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An Extensive Interaction Interface Between Thrombin andFactor V is Required for Factor V Activation*

Timothy Myles†, Thomas H. Yun, Scott W. Hall and Lawrence L. K. Leung†.

Division of Hematology , Stanford University School of Medicine, 269 Campus Drive, CCSR

(Room 1155), Stanford, CA 94305, USA.

†To whom correspondence should be addressed

Division of Hematology,

Stanford University School of Medicine,

CCSR (Rm 1155),

Stanford,

CA 94305-5156.

Phone: 1 (650) 725 40 43

Fax: 1 (650) 736 09 74

Email: lawrence.leung@stanford.edu, tmyles@stanford.edu

RUNNING TITLE Mapping the Thrombin-Factor V Interaction Interface

KEYWORDS Serine Protease, Thrombin, Factor V, Site Directed Mutagenesis

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 18, 2001 as Manuscript M011324200 by guest on M

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ABSTRACT

The interaction interface between human thrombin and human factor V (FV), necessary for

complex formation and cleavage to generate factor Va, was investigated using a site directed

mutagenesis strategy. Fifty-three recombinