyogenes is mediated by the surface proteins, SclA and SclB. In
chapter 4, we characterized the TAFI binding region that is involved in this interaction.
In chapters 5 and 6 we investigated whether TAFI is involved in the course and the outcome
of experimental murine S. pyogenes infection in vivo. To this end, humanized-TAFI and TAFI-
deficient mice have been used.
Finally, in chapter 7 the data of this thesis are summarized and discussed.
Chapter 1
18
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41 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable
fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-like proteins A
and B. J Biol Chem 2007; 282: 24873-81.
42 Bengtson SH, Sanden C, Morgelin M, Marx PF, Olin AI, Leeb-Lundberg LM, et al. Activation of
TAFI on the surface of Streptococcus pyogenes evokes inflammatory reactions by modulating the
kallikrein/kinin system. J Innate Immun 2008; 1: 18-28.
43 Perry RD, Fetherston JD. Yersinia pestis--etiologic agent of plague. Clin Microbiol Rev 1997; 10:
35-66.
44 Duplantier JM, Duchemin JB, Chanteau S, Carniel E. From the recent lessons of the Malagasy foci
towards a global understanding of the factors involved in plague reemergence. Vet Res 2005; 36:
437-53.
45 Sebbane F, Jarrett CO, Gardner D, Long D, Hinnebusch BJ. Role of the Yersinia pestis plasminogen
activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague. Proc
Natl Acad Sci U S A 2006; 103: 5526-30.
46 Sebbane F, Lemaitre N, Sturdevant DE, Rebeil R, Virtaneva K, Porcella SF, et al. Adaptive response
of Yersinia pestis to extracellular effectors of innate immunity during bubonic plague. Proc Natl
Acad Sci U S A 2006; 103: 11766-71.
47 Ferber DM, Brubaker RR. Plasmids in Yersinia pestis. Infect Immun 1981; 31: 839-41.
48 Wren BW. The yersiniae--a model genus to study the rapid evolution of bacterial pathogens. Nat
Rev Microbiol 2003; 1: 55-64.
49 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the
invasive character of plague. Science 1992; 258: 1004-7.
50 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically
controls the development of primary pneumonic plague. Science 2007; 315: 509-13.
51 Raetz CR, Whitfield C. Lipopolysaccharide endotoxins. Annu Rev Biochem 2002; 71: 635-700.
52 Kukkonen M, Suomalainen M, Kyllonen P, Lahteenmaki K, Lang H, Virkola R, et al. Lack of O-
antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol
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53 Kramer RA, Brandenburg K, Vandeputte-Rutten L, Werkhoven M, Gros P, Dekker N, et al.
Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane
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54 Prior JL, Parkhill J, Hitchen PG, Mungall KL, Stevens K, Morris HR, et al. The failure of different
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55 Skurnik M, Peippo A, Ervela E. Characterization of the O-antigen gene clusters of Yersinia
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plague bacillus is most closely related to and has evolved from Y. pseudotuberculosis serotype
O:1b. Mol Microbiol 2000; 37: 316-30.
56 Lahteenmaki K, Virkola R, Saren A, Emody L, Korhonen TK. Expression of plasminogen activator
pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix.
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60 Gorvel JP, Meresse S. Maturation steps of the Salmonella-containing vacuole. Microbes Infect
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Recent developments in thrombin-activatable fibrinolysis inhibitor research Pauline F. Marx, Chantal J.N. Verkleij, Mercedes Valls Serón and Joost C.M. Meijers Mini Reviews in Medicinal Chemistry, 2009; 9 (10): 1165-73
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Abstract
Thrombin-activatable fibrinolysis inhibitor (TAFI) provides an important molecular link
between the coagulation and fibrinolytic systems. In this review, recent major advances in
TAFI research, including the elucidation of crystal structures, the development of small
inhibitors and the role of TAFI in systems other than hemostasis, are described and discussed.
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The basics about TAFI
The coagulation system is a strictly regulated series of enzymatic reactions that prevents
blood loss after vascular injury. The reactions ultimately lead to the formation of thrombin.
Thrombin converts soluble fibrinogen into a fibrin network, which is subsequently removed
by the fibrinolytic system during the healing process.
Thrombin-activatable fibrinolysis inhibitor (TAFI, recent reviews include: [1-10]) is a
glycoprotein with a molecular mass of 55 kDa that is synthesized in the liver and secreted into
the bloodstream in a zymogen form. TAFI is best known for its function in bridging the
coagulation and fibrinolytic cascades. TAFI is activated by the key component of the
coagulation system thrombin, either free or – more likely [11] – in complex with
thrombomodulin [12]. Alternative activators are plasmin [13-15] and neutrophil-derived
elastase [16]. The active form, the enzyme TAFIa, attenuates premature breakdown of the
fibrin clot. Hence its name with the accompanying acronym TAFI was chosen.
TAFIa functions by removing C-terminal lysine residues from partially degraded fibrin, which
act as binding sites for plasminogen and tissue-type plasminogen activator. This binding
facilitates the conversion of plasminogen into plasmin, the enzyme that degrades the fibrin
network of the blood clot.
Besides a function in fibrinolysis, TAFI also plays a role in inflammatory processes by
hydrolysis of bradykinin, osteopontin and the anaphylotoxins C3a and C5a (reviewed
elsewhere [10]). An overview of TAFI activation and TAFIa’s substrates, functions and
inactivation process is provided in Figure 1.
Due to more or less simultaneous discovery in various laboratories, the enzyme TAFIa was
also given different names, based on the biochemical features of the protein. TAFIa is a
member of the metallocarboxypeptidase subfamily, which is characterized by the presence of
a zinc atom in the active site that is required for the catalytic mechanism of the enzyme.
Metallocarboxypeptidases are further divided according to their substrate specificity into the
carboxypeptidases A (CPA), which preferentially hydrolyze aliphatic residues, and
carboxypeptidases B (CPB), which preferentially hydrolyze basic residues. TAFIa belongs to
the latter subfamily and is therefore sometimes referred to as plasma carboxypeptidase B.
The finding that TAFIa prefers to hydrolyse arginine residues, prompted other researchers to
call it carboxypeptidase R, where R stands for Arginine. Finally, TAFIa is a very labile enzyme,
hence it is also known as carboxypeptidase U, where the U stands for unstable.
The auto-regulation mechanism of TAFIa
Notwithstanding the high degree of homology between TAFIa and other members of the
carboxypeptidase B family (approximately 45%), TAFIa distinguishes itself clearly via an auto-
regulation mechanism which accounts for the enzyme’s short half-life. One of the first
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observations regarding TAFIa inactivation was that TAFIa is not inactivated by proteolysis
[17,18], and second that the catalytic zinc ion is not released in the inactivation process [19].
A third possibility was that the bond between the activation peptide and the catalytic domain,
amino acids 92 and 93, is cleaved during activation, but that the activation peptide remains
attached to the remainder of the protein. The actual release of the activation peptide could
then account for loss of activity. However, recently we were able to shown that the activation
peptide is not required for TAFIa activity and is not involved in stabilization of TAFIa, excluding
a role for the activation peptide in the inactivation mechanism [20].
Figure 1. Diagram of TAFI activation, TAFIa substrates, TAFIa functions and TAFIa inactivation. TAFI is
activated (closed arrows) by thrombin generated by the coagulation cascade, plasmin generated by the
fibrinolytic system, and elastase, that is released from neutrophils during inflammation, into TAFIa.
TAFIa, the active enzyme, converts several substrates (partially degraded fibrin, C3a, C5a, bradykinin and
osteopontin) to attenuate (open arrows) fibrinolysis or inflammatory processes. TAFIa inactivates
rapidly into TAFIai due to its structural instability after which it is proteolytically broken down and prone
to aggregation.
In the past decade, numerous studies were conducted to reveal the mechanism of TAFIa
inactivation by engineering more stable variants [17,18,21-26]. Extensive mutagenesis studies
revealed that all mutations that influence TAFIa stability are located in one segment of the
protein covering β-sheet 9 and α-helix 11 (residues 297-335). The most stable variant
generated today contains five point mutations (T325I, T329I, S305C, H333Y and H335Q) and it
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has a half-life of 180 times that of wild type TAFIa [25]. Remarkably, several of the more
stable mutants have an anti-fibrinolytic capacity that is less than expected from their increase
in half-life [21,25,26]. Although the reason for this observation is unknown, suboptimal
fitting of larger substrates due to structural changes distinct from the active site, may explain
this phenomenon.
Crystal structures explain the mechanism of TAFIa auto-regulation
Recently a breakthrough in the understanding of the auto-regulatory mechanism was made
when the crystal structures of various TAFI forms were solved [27-29]. Obtaining crystals
suitable for structure determination was a time consuming process due to the glycosylation
extent of the protein. TAFI has five putative N-glycosylation sites which account for the
heterogeneous appearance of the protein. Expression of TAFI in a particular cell line –
HEK293ES, which lacks N-acetylglucosaminoyltransferase-I – yielded a recombinant TAFI form
with homogeneous N-linked glycans. This engineering trick made it possible to grow properly
diffracting crystals and to solve the TAFI structure [27].
Similar to other members of the procarboxypeptidase A and B families [30-34], TAFI consists
of two structural domains, the activation peptide (first 92 amino acid residues) and the
catalytic domain [27]. In the zymogen, the activation peptide covers the active site preventing
substrates to enter the catalytic cavity and stabilizing a dynamic segment of the enzyme
moiety (residues 296-350). Proteolytic activation by for example thrombin, results in release
of the activation peptide and concomitant increase in dynamic segment mobility. The
increased dynamics lead to conformational changes that disrupt the catalytic site and
exposure of a cryptic thrombin-cleavage site at Arg302. An overall structure of TAFI is given in
Figure 2.
In agreement with this model, introduction of the stabilizing mutations T325I, T329I, H333Y
and H335Q, which results in a 70-fold more stable TAFIa form, or binding of the reversible
inhibitor GEMSA, which also stabilizes TAFIa, reduced the mobility of the dynamic segment
(Figure 3). Earlier research had already shown that Arg302 is the main site for proteolytic
breakdown of the enzyme moiety after it had lost the active conformation [17,18]. Our
structural data [27] were confirmed by two other studies published shortly after on the
crystal structures of bovine TAFI and TAFIa [28,29]. Recently, we observed that the
inactivated species, TAFIai, is prone to aggregation, forming large, insoluble protein
aggregates that are easily removed by centrifugation [20].
Chapter 2
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Figure 2. Ribbon drawing of TAFI. TAFI (401 amino acid residues) has two structural domains, the activation peptide (blue) and the catalytic domain (green), including the catalytic zinc ion (magenta sphere) and the highly dynamic ‘flap’ (residues 296-350, orange).The dynamic region provides an explanation for the instability of the enzyme TAFIa. As a result of proteolytic activation of TAFI at Arg92 and the ensuing release of the activation peptide, the activation peptide is no longer capable to restrict the dynamics of the flap. Increased dynamics lead to loss of structural integrity and consequently to TAFIa inactivation. Inactivated TAFIa (TAFIai) is prone to proteolytic breakdown at Arg302 and aggregation
Figure 3. Inhibitor binding stabilizes TAFIa. Crystallographic data provided an explanation for the
stabilizing effect of reversible inhibitors, like 2-guanidino-ethyl-mercaptosuccinic acid (GEMSA), on
TAFIa. GEMSA binds in the catalytic cleft S1’ pocket where the carboxy-terminal arginine or lysine
residue of the substrate would bind. One carboxylate group of GEMSA coordinates the catalytic zinc ion
and the second carboxylate is coordinated by catalytic site residues Arg217 and Arg235. Additional
hydrogen bonds are formed with Asp348 and Asp349, while hydrophobic interactions are formed with
residues 299 and 340-349 that are all part of the dynamic flap of TAFI. The stabilizing effect of GEMSA
on the flap region supports the idea that flap dynamics and the instability of TAFIa are directly linked.
Dynamic flap, orange; catalytic domain, green; catalytic zinc ion, magenta sphere; GEMSA, cyan; oxygen,
red; nitrogen blue.
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Sugars: important post-translational modifications
The crystal structures also provided more information on the glycosylation status of TAFI.
Four N-linked glycans were observed in the structure, all located in the activation peptide, e.g.
Asn22, Asn51, Asn63, and Asn288. The fifth reported N-linked glycosylation site at Asn219
[35] is entirely buried, excluding glycosylation. Usage of different sources of TAFI,
recombinant or plasma-derived, may explain differences in glycosylation pattern, although
the fact that Asn219 was completely buried within the structure indicates that the
physiological significance of glycosylation of this residue is most likely limited. The role of the
four glycans is probably to increase the solubility of the protein, as non-glycosylated TAFI, as
well as TAFIa, which contains no sugars, have a poor solubility [35], and to ensure proper
folding and secretion of the protein. In addition, contacts between the glycans could play a
role in stabilizing the dynamic flap. In particular a complex N-glycan attached to Asn22 seems
sufficiently close to the dynamic flap to establish direct interactions [27]. A recent study
showed that the replacement of this Asn22 resulted in an increased intrinsic activity of the
zymogen [36], indicating that this particular glycan indeed forms interactions within the TAFI
molecule. It is however unclear if it interacts with the dynamic flap directly. Slight changes in
catalytic efficiency of the active form and anti-fibrinolytic potential of this glycosylation
mutant as well as one other, Asn63Gln, were also reported [36].
Intrinsic activity of the zymogen: no role in fibrinolysis
As mentioned earlier, the glycosylated activation peptide is cleaved off during the activation
process, but a recent paper suggests that not only the enzyme TAFIa, but also the zymogen
TAFI, exerts catalytic activity [37]. However, although it was shown that the zymogen displays
enzymatic activity towards small molecular substrates [37,38], it does not add significantly to
the attenuation of fibrinolysis [39,40].
TAFI as therapeutic target: inhibition versus stabilization
The crystal structure provides not only information on the TAFIa inactivation process, it also
paves the way for the development of rationally designed inhibitors and stabilizers for TAFIa
that can be used in a clinical setting in the future. Inhibition of TAFIa is an attractive new
concept of antithrombotic therapy as it is based on enhancing fibrinolysis rather than direct
inhibition of the coagulation cascade, thus limiting the adverse hemorrhagic side effects seen
with anticoagulant drugs. It may also find application as an adjunct to thrombolytics. Ideally, a
useful inhibitor is not only a potent inhibitor of TAFIa, but is also strongly selective for this
particular enzyme. The major blood component of interest in this respect is carboxypeptidase
N (CPN). CPN is a constitutively active enzyme circulating in the bloodstream that shares
TAFIa’s specificity for C-terminal basic residues. Although commonly regarded as an inhibitor
Chapter 2
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of inflammatory processes – among CPN substrates are the anaphylotoxins C5a and C3a – an
anti-fibrinolytic function was recently ascribed to CPN [41]. Simultaneous inhibition of both
TAFIa and CPN may have adverse effects.
Some encouraging efforts were made in developing TAFIa inhibitors as well as in in vitro and
in vivo testing of the efficacy of TAFIa inhibitor therapy [42-48]. An alternative to the
structure-based design of small inhibitory molecular component is the production of
inhibitory antibodies and fragments thereof [49].
Among the inhibitors commonly used in in vitro experiments, and some also in animal studies,
are the carboxypeptidase inhibitor derived from potatoes (CPI), ε-amino caproic acid (ε-ACA),
guanidinoethyl-mercaptosuccinic acid (GEMSA), dithiothreitol (DTT), DL-2-mercaptomethyl-
3guanidino-ethylthiopropanoic acid (MERGEPTA) and zinc-chelators. One of the major
findings on TAFIa inhibitors is that reversible TAFIa inhibitors can both stimulate and inhibit
fibrinolysis [50,51]. A potential mechanism explaining this observation is that substrate-bound
TAFIa inactivates at a lower rate than free TAFIa [50,51] as the flexibility of the dynamic loop
is limited by interactions of the inhibitor with the dynamic flap, the catalytic residues and the
zinc ion [27] (Figure 3).
In contrast to thrombotic episodes, in the case of excessive bleeding, stabilizers of TAFIa
would increase the stability of a blood clot and prevent premature lysis. With the discovery of
TAFIa’s threshold mechanism of action [52,53] and the TAFI-325 polymorphism – either an Ile
residue or a Thr residue at position 325 – the impact of TAFIa stability became apparent
[54,55]. The TAFIa-325Ile variant is more stable than the more common TAFIa-325Thr variant
and this is also reflected in its antifibrinolytic potential [55]. Stabilization of TAFIa would
therefore potentially be a good therapeutic strategy for the
treatment of bleeding disorder.
The TAFI gene, polymorphisms and TAFI expression
Besides the TAFI-325 polymorphism (1057C/G), two other polymorphic sites in the coding
sequence of TAFI have been identified, TAFI-147 (505A/G, Ala or a Thr residue), which has no
known functional consequences, and a silent variation at position 678. In contrast, in the
promoter region of the TAFI gene and the 3’UTR, numerous additional single nucleotide
polymorphisms (SNPs) have been identified [56], many of which are in strong linkage
disequilibrium and some are in or in the proximity of potential transcription factor binding
sites [56,57]. Some polymorphisms are associated with clinical outcome, such as blood
pressure [58], angina pectoris [59], meningococcal disease [60], splanchnic vein thrombosis
[61], recanalization resistance [62] and recurrent pregnancy loss [63].
The gene encoding the 423 amino acids of pre-TAFI, CPB2, is located on chromosome
13q14.11 [64,65]. The 11 encoding exons stretch over approximately 48 kb of DNA [66] TAFI is
produced in the liver and seems to be under control of liver-specific transcription factors.
Research using the liver cell line HepG2 showed the importance of binding of nuclear factor Y
Recent developments in TAFI research
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and hepatocyte nuclear factor α for TAFI expression [67]. In mice, mRNA was detectable in
the liver, in a hepatocyte-specific, pericentral lobular distribution pattern [68]. TAFI was also
detected in human platelets and may have been produced in megakaryocytes rather than
taken up from the plasma [69].
A large number of studies over the years have been dedicated to the determination of the
plasma TAFI concentration in health and disease. In normal individuals, mean TAFI levels were
reported between approximately 75-275 nM [70-73], with a considerable variation of 45%-
150% of the mean value. The variation can in part be explained by the presence of different
genotypes, both because the variants are expressed at different levels and because some of
the polymorphisms affect the assays to detect TAFI. Because of the latter phenomenon –
discussed in more detail below – also the data on the influence of age, gender, ethnicity,
disease etc. and TAFI levels, is confusing and warrants further analysis in the near future. For
now it seems that approximately 25% of the variation can be explained by SNPs in the TAFI
gene [74], leaving a large percentage for non-genetic factors. Glucocorticoids were shown to
upregulate TAFI expression in vitro, whereas the interleukins IL-1β and IL-6 could down
regulate expression [75]. Since there are essential differences in the promoter sequences of
the human and mouse TAFI gene the mouse is not an optimal model system to study TAFI
gene regulation [2]. This may hamper progress in revealing the role of inflammation in
regulation of the CPB2 gene.
TAFI assays: not all assays measure the same
Although it is not quite clear yet what the impact of the polymorphisms is on development
and progression of various disorders, it is fact that many assays to measure TAFI and/or TAFIa
are compromised by the presence of various TAFI forms, especially the 325 polymorphism, in
the general and patient population [76,77]. Antibody-dependent assays suffer from affinity
differences between the TAFI-325-Thr and Ile form, and activity-based assays from a
difference in half-life. Nevertheless progress in this area over the past few years resulted in a
polymorphism-independent activity-based assay [78,79], an assay for the direct measurement
of functional TAFIa in plasma [80], the development of various new ELISAs specific for the
various TAFI fragments (zymogen, activation peptide, TAFIa/TAFIai) [81] and TAFI from
different species (human, mouse, rat) [82-84], and (global) fibrinolytic assays [85-87]. Also,
testing of novel substrates resulted in more selective TAFIa substrates that distinguish
between TAFIa and CPN [88]. Although not all these techniques are widely available, they are
valuable tools in TAFI research.
Chapter 2
32
TAFI from different species
The above mentioned assays for measuring TAFI of animal origin are important since the use
of experimental animals can yield valuable information. Also, TAFI from different species has
been cloned and characterized. The deduced amino acid sequence of rat TAFI is 83% [82]
identical to human TAFI. For mice this is 85% [68] compared to human, whereas the protein
sequences of rat and mice share 95% identity [82]. Although rat, mouse and human TAFI
share similar biochemical properties, the half-life of the rodents’ TAFI is shorter than the half-
life of their human counter part [68,82,89]. Also the plasma concentration of TAFI is lower in
these animals [68,82,90].
Some of the crystal structures of TAFI were solved using bovine TAFI, which has a sequence
identity of 77% with human TAFI, although little characterization of the functionality of this
protein in cows has been reported so far. Furthermore, the presence of TAFI was established
in pig, guinea pig, rabbit, dog, and baboon [90].
Functions: the interface between coagulation, fibrinolysis, inflammation and more
Besides in experimental animals, relations between TAFI levels and diseases were
investigated in human subjects. Recent advances on the role of TAFI in bleeding and
thrombotic disorders included the discovery that increased plasma TAFI concentrations are
associated with an increased risk for venous thrombosis and coronary artery disease [91,92]
and associations were found between TAFI levels and a number of disorders such as type-2
diabetes mellitus [93-96], hypertension [97,98], obesity [99], stroke [100-106], sepsis
[103,107-109], liver cirrhosis [110] and glomerulonephritis [111].
Patients with type 2 diabetes mellitus showed significantly higher TAFI levels compared to
non-diabetics [93] and TAFI levels were correlated with the urinary albumin excretion rate in
normotensive diabetes mellitus patients [94,96]. However, fasting TAFI levels were decreased
in normotriglyceridemic patients with type 2 diabetes compared to non-diabetes patients and
TAFI levels decreased postprandially in both groups [112].
The risk for ischemic stroke was also associated with elevated TAFI levels [101,102,105].
These patients showed elevated TAFI levels during the acute phase [100,106] and significantly
higher levels of TAFI were observed in stroke patients after recanalization by tissue-type
plasminogen activator infusion [103]. The baseline levels of TAFI, together with plasminogen
activator inhibitor 1, can predict the risk of symptomatic intracranial hemorrhage after tissue-
type plasminogen activator infusion [113].
In contrast, septic (both severe sepsis and septic shock) patients had significantly decreased
TAFI levels compared to controls [108]. Moreover, associations were found with TAFI and
mortality of meningococcal sepsis [107].
Recent developments in TAFI research
33
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With the engineering of TAFI knockout animals [84,114-116], the in vivo role of TAFI advanced
rapidly in the past few years. Compared to wild type animals, these mice were normal in
many respects, including survival, development, and fertility. Mao et al. reported that mice
lacking TAFI indeed have an enhanced endogenous fibrinolysis [117] and Wang et al. [116]
demonstrated the protective effect of TAFI deficiency in a ferric choride-induced occlusion
model of the vena cava. Similar results were obtained when TAFIa was inhibited by treatment
with carboxypeptidase inhibitor (CPI) [48,116]. Previously, enhanced in vivo thrombolysis was
already observed for TAFI deficiency in a background of plasminogen deficiency [118].
Besides the anti-fibrinolytic function of TAFIa, TAFI is also involved in inflammation and
wound healing [84]. The role of TAFI in inflammation is for example illustrated by the
observation that TAFI knock out mice, in contrast to control mice, were protected from liver
necrosis after intra peritoneal injection with Escherichia coli [119]. In another inflammation
model, this time with TAFI/plasminogen double knock out mice, the migration of leukocytes
towards the peritoneum was increased in the deficient animals compared to the wild types
showing the importance for TAFI in (plasminogen-dependent) cell migration in vivo [118].
Lately, we reported the binding of TAFI to the surface of a group A streptococci (M41
serotype) and subsequent activation at the bacterial surface via plasmin and thrombin-
thrombomodulin [120]. Furthermore, activation of TAFI on the surface of Streptococcus
pyogenes evoked inflammatory reactions by modulating the kallikrein/kinin system [121].
Additional in vivo experiments showed that the TAFI-deficient mice have a wound healing
problem [84], which may be related to the cell migration process mentioned above. In a skin
wound model [84], the keratinocyte migration pattern was disturbed, again pointing to a role
for TAFI in cell migration. Subsequent in vitro studies showed that TAFI inhibits endothelial
cell movement and tube formation [122].
However, it will take another while before the exact (patho)physiological roles of TAFI are
revealed, partly because interpretation of the data is difficult due to the genotype sensitivity
of many assays used in the past and still in use at the moment, and partially because some
studies were contradictory.
Concluding remarks
As outlined above, the TAFI research field has developed swiftly in the past few years and
now expands beyond hemostasis. The interest for the protein has increased since it is
recognized as a potential therapeutic target for novel intervention strategies. Inhibition of
TAFIa is expected to increase the efficacy of fibrinolytic therapy in thrombotic disorders.
Conversely, agents that improve or stabilize TAFIa, thereby down-regulating fibrinolysis, may
be useful for the treatment of bleeding disorders. In addition it may prove to be a target to
treat inflammatory or wound healing disorders.
Chapter 2
34
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thrombin-activatable fibrinolytic inhibitor (TAFI) among healthy subjects and patients with
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104 Ribo, M.; Montaner, J.; Molina, C. A.; Arenillas, J. F.; Santamarina, E.; Quintana, M.; varez-Sabin,
J. Admission fibrinolytic profile is associated with symptomatic hemorrhagic transformation in
stroke patients treated with tissue plasminogen activator. Stroke, 2004, 35, 2123-7.
105 Santamaria, A.; Oliver, A.; Borrell, M.; Mateo, J.; Belvis, R.; Marti-Fabregas, J.; Ortin, R.; Tirado, I.;
Souto, J. C.; Fontcuberta, J. Risk of ischemic stroke associated with functional thrombin-
activatable fibrinolysis inhibitor plasma levels. Stroke, 2003, 34, 2387-91.
106 Montaner, J.; Ribo, M.; Monasterio, J.; Molina, C. A.; Alvarez-Sabin, J. Thrombin-Activable
Fibrinolysis Inhibitor Levels in the Acute Phase of Ischemic Stroke. Stroke, 2003, 1038-40.
107 Emonts, M.; de Bruijne, E. L.; Guimaraes, A. H.; Declerck, P. J.; Leebeek, F. W.; de Maat, M. P.;
Rijken, D. C.; Hazelzet, J. A.; Gils, A. Thrombin-activatable fibrinolysis inhibitor is associated with
severity and outcome of severe meningococcal infection in children. J. Thromb. Haemost., 2008,
6, 268-76.
108 Zeerleder, S.; Schroeder, V.; Hack, C. E.; Kohler, H. P.; Wuillemin, W. A. TAFI and PAI-1 levels in
human sepsis. Thromb. Res., 2006, 118, 205-12.
109 Watanabe, R.; Wada, H.; Watanabe, Y.; Sakakura, M.; Nakasaki, T.; Mori, Y.; Nishikawa, M.;
Gabazza, E. C.; Nobori, T.; Shiku, H. Activity and antigen levels of thrombin-activatable fibrinolysis
inhibitor in plasma of patients with disseminated intravascular coagulation. Thromb. Res., 2001,
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110 Gresele, P.; Binetti, B. M.; Branca, G.; Clerici, C.; Asciutti, S.; Morelli, A.; Semeraro, N.; Colucci, M.
TAFI deficiency in liver cirrhosis: relation with plasma fibrinolysis and survival. Thromb. Res.,
2008, 121, 763-8.
111 Bruno, N. E.; Yano, Y.; Takei, Y.; Qin, L.; Suzuki, T.; Morser, J.; essandro-Gabazza, C. N.; Mizoguchi,
A.; Suzuki, K.; Taguchi, O.; Gabazza, E. C.; Sumida, Y. Immune complex-mediated
glomerulonephritis is ameliorated by thrombin-activatable fibrinolysis inhibitor deficiency.
Thromb. Haemost., 2008, 100, 90-100.
112 Rigla, M.; Wagner, A. M.; Borrell, M.; Mateo, J.; Foncuberta, J.; de, L. A.; Ordonez-Llanos, J.;
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113 Ribo, M.; Montaner, J.; Molina, C. A.; Arenillas, J. F.; Santamarina, E.; varez-Sabin, J. Admission
fibrinolytic profile predicts clot lysis resistance in stroke patients treated with tissue plasminogen
activator. Thromb. Haemost., 2004, 91, 1146-51.
114 Nagashima, M.; Yin, Z. F.; Zhao, L.; White, K.; Zhu, Y.; Lasky, N.; Halks-Miller, M.; Broze, G. J., Jr.;
Fay, W. P.; Morser, J. Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible
with murine life. J. Clin. Invest, 2002, 109, 101-10.
115 Asai, S.; Sato, T.; Tada, T.; Miyamoto, T.; Kimbara, N.; Motoyama, N.; Okada, H.; Okada, N.
Absence of procarboxypeptidase R induces complement-mediated lethal inflammation in
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116 Wang, X.; Smith, P. L.; Hsu, M. Y.; Tamasi, J. A.; Bird, E.; Schumacher, W. A. Deficiency in
thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced
vena cava thrombosis. J. Thromb. Thrombolysis., 2007, 23, 41-9.
117 Mao, S. S.; Holahan, M. A.; Bailey, C.; Wu, G.; Colussi, D.; Carroll, S. S.; Cook, J. J. Demonstration
of enhanced endogenous fibrinolysis in thrombin activatable fibrinolysis inhibitor-deficient mice.
Blood Coagul. Fibrinolysis, 2005, 16, 407-15.
118 Swaisgood, C. M.; Schmitt, D.; Eaton, D. L.; Plow, E. F. In vivo regulation of plasminogen function
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119 Renckens, R.; Roelofs, J. J.; ter Horst, S. A.; van, '., V; Havik, S. R.; Florquin, S.; Wagenaar, G. T.;
Meijers, J. C.; van der, P. T. Absence of thrombin-activatable fibrinolysis inhibitor protects against
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120 Pahlman, L. I.; Marx, P. F.; Morgelin, M.; Lukomski, S.; Meijers, J. C.; Herwald, H. Thrombin-
activatable fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-
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121 Bengtson, S.; Sadén, C.; Mörgelin, M.; Marx, P. F.; Olin, A.; Leeb-Lundberg, L.; Meijers, J. C. M.;
Herwald, H. Activation of TAFI on the Surface of Streptococcus pyogenes Evokes Inflammatory
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122 Guimaraes, A. H.; Laurens, N.; Weijers, E. M.; Koolwijk, P.; van, H., V; Rijken, D. C. TAFI and
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Thrombin-activatable fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis Mercedes Valls Serón, Johanna Haiko, Philip G. de Groot, Timo K. Korhonen, and Joost C.M. Meijers Journal of Thrombosis and Haemostasis, 2010; 8: 2232-40
Chapter 3
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Abstract
Pathogenic bacteria modulate the host coagulation system to evade immune responses or to
facilitate dissemination through extravascular tissues. In particular, the important bacterial
pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic
system. Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) has anti-fibrinolytic properties as
the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating
accelerated plasmin formation. Here, we demonstrate inactivation and cleavage of TAFI by
homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show
that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin
and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli
expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI
by the omptins. Our results indicate that TAFI is degraded by the omptins PgtE of S. enterica
and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue
barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during
infection.
S. enterica and Y. pestis proteases degrade TAFI
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Introduction
Several bacterial infectious diseases are associated with an imbalance of the mammalian
fibrinolytic/coagulation systems, however, the mechanistic details and pathogenetic roles of
these processes have remained largely unexplored. Fibrinogen-binding proteins have been
mainly identified in Gram-positive bacterial pathogens, such as Staphylococcus aureus,
Staphylococcus epidermis, and group A, C, and G Streptococci [1]. They interact with
fibrinogen and are thought to protect invading bacteria against host immune response and to
enhance survival of bacteria inside the host [2]. On the other hand, enhanced fibrinolytic
activity has been associated with several bacterial infectious diseases [3-6].
Plasmin is a key player in fibrinolysis, and bacterial pathogens have been observed to enhance
fibrinolysis either by directly activating plasminogen, by inactivating the main plasmin
inhibitor α2-antiplasmin, or by immobilizing plasminogen/plasmin on the bacterial surface [6].
The relationships between bacteria and the coagulation/fibrinolysis pathways appear
complex, as recent reports have identified that some serious bacterial pathogens, such as S.
aureus, Yersinia pestis and Salmonella enterica exhibit in vitro capacities which promote
either procoagulant or anticoagulant activities that lead to enhanced or decreased formation
of fibrin clots [5,7-10].
Y. pestis is the causative agent of plague, a highly fatal zoonotic infection transmitted to
humans through an intradermal bite by an infected flea in bubonic plague, or via respiratory
tract in aerosols from individuals with pneumonic plague. The importance of plasminogen
activation in pathogenesis of plague is well established. The plague bacterium harnesses the
plasminogen system for migration from intradermal infection sites at the early stages of
bubonic plague [5]. Altered fibrinolysis and importance of plasminogen activation have been
detected in both bubonic and pneumonic plague [10-12] and plasminogen-deficient mice are
more resistant than normal mice to infections by Y. pestis [5].
Plasminogen activation by Y. pestis is mediated by the transmembrane -barrel surface
protease Pla, which also inactivates α2-antiplasmin, thus creating uncontrolled plasmin
activity at the infection site [12,13]. Deletion or inactivation of the pla gene attenuates Y.
pestis both in bubonic [11,12] and pneumonic plague [10], and the pla gene is highly
transcribed by Y. pestis from buboes in infected rats [14].
Pla belongs to the omptin family of bacterial outer membrane proteases whose members
occur in several Gram-negative bacterial pathogens [15-17]. PgtE is the Pla ortholog in S.
enterica serovar Typhimurium, an intracellular pathogen that causes gastroenteritis. PgtE is
highly expressed by S. enterica inside infected mouse macrophages [4] and its genetic
deletion attenuates S. enterica in infected mice [18]. Fibrinolysis has not been earlier
associated with salmonellosis, and PgtE is a poor plasminogen activator [8] but it efficiently
inactivates α2-antiplasmin and activates procollagenases and progelatinases [4,18,19] which
can increase proteolysis and motility of macrophages and bacteria during salmonellosis [17].
Chapter 3
48
Thombin-activatable fibrinolysis inhibitor (TAFI) is a regulatory, anti-fibrinolytic protein linking
the coagulation and fibrinolytic systems. The protein is synthesized in the liver and secreted
into plasma as a 56-kDa procarboxypeptidase with 2 domains: the first 92 amino acids form
the activation peptide; the next 309 amino acids form the catalytic domain. During
coagulation, the proenzyme is activated to TAFIa by thrombin. TAFIa has anti-fibrinolytic
properties as it inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine
residues from partially degraded fibrin that are required for positive feedback in tissue-type
plasminogen activator dependent plasmin generation. Apart from thrombin, the serine
proteases plasmin, trypsin and neutrophil elastase have been reported to function as TAFI
activators [20-22]. TAFI activation by thrombin is inefficient but the process can be stimulated
approximately 1250-fold by the cofactor thrombomodulin [23]. TAFIa is unstable at 37 °C and
upon activation by thrombin it is inactivated (TAFIai) by a conformational change in a
temperature-dependent manner [24]. In contrast, in the presence of plasmin, TAFIa is
inactivated by plasmin proteolysis [20].
No direct physiological inhibitors of TAFIa have been identified to date. The cleavage of TAFI
by human proteinases has been well described biochemically and functionally [20,25],
whereas its possible activation or inactivation by bacteria has not been described.
As TAFIa regulates plasmin formation, invasive microorganisms that use the plasminogen
system to spread may target TAFI to enhance fibrinolysis. Therefore the aim of this study was
to investigate the effects of Y. pestis and S. enterica proteases Pla and PgtE on TAFI in order to
provide novel insights in the complex relationships between bacteria, coagulation and
fibrinolysis.
Materials and methods
Reagents
Hippuryl-arginine and H-D-Phe-Pro-Arg-chloromethylketone (PPACK) were purchased from
Bachem (Bubendorf, Switzerland), and ε-amino-n-caproic acid was purchased from Sigma-
Aldrich (St Louis, MO, USA). Rabbit lung thrombomodulin was purchased from American
Diagnostica (Stamford, CT, USA). Phosphoenolpyruvate (PEP), ATP, NADH, pyruvate
kinase/dehydrogenase (PK/LDH), and recombinant tissue-type Plasminogen Activator (t-PA,
Actilyse) were purchased from Biopool AB (Umeå, Sweden). Thrombin was a generous gift
from Dr. W. Kisiel (University of New Mexico, Albuquerque, NM, USA). TAFI was purified as
previously described [25], except that proteins were eluted from the Nik-9H10-Sepharose
column with 0.1 M glycine (pH 4.0) and that the fractions were collected in 20 mM Tris, 200
mM NaCl (pH 7.4). TAFI was stored at -80 °C after addition of Tween-20 (0.01%). Fibrinogen
was purchased from Kordia (Leiden, The Netherlands). A polyclonal antibody specific for TAFI
was obtained in rabbits as described elsewhere [27]. Plasminogen was purified from frozen
human plasma as previously described [28]. Alkaline-phosphatase-conjugated anti-rabbit
immunoglobulin G (IgG) was purchased from Dako (Glostrup, Denmark).
S. enterica and Y. pestis proteases degrade TAFI
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Bacteria and plasmids
The bacterial strains and plasmids used in this study are listed in Table 1. pla, pgtE and their
derivatives have been cloned in the inducible pSE380 vector in Escherichia coli XL1-Blue MRF’
and described before [13,29]. The recombinant E. coli strains were cultivated overnight at 37
°C in Luria broth (10 ml) supplemented with glucose (0.2% wt/vol), ampicillin (100 µg/ml) and
tetracycline (12.5 µg/ml). For protein expression, the culture was pelleted, suspended in 150
μl of phosphate-buffered saline (PBS, pH 7.1) and plated on Luria plates containing 5 µM
isopropyl-β-D-thiogalactopyranoside (IPTG) and antibiotics as above. Y. pestis strains were
cultivated over two nights at 37 °C on brain-heart infusion (BHI) plates and then inoculated in
10 ml BHI broth supplemented with hemin (40 µg/ml) and cultivated twice over two nights at
37 °C. S. enterica 14028R and 14028R-1 were cultivated overnight at 37 °C in 10 ml PhoP/Q-
inducing medium (N-minimal medium, pH 7.4, supplemented with 38 mM glycerol, 0.1%
casamino acids, 2 mg/ml thiamine, and 8 mM MgCl2). S. enterica 14028R-1 complemented
with pSE380 or pMRK3 were cultivated overnight at 37 °C in 10 ml Luria broth supplemented
with 5 µM IPTG and ampicillin (100 µg/ml). For the assays, bacteria were collected in PBS or in
Tris-buffered saline (TBS, pH 7.4), pelleted, and adjusted to OD600 value of 2.0 (corresponding
to c. 2 × 109 cells/ml).
TAFIa activation and activity assay
TAFI (54 nM for Y. pestis and S. enterica; for E. coli, 179 nM or 714 nM in dose- and time-
dependent assays; all concentrations are final concentrations) was incubated with the
bacteria (2 × 107
or 4 × 107 in TBS) in 100 mM Hepes/0.01% Tween-20, pH 8.0. In some
experiments, ε-ACA (2.5 mM) was added. Incubations were performed at 37 °C shaking. After
incubation, the samples were centrifuged, and the supernatants were further analyzed. TAFIa
activity was measured as previously described [26,30]. TAFI (40 nM) was added to a premix of
thrombin (8 nM) and thrombomodulin (16 nM) in the presence of CaCl2 (5 mM) in 100 mM
Hepes/0.01% Tween-20, pH 8.0 for 15 min at room temperature. Subsequently, a reaction
mixture containing MgSO4 (2.7 mM), KCl (10.9 mM), PPACK (30 µM), PEP (2.4 mM), NADH (0.5
mM), ATP (2.7 mM), hippuryl-arginine (6 mM), PK (45 µg/ml), LDH (15 µg/ml), and excess of
arginine kinase was added in 100 mM Hepes/0.01% Tween-20, pH 8.0.
When plasma was used, 20 µl TAFI-deficient plasma (three times diluted) was reconstituted
with TAFI to a final concentration of 165 nM and incubated with 20 µl bacteria (4 × 109).
Incubations and TAFIa activity measurement were performed as described above. Prewarmed
reaction mixture (90 μl) was transferred to a prewarmed microtiter plate, and 10 μl of TAFI
activated as described above was added.
TAFI activation was followed over time as a loss of NADH absorbance at 340 nm in a
Thermomax microplate reader (MolecularDevices Corp., Menlo Park, CA, USA) or in a
Multiskan EX reader (Thermo, Waltham, MA, USA).
Chapter 3
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Table 1. Bacterial strains and plasmids used in this study
Bacterial strain or plasmid Description Reference
Escherichia coli XL1 Blue MRF’ Δ(mcrA) 183 Δ(mcrCB-hsdSMR-mrr) 173 endA1
supe44 thi-1 recA1 gyrA96 relA1 lac *F’ proAB
lacIqZΔ M15 Tn 10 (tet)]
Stratagene
Yersinia pestis KIM D27 pPCP1+ Δpgm pYV
+ derivative of Y. pestis KIM-10 [52,53]
Yersinia pestis KIM D34 pPCP1-, otherwise identical to KIM D27 [52,53]
Salmonella enterica
serovar Typhimurium 14028R
Rough lipopolysaccharide derivative of 14028 [54]
Salmonella enterica
serovar Typhimurium 14028R-1
ΔpgtE derivative of 14028R [4]
pSE380 Expression vector, trc promoter, lacO operator,
lacI, bla
Invitrogen
pMRK1 pla in pSE380 [13]
pMRK3 pgtE in pSE380 [29]
pMRK111 Pla D206A [13]
pMRK31 PgtE D206A [29]
Clot-lysis assay
TAFI (179 nM or 714 nM in dose- and time-dependent assays) was incubated with the
bacteria (2 × 107
or 4 × 107 in TBS) in 100 mM Hepes/0.01% Tween-20, pH 8.0, at 37 °C with
continuous shaking. After incubation, the samples were centrifuged and the supernatants
were further analysed. The clot-lysis times were determined in a purified system. Briefly, 10
nM thrombin, 0.3 μg/ml recombinant t-PA (Actilyse), 20 mM CaCl2 and 5 nM thrombomodulin
were mixed with 40 nM preincubated TAFI (all concentrations are final concentrations in the
assay) in a 96-well microtiter plate. The volumes were adjusted to 30 μl with HBS (25 mM
Hepes, 137 mM NaCl, 3.5 mM KCl, pH 7.4) containing 0.1% (wt/vol) bovine serum albumin. A
mixture (20 μl) of fibrinogen (4.5 µM), plasminogen (90 nM) in HBS/0.1% bovine serum
albumin was added and turbidity was measured in time at 37 °C at 405 nm in a Thermomax
microplate reader. The clot-lysis time was defined as the time difference between half-
maximal lysis and half-maximal clotting.
Degradation of TAFI
For detecting the degradation of TAFI with anti-TAFI antibody, TAFI (820 nM) was incubated
with the bacteria (4 × 107 in PBS) in a volume of 12.5 µl for 2 h at 37 °C shaking. The bacteria
were pelleted, and the supernatant was boiled with sodium dodecylsulfate polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer containing β-mercaptoethanol. The samples were
run in a 12% (wt/vol) SDS-PAGE gel, transferred onto a nitrocellulose membrane and detected
S. enterica and Y. pestis proteases degrade TAFI
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with polyclonal anti-TAFI antibody (1:2000), followed by alkaline-phosphatase-conjugated
anti-rabbit IgG (1:1000), and phosphatase substrate.
To detect the degradation of TAFI by SDS-PAGE, TAFI (1.9 µM) was incubated with bacteria
(4×107 in TBS) in 100 mM Hepes/0.01% Tween-20, pH 8.0. In some experiments, ε-ACA (5
mM) was added. The samples were prepared as above, subjected to SDS-PAGE (10%) and
stained with Coomassie Brilliant Blue.
Determination of TAFI cleavage site
Degraded TAFI samples with E. coli expressing Pla or PgtE were prepared as for degradation
assay by Western blotting with the exception that Pla-expressing bacteria were incubated for
5 h. For N-terminal sequencing, the samples were run in a 12 % SDS-PAGE, blotted and
sequenced by Edman degradation. For MALDI-TOF-MS, the samples were run on a 12 % SDS-
PAGE, cut from the gel, digested with trypsin, and exposed to matrix-assisted laser desorption
ionization - time of flight mass spectrometry (MALDI-TOF-MS). The obtained peptides were
analyzed with Mascot search engine (http://www.matrixscience.com).
Results
Decreased activatability of TAFI by bacteria expressing PgtE or Pla.
To determine the effect of omptins on TAFIa activity, TAFI was pre-incubated with S. enterica
and Y. pestis strains differing in possession of pgtE or pla genes, and subsequently activated
with the thrombin-thrombomodulin complex. The PgtE-positive S. enterica 14028R and the
Pla-positive Y. pestis KIM D27 decreased the activatability of TAFI to TAFIa (by 85% and 97%,
respectively) (Fig. 1A). In contrast, the pgtE deletion mutant S. enterica 14028R-1 had no
detectable effect on TAFIa activity, and Y. pestis KIM D34, cured of the pla-encoding plasmid
pPCP1, reduced TAFIa activity by 59% (Fig. 1A). Inhibition of TAFIa activity was seen with S.
enterica 14028R-1 (pMRK3) complemented with the pgtE-encoding plasmid but not after
complementation with the expression vector, S. enterica 14028R-1 (pSE380), further
indicating that reduction of TAFIa activity by 14028R resulted from the expression of PgtE.
To further confirm the role of PgtE and Pla on TAFI activity, we studied the activatability of
TAFI with recombinant E. coli XL1 expressing PgtE or Pla. E. coli XL1 (pMRK3) with PgtE and E.
coli XL1 (pMRK1) with Pla reduced TAFI activatability (by 80% and 61%, respectively). E. coli
expressing PgtE or Pla with the catalytic site substitution D206A (pMRK31 or pMRK111,
respectively) decreased TAFI activatability by approximately 32% and 31%, respectively (Fig.
1B).
The recombinant E. coli expressing Pla or PgtE attenuated TAFIa activity in a dose-dependent
manner (Fig. 1C). Moreover, when the bacteria were pre-incubated with TAFI for different
time periods (0.5-2 h), we observed a progressive reduction in TAFIa activity (Fig. 1D).
Chapter 3
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Figure 1. Effect of PgtE- or Pla-expressing bacteria on TAFIa activity. (A, B) Loss of TAFIa activity with S.
enterica, Y. pestis (A) and recombinant E. coli (B). Purified TAFI was incubated for 2 hours with the
bacterial strains indicated below the columns. After incubation, TAFI was activated by the thrombin-
thrombomodulin complex and TAFIa activity was measured. (C, D) Dose-dependent (C) and time-
dependent (D) reduction in TAFIa activity. TAFI was incubated in buffer (▲) or with recombinant E. coli
XL1 expressing PgtE (pMRK3) (■), Pla (pMRK1) (●), or the catalytic-site mutants PgtE D206A (pMRK31)
(□) and Pla D206A (pMRK111) (○).Data are expressed as a percentage of the maximal TAFIa activity in
buffer. For S. enterica and Y. pestis strains, a representative experiment is shown, and for recombinant
bacteria, mean ± SD, n = 3 is shown.
Next, we investigated the effect of recombinant E. coli XL1 expressing PgtE or Pla on TAFI in
plasma. TAFI was added to TAFI-depleted plasma and incubated for 2 hours at 37°C with
recombinant E. coli XL1 expressing PgtE, Pla or the catalytic-site mutants PgtE D206A or Pla
D206A . After incubation, TAFI was activated with the thrombin-thrombomodulin complex
and TAFIa activity was measured. Our results indicated that E. coli XL1 (pMRK3) with PgtE and
E. coli XL1 (pMRK1) with Pla reduced TAFI activatability by 49% and 60%, respectively. E. coli
expressing PgtE or Pla with the catalytic site substitution D206A (pMRK31 or pMRK111,
respectively) both decreased TAFI activatability by 31%. The omptins did not activate TAFI
directly. Also, the omptins had no effect on the half-life of activated TAFI (data not shown).
S. enterica and Y. pestis proteases degrade TAFI
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The reduction in TAFIa activity by PgtE and Pla expressing bacteria could be due to an effect of
omptins on the TAFI activator thrombin-thrombomodulin. However, we could exclude this
possibility since the activation of another substrate of thrombin-thrombomodulin, protein C,
was not affected by the omptins (data not shown).
Taken together, these results indicated that PgtE of S. enterica and Pla of Y. pestis prevented
TAFI to become activated by thrombin-thrombomodulin and that the proteolytic activity of
these omptins was essential for the inhibition.
Reduction of the anti-fibrinolytic potential of TAFIa by recombinant E. coli.
The anti-fibrinolytic function of TAFIa was studied in a clot-lysis assay using purified
components and recombinant E. coli. TAFI-dependent prolongation of clot-lysis time was
reduced when TAFI was incubated with PgtE- or Pla-expressing E. coli XL1, in comparison to
bacteria expressing proteolytically inactive PgtE or Pla (Fig. 2A). These data indicate that
proteolytically active PgtE and Pla are responsible for functional inactivation of TAFI.
Incubation of TAFI with E. coli XL1 (pMRK3) or E. coli XL1 (pMRK1) showed that the TAFI-
dependent prolongation of clot-lysis time was dependent on bacterial numbers (Fig. 2B) and
on the incubation time of TAFI with the bacteria (Fig. 2C). Thus, PgtE- and Pla-mediated
proteolysis had negative effect on TAFIa function.
Proteolytic cleavage of TAFI by PgtE of S.enterica and by Pla of Y. pestis.
To determine why the activatability of TAFI was diminished by PgtE- or Pla-expressing
bacteria, we incubated TAFI with the bacteria and analyzed the supernatants by Western
blotting with an anti-TAFI antibody (Fig. 3A) and SDS-PAGE (Fig. 3B).
TAFI was degraded by PgtE- or Pla-expressing S. enterica and Y. pestis but not by bacteria
lacking the omptins (Fig. 3A). E. coli XL1 (pMRK3) with PgtE and XL1 (pMRK1) with Pla
degraded TAFI into smaller molecular weight peptides in a 2-h incubation, whereas XL1
(pMRK31) and XL1 (pMRK111) with mutated omptins were much weaker in TAFI degradation
(Fig. 3B). These results suggest that the decreased activatability of TAFI after its incubation
with omptin-expressing bacteria is a result of proteolytic degradation of TAFI by these
bacterial proteases.
Chapter 3
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Figure 2. Effect of PgtE- or Pla-expressing bacteria on the anti-fibrinolytic potential of TAFIa. (A) Loss of
TAFIa anti-fibrinolytic potential. TAFI was incubated for 2 hours with the E. coli XL1 strains indicated
below the columns. (B, C) Dose-dependent (B) and time-dependent (C) reduction in the fibrinolytic
potential of TAFIa. TAFI was incubated in the presence or absence of recombinant bacteria, after
incubation, bacteria were removed and the anti-fibrinolytic potential of TAFIa was determined in a clot-
lysis assay using purified components. TAFI was incubated in buffer (▲) or in the presence of XL1
(pMRK3) (■), XL1 (pMRK1) (●), XL1 (pMRK31) (□) or XL1 (p MRK111) (○). The TAFIa-dependent
prolongation was defined as the difference in clot-lysis time in the presence or absence of TAFI. Data are
expressed as a percentage (mean ± SD, n = 3) of TAFIa dependent prolongation of TAFIa in buffer.
S. enterica and Y. pestis proteases degrade TAFI
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Figure 3. Degradation of TAFI by bacteria expressing PgtE or Pla. TAFI degradation by PgtE-expressing S.
enterica and Pla-expressing Y. pestis (A) and PgtE- or Pla-expressing E. coli XL1 (B) is shown. TAFI was
incubated for two hours with the bacterial strains indicated above the lanes. Samples were analyzed by
Western blot with anti-TAFI antibody (A) or subjected to SDS-PAGE and Coomassie staining (B).
Migration distance of the molecular weight standard (in kilodaltons) is indicated on the left. For plasmid
constructs, see Table 1.
The fuzzy migration of the TAFI-degradation product suggested that the activation peptide,
where all the four N-linked glycosylation sites are located, was still present [31], suggesting a
C-terminal cleavage of TAFI. N-terminal sequencing and peptide mass fingerprint analysis of
trypsin-digested omptin-cleaved TAFI confirmed C-terminal cleavage of TAFI (data not
shown).
The lysine analogue ε-ACA is considered a TAFIa inhibitor [32], but it also stabilizes TAFIa
activity and prevents the formation of the no longer activatable 44.3-kDa TAFI fragment
generated after C-terminal cleavage by plasmin [20]. In order to assess if the C-terminal TAFI
cleavage by omptins was prevented by ε-ACA, the lysine analogue was added during
incubation of TAFI with recombinant E. coli XL1 expressing PgtE or Pla. Addition of ε-ACA
prevented the proteolytic cleavage of TAFI (Fig. 4A) and led to full TAFIa activity even in the
presence of proteolytically active PgtE and Pla (Fig. 4B). TAFIa activity in the presence of ε-
ACA was increased by < 10% (data not shown). This indicated that TAFI cleavage by omptins is
dependent on lysine residues, since ε-ACA acted as a competitive inhibitor of TAFI
degradation by omptins.
Chapter 3
56
Figure 4. Influence of ε-ACA on TAFI proteolysis and on TAFIa activity by PgtE-expressing (A) or Pla-
expressing (B) E. coli XL1. TAFI was incubated with recombinant E. coli XL1 in the presence or absence of
ε-ACA. After incubation, bacteria were removed and samples subjected to SDS-PAGE and Coomassie
staining. Migration distance of the molecular weight standards (in kilodaltons) is indicated on the left.
TAFI incubated with the bacteria was activated by the thrombin-thrombomodulin complex and TAFIa
activity was measured (lower panel). Data are expressed as a percentage (mean ± SD, n = 2) of maximal
TAFIa activity of TAFI incubated without bacteria. For plasmid constructs, see Table 1.
Discussion
TAFI is an important regulator that participates in maintaining the balance of coagulation and
fibrinolysis. Activated TAFI removes lysine residues from partially degraded fibrin, thus
reducing the binding of t-PA and plasminogen to the fibrin clot and subsequently diminishing
plasmin generation and fibrinolysis. TAFIa is regulated by its intrinsic, temperature-dependent
instability but no physiological inhibitors are known to date. Several inhibitory molecules for
TAFIa have however been characterized: the carboxypeptidase inhibitor from potato tubers
(CPI) [33], chelating and reducing agents [22,23], arginine and lysine analogues [34,35],
synthetic inhibitors [36-41], monoclonal antibodies [42-44] and nanobodies [45] against TAFI.
This is the first report in which two bacterial proteases, PgtE of S. enterica serovar
Typhimurium and Pla of Y. pestis, interfere with the activation of TAFI to TAFIa. Both bacterial
species are highly invasive pathogens, and PgtE and Pla have been identified as virulence
factors [5,10-12,18] Here we show that PgtE and Pla degrade TAFI via proteolytic breakdown.
Degraded TAFI can no longer be activated by the thrombin-thrombomodulin complex and
S. enterica and Y. pestis proteases degrade TAFI
57
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therefore the ability of TAFIa to remove C-teminal lysine residues from partially degraded
fibrin is abrogated.
The biological significance of the TAFI degradation remains to be elucidated but it is
important to note that decrease in TAFIa activity by PgtE and Pla also occurred in a plasma
environment where several putative omptin substrates, such as plasminogen, serpins, and
complement proteins, are present [13,46,47].
Plasmin can activate TAFI and subsequently inactivate TAFIa. However, plasmin can also
cleave TAFI to a form that cannot be activated anymore, and the TAFIa activity in the
presence of plasmin is low. Besides the direct effect of the omptins on TAFI degradation, the
plasmin generated by Pla or PgtE may indirectly cause TAFI degradation. The functional
heterogeneity of omptins also concerns TAFI degradation as we have observed that OmpT, an
omptin of E. coli, does not interact with TAFI (M. Valls Serón, J. Haiko, unpublished
observations). As a pathogen, E. coli is less invasive than Y. pestis and S. enterica.
The observation that the S. enterica pgtE-deletion mutant was completely inactive in
decreasing TAFIa activity whereas KIM D34 caused an approximately 59% inhibition, indicate
that Y. pestis might have other so far unidentified protease(s) involved in the TAFI
degradation. The recombinant E. coli XL1 expressing the catalytic-site substitutions D206A in
PgtE or Pla were clearly weaker in reducing TAFIa activity but still a low degree of TAFI
degradation was detectable with E. coli XL1 expressing the substituted omptins, which
indicates that omptin proteolysis is critical for the observed decrease in TAFI activatability.
Single substitutions rarely abolish the proteolytic activity completely, which may explain the
activity remaining with the substituted Pla and PgtE. On the other hand, our results do not
rule out the possibility that additional proteases in E.coli may target TAFI.
We observed that lysine analogue ε-ACA also inhibits the C-terminal cleavage of TAFI by PgtE
and Pla, thus indicating that lysine residues are critical for the cleavage of TAFI by these
omptins. However, it is also possible that ε-ACA prevents the interaction of the bacterial
proteases with TAFI. OmpT preferentially cleaves its substrates after arginine or lysine [48],
and Pla prefers Arg-Val bond present in its plasminogen substrate, so it is possible that the
cleavage occurs at arginine or lysine residues.
Many invasive bacterial pathogens interact with the haemostatic system in order to
disseminate within the host or avoid the host inflammatory immune response. In particular,
the Gram positive Streptococcus pyogenes have developed a variety of strategies to
circumvent the host defense such as the expression of streptokinase, a plasminogen-
activating protein. The streptokinase-plasmin(ogen) complex is protected against enzymatic
inactivation by the plasmin inhibitor α2-antiplasmin [49]. Recent studies have shown that TAFI
binds to the bacterial surface by interacting with the streptococcal collagen-like surface
proteins A and B (SclA and SclB) [50]. Bacteria-bound TAFI was activated by its natural
activators, plasmin and thrombin, which are also recruited to the streptococcal surface. Other
studies revealed that TAFI was used to redirect inflammation from a transient to a chronic
state by modulation of the kallikrein/kinin system [51]. Our results thus show, for the first
Chapter 3
58
time, an interaction of TAFI with Gram negative bacteria, indicating that TAFI might be a
general target for bacteria.
Pla, PgtE, and OmpT have been recently shown to inhibit tissue-factor pathway inhibitor
(TFPI), which inhibits coagulation [9]. This function is seemingly contrary to our present and
other previously described results [4,8,12,13] showing enhanced plasminogen activation and
fibrinolysis in vitro and in vivo. The in vivo significance of the procoagulation effects of
omptins remain to be determined, these observations however indicate that the interactions
of omptin proteases with the haemostatic system are complex and perhaps lead to opposite
directions at different stages of the infectious processes. S. enterica and Y. pestis employ
omptins to advance fibrinolysis by plasminogen activation and α2-antiplasmin degradation,
and inhibition of TAFIa function is still another way for these pathogens to influence
fibrinolysis and inflammation and promote their spread during infection.
S. enterica and Y. pestis proteases degrade TAFI
59
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Binding characteristics of Thrombin-Activatable Fibrinolysis Inhibitor to streptococcal surface collagen-like proteins A and B Mercedes Valls Serón, Tom Plug, J. Arnoud Marquart, Pauline F. Marx, Heiko Herwald, Philip G. de Groot and Joost C.M. Meijers Thrombosis and Haemostasis, 2011; Sep 27; 106(4): 609-16
Chapter 4
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Abstract
Streptococcus pyogenes is the causative agent in a wide range of diseases in humans.
Thrombin-Activatable Fibrinolysis inhibitor (TAFI) binds to collagen-like proteins SclA and SclB
at the surface of S. pyogenes. Activation of TAFI at this surface redirects inflammation from a
transient to chronic state by modulation of the kallikrein/kinin system. We investigated TAFI
binding characteristics to SclA/SclB. 34 overlapping TAFI peptides of approximately 20 amino
acids were generated. Two of these peptides (P18: residues G205-S221, and P19: R214-D232)
specifically bound to SclA/SclB with high affinity, and competed in a dose-dependent manner
with TAFI binding to SclA/SclB. In addition, the glycosaminoglycan consensus repeats in P18
and P19 were critical for the interaction of the TAFI peptides with SclA/SclB. In another series
of experiments, the binding properties of activated TAFI (TAFIa) to SclA/SclB were studied
with a quadruple TAFI mutant (TAFI-IIYQ) that after activation is a 70-fold more stable enzyme
than wild-type TAFIa. TAFI and TAFI-IIYQ bound to the bacterial proteins with similar affinities.
The rate of dissociation was different between the proenzyme (both TAFI and TAFI-IIYQ) and
the stable enzyme TAFIa-IIYQ. TAFIa-IIYQ bound to SclA/SclB, but dissociated faster than TAFI-
IIYQ. In conclusion, the bacterial proteins SclA and SclB bind to a TAFI fragment encompassing
residues G205-D232. Binding of TAFI to the bacteria may allow activation of TAFI, whereafter
the enzyme easily dissociates.
TAFI binding site to SclA and SclB
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Introduction
Streptococcus pyogenes is an important human Gram-positive pathogen that mainly causes
throat and skin infections, such us pharyngitis, impetigo and cellulitis. Although the majority
of streptococcal infections are superficial, some cases may progress into invasive and life
threatening diseases with an extremely rapid progression, such as streptococcal septic shock
and necrotizing fasciitis [1]. In order to infect the human host, S. pyogenes expresses a
number of virulence factors that mediate adhesion to host tissues, enable dissemination of
bacteria, and/or that modulate the immune response [2-4].
The streptococcal collagen-like surface proteins A and B (SclA and SclB), also known as Scl1
and Scl2, are two related proteins with a similar structure motif, including a C-terminal region
that is attached to the cell wall-membrane (CWM) via a LPATG anchor. This is followed by a
central region composed of a collagen-like domain (CL) with long segments of repeated GXY
amino acids, a sequence considered a defining feature of collagens. Next to the CL domain, an
amino terminal variable domain (V) is located. In addition, SclA, but not SclB, contains a linker
(L) region between the CWM and the CL domain. Both proteins are organized into a “lollipop-
like” structure, where the CL domain forms a triple helical-stalk and the V domain folds into a
globular head. The genes encoding SclA and SclB are located at different sites of the bacterial
chromosome and are differently regulated. While SclA is up-regulated by the Mga regulon,
the transcription of SclB is down-regulated by the same protein [5-10].
Recently, we reported the binding of Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) to the
surface of a group A streptococci (M41 serotype) and its subsequent activation at the
bacterial surface via plasmin and thrombin-thrombomodulin [11]. Furthermore, activation of
TAFI on the surface of S. pyogenes evoked inflammatory reactions by modulating the
kallikrein/kinin systems [12]. Identifying the molecular determinants of Scls-TAFI interaction
may well offer possibilities for prevention of diseases caused by inflammatory reactions
induced by S. pyogenes.
TAFI is a zinc-dependent procarboxypeptidase that is synthesized in the liver [13]. It is
thereafter released into the bloodstream, where it circulates at a concentration in the range
of 70-275 nM [14]. Cleavage of the TAFI zymogen by enzymes such as thrombin or plasmin
results in the formation of activated TAFI (TAFIa), which attenuates fibrinolysis. TAFIa
prevents accelerated plasmin formation by removing C-terminal lysine residues from partially
degraded fibrin that augment the efficacy of plasminogen activation. Besides a function in
fibrinolysis, TAFIa also plays a role in inflammatory processes by hydrolysis of bradykinin,
osteopontin and the anaphylotoxins C3a and C5a [15].
The present study was undertaken to 1) identify the TAFI binding site involved in the
interaction with the SclA, SclB expressed by S. pyogenes and 2) establish the binding
properties of TAFIa to SclA and SclB. Using synthetic TAFI peptides, we demonstrated that
TAFI binds to both Scl proteins via residues G205 to D232. In addition, we determined that
TAFIa is able to bind SclA and SclB but that it rapidly dissociates from the bacterial proteins.
Chapter 4
68
Matherials and methods
Proteins
34 overlapping TAFI peptides and the TAFI mutant peptides were synthesized by Peptide 2.0
Inc. (Chantilly, VA). Surface Plasmon Resonance (SPR) experiments with all 34 TAFI peptides
and with P18 and P19 mutants (P18.1, P18.2, P18.3, P18.4; P19.1, P19.2) were performed
with crude peptides. In SPR experiments with peptide 18 (P18), and peptide 19 (P19) and in
solid-phase binding assays, peptides were 95 % pure as determined by high-pressure liquid
chromatography analysis, and their identity was confirmed by mass spectrometry.
Recombinant SclA and SclB from the S. pyogenes AP41 strain were produced and purified as
described elsewhere [11]. TAFI was purified as previously described [16]. The rTAFI-IIYQ
mutant, which harbours the Thr325Ile, Thr329Ile, His333Tyr and His335Gln
mutations, and the active form of the mutant (rTAFIa-IIYQ) were generated and expressed as
described previously [17,18]. A polyclonal antibody specific for TAFI was obtained in rabbits as
described elsewhere [19]. HRP (horseradish peroxidise)-labeled swine anti-rabbit
immunoglobulin G (IgG) was purchased from Dako.
SPR binding assays
All SPR measurements were performed at 25°C using a BIAcore 2000 biosensor System (GE
Healthcare). Recombinant AP41 SclA and SclB were immobilized to a CM5 sensor chip using
the amine coupling kit according to the supplier’s recommendation (GE Healthcare). SclA and
SclB were applied in 10 mM NaAc (pH 3.1). Immobilization of SclA on the chip resulted in an
increase of the resonance signal by ~400 RU (resonance units) and with SclB of ~440 RU.
Binding studies were done in 10 mM Hepes, 150 mM NaCl, 0.005% Sulfactant P20 (pH 7.4), at
a flow rate of 30 µl/min. Different concentrations of rTAFI-IIYQ, rTAFIa-IIYQ (0-200 nM) or 5
µM TAFI peptides were injected for 2 or 3 minutes. The dissociation was followed for a period
of 1 or 10 minutes and the ligand surface was regenerated with a 30-s injection of 1/3 or 1/5
ionic buffer (92 mM KSCN, 366 mM MgCl2, 184 mM urea, 366 mM guanidine) followed by
equilibration with flow buffer at the end of each binding cycle. The data sets were fit to a
simple interaction model to obtain rate constants. When appropriate, data sets were also fit
to a heterogeneous analyte model. The association (ka) and dissociation (kd) rate constants
were determined using the BIAEvaluation Software (version 4.1; Biacore). The equilibrium
dissociation constants (KD) were calculated from the ratio of the measured kinetic rate
constants kd/ka. To generate the KD in binding studies with P18, P19 and P18 and P19 mutants,
the responses at equilibrium were fitted by non-linear regression using the Scrubber software
( version 2.0; Biologic Software).
Solid-phase binding assays
Ninety-six-well NUNC MaxiSorpTM
plates (Nalge Nunc International) were coated overnight
with 100 µl of recombinant SclA or SclB (46 nM) in NaHCO3 (pH 9.6) at 4°C. Following three
TAFI binding site to SclA and SclB
69
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washes with Tris-buffered saline supplemented with Tween [50 mM Tris, 150 mM NaCl, 0.1%
Tween-20 (pH 7.4)], plates were blocked with 150 µl of blocking solution [1.5 % w/v bovine
serum albumin (BSA) in Tris-buffered saline] for 1 hour at 37ºC. Wells were washed as
described above, and 100 nM TAFI in the presence or absence of P18, P19 or P29 (0 to 150
µM) diluted in blocking buffer was applied to the wells. The assay mixture was incubated for 1
hour at room temperature. Following the competition step, microtiter plates were washed
three times, and 100 µl of anti-TAFI antibody was incubated in blocking buffer for 1 hour at
room temperature. Next, the plates were washed three times and incubated with HRP-
labeled swine anti-rabbit IgG diluted 1:5000 with blocking buffer. After four washes, the
reactions were developed by the addition of 100 µl o-phenylenediamine (Sigma-Aldrich)
substrate [8 mM Na2HPO4 (pH 5.0) 2.2 mM o-phenylenediamine, 3% H2O2]. Colour
development was stopped by the addition of 50 µl of 1 M H2SO4, and the plates were read at
490 nm using a Thermomax microplate reader (Molecular Devices Corp.). Data were
corrected for binding to empty microtiter wells.
Results
Characterization of TAFI binding to SclA and SclB
We have previously shown that TAFI binds to SclA and SclB [11]. In the present study, we
examined the TAFI region involved in SclA and SclB binding. To this end, 34 overlapping TAFI
peptides of approximately 20 amino acids comprising the complete TAFI molecule were
generated (Table 1).
SPR measurements were carried out to determine binding between TAFI peptides and the
Scls. The recombinant bacterial proteins, and BSA as a negative control, were immobilized on
the surface of the biosensor chip in three separate flow cells. As shown in Figure 1, only two
synthetic peptides (Gly205
-Ser221
, P18, and Arg214
-Asp232
, P19) were able to bind immobilized
Scls. The other synthetic peptides did not show appreciable binding to the bacterial proteins.
Chapter 4
70
Table 1. Overlapping synthetic peptides of TAFI.
The one-letter code for amino acid residues is used for the sequence. The positions of the amino acids in
TAFI protein are shown.
Peptide Sequence Position
1 FQSGQVLAALPRTSRQVQ F1 - Q18
2 RTSRQVQVLQNLTTTYE R12 - E26
3 TYEIVLWQPVTADLIVKKKQ T26 - Q45
4 TADLIVKKKQVHFFVNAS T36 - S53
5 HFFVNASDVDNVKAHLN H47 - N63
6 DNVKAHLNVSGIPCSVLLA D56 - A74
7 IPCSVLLADVEDLIQQQISN I67 - N86
8 DVEDLIQQQISNDTVSPR D75 - R92
9 DTVSPRASASYYEQYHSLNE D87 - E106
10 EQYHSLNEIYSWIEFITERH E99 - H118
11 TERHPDMLTKIHIGS T115 - S129
12 SFEKYPLYVLKVSGKEQTAK S130 - K149
13 GKEQTAKNAIWIDCGIHARE G143 - E162
14 HAREWISPAFCLWFIGH H159 - H175
15 GHITQFYGIIGQYTN G174 - N188
16 GQYTNLLRLVDFYVM G184 - M198
17 DFYVMPVVNVDGYDYSWKKN D194 - N213
18 GYDYSWKKNRMWRKNRS G205 - S221
19 RMWRKNRSFYANNHCIGTD R214 - D232
20 ANNHCIGTDLNRNFASKHW A224 - W242
21 DLNRNFASKHWCEEGASSSS D232 - S251
22 SETYCGLYPESEPEVKAVA S253 - A271
23 ESEPEVKAVASFLRRNINQ E262 - Q280
24 RRNINQIKAYISMHSYSQH R275 - H293
25 HSYSQHIVFPYSYTRSKSKD H288 - D307
26 PYSYTRSKSKDHEELSLVAS P297 - S316
27 KDHEELSLVASEAVRAIEKT K306 - T325
28 EAVRAIEKTSKNTRYTHGHG E317 - G336
29 SKNTRYTHGHGSETLYLAPG S326 - G345
30 SETLYLAPGGGDDWIYDLGI S337 - I356
31 GGDDWIYDLGIKYSFTIELR G346 - R365
32 KYSFTIELRDTGTYGFLLPE K357 - E376
33 DTGTYGFLLPERYIKPTCRE D366 - E385
34 CREAFAAVSKIAWHVIRNV C383 - V401
TAFI binding site to SclA and SclB
71
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Figure 1. Binding of TAFI peptides to immobilized SclA and SclB. 34 overlapping TAFI peptides were
used to assess the binding to immobilized SclA and SclB by SPR-based assay. The bars represent the
responses in resonance units (RU) corrected for non-specific binding to BSA.
To further analyze the relative affinity of TAFI for SclA and SclB compared to the synthetic
peptides, we tested P18 and P19 for their ability to interfere with the interactions between
TAFI and the immobilized Scls. TAFI binding to SclA and SclB was competed with various
concentrations of P18 or P19. P18 and P19 displayed a dose-dependent inhibition of TAFI
binding to immobilized SclA (Fig. 2A) and SclB (Fig. 2B).
Figure 2. Competitive binding of TAFI to immobilized recombinant SclA and SclB with TAFI peptides
P18 and P19. Binding of TAFI to SclA (A) or SclB (B) onto 96-well plate was measured in the presence of
various concentrations of P18, P19 and P29. The assays were repeated three times, and results of a
representative experiment with duplicate samples are shown.
Chapter 4
72
In addition, P18 and P19 bind to SclA and SclB in a dose-dependent fashion (Fig. 3). Next, we
evaluated the affinity of P18 and P19 towards the Scls. For SclA we calculated a KD of 230 nM
(P18) and 520 nM (P19) and for SclB we calculated a KD of 230 nM (P18) and 530 nM (P19).
These results suggest that the TAFI sequence Gly205
- Asp232
contains important residues
involved in the interactions with SclA and SclB.
These data imply that P18 and P19 have a similar binding region as TAFI for binding to SclA
and SclB.
Peptides 18 and P19 contain a number of positively charged amino acids (Table 1), that could
contribute to binding. In fact, P18 and P19 contain respectively two and one
glycosaminoglycan consensus repeats [20] that have been shown in many other proteins to
be involved in binding to proteins and heparin [21]. The consensus sequence of such a repeat
is XBBXBX, where B stands for a basic residue and X is a non-basic residue. We next
investigated if the glycosaminoglycan consensus motif is involved in the interaction of the
peptides with SclA and B: peptide mutants were generated in which several basic amino acids
were changed to glutamine (Table 2).
Table 2. Binding of peptides 18, 19 and mutants thereof to SclA and SclB. The one-letter code for
amino acid residues is used for the sequence. The equilibrium responses of SPR were fitted by non-
linear regression to generate KD values. Values are means ± S.D. ND, no detectable binding (KD > 50,000
nM).
Peptide Sequence KD (nM) for
binding to SclA
KD (nM) for
binding to SclB
18 GYDYSWKKNRMWRKNRS 230 ± 20 230 ± 20
18.1 GYDYSWQQNRMWRKNRS 1220 ± 5 1280 ± 5
18.2 GYDYSWKKNRMWQQNRS 1530 ± 4 1530 ± 8
18.3 GYDYSWQQNRMWQQNRS ND ND
18.4 GYDYSWQQNQMWQQNQS ND ND
19 RMWNRRSFYANNHCIGTD 520 ± 10 530 ± 10
19.1 RMWQQNRSFYANNHCIGTD 1090 ± 3 1030 ± 3
19.2 RMWQQNQSFYANNHCIGTD ND ND
These peptide mutants were tested for binding to immobilized SclA and B using surface
plasmon resonance (Fig. 3). Our results indicated that replacement of basic residues in one of
the two glycosaminoglycan consensus repeats of P18 (P18.1 and P18.2) resulted in lower
binding affinity for both bacterial proteins. In addition, when basic residues in both
TAFI binding site to SclA and SclB
73
Ch
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glycosaminoglycan consensus repeats were replaced, the peptides did not bind anymore to
the bacterial proteins. In analogy, replacement of some of the basic residues in the consensus
repeat of P19 (P19.1) resulted in decreased binding to SclA and SclB and replacement of all
the basic residues (P19.2) resulted in no binding to the bacterial proteins. KD values for the
binding of the peptides to the bacterial proteins are shown in Table 2.
Together, these data suggested that at least one intact glycosaminoglycan consensus motif
XBBXBX is critical for the interaction of the TAFI peptides with SclA or B.
Figure 3. Binding of TAFI peptides 18 and 19 and mutants thereof to immobilized SclA and SclB.
Peptides 18 (A, C), 19 (B, C) and mutants thereof were used to assess the binding to immobilized SclA (A,
B) and SclB (C, D) by SPR-based assay. Values at equilibrium were plotted as a function of the TAFI
peptides.
Chapter 4
74
Binding analysis of TAFIa to SclA and SclB
Next we used SPR to investigate the binding characteristics of TAFIa to SclA and SclB. Because
TAFIa is a labile enzyme that is inactivated by a conformational change in a temperature-
dependent way [16] we used a TAFI quadruple mutant, rTAFI-IIYQ, which has a 70-fold more
stable active form (rTAFIa-IIYQ) than the wild type.
Both SclA and SclB were immobilized onto the sensor chip surface, and binding curves were
recorded for both rTAFI-IIYQ and rTAFIa-IIYQ variants at 6 different concentrations between 0
and 200 nM. Representative curves for rTAFI-IIYQ and rTAFIa-IIYQ binding to SclA and B are
shown in Figure 4.
Figure 4. Binding of TAFI-IIYQ and TAFIa-IIYQ to immobilized SclA and SclB. SPR analysis of binding of
TAFI-IIYQ to SclA (A) and SclB (B) and binding of TAFIa-IIYQ to SclA (C) and SclB (D). SclA and SclB (~400
RU for SclA, 440 RU for SclB) were immobilized to a CM5 sensor chip and increasing concentrations (0, 5,
20, 50, 100, and 200 nM) of TAFI-IIYQ or TAFIa-IIYQ were applied to the chip.
TAFI binding site to SclA and SclB
75
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Fitting the SPR profiles to interaction models yielded kinetic and affinity information for the
different TAFI-Scls interactions. The responses for rTAFI-IIYQ were well described by a 1:1
interaction model, however, the responses for rTAFIa-IIYQ were not. Instead, the profiles for
the active form could be better described by a heterogeneous analyte model, which assumed
two or more different classes of binding sites. We found similar association rates of rTAFI-IIYQ
and rTAFIa-IIYQ towards the bacterial proteins. In contrast, the dissociation curves for rTAFI-
IIYQ and rTAFIa-IIYQ were different. rTAFIa-IIYQ displayed ~ 3 to 58 fold faster dissociation
phase towards SclA (kd1 = 4.3 ± 0.2 x 10-4
; kd2 = 9.8 ± 0.1 x 10-3
), and a ~ 2 to 46 fold faster
dissociation phase towards SclB (kd1 = 3.0 ± 0.0 x 10-4
; kd2 = 7.3 ± 0.1 x 10-3
).
Based on the best data fit, rTAFI-IIYQ bound the Scls with affinities in the nanomolar range
(rTAFI-IIYQ-SclA: KD = 3.5 nM. rTAFI-IIYQ-SclB: KD = 4.0 nM). These values were lower than
previously established for plasma TAFI [11]. We also investigated binding of plasma TAFI to
SclA and B and found KD’s of 5.4-6.6 nM respectively (data not shown). This indicated that
rTAFI-IIYQ and plasma TAFI had similar binding kinetics. However, it is unknown why the
values for plasma TAFI were lower than in our earlier study.
Compared to rTAFI-IIYQ, a lower affinity was measured for rTAFIa-IIYQ, suggesting that the
conformation of the Scl-recognition domain had been slightly changed upon TAFI activation.
Kinetic and affinity values obtained for the bacterial proteins with both TAFI variants are
shown in Table 3. Taken together, the results imply that rTAFIa-IIYQ dissociates faster from
SclA and SclB compared to rTAFI-IIYQ.
Table 3. Kinetic and affinity parameters for interactions between TAFI-IIYQ, TAFIa-IIYQ and SclA, SclB.
The parameters were determined by surface plasmon resonance measurements using immobilized SclA
and SclB as the ligand, and TAFI-IIYQ or TAFIa-IIYQ as the analyte. ka, association rate constant; kd,
dissociation constant; KD, equilibrium dissociation constant. Values are means ± S.D.
Analyte/ligand ka1 (M-1
s-1
) kd1 (s-1
) KD1 (nM) ka2 (M-1
s-1
) kd2 (s-1
) KD2 (nM)
TAFI-IIYQ/SclA 4.5±0.0x104 1.6±0.1x10
-4 3.5±0.3
TAFI-IIYQ/SclB 4.0±0.1x104 1.6±0.0x 0
-4 4.0±0.0
TAFIa-IIYQ/SclA 3.4±0.1x104 4.3±0.2x10
-4 12.6±0.3 2.0±0.0x10
6 9.8±0.1x10
-3 4.9±0.1
TAFIa-IIYQ/SclB 2.7±0.0x104 3.0±0.0x10
-4 11.1±0.2 1.1±0.0x10
6 7.3±0.1x10
-3 6.6±0.0
Chapter 4
76
Discussion
In the present study we investigated the binding interactions of TAFI and activated TAFI to
streptococcal collagen-like surface protein A and B. By using TAFI peptides we identified the
binding region involved in the interaction with SclA and B within amino acids 205 to 232
(partially overlapping peptides 18 and 19) of TAFI. P18 bound to the same extent to SclA (KD =
230 nM) and SclB (KD = 230 nM). Affinity of P19 to SclA and SclB was similar (KD = 520 nM and
KD = 530 nM, respectively). In addition, SclA/B-TAFI interaction was mediated by the
glycosaminoglycan-binding site suggesting that this consensus motif may play an important
role in the binding of TAFI to other proteins.
The region of Gly205
to Asp232
is located distally from the TAFI catalytic site (Fig. 5) within helix
α-4 and is surface exposed and does not interfere with the region known to influence TAFIa
stability (Arg302
, Arg320
, Arg330
, and Thr/Ile325
) [22-24], neither with the residues involved in
substrate binding (Gly336
, Tyr341
, and Glu363
) [25].
Figure 5. P18 and P19 shown in the overall structure of TAFI. Ribbon drawing (A) and space-filling
representation (B) of TAFI with the activation peptide shown in blue, the catalytic domain in green, and
residues 205-232 representing the partially overlapping peptides 18 and 19 in red.
Although different binding partners have been identified for TAFI, such as plasminogen and
fibrinogen [26], the region described here as involved in SclA and SclB binding has not been
shown to be overlapping with another TAFI-protein interaction.
We demonstrated that P18 and P19 have the potential to compete with TAFI for binding to
the streptococcal proteins. TAFI binding to SclA was inhibited 55% and 76% by P18 and P19
respectively. In contrast, TAFI binding to SclB was inhibited 44% and 26% by P18 and P19
respectively. The partial contribution of P18 and P19 to inhibit TAFI-SclB interaction may be
attributed to the fact that SclA and SclB contain different sizes of the variable region. In
TAFI binding site to SclA and SclB
77
Ch
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addition, it is likely that the recognition between the bacterial proteins and TAFI is dependent
on the three-dimensional conformation of the protein, which is not optimally represented by
the linear peptides.
Recent findings that the physiological activity of TAFIa is not inhibited by SclA or SclB [11], and
the ability of activated TAFI on the surface of S. pyogenes to evoke inflammatory reactions by
modulating the kallikrein/kinin system [12], prompted us to investigate the TAFIa binding
properties to SclA and SclB. Here we provide evidence that activated TAFI binds to SclA and
SclB and, in contrast to the TAFI proenzyme, is rapidly dissociated from the bacterial proteins.
It is tempting to speculate that this constitutes a mechanism whereby the bacteria attract
TAFI to their surface and localize it there. After activation however, the activated TAFI can
dissociate and act on substrates elsewhere. These findings suggested that the activation
peptide may play a role in the TAFI binding to SclA and SclB. However, we found that the TAFI
binding to SclA and SclB was mediated by a region within the TAFI catalytic domain. The
crystal structure of TAFI showed that TAFIa stability is directly related to the dynamics of a 55-
residue segment (residues 296-350) of the active site [17, 27]. Release of the activation
peptide increases dynamic flap mobility and in time this leads to conformational changes that
expose the cleavage site at Arg302
. It cannot be ruled out that binding of TAFIa to SclA and SclB
is influenced by the conformational change in TAFIa.
In summary, this study demonstrates that binding of TAFI to SclA and SclB can be inhibited by
TAFI peptides. We have identified the region on TAFI, encompassing residues 205-232, that
bind to the bacterial proteins. In addition, it was shown that TAFIa rapidly dissociates from
SclA and SclB. A better understanding of the molecular mechanisms behind host/bacteria
interactions has the potential to discover important targets in the human host and ultimately
new therapeutic approaches for treatment of severe infectious diseases.
Chapter 4
78
Reference List
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2 Hertzen E, Johansson L, Wallin R, et al. M1 protein-dependent intracellular trafficking promotes
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3 Maamary PG, Sanderson-Smith ML, Aziz RK, et al. Parameters governing invasive disease
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5 Lukomski S, Nakashima K, Abdi I, et al. Identification and characterization of the scl gene
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6 Lukomski S, Nakashima K, Abdi I, et al. Identification and characterization of a second
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7 Rasmussen M, Bjorck L. Unique regulation of SclB - a novel collagen-like surface protein of
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9 Whatmore AM. Streptococcus pyogenes sclB encodes a putative hypervariable surface protein
with a collagen-like repetitive structure. Microbiology 2001; 147: 419-429.
10 Xu Y, Keene DR, Bujnicki JM, et al. Streptococcal Scl1 and Scl2 proteins form collagen-like triple
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11 Pahlman LI, Marx PF, Morgelin M, et al. Thrombin-activatable fibrinolysis inhibitor binds to
Streptococcus pyogenes by interacting with collagen-like proteins A and B. J Biol Chem 2007;
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12 Bengtson SH, Sanden C, Morgelin M, et al. Activation of TAFI on the surface of Streptococcus
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13 Marx PF, Bouma BN, Meijers JC. Role of zinc ions in activation and inactivation of thrombin-
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14 Bouma BN, Meijers JC. Thrombin-activatable fibrinolysis inhibitor (TAFI, plasma
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2003; 1: 1566-1574.
15 Leung LL, Nishimura T, Myles T. Regulation of tissue inflammation by thrombin-activatable
carboxypeptidase B (or TAFI). Adv Exp Med Biol 2008; 632: 61-69.
16 Valls Serón M, Haiko J, de Groot PG, et al. Thrombin-activatable fibrinolysis inhibitor is degraded
by Salmonella enterica and Yersinia pestis. J Thromb Haemost 2010; 8: 2232-2240.
17 Marx PF, Brondijk TH, Plug T, et al. Crystal structures of TAFI elucidate the inactivation
mechanism of activated TAFI: a novel mechanism for enzyme autoregulation. Blood 2008; 112:
2803-2809.
18 Marx PF, Plug T, Havik SR, et al. The activation peptide of thrombin-activatable fibrinolysis
inhibitor: a role in activity and stability of the enzyme? J Thromb Haemost 2009; 7: 445-452.
19 Mosnier LO, von dem Borne PA, Meijers JC, et al. Plasma TAFI levels influence the clot lysis time
in healthy individuals in the presence of an intact intrinsic pathway of coagulation. Thromb
Haemost 1998; 80: 829-835.
20 Cardin AD, Weintraub HJ. Molecular modeling of protein-glycosaminoglycan interactions.
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21 Hileman RE, Fromm JR, Weiler JM, et al. Glycosaminoglycan-protein interactions: definition of
consensus sites in glycosaminoglycan binding proteins. Bioessays. 1998; 20: 156-67.
22 Boffa MB, Bell R, Stevens WK, et al. Roles of thermal instability and proteolytic cleavage in
regulation of activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2000; 275: 12868-
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23 Marx PF, Hackeng TM, Dawson PE, et al. Inactivation of active thrombin-activable fibrinolysis
inhibitor takes place by a process that involves conformational instability rather than proteolytic
cleavage. J Biol Chem 2000; 275: 12410-12415.
24 Schneider M, Boffa M, Stewart R, et al. Two naturally occurring variants of TAFI (Thr-325 and Ile-
325) differ substantially with respect to thermal stability and antifibrinolytic activity of the
enzyme. J Biol Chem 2002; 277: 1021-1030.
25 Eaton DL, Malloy BE, Tsai SP, et al. Isolation, molecular cloning, and partial characterization of a
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26 Marx PF, Havik SR, Marquart JA, et al. Generation and characterization of a highly stable form of
activated thrombin-activable fibrinolysis inhibitor. J Biol Chem 2004; 279: 6620-6628.
27 Anand K, Pallares I, Valnickova Z, et al. The crystal structure of thrombin-activable fibrinolysis
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Susceptibility of humanized TAFI-transgenic mice to Streptococcus pyogenes Mercedes Valls Serón, Stefan R. Havik, Heiko Herwald Philip G. de Groot, and Joost C.M. Meijers Submitted for publication
Chapter 5
82
Abstract
Streptococcus pyogenes mainly causes throat and skin infections. Although the majority of
streptococcal infections are superficial, some cases may progress into invasive and life
threatening diseases such as sepsis and necrotizing fasciitis. S. pyogenes is highly host-specific
and normally only infects humans. Thrombin-Activatable Fibrinolysis inhibitor (TAFI) binds to
the collagen-like proteins SclA and SclB found at the surface of S. pyogenes. To test the
relative contribution of human TAFI to the outcome of S. pyogenes, we generated TAFI
knockout mice expressing human TAFI (humanized-TAFI). Wild-type and humanized-TAFI
transgenic mice were infected with S. pyogenes using a subcutaneous air-pouch model. Mice
were sacrificed after 24 or 48 h for determination of bacterial load, coagulation and
fibrinolysis activation, histopathology, inflammatory parameters, or were observed in a
survival study. Bacterial outgrowth was equal between groups. Levels of thrombin-
antithrombin complexes in spleen and lung were increased in humanized-TAFI transgenic
mice at 24 h and D-dimer was elevated in liver tissue in humanized-TAFI transgenic mice at 24
h after injection. Expression of human TAFI in mice had no effect on lung, liver, spleen and
kidney histopathology or cytokine levels. Remarkably, S. pyogenes inoculation led to a 65%
mortality rate in humanized-TAFI mice 120 h postinfection, in contrast to a 20% mortality rate
in wild-type mice (p=0.006). These findings suggest that introduction of human TAFI in mice
leads to an increased susceptibility to S. pyogenes infection.
Susceptibility of humanized TAFI mice to S. pyogenes
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Introduction
Streptococcus pyogenes (group A streptococcus, GAS) is an important gram-positive pathogen
that preferably colonizes humans. For the past two decades, several studies have reported an
increase in the severity and incidence of GAS infections [1]. S. pyogenes is responsible for a
wide variety of uncomplicated but also life-threatening infections in skin and throat.
Erysipelas, impetigo, pharyngitis and tonsillitis are considered rather harmless conditions
[2,3], whereas streptococcal toxic shock and necrotizing fasciitis are associated with invasive
and systemic infections [4]. Two serious complications are acute rheumatic fever and acute
glomerulonephritis [5,6].
S. pyogenes infection involves interactions with several components of the host hemostatic
system. The bacteria have been shown to bind and modulate the function of several
important factors involved in coagulation and fibrinolysis. Fibrinogen, factor V, XI, XII and
high-molecular-weight-kininogen are assembled at the surface of S. pyogenes through specific
interactions with bacterial surface proteins to promote formation of the fibrin network [7,8].
Plasminogen binding and its activation to the serine protease plasmin has been implicated as
a contributing mechanism of invasion for S. pyogenes and a variety of other bacterial species
[9,10]. Another hemostatic protein that interacts with S. pyogenes is TAFI [11].
TAFI is synthesized in the liver and secreted into plasma as a 56-kDa procarboxypeptidase.
During coagulation, the proenzyme is activated to TAFIa by thrombin and the thrombin-
thrombomodulin complex. TAFIa has anti-fibrinolytic properties as it inhibits plasmin-
mediated blood clot lysis by removing C-terminal lysine residues from partially degraded
fibrin that are required for positive feedback in tissue-type plasminogen activator dependent
plasmin generation. Apart from thrombin, the serine proteases plasmin, trypsin and
neutrophil elastase have been reported to function as TAFI activators [12-14]. TAFIa is
unstable at 37 ºC and upon activation by thrombin it is inactivated (TAFIai) by a
conformational change in a temperature-dependent manner [15].
TAFI binds to the surface of S. pyogenes (M41 serotype) and is subsequently activated at the
bacterial surface via plasmin and thrombin-thrombomodulin [11]. Furthermore, activation of
TAFI on the surface of Streptococcus pyogenes evoked inflammatory reactions by modulating
the kallikrein/kinin system [16].
The highly host-specificity nature of GAS in combination with the binding of TAFI to SclA and
SclB and subsequent activation at the bacterial surface, prompted us to investigate the
susceptibility of mice expressing human TAFI to S. pyogenes infection. To this end, mice
expressing only human TAFI were generated and infected with S. pyogenes serotype AP41.
The study suggests that introduction of human TAFI in mice leads to an increased
susceptibility for S. pyogenes infection.
Chapter 5
84
Materials and Methods
Animals
C57BL/6 mice were supplied by Harlan (Venray, The Netherlands). TAFI-transgenic mice were
generated by introduction of the human TAFI cDNA. The mouse albumin enhancer/promoter
was chosen to drive liver-specific expression of the human TAFI cDNA. The transgenic
construct containing the human TAFI cDNA was micro injected into fertilized oocytes of
FVB/NAU strain mice. Founders were bred with FVB/NAU strain mice. Transgenic mice were
identified by PCR analysis, and back-crossed to C57BL/6 background. TAFI-humanized
transgenic mice were generated by crossing TAFI-transgenic mice with TAFI- deficient mice.
Mice exclusively expressing human TAFI were selected. TAFI-deficient mice on a C57BL/6
background have been characterized previously [17-20]. Animals were bred in the animal
facility at the Academic Medical Center, The Netherlands and kept on a controlled 12 h
light/dark cycle and food and water were provided ad libitum. Experiments were carried with
8-12 weeks old male mice and all procedures were approved by the Institutional Animal Care
and Use Committee of the Academic Medical Center.
Experimental model of S. pyogenes infection
The S. pyogenes strain AP 41 (M41 type) was obtained from the Institute of Hygiene and
Epidemiology (Prague, Czech Republic). Bacteria were grown in Todd-Hewitt broth (BD
Bioscience) at 37°C, an aliquot of these cells were added to fresh media and grown up to the
exponential phase of growth (OD620=0.5). The cells were washed three times with saline (0.9
% NaCl) and diluted to 1 to 3.5 x 108/ml in saline. Wild-type and TAFI-humanized transgenic
mice were subjected to an air-pouch infection. Briefly, 0.8 ml of air and 0.2 ml of S. pyogenes
diluted were injected with a 25-gauge needle subcutaneously on the neck of the mouse. Mice
were sacrificed 24 and 48 hours after infection or were observed in a survival study for 5 days.
During the survival experiment, the animals were daily monitored for signs and symptoms of
infection.
Sample harvesting
At the time of sacrifice (24 and 48 hours), mice were first anesthetized by inhalation of
isoflurane (Abbott Laboratories). Blood was drawn from the vena cava inferior into a sterile
syringe containing 3.2% sodium citrate (1/10 vol) and immediately placed on ice. Thereafter,
spleen, kidney, lung and liver were harvested and processed for measurements of CFU,
histology, cytokines.
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Determination of bacterial outgrowth
After 24 and 48 hours postinfection, spleen, kidney, lung and liver were homogenized at 4°C
in 4 volumes of sterile isotonic saline with a tissue homogenizer (Biospect Products) which
was carefully cleaned and disinfected with 70% ethanol after each homogenization. Serial 10-
fold dilutions in sterile saline were made from these homogenates and blood. Thereafter, 50-
µl volumes were plated onto sheep-blood agar plates and incubated at 37°C. CFU were
counted after 16 h.
Histology
Organs collected from both mice groups 24 and 48 hours after infection were fixed in formalin
for 24 h and then embedded in paraffin. Sections 5- µm thick were cut, stained with
hematoxylin and eosin following standard procedures and then examined by light microscopy.
Assays
Interferon-γ, TNF-α, IL-6, IL-12, IL-10 and MCP-1 were measured by cytometric bead array
multiplex assay (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s
recommendations. For these cytokine measurements, harvested organs were homogenized
as described above. Sample homogenates were lysed in 1.25 volumes lysis buffer (300 mM
NaCl, 15 mM Tris [tris(hydroxymethyl) aminomethane], 2 mM MgCl2, 1% Triton X-100,
pepstatin A, leupeptin, and aprotinin (each at 20 ng/ml) (pH 7.4)) on ice for 20 min and spun
at 3600 rpm at 4°C for 10 min. The supernatant was frozen at -80°C until assayed. Thrombin-
antithrombin complexes (TATc; Siemens Healthcare Diagnostics, Marburg, Germany) and D-
dimer (Diagnostica Stago, Asnieres, France) were measured by ELISA.
Statistical analysis
Differences between groups were calculated by using the Mann-Whitney U test. Values were
expressed as means ±SE. A p value of < 0.05 was considered statistically significant. Survival
curves were analyzed by the Kaplan-Meyer log-rank test. All statistics were performed using
GraphPad Prism, version 5.01 (GraphPad Software, San Diego, CA, USA).
Results
Bacterial outgrowth
To investigate if human TAFI influences S. pyogenes dissemination, we compared the bacterial
load in spleen, kidney, lung, liver and blood of TAFI humanized and wild-type mice after 24
and 48 h of initiation of infection (Fig. 1). At 24 h, there were no differences in bacterial
outgrowth in spleen, lung or kidney between mouse strains. However, bacterial loads in liver
and blood from wild-type mice were slightly higher. After 48 h, bacterial loads in spleen, lung,
and blood from TAFI humanized and wild-type mice were equal, whereas the bacterial loads
in kidney and liver tended to be higher but not significant in TAFI-humanized transgenic mice.
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Figure 1. Bacterial dissemination. Wild-type (●) and humanized-TAFI mice (○) were injected with S.
pyogenes AP41 (3.5 x108/ml) administrated by subcutaneous injection. After 24 h or 48 h mice were
sacrificed and the bacterial load in the spleen, kidney, liver, lung and blood was determinated. Data are
expressed as CFU/ml of organ homogenate or blood from each animal with lines drawn at the median
for each group. The dotted horizontal line represents the detection limit. There were no statistically
significant differences between groups (Mann-Whitney test).
Activation of coagulation and fibrinolysis
To determine whether human TAFI impacts on local or systemic activation of coagulation
and/or fibrinolysis we determined levels of TATc and D-dimer after 24 and 48 h S. pyogenes
AP41 infection in organs and plasma.
At 24 h TATc levels were significantly elevated in spleen and lung of TAFI-humanized
transgenic (Fig. 2A, D), however after 48 h, TATc levels were not different between mouse
strains. In addition, TATc concentrations in kidney, liver, and plasma were not different
between the groups at both time points (Fig. 2B, C, E). To investigate if the introduction of
human TAFI influenced the fibrinolytic activity, we measured D-dimer (Fig. 3). D-dimer
concentrations were significantly increased in liver of TAFI-humanized transgenic mice after
24 h compared to wild-type and tended to be elevated (but not significant) after 48 h S.
pyogenes infection (Fig. 3C). D-dimer levels in spleen, kidney, and lung were not different
between the groups at both time points (Fig. 3A, B, D). Plasma D-dimer decreased after 48 h
but levels between strains remained similar at both time points (Fig. 3E).
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Figure 2. Activation of coagulationq. Levels of TATc in spleen (A), kidney (B), liver (C), lung (D) and
plasma (E) at 24 and 48 hours after inoculation of S. pyogenes in wild-type mice (black bars) and
humanized-TAFI mice (white bars). Data are expressed as means ± SE of eight mice per time point
(except at 48 h for all organs where n=7 and at 48 h for blood where n=6 in the wild-type group or n=4
in the humanized-TAFI transgenic group). Differences between groups were calculated using Mann-
Whitney U test. p values of humanized-TAFI mice vs wild-type mice of the same time point.
Local and systemic inflammation
Levels of various cytokines and chemokines were analyzed in organ homogenates and plasma
from TAFI-humanized transgenic and wild-type mice after 24 and 48 h infection. Levels of
interferon-γ, TNF-α, IL-6, IL-12, IL-10 and MCP-1 in organs and in plasma were below
detection in this model (data not shown). Histological examination of the organs showed no
differences between groups at both time points (data not shown).
Survival
To determine if introduction of human TAFI impacts on mortality during S. pyogenes infection
we performed a survival study. Despite the absence of clear differences in bacterial loads,
coagulation, fibrinolysis and inflammation, remarkably, introduction of human TAFI resulted
in significantly increased mortality in response to S. pyogenes compared to wild-type mice
(Fig. 4; p = 0.006). The mortality rates of the 5-day observation period were 20% for wild-type
and 65% for humanized-TAFI transgenic mice
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.
Figure 3. Activation of fibrinolysis. Levels of D-dimer in spleen (A), kidney (B), liver (C), lung (D) and
plasma (E) at 24 and 48 hours after inoculation of S. pyogenes in wild-type mice (black bars) and
humanized-TAFI mice (white bars). Data are expressed as means ± SE of eight mice per time point
(except at 48 h for all organs where n=7 and at 48 h for blood where n=6 in the wild-type group or n=4
in the humanized-TAFI transgenic group). Differences between groups were calculated using Mann-
Whitney U test. p values of humanized-TAFI mice vs wild-type mice of the same time point.
Discussion
In vitro experiments revealed that TAFI binds to S. pyogenes via SclA and SclB and that it can
be converted into its active fragment by recruitment of the natural activators, plasmin and
thrombin [11]. Analysis of the in vivo role of TAFI in S. pyogenes infection is complicated by
the fact that S. pyogenes is a human pathogen, limiting the use of animal models. Although ex
Figure 3. Survival rates. Wild-type (●) and
humanized-TAFI mice (○) were infected with 1
or 3 x108 CFU/ml of S. pyogenes AP41
subcutaneously. Data were pooled from two
independent experiments, each of which was
conducted with 10 mice per group. Survival
data are presented as a Kaplan-Meier plot.
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vivo [21] and primate models [22] are available, both have limitations and the mouse remains
the system of choice.
Fibrinogen is the major clotting protein of human plasma that is converted to fibrin by
thrombin cleavage upon vascular injury. Fibrin(ogen) plays multiple roles in the GAS/host
interaction. During infection, the host generates fibrin at the local site of infection that can be
used to wall off the site of infection and limit pathogen invasion and spread. However,
bacteria within a fibrin network could be protected from the host defense machinery. To be
able to circumvent the thrombotic host defense, S. pyogenes expresses a number of
molecules [23-27] which confer the bacterium ability to dissolve formed fibrin clots to
facilitate bacterial spread. Thus, at different stages of the infectious process, S. pyogenes may
recruit either thrombotic or thrombolytic factors to meet the demand for bacterial survival
and proliferation.
In this study, we examined the contribution of human TAFI to virulence in a murine model of
S. pyogenes infection. Using S. pyogenes M41 serotype strain at a dose of 1 to 3 x108 CFU/ml,
we observed 20% mortality in wild-type mice after 5 days. However, introduction of human
TAFI markedly increased mortality to 65%. Our results suggest that S. pyogenes may gain
additional advantages by binding and activating TAFI. For instance, S. pyogenes can use TAFI
to prevent fibrinolysis of the surrounding fibrin network, which may protect against
phagocytosis. Furthermore, activated TAFI is able to inactivate inflammatory peptides such as
complement factors C3a and C5a [28], which should lead to an impaired chemotactic activity
of these peptides.
Altered haemostasis and massive inflammation are common features in severe S. pyogenes
infection [29]. Our results demonstrated local activation of coagulation and fibrinolysis as
shown by increased TATc in spleen and lung and elevated d-dimer in liver after 24 h infection
in humanized-TAFI transgenic mice compared to wild-type mice. However, we were unable to
demonstrate an altered local or systemic inflammatory response in humanized-TAFI mice, as
evidenced by unaltered cytokine levels after 24 and 48 h S. pyogenes infection. In addition,
after 24 and 48 h S. pyogenes infection bacterial loads were not consistently altered. By the
design of our study we can not underline the mechanism(s) responsible for the increased
susceptibility of humanized-TAFI mice. It seems likely that acceleration of the disease
progression may have occurred later than for instance 48 h after S. pyogenes infection.
S. pyogenes has a repertoire of pathogenic mechanisms that assure its success as colonizing
and invading microorganism. This paper reports the first application of humanized-TAFI mice
in a model of infection. Although our data can not establish the etiology of the infection, it is
striking that mice expressing human TAFI are more susceptible to S. pyogenes infection. More
research is warranted to investigate the mechanisms by which TAFI contributes to S.
pyogenes infection.
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8 Kantor FS. Fibrinogen precipitation by streptococcal M protein. I. Identity of the reactants, and
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9 Lahteenmaki K, Kuusela P, Korhonen TK. Bacterial plasminogen activators and receptors. FEMS
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11 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable
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12 Marx PF, Dawson PE, Bouma BN, Meijers JC. Plasmin-mediated activation and inactivation of
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16 Bengtson SH, Sanden C, Morgelin M, Marx PF, Olin AI, Leeb-Lundberg LM, Meijers JC, Herwald H.
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17 te Velde EA, Wagenaar GT, Reijerkerk A, Roose-Girma M, Borel Rinkes IH, Voest EE, Bouma BN,
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20 Nagashima M, Yin ZF, Zhao L, White K, Zhu Y, Lasky N, et al. Thrombin-activatable fibrinolysis
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Murine TAFI improves survival against Streptococcus pyogenes Mercedes Valls Serón, Stefan R. Havik, Joris J.T.H. Roelofs, Charlotte Spaendonk, Heiko Herwald, Philip G. de Groot and Joost C.M. Meijers
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Abstract
Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized in the liver and secreted into
the bloodstream in a zymogen form. TAFI is activated by thrombin, either free or in complex
with thrombomodulin. Active TAFI (TAFIa) is involved in down regulation of plasmin formation
and plays a role in inflammatory processes by inactivation of inflammatory mediators. We
investigated the role of TAFI in the haemostatic and immune response during infection of
mice with Streptococcus pyogenes. Wild-type and TAFI-knockout mice were infected using a
subcutaneous air-pouch model with S. pyogenes. Mice were terminated after 24 or 48 hours
or observed in a 5-day survival study. Our results showed that absence of TAFI did not have a
consistent effect on bacterial dissemination into systemic organs in the first 48 hours. In
addition, wild-type and TAFI-knockout presented no differences in activation of coagulation
or fibrinolysis, as reflected by comparable plasma levels of thrombin-antithrombin complexes
and D-dimers. TAFI-deficiency had no effect on lung, liver, spleen or kidney histopathology, or
cytokine levels in plasma. Remarkably, TAFI-knockout mice had increased susceptibility to
infection, with a 35% survival of TAFI-knockout compared to 87% of wild-type mice.
Immunohistochemistry revealed that both wild-type and TAFI-knockout mice accumulated
megakaryocytes in spleen during the 5-day infection. Megakaryocyte levels were significantly
lower in TAFI-knockout compared to wild-type mice, which was associated with mortality.
These data suggest that murine TAFI is necessary for survival after S. pyogenes infection and
that TAFI plays a role in megakaryopoiesis in spleen. In addition, megakaryocytes in spleen
may contribute to host defense towards bacteria.
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Introduction
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 56-kDa metallo-carboxypeptidase that is
predominantly synthesized in the liver and circulates in plasma as a zymogen. Upon activation
during coagulation by thrombin or the complex of thrombin with thrombomodulin, TAFI
becomes active (TAFIa) and modulates fibrinolysis by cleaving off the C-terminal lysine
residues from partially degraded fibrin. Plasminogen and tissue tissue-type plasminogen
activator (t-PA) bind to those lysine residues [1] and therefore removal of the lysines
attenuates the fibrin cofactor function of t-PA-mediated plasminogen activation, resulting in
less plasmin formation and reduced fibrinolysis [2]. TAFI can also be activated by plasmin,
trypsin, neutrophil elastase and meizothrombin [3-6].
TAFI activation occurs after proteolytic cleavage of the Arg92
-Ala93
bond. TAFIa is
characterized by a temperature-dependent instability, showing a spontaneous decay with a
half-life less than 10 minutes at 37°C or several hours at 22°C [7]. TAFIa’s self-destruction
mechanism was elucidated by the crystal structure [8,9], in which it was shown that a highly
dynamic structure is responsible for its own regulation.
Besides fibrin, a number of other TAFIa substrates have been described. TAFIa inactivates the
pro-inflammatory mediators bradykinin (BK), complement factors C3a and C5a, thrombin-
cleaved osteopontin and fibrinopeptide B [10-13].
TAFIa can therefore have two different effects during infection: regulating plasmin
generation, and inactivating inflammatory mediators. This dual function makes TAFI an
interesting protein to study in relation to infection.
Streptococcus pyogenes is a common bacterial human pathogen that causes a variety of
diseases affecting the skin or the upper respiratory tract [14,14]. These pathological
conditions are relatively mild such as impetigo and pharyngitis [14,15]. However, S. pyogenes
can cause invasive diseases such as toxic shock syndrome and necrotizing fasciitis which over
the last twenty years have been reported to increase world-wide [16]. Serious sequeale
following S. pyogenes infections are rheumatic fever and glomerulonephritis.
Although several studies have investigated the role of TAFI after LPS challenge [17,18] or
during sepsis induced by Gram-negative intact bacteria [19], the in vivo role of TAFI after
Gram-positive bacterial infection has not been investigated. In the present study, we analyzed
the role of TAFI during infection caused by S. pyogenes in TAFI-deficient mice.
Materials and Methods
Animals
C57BL/6 mice (wild-type) were supplied by Harlan (Venray, The Netherlands). TAFI-KO mice
(backcrossed at least 10 times to a C57BL/6 background) [20-22] were bred in the animal
facility at the Academic Medical Center, The Netherlands. Animals were kept on a controlled
12 h light/dark cycle and food and water were provided ad libitum. Experiments were carried
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with 8-12 weeks old male mice and all procedures were approved by the Institutional Animal
Care and Use Committee of the Academic Medical Center.
Experimental model of S. pyogenes infection
The S. pyogenes strain AP 41 (M41 type) was obtained from the Institute of Hygiene and
Epidemiology (Prague, Czech Republic). Bacteria were grown in Todd-Hewitt broth (BD
Bioscience, San Jose, CA, USA) at 37°C and an aliquot of these cells were added to fresh media
and grown up to the exponential phase of growth (OD620=0.5). The cells were washed three
times with saline and diluted to 0.5 to 3.7 x 108/ml in saline.
Wild-type and TAFI-KO mice were subjected to an air-pouch infection model. Briefly, 0.8 ml of
air and 0.2 ml of S. pyogenes in saline were injected with a 25-gauge needle subcutaneously
on the back of the mouse.
Mice were sacrificed 24 and 48 hours (16 per group) after infection or were observed in a
survival study for 5 days (40 per group). During the 5 days, animal survival was recorded in 40
animals per group; animals were monitored (4-6 times a day) for signs of disease such as
hunched posture, ruffed fur, and decreased responsiveness to stimuli. When severe disease
signs were observed (moribund), mice were humanely euthanized. Animals (n=20) terminated
at day 5 (survivors) or those that were terminated within the 5 day observation (non-
survivors) were included for analysis of histopathology. Eight mice injected with saline were
used as controls.
Sample harvesting
At the time of sacrifice (24 hours, 48 hours or during the 5-days), mice were first anesthetized
by inhalation of isoflurane (2L/min O2/2% volume isoflurane, Abbott Laboratories). Blood was
drawn after cardiac puncture into a sterile syringe containing citrate and immediately placed
on ice. Blood was centrifuged for 20 minutes at 2000 g. Plasma was stored at -80°C until use.
Thereafter, spleen, kidney, lung and liver were harvested and processed for measurements of
CFU, histology.
Determination of bacterial outgrowth
Spleen, kidney, lung and liver were homogenized at 4°C in 4 volumes of sterile isotonic saline
with a tissue homogenizer (Biospec Products, Bartlesville, OK, USA) which was carefully
cleaned and disinfected with 70% ethanol after each homogenization. Serial 10-fold dilutions
in sterile saline were made from these homogenates and blood. Thereafter, 50-µl volumes
were plated onto sheep-blood agar plates and incubated at 37°C. CFU were counted after
16 h.
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Assays
Interferon-γ, TNF-α, IL-6, IL-12, IL-10 and MCP-1 were measured by cytometric bead array
multiplex assay (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s
recommendations. For these cytokine measurements, harvested organs were homogenized
as described above. Sample homogenates were lysed in 1.25 volumes lysis buffer (300 mM
NaCl, 15 mM Tris [tris(hydroxymethyl) aminomethane], 2 mM MgCl2, 1% Triton X-100,
pepstatin A, leupeptin, and aprotinin (each at 20 ng/ml) (pH 7.4)) on ice for 20 min and spun
at 3600 g at 4°C for 10 min. The supernatant was frozen at -80°C until assayed. Thrombin-
antithrombin complexes (TATc; Siemens Healthcare Diagnostics, Marburg, Germany) and D-
dimer (Diagnostica Stago, Asnieres, France) were measured by ELISA.
Histopathology of mouse tissue samples
Directly after sacrifice spleen, kidney, liver and lung were fixed in 4% formalin. Tissue samples
were embedded in paraffin, sectioned (5-µm) and stained with haematoxylin and eosin (H-E)
using standard methods. Megakaryocytes were detected in spleen by immunohistochemistry
staining using an antibody against human von Willebrand factor (anti-vWF; Dakocytomation,
Glostrup, Denmark) as primary antibody. Briefly, sections were deparaffined and then
immersed in 3% hydrogen peroxide in methanol to block endogenous peroxidase for 20
minutes. Sections were then rinsed with PBS, blocked with 10% normal goat serum in PBS for
30 minutes and incubated overnight with the primary antibody in PBS.
After the overnight incubation, the sections were incubated with a secondary antibody (HRP
conjugated). Immune complexes were revealed via incubation with 3, 3’-diaminobenzidine
(DAB) and sections were counterstained with haematoxylin. Sections were examined using an
Olympus BX51 microscope and microscope fields were photographed with an Olympus DP70
camera. To quantify the number of megakaryocytes in spleen from wild-type and TAFI-KO
mice, vWF positive megakaryocytes were counted under the microscope in 10 randomly
chosen X 4 observation fields. For image analysis the freeware ImageJ v1.45a was used.
Statistical analysis
Differences between groups were calculated using the Mann-Whitney U test. Values were
expressed as means ±SE. A p value of < 0.05 was considered statistically significant. Survival
curves were analyzed by the Kaplan-Meyer log-rank test. All statistics were performed using
GraphPad Prism, version 5.01 (GraphPad Software, San Diego, CA, USA).
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Results
Bacterial outgrowth
To determine whether TAFI deficiency influences S. pyogenes dissemination, we compared
the bacterial load in spleen, kidney, lung, liver and blood of TAFI-KO and wild-type mice after
24 and 48 h induction of infection (Fig.1). After 24 and 48 h there were no differences in
bacterial loads in organs or blood between mouse strains, suggesting that at early time of
infection TAFI deficiency does not influence S. pyogenes AP41 spread.
Figure 1. Bacterial dissemination. Wild-type (●) and TAFI-KO mice (○) were injected with S. pyogenes
AP41 (1.4 to 3.7 x108/ml) administrated by subcutaneous injection. After 24 or 48 h mice were sacrificed
and the bacterial load in the spleen (A), kidney (B), liver (C), lung (D) and blood (E) was determined. Data
are expressed as CFU/ml of homogenated organ or blood from each animal with lines drawn at the
median for each group (n=16). The dotted horizontal line represents the detection limit. There were no
statistically significant differences between groups (Mann-Whitney test).
Activation of coagulation and fibrinolysis
To determine whether TAFI plays a role in the coagulant and fibrinolytic host responses, we
determined levels of TATc and D-dimer after 24 and 48 h S. pyogenes AP41 infection in
plasma. At 24 h TATc levels in TAFI-KO mice tended to be higher, although not significant,
compared to wild-type mice. After 48 h, TATc levels were not different between the mouse
strains (Fig.2A). D-dimer concentrations were increased after 24 h and 48 h compared to the
saline control, but were not different between wild-type and knockout animals (Fig. 2B).
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Fiure 2. Activation of coagulation and fibrinolysis. Levels of TATc (A) or D-dimer (B) in plasma at 24 and
48 hours after inoculation of S. pyogenes in wild-type mice (black bars) and TAFI-KO mice (white bars).
Data are expressed as means ± SE of n= 8 (saline group) or n=16 (infected group). There were no
statistically significant differences between groups (Mann-Whitney test).
Survival
To determine if TAFI has an impact on mortality during S. pyogenes infection, we performed a
survival study. The survival of the mice in the two groups over time is illustrated in Figure 3.
Despite having similar responses to infection at earlier time points (24, 48 h) as shown by
analysis of bacterial loads, coagulation, fibrinolysis and inflammation, TAFI-KO mice exhibited
significantly increased mortality towards S. pyogenes (p < 0.0001). At day 5 post-infection,
35% TAFI-KO animals had survived infection in contrast to 87% of wild-type mice.
Histopathology and inflammation
We examined the influence of TAFI deficiency on tissue damage after 24 h, 48 h and during a
5-day survival experiment where survivors and non-survivors were included for analysis.
Histopathology examination of organs from infected wild-type and TAFI-KO mice after 24 and
48 h showed no relevant inflammation (data not shown). In addition, cytokines and
Figure 3. Survival rates. Wild-type (●) and TAFI-KO
mice (○) were infected with 0.5 to 3 x108 CFU/ml
of S. pyogenes AP41 subcutaneously. Data are
pooled from three independent experiments, each
of which was conducted using 20, 10 and 10 mice
per group. Survival data are presented as a Kaplan-
Meier plot.
Chapter 6
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chemokines in plasma were measured. Levels of interferon-γ, TNF-α and IL-12 were below the
detection limit in this model, while levels of IL-6, IL-10 and MCP-1 did not differ between wild-
type and TAFI-KO mice (data not shown).
Histopathology examination of kidney, liver and lung from infected wild-type and TAFI-KO
mice during the 5-day infection showed no relevant changes compared to the saline control
group (data not shown). In contrast, as shown by haematoxylin and eosin staining of paraffin
sections, the spleens from S. pyogenes AP 41 infected wild-type and TAFI-KO mice showed
evidence of an infiltration with large, multinucleated cells (Fig. 4), that were present in
occasional clusters within the red pulpa in both groups and morphologically appeared to be
megakaryocytes.
Figure 4. Histopathology of wild-type and TAFI-KO mice. Representative H-E stained sections of spleen
tissue from S. pyogenes-infected wild-type (A, B) and TAFI-KO spleen tissue (C, D). Infected animals were
injected with 1 to 3 x108 CFU/ml and followed for 5 days as indicated in methods. Magnifications are
indicated in each panel. Data were from two independent experiments, each of which was conducted
with 10 mice per group.
Immunohistochemical staining with antibodies against von Willebrand factor confirmed these
cells to be megakaryocytes (Fig. 5). The presence of megakaryocytes in spleen was suggestive
for extramedullary hematopoiesis. Spleens from TAFI-KO mice showed decreased numbers of
megakaryocytes (Fig. 5A,B), in comparison to spleens of wild-type mice (Fig. 5C,D). In spleens
from wild-type and TAFI-KO mice injected with saline (controls), megakaryocytes were not
observed (data not shown). Quantification of the megakaryocytes in spleen (Fig. 5E)
confirmed that TAFI-KO have lower MK numbers in spleen after S. pyogenes infection,
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suggesting that TAFI could play a role in extramedullary hematopoiesis. Furthermore, non-
survivors from both infected groups had lower megakaryocyte numbers in spleen compared
to survivors (Fig. 5E), indicating that extramedullary hematopoiesis may play an important
role in survival against S. pyogenes.
Figure 5. Immunohistochemical staining of spleens of wild-type and TAFI-KO mice. Representative
immunohistochemical staining for vWF of sections from S. pyogenes-infected wild-type spleen tissue (A,
B) and TAFI-KO mice (C, D). Infected animals were injected with 1 to 3 x108 CFU/ml and followed for 5
days. Original Magnifications are indicated in each panel. Arrows indicate megakaryocytes. (E)
Quantification of vWF positive megakaryocytes in spleen from wild-type (●) and TAFI-KO (■) mice after
S. pyogenes AP41 injection. Horizontal lines in the graphs represent the medians of the groups.
Representative images of two independent experiments, each of which was conducted with 10 mice per
group. Non survivors (open symbols) are indicated for both groups.
Discussion
Upon infection, many complex processes such as inflammation and activation of both the
coagulation and the fibrinolytic systems are compromised. TAFI represents a link between
coagulation and fibrinolysis and inactivates pro-inflammatory mediators, therefore it can be
expected that TAFI is involved in the response to infection. The present study was performed
to elucidate the role of endogenous TAFI during infection of the Gram-positive bacteria, S.
pyogenes.
Creation of TAFI-KO mice has enabled the possibility of studying the relevance of endogenous
TAFI in different experimental settings. Compared to wild type animals, the TAFI-KO mice
were normal in many respects, including survival, development, and fertility. In addition,
TAFI-KO displayed no bleeding disorders but as expected prolonged clot lysis time [20,23].
Chapter 6
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Using S. pyogenes M41 serotype strain at a dose of 0.5 to 3 x108 CFU/ml, we observed 87%
survival in wild-type mice after 5 days. TAFI deficiency markedly decreased survival to 35%.
Our results suggested that the increased mortality observed in TAFI-KO might be due to
impaired regulation of the inflammatory mediators C3a and C5a leading to uncontrolled
inflammation. C5a has been suggested to play a role in the increased mortality when TAFI-KO
mice were challenged with both LPS and cobra venom factor [17]. However, we were unable
to demonstrate an altered local or systemic inflammatory response in TAFI-KO mice, as
evidenced by unchanged cytokine levels after 24 and 48 h S. pyogenes infection. In addition,
bacterial loads were similar after 24 and 48 h S. pyogenes infection between wild-type and
TAFI-KO mice. It seems likely that acceleration of the disease progression may have occurred
later than 48 h after S. pyogenes infection.
Upon infection, various hematopoietic factors promote hematopoiesis mainly in liver and
spleen (extramedullary hematopoiesis) [24]. It has been suggested that inadequate
extramedullary hematopoiesis leads to insufficient production or maturation of blood cells
[24]. Extramedullary hematopoiesis can occur as long as there are local production of
hematopoietic factors that maintain and induce differentiation of the hematopoietic stem
and progenitor cells (HSPCs). In our mouse model, megakaryocytes appearance correlated
with reduced mortality in both wild-type and TAFI-KO mice. Surprisingly, in the 20 animals in
which immunohistopathology was performed (Fig. 5), the TAFI-KO survivors only represented
25% compared to 80% in the wild-type mice, suggesting that TAFI may be involved in
extramedullary hematopoiesis leading to megakaryocyte migration and/or differentiation. To
our knowledge, a role for TAFI in these processes has never been described. A potential target
for TAFI in this process may be stromal cell-derived factor-1α (SDF-1α). SDF-1α also known as
C-X-C type chemokine ligand 12 (Cxcl12) is produced by various cell types including bone
marrow stromal cells, inflammatory cells, endothelial cells and osteoblasts [25]. SDF-1α plays
a role in regulating the retention of progenitor cells in hematopoietic tissues [26]. It has been
suggested that perturbation of the SDF-1α chemoattractant gradient by cleavage of the C-
terminal lysine residue of SDF-1 α by carboxypeptidase M can lead to mobilization of HSPCs
from bone marrow to peripheral blood [27]. This might facilitate entrance of megakaryocyte
precursors in peripheral organs such as the liver and spleen leading to megakaryocyte
differentiation. Therefore, TAFI activation during S. pyogenes infection, additionally to other
carboxypeptidases, could allow colonization of the spleen with bone marrow-derived stromal
cells by cleaving the C-terminal lysine of SDF-1 α. Enhanced TAFI activation could be the result
of increased TAFI levels upon infection. Both, TAFI mRNA and TAFI protein have been
reported to be upregulated during Escherichia coli-induced abdominal sepsis [19]. Moreover,
a local increased level of TAFI in the bone marrow can be achieved by a pool of TAFI present
in megakaryocytes [28,29].
The spleens of survivors from TAFI-KO and wild-type mice contained higher numbers of
megakaryocytes, suggesting that they are important for host defense against S. pyogenes. A
role for toll-like receptors (TLR) in megakaryocytes has recently been demonstrated. The TLR2
mTAFI improves survival against S. pyogenes
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that recognizes the lipoteichoic acid of gram-positive bacteria has been detected in human
megakaryocytes and lately, platelets which derive from megakaryocytes were shown to bind
and internalize pathogens and release microbial proteins [30,31]. In addition, many studies
have identified TLRs on platelets [32-36]. In line with our observation, extramedullary
hematopoiesis has been reported before in direct association with infection [37,38].
Recently, we reported the binding of human TAFI to two surface proteins of S. pyogenes, and
its subsequent activation at the bacterial surface via plasmin and the thrombin-
thrombomodulin complex [39]. The TAFI region involved in binding to the streptococcal
collagen-like surface proteins A and B (SclA and SclB) is Gly205
to Asp232
which is 99%
conserved in mouse TAFI compared to human TAFI (chapter 4). In addition, mouse TAFI also
binds the S. pyogenes serotype used in this study (data not shown). Because both mouse and
human TAFI interact with S. pyogenes, it is tempting to speculate that mouse TAFI can also be
used by S. pyogenes to its own benefit. However, TAFI-KO mice but not wild-type mice were
susceptible to S. pyogenes AP41 infection, suggesting that mouse TAFI is of importance for
defense during S. pyogenes infection.
In conclusion, we have shown that TAFI-KO mice are susceptible to S. pyogenes infection.
However, the etiology of the disease could not be clarified. It seems likely that TAFI could
contribute to protection of infection by facilitating extramedullary hematopoiesis that might
be used to counteract the infection.
Chapter 6
104
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39 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable
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Summary and general discussion
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The coagulation and fibrinolytic systems are tightly regulated and protected against
dysfunction by various activators and inhibitors. During bacterial infections, both systems in
cooperation with the inflammatory system act in a very efficient way to contain and eliminate
the infection. However, bacteria have developed mechanisms during evolution to target such
systems in a specific manner to protect themselves against the host hemostatic and immune
response. In addition, bacteria are capable to benefit from components of the hemostatic
system in order to promote invasion.
In this thesis we have investigated novel interactions between TAFI and Gram-negative
bacteria, and we elaborated in molecular detail on how TAFI binds to the Gram-positive
Streptococcus pyogenes and how these interactions could influence the outcome of infection.
As an introduction to the thesis, in chapter 1 we describe how the pathogenic bacteria S.
pyogenes, Yersinia pestis and Salmonella enterica interact with the coagulation and the
fibrinolytic systems in order to confer the bacteria ability to establish major infection. Next, in
chapter 2 we extensively review one component of the fibrinolytic system, thrombin-
activatable fibrinolysis inhibitor (TAFI). The glycoprotein TAFI circulates in plasma as zymogen
and once activated acts as a carboxypeptidase B-like enzyme and cleaves C-terminal lysine
and arginine residues. Activated TAFI plays a role in down-regulating fibrinolysis but also has a
function in regulating inflammatory processes.
Interaction of TAFI with Y. pestis and S. enterica
Proteolysis plays an important role in the pathogenesis of bacterial infections. Bacterial
proteases target several host factors during infection to gain pathogenesis. Both, Y. pestis and
S. enterica express outer membrane proteases, omptins such as Pla and PgtE, on their
surface. Both bacterial species are highly invasive pathogens, and Pla and PgtE have been
identified as virulence factors [1-4]. In chapter 3 we investigated the effect of the omptins Pla
of Y. pestis and PgtE of S. enterica on TAFI.
We observed that adding recombinant E.coli expressing the omptins or live Y. pestis and S.
enterica bacteria to purified TAFI or plasma, resulted in reduced TAFI antifibrinolytic activity.
This prompted us to investigate the underlying mechanism. We showed that Pla and PgtE
degrade TAFI via proteolytic breakdown. TAFI was degraded at the C-terminal region and the
lysine analogue ε-ACA prevented cleavage suggesting that lysine residues are critical for the
cleavage of TAFI by these omptins. OmpT, an omptin of Escherichia coli, preferentially cleaves
its substrates after arginine or lysine thus it is possible that the cleavage occurs at lysine
residues.
Besides TAFI, Y. pestis and S. enterica target other components of the fibrinolytic system to
increase plasmin formation. Pla and PgtE activate plasminogen, degrade α2-antiplasmin and
inactivate PAI-1, and this may be beneficial for the bacteria to penetrate through tissues and
spread to distant organs.
Summary and discussion
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This is the first report demonstrating that bacterial proteases degrade TAFI and thus interfere
with TAFI activation. In line with our findings, it was shown recently that the protease InhA
secreted by Bacillus anthracis degraded TAFI in vitro. Injection of InhA to mice reduced TAFIa
activity in plasma [5].
Interaction of TAFI with S. pyogenes
TAFI binds to the surface of group A streptococci of an M41 serotype [6]. The interaction is
mediated by the streptococcal collagen-like surface proteins A and B (SclA and SclB), and the
streptococcal-associated TAFI can then be activated at the bacterial surface by plasmin or
thrombin-thrombomodulin. In chapter 4 we further investigated the TAFI binding
characteristics to SclA and SclB.
Using TAFI peptides, we identified the binding region involved in the interaction with SclA and
B between amino acids 205 to 232 of TAFI which are located distally from the TAFI catalytic
site. The Gly205 to Asp232 region is surface exposed and does not interfere with the region
known to influence TAFIa stability (Arg302, Arg320, Arg330, and Thr/Ile325), neither with the
residues involved in substrate binding (Gly336, Tyr341, and Glu363).
The amino acids in the Gly205 to Asp232 region responsible for the interaction were those
belonging to the so called glycosaminoglycan consensus motifs. The consensus sequence of
such a repeat is XBBXBX, where B stands for a basic residue and X is a non-basic residue.
The glycosaminoglycan consensus repeats (XBBBXXBX and XBBXBX) are found in some
proteins that bind glycosaminoglycans such as vitronectin, laminin and protein C inhibitor.
Therefore, it is reasonable to propose that S. pyogenes might target proteins with
glycosaminoglycan-binding sites as a conserved mechanism to recruit proteins for its own
benefit.
Additionally, we demonstrated that not only the TAFI zymogen bound to bacterial proteins
but also the active enzyme. Interestingly, TAFIa is easily dissociated from the bacterial
proteins. These results suggested that the conformation of the Scl-recognition domain had
been slightly changed upon TAFI activation. It is tempting to speculate that this constitutes a
mechanism whereby the bacteria attract TAFI to their surface, localize it there and
subsequently may allow activation of TAFI, whereafter the active enzyme easily dissociates.
Role of TAFI during S. pyogenes infection
In chapter 5 and chapter 6 we investigated the role of TAFI during S. pyogenes infection.
The highly host-specific nature of S. pyogenes in combination with the binding of TAFI to SclA
and SclB and subsequent activation at the bacterial surface, prompted us to investigate the
susceptibility to S. pyogenes infection of mice expressing human TAFI (chapter 5). To this end,
mice expressing only human TAFI were generated and infected with S. pyogenes serotype
M41. Introduction of human TAFI resulted in significantly increased mortality in response to S.
Chapter 7
112
pyogenes compared to wild-type mice. Our results demonstrated increased local activation of
coagulation and fibrinolysis in some organs after 24 h infection in TAFI-humanized transgenic
mice compared to wild-type mice. However, we were unable to demonstrate an altered local
or systemic inflammatory response in humanized-TAFI mice, as evidenced by unaltered
cytokine levels after 24 and 48 h S. pyogenes infection. In addition, after 24 and 48 h S.
pyogenes infection bacterial loads were not consistently altered.
Although our data cannot establish the etiology of the infection, it is striking that mice
expressing human TAFI are more susceptible to S. pyogenes infection. More research is
warranted to investigate the mechanisms by which TAFI contributes to S. pyogenes infection.
The study in chapter 6 was performed to elucidate the role of endogenous TAFI during
infection of S. pyogenes. Upon induction of infection, the mortality of TAFI deficiency
markedly increased compared to wild-type mice. However, there were no clear differences in
bacterial loads, coagulation, fibrinolysis and inflammation at early phase of infection. These
data indicate that endogenous TAFI did not modify host defense at early stage, but is of
importance for the final outcome of infection.
Interestingly, immunohistochemistry revealed that both wild-type and TAFI-knockout mice
accumulated megakaryocytes in spleen during a 5-day infection. Megakaryocyte levels were
significantly lower in TAFI-knockout compared to wild-type mice, suggesting that TAFI could
play a role in promoting hematopoiesis in spleen (extramedullary hematopoiesis).
Furthermore, non-survivors from both infected groups had lower megakaryocyte numbers in
spleen compared to survivors, indicating that megakaryocytes may play an important role in
survival against S. pyogenes.
It is unclear why we failed to observe clear differences in host defense after S. pyogenes
infection in both TAFI-humanized transgenic and TAFI-KO mice. A reason may include that
acceleration of the disease progression may have occurred later than for instance 48 h after S.
pyogenes infection.
Discussion
In this thesis we studied TAFI interactions with Gram-positive and Gram-negative bacteria.
Here we show that S. pyogenes targets TAFI through binding to glycosaminoglycan consensus
repeats. Whether this interaction is crucial for the increased susceptibility of TAFI-humanized
mice compared to wild-type mice is unclear since murine TAFI which has 85% homology with
human TAFI was found to bind S. pyogenes AP41. In addition, the glycosaminoglycan
consensus motifs involved in binding to SclA and SclB are 100% conserved in mouse TAFI
compared to human TAFI. Having established that both mouse and human TAFI interact with
S. pyogenes, it was to our surprise that TAFI-KO mice have reduced survival towards S.
pyogenes infection. An explanation could be that mouse TAFI is not as well activated as
human TAFI at the bacterial surface. This would also explain why transgenic mice expressing
both mouse and human TAFI had similar susceptibility to S. pyogenes during a 5-day survival
Summary and discussion
113
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experiment than TAFI-humanized mice (Figure 1). In this experiment we analyzed survival of
TAFI-transgenic mice expressing both mouse and human TAFI. The mortality rates of the 5-
day observation period were 5% for wild-type and 55% for TAFI transgenic mice. Therefore
we suggest that contribution of human TAFI to infection was predominant to the protective
effects of mouse TAFI (chapter 6) during S. pyogenes infection.
In chapter 5 we observed that human TAFI contributes to S. pyogenes infection. In contrast, in
chapter 6 we showed that murine TAFI protects against S. pyogenes infection. Our results
suggested that human and murine TAFI contribute differently to the infection. S. pyogenes
might gain survival advantage by interacting with human TAFI. Contrary, murine TAFI could
contribute to protection of infection by facilitating extramedullary hematopoiesis that might
be used to counteract against infection.
One of the most striking findings in Chapter 6 was that TAFI deficiency can impair survival in
mice with S. pyogenes. Recently, it was shown that TAFI-KO mice exhibited significantly
reduced survival following inoculation with the human bacterium Yersinia enterocolotica,
which is responsible of self-limiting enterocolitis but can also cause extraintestinal disorders,
including sepsis [7]. Bacterial counts in liver on days 3 and 5 were similar between wild-type
and TAFI-KO mice, but were increased on day 7 in spleen from TAFI-KO compared to wild-type
mice. In addition, levels of plasma D-dimer did not increase significantly in wild-type or TAFI-
KO mice infected with Y. enterocolotica. Interestingly, mice lacking both plasminogen
activator inhibitor-1 (PAI-1) and TAFI displayed the greatest susceptibility, followed
sequentially by PAI-1-KO and TAFI-KO mice. These findings suggested that both TAFI and PAI-1
deficiency can impair host defense against Gam-negative bacteria.
Similar to many other procarboxypeptidases, the TAFI zymogen is activated through cleavage
of an activation peptide to form activated TAFI (TAFIa), which is involved in downregulation of
plasmin formation. However, TAFIa is unique among carboxypeptidases in that it
spontaneously inactivates with a short half-life, a property that is essential for its function in
controlling blood clot lysis. The recent determination of the TAFI crystal structure revealed
Figure 1. Survival rates. Wild-type (■) and
TAFI-transgenic mice (□), expressing both
mouse and human TAFI were infected with
0.5 x108 CFU/ml of S. pyogenes AP41
subcutaneously. Survival data (n=20) are
presented as a Kaplan-Meier plot.
Chapter 7
114
the mechanism by which TAFI is able to autoregulate its activity [8,9]. The crystal structures
showed that TAFIa stability is directly related to the dynamics of a 55-residue segment
(dynamic flap). The dynamic flap is stabilized in the TAFI zymogen by interactions with the
activation peptide. Upon activation, release of the activation peptide increases dynamic flap
mobility and in time this leads to conformational changes that disrupt the catalytic site and
make TAFIa prone to inactivation and aggregation. This novel mechanism for enzyme
regulation may be an advantage to S. pyogenes which could benefit from both TAFI activity
and inactivation.
If SclA and SclB do indeed bind to and facilitate TAFI activation in vivo, this would seem to
counteract the pro-fibrinolytic effects of plasminogen activation by streptokinase. How can
these two opposite functions be reconciled? Perhaps S. pyogenes employs a strategy whereby
it initially promotes activation of the clotting system [10] to generate a protective fibrin
barrier around the infected area, and uses active TAFI to prevent fibrinolysis of the
surrounding fibrin network, thereby decreasing the chance of elimination by host
inflammatory cells at the site of infection (and possibly preventing premature dissemination
of the bacteria into the bloodstream). This period of safety from immune attack could then be
used by S. pyogenes to allow for multiplication of bacterial numbers and upregulation of
genes to overcome subsequent immune attack, followed by generation of a sufficiently high
local TAFI concentration. A similar argument could be made for Y. pestis where Pla activates
factor VII and degrades TFPI in vitro [11] leading to clot formation but on the other hand, in
this thesis we showed that Pla degrades TAFI which together with other Pla targets such as
plasminogen activation, α2-antiplasmin inactivation and PAI-1 degradation may promote
uncontrolled fibrinolysis. This scenario is supported by a recent study where B. anthracis, the
anthrax-causing pathogen, targets the host fibrinolytic system [5]. Such effects were achieved
by the bacterial proteases InhA and NprB which respectively degraded TAFI in vitro and in
mice, and activated human pro-urokinase plasminogen activator. The activation of fibrinolysis
by NprB and InhS may contribute to bleeding seen in anthrax disease. In contrast, another
study demonstrated that B. anthracis is able to initiate coagulation [12], and clustering of B.
anthacis was capable of directly activating prothrombin and factor X, thus bypassing the
initiators of the intrinsic (FXII) or extrinsic (TF-FVII) pathways. The molecular component
responsible for activating prothrombin and FX coagulation factors was InhA.
Taken all together, pathogenic bacteria may exploit pro-coagulant and pro-fibrinolytic factors
for its convenience to meet the demand for bacterial survival and proliferation. Importantly,
we underscored multiple ways that bacteria interact with the anti-fibrinolytic/anti-
inflammatory protein TAFI. However, we cannot exclude the possibility that other (unknown)
TAFI functions play a role during bacterial infection.
Summary and discussion
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Reference List
1 Degen JL, Bugge TH, Goguen JD. Fibrin and fibrinolysis in infection and host defense. J Thromb Haemost 2007; 5 Suppl 1: 24-31.
2 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007; 315: 509-13.
3 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the invasive character of plague. Science 1992; 258: 1004-7.
4 Ramu P, Lobo LA, Kukkonen M, Bjur E, Suomalainen M, Raukola H, et al. Activation of pro-matrix metalloproteinase-9 and degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium. Int J Med Microbiol 2008; 298: 263-78.
5 Chung MC, Jorgensen SC, Tonry JH, Kashanchi F, Bailey C, Popov S. Secreted Bacillus anthracis proteases target the host fibrinolytic system. FEMS Immunol Med Microbiol 2011; 62: 173-81.
6 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-like proteins A and B. J Biol Chem 2007; 282: 24873-81.
7 Luo D, Szaba FM, Kummer LW, Plow EF, Mackman N, Gailani D, et al. Protective Roles for Fibrin, Tissue Factor, Plasminogen Activator Inhibitor-1, and Thrombin Activatable Fibrinolysis Inhibitor, but Not Factor XI, during Defense against the Gram-Negative Bacterium Yersinia enterocolitica. J Immunol 2011.
8 Marx PF, Brondijk TH, Plug T, Romijn RA, Hemrika W, Meijers JC, et al. Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation. Blood 2008; 112: 2803-9.
9 Anand K, Pallares I, Valnickova Z, Christensen T, Vendrell J, Wendt KU, et al. The crystal structure of thrombin-activable fibrinolysis inhibitor (TAFI) provides the structural basis for its intrinsic activity and the short half-life of TAFIa. J Biol Chem 2008; 283: 29416-23.
10 Herwald H, Morgelin M, Dahlback B, Bjorck L. Interactions between surface proteins of Streptococcus pyogenes and coagulation factors modulate clotting of human plasma. J Thromb Haemost 2003; 1: 284-91.
11 Yun TH, Cott JE, Tapping RI, Slauch JM, Morrissey JH. Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins. Blood 2009; 113: 1139-48.
12 Kastrup CJ, Boedicker JQ, Pomerantsev AP, Moayeri M, Bian Y, Pompano RR, et al. Spatial localization of bacteria controls coagulation of human blood by 'quorum acting'. Nat Chem Biol 2008; 4: 742-50.
Nederlandse samenvatting
Acknowledgements
List of publications
Nederlandse samenvatting
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Nederlandse samenvatting Na beschadiging van de vaatwand treedt een bloeding op. Voordat het wondgenezingsproces
kan starten moet het bloeden ophouden. Het mechanisme waardoor bloedingen worden
voorkomen is de bloedstolling, en de primaire functie van het stollingsproces is om stolsels te
vormen om zodoende verder bloeden te voorkomen. Het fibrinolytische systeem kan op zijn
beurt de stolsels weer oplossen zodat wondgenezing kan plaatsvinden. Zowel het
bloedstollingssysteem als het fibrinolytische systeem (samen worden ze ook wel de
hemostase genoemd) zijn krachtige enzymatische systemen en worden gereguleerd door
verschillende activatoren en remmers.
Tijdens bacteriële infecties worden beide systemen net zoals het immuunsysteem in werking
gesteld om de infectie op een efficiënte wijze te bestrijden. Echter, de bacteriën hebben
tijdens de evolutie allerlei mechanismen ontwikkeld die aangrijpen op deze systemen om zich
zodoende te beschermen tegen de hemostatische en immuunreactie van de gastheer. Sterker
nog, de bacterien zijn in staat om gebruik te maken van verschillende componenten van het
hemostatische systeem om verdere verspreiding te vergemakkelijken.
In dit proefschrift hebben we nieuwe interacties bestudeerd tussen een component van het
fibrinolytische systeem, de zogenaamde trombine-activeerbare fibrinolyse inhibitor of TAFI,
en bacteriën. De binding van TAFI aan een specifieke bacterie, Streptococcus pyogenes, werd
gekarakteriseerd, en vooral hoe deze interactie de uitkomst van infectie door de bacterie
beinvloedt.
Het proefschrift begint met een introductie: in hoofdstuk 1 wordt de interactie beschreven
tussen de stollings- en fibrinolytische sytemen en de bacteriën S. pyogenes, Yersinia pestis en
Salmonella enterica met de nadruk op hoe de bacteriën gebruik maken van de systemen van
de gastheer om infectie te bevorderen. Vervolgens wordt in hoofdstuk 2 een overzicht
gegeven over TAFI. TAFI is een zogenaamd proenzym, wat betekent dat het circuleert in
plasma in een inactieve vorm. Voordat het zijn functie kan utivoeren moet TAFI geactiveerd
worden door enzymen uit de stolling (trombine) of fibrinolyse (plasmine). Als TAFI eenmaal
geactiveerd is (TAFIa) dan speelt het een rol om fibrinolyse te verminderen, doordat het
eindstandige lysine en arginine residuen afknipt van fibrine. TAFIa heeft ook een regulerende
functie bij ontstekingen.
Interactie van TAFI met Y. pestis en S. enterica
Proteolyse of enzymatische activiteit speelt een belangrijke rol bij bacteriële infecties. De
bacteriën maken gebruik van verschillende gastheer eiwitten om verdere verspreiding
mogelijk te maken. Beide bacterien, Y. pestis en S. enterica, expresseren eiwitten op hun
membraan die omptins worden genoemd. Y. pestis heeft het Pla eiwit en S. enterica PgtE.
Beide soorten bacteriën zijn zeer infectieus en de Pla en PgtE eiwitten zijn belangrijke
virulentiefactoren [1-4]. In hoofdstuk 3 hebben we de effecten onderzocht van de omptins
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Pla en PgtE op TAFI. Door toevoeging van recombinante E. coli bacterien die de omptins op
hun oppervlak expresseren, of door gebruik te maken van levende Y. pestis en S. enterica
bacteriën werd duidelijk dat de omptins de TAFI activiteit en antifibrinolytische functie
remden doordat ze het TAFI eiwit degraderen. Dit was de eerste beschrijving in de literatuur
dat bacteriële eiwitten TAFI kunnen degraderen en op deze wijze konden interfereren met de
activatie en activiteit van TAFI.
Interactie van TAFI met S. pyogenes
TAFI bindt aan het oppervlak van groep A streptococcen van het M41 serotype [5]. Deze
interactie wordt bewerkstelligd door de streptococcal collageen-achtige oppervlakte eiwitten
SclA en SclB. Aan het oppervlak gebonden TAFI kan geactiveerd worden door plasmine of het
trombine-trombomoduline complex. In hoofdstuk 4 hebben we de binding van TAFI aan SclA
en SclB verder gekarakteriseerd.
Met behulp van peptiden van de TAFI sequentie hebben we de bindingsplaats voor de
interactie van SclA en SclB met TAFI vastgesteld tussen de aminozuren glycine 205 en
asparaginezuur 232. Deze bindingsplaats is op enige afstand van de actieve site van TAFIa.
Deze regio zit aan het oppervlak van het molecuul en heeft geen invloed op de aminozuren
die de stabiliteit van het geactiveerde TAFI bepalen (arginine 302, arginine 330, en
threonine/isoleucine 325) noch met de residuen die betrokken zijn bij substraat binding
(glycine 336, tyrosine 341 en glutaminezuur 363).
Interessant was dat TAFI niet alleen als proenzym aan het oppervlak van bacterien kan
binden, maar ook in de geactiveerde vorm. TAFIa dissocieerde wel makkelijker van de
bacteriële eiwitten vergeleken met TAFI. Deze resultaten suggereren dat de conformatie van
het domein wat aan de bacteriële eiwitten bindt iets veranderd bij activatie van TAFI.
Hierdoor ontstaat een aantrekkelijke hypothese dat de bacteriën TAFI aantrekken, binden en
activatie mogelijk maken van TAFI. Het actieve enzyme kan echter elders zijn functie gaan
uitoefenen omdat het ook weer makkelijk van de bacteriën loslaat.
Rol van TAFI tijdens S. pyogenes infectie
In hoofdstuk 5 en hoofdstuk 6 hebben we de rol van TAFI tijdens S. pyogenes infectie
onderzocht. Deze bacterie is nogal kieskeurig voor zijn gastheer en infecteert voornamelijk
mensen. Aangezien er binding is van eiwitten van de bacterie en het humane TAFI eiwit
hebben we onderzocht of het hebben van humaan TAFI in een muis model bijdraagt aan de
gevoeligheid voor infectie met S. pyogenes (hoofdstuk 5). Hiervoor werden muizen
gegenereerd die in plaats van het normale muis TAFI uitsluitend humaan TAFI expresseren.
Na infectie met het serotype M41 van S. pyogenes werd duidelijk dat de humaan TAFI
expresserende muizen in vergelijking met normale muizen veel gevoeliger waren voor infectie
wat leidde tot een verhoogde mortaliteit. Dit ging gepaard met een verhoogde locale
Nederlandse samenvatting
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activiteit van de stollings- en fibrinolytische systemen in sommige organen na 24 uur infectie.
Echter, we waren niet in staat om een veranderde locale of systemische ontstekingsresponse
te bepalen in de humaan TAFI expresserende muizen, en de cytokine niveaus waren na 24 en
48 uur vergelijkbaar aan normale muizen. Bovendien waren er geen verschillen in aantallen
bacteriën na 24 of 48 uur infectie.
Hoewel onze data niet kunnen aantonen waarom de muizen een verhoogde mortaliteit lieten
zien, is de bevinding van een verhoogde sterfte in humaan TAFI expresserende muizen zeker
interessant. Meer onderzoek is nodig om het precieze mechanisme te ontrafelen.
De studie die in hoofdstuk 6 is beschreven werd uitgevoerd om de rol van het muis TAFI
tijdens infectie door S. pyogenes vast te stellen. Hiervoor werden normale muizen vergeleken
met muizen waarin het TAFI gen was uitgeschakeld, zogenaamde TAFI knockout muizen. Na
infectie met S. pyogenes was de mortaliteit van TAFI knockout muizen hoger dan van normale
muizen. Echter, er waren geen duidelijke verschillen in aantallen bacteriën en stollings-,
fibrinolytische of ontstekingsparameters bij de vroege fase van infectie. Hieruit bleek dat het
normale muis TAFI geen rol speelt bij de bescherming van de gastheer bij vroege infectie,
maar wel van belang is bij de uiteindelijke uitkomst van infectie.
Een interessante bevinding tijdens het onderzoek kwam bij het bestuderen van de organen
van de muizen die geïnfecteerd waren. In de milt van geïnfecteerde muizen werd een
ophoping gezien van megakaryocyten. Deze cellen zijn normaal betrokken bij de productie
van bloedplaatjes in het beenmerg. De niveaus van megakaryocyten in de milt waren
significant lager in de TAFI knockout muizen in vergelijking met normale muizen. Dit
suggereert dat TAFI een rol kan spelen bij de bevordering van hematopoiese in de milt, ook
wel extramedullaire hematopoiese genoemd. Bovendien hadden de muizen die de infectie
niet overleefden lagere aantallen megakaryocyten in de milt in vergelijking met muizen de de
infectie wel overleefden. Hieruit blijkt dat megakaryocyten een belangrijke rol kunnen spelen
bij de overleving na een infectie met S. pyogenes.
Het is nog onduidelijk waarom er geen duidelijke verschillen in ontstekingsresponse werden
waargenomen na infectie van humaan TAFI expresserende muizen en TAFI knockout muizen.
Een mogelijke verklaring is dat de verslechtering van de gezondheid van de muizen vooral
optreedt na 2 dagen, het laatste tijdstip waarop de response op infectie werd beoordeeld.
Tenslotte, werden in hoofdstuk 7 de bevindingen in dit proefschrift samengevat en
bediscussieerd.
Chapter 8
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Referenties
1 Degen JL, Bugge TH, Goguen JD. Fibrin and fibrinolysis in infection and host defense. J Thromb Haemost 2007; 5 Suppl 1: 24-31.
2 Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007; 315: 509-13.
3 Sodeinde OA, Subrahmanyam YV, Stark K, Quan T, Bao Y, Goguen JD. A surface protease and the invasive character of plague. Science 1992; 258: 1004-7.
4 Ramu P, Lobo LA, Kukkonen M, Bjur E, Suomalainen M, Raukola H, et al. Activation of pro-matrix metalloproteinase-9 and degradation of gelatin by the surface protease PgtE of Salmonella enterica serovar Typhimurium. Int J Med Microbiol 2008; 298: 263-78.
5 Pahlman LI, Marx PF, Morgelin M, Lukomski S, Meijers JC, Herwald H. Thrombin-activatable fibrinolysis inhibitor binds to Streptococcus pyogenes by interacting with collagen-like proteins A and B. J Biol Chem 2007; 282: 24873-81.
Ackowledgements
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Acknowledgements
Hereby, I would like to take the chance to thank everyone who contributed to the journey of
my PhD research. I am most lucky to have had enormous support along the way.
To my promotors: prof. dr. Joost Meijers and prof. dr. Philip de Groot. Beste Joost, thank you
for your guidance and for always being so enthusiastic and available for discussion of
(sometimes) challenging data. Beste Flip, I very much appreciate your supervision of this
thesis.
Bedank voor jullie (vele) hulp, steun bij het bedenken en opschrijven van dit proefschrift. Ik
heb veel van jullie geleerd!
My gratitude also goes to the members of the supervisory committee: Prof.dr. F. Leebeek,
Prof. dr. C. J. F van Noorden, Prof. dr. T. van der Poll, and Prof. dr. A. J. Verhoeven and in
particular to Prof. Erik Hack, for giving me the opportunity to start science in The Netherlands
(7 years ago).
To my colleagues at G1:
Thank you to Pauline who properly introduced me to the subject and has always been
available when I needed help and provided useful critique and guidance when it was most
necessary. Your depth of knowledge on both experimental and writing details is very much
appreciated.
Stefan, a big thank you for all your help but in particular for doing the animal experiments
with me. I enjoyed being “on vacation” at the AMC with you! 5 star room, delicious meals and
the latest movie at the cinema! What else did we want, ehh??
Tom, you always had an answer to ALL my questions. It was a pleasure to share our “TAFI-
issues”! Things were much easier thanks to your help. I wish you all the best!
Chantal, I enjoyed being your TAFI-PhD colleague and sharing all the conferences we attended
together!
Arnoud, I really appreciate your invaluable assistance in all laboratory related matters. Always
very enthusiastic to solve the unsolvable “challenges”!
Gwen and Çetin, we all started together and learned from each other. It was a pleasure to
share this journey with you.
A las latinas, Chiara y Joana. Muchas grácias (sobretodo) por esos momentos fuera del
laboratorio.
To Mirella, always up for a chat. Thank you for your support!
Jorge, Alinda, Han, Mauritz and Joram: thank you for all the fun in and outside the laboratory,
you created a great working atmosphere! And also to the rest of my colleagues from the EVG,
MEVG and DEVG, in particular to Wil who was always kind to help me with my experiments.
Chapter 8
124
Ad and Agnes: thank you for making the “non-lab work” easier. And in particular to Agnes at
the end, with all the forms and procedures linked to finishing my PhD, you were a great help!
I also thank the students (Joslyn Lardy, Sofie Verschueren and Charlotte van Spaendonck) for
their help.
Besides in the EVG department, my PhD has exposed me to so many other people that greatly
enriched the experience of working on my PhD thesis. Marcel Schouten, thank you for
dedicating your time to help me with setting up the animal experiments. From the
(Neuro)Pathology department: Rene Sersansie and Jasper Anik: you always found time to help
me. I learned a lot from you! Emanuele Zurolo and Anand Iyer: it was always fun to work
around you. To all the people from the University Medical Center in Utrecht: thank you for
your hospitality whenever I had to perform experiments there and for the fun times during
the conferences.
In addition, I would like to thank my collaborators and in particular to prof. dr. Heiko Herwald,
thank you for your hospitality and guidance throughout my doctoral project. Monica
Heidenholm: always so kind to help me. And I also would like to thank prof. dr. Artur
Schmidtchen and Martina Kalle for their hospitality and help. It was fun to work with you
Martina!
To Regina, Alida, Seema, Wing, Melissa and Ya-hui, thank you for being patient with my
schedules and adjusting always your times for our gezellige dinners. Thank you for
understanding!
To my paranimfs Stefi and Marta, thank you for being with me during these years but
specially for being with me during my thesis defense. Your help is very much appreciated!
A mis amigos Toni, Claudia, Douwe, Noelia, Olivier, Marta, Santi, Ester (y Sabina!), Rubén y
Yolanda MUCHAS GRÁCIAS por las cenas, los domingos “a la española” y por esas risas y
buenos momentos que me han hecho desconectar y empezar los lunes con más ganas de
trabajar! Vane, Marta y Noemi: muchas grácias por estar SIEMPRE a mi lado.
A mis herman@s, cuñad@s, cognati, suocero, mi schoonmoeder, tíos y primos, especialmente
a aquellos que han podido venir a mi presentación de tesis: muchas grácias por compartir este
día conmigo!
A mis padres, grácias por apoyarme siempre en mis decisions. Grácias a vosotros siempre he
tenido la voluntad de iniciar proyectos nuevos.
Y sobretodo muchas grácias a tí, Orlando, a pesar de no estar familiarizado con los términos
científicos siempre me has entendido y apoyado. Me allegro de tenerte día tras día a mi lado!
List of publications
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List of publications
Valls Serón M, Plug T, Marquart JA, Marx PF, Herwald H, de Groot PG, Meijers JCM.
Binding characteristics of Thrombin-Activatable Fibrinolysis Inhibitor to streptococcal
surface collagen-like proteins A and B. Thromb Haemost. 2011. Sep 27;106(4):609-16
Valls Serón M, Haiko J, de Groot PG, Korhonen TK, Meijers JC. Thrombin-activatable
fibrinolysis inhibitor is degraded by Salmonella enterica and Yersinia pestis. J Thromb
Haemost. 2010 Oct; 8 (10): 2232-40
Marx PF, Verkleij CJ, Valls Serón M, Meijers JMC. Recent developments in thrombin-
activatable fibrinolysis inhibitor research. Mini Rev Med Chem. 2009 Sep; 9 (10): 1165-73
Padilla ND, van Vliet AK, Schoots IG, Valls Serón M, Maas MA, Peltenburg EE, de Vries A,
Niessen HW, Hack CE, van Gulik TM. C-reactive protein and natural IgM antibodies are
activators of complement in a rat model of intestinal ischemia and reperfusion. Surgery.
2007 Nov; 142 (5): 722-33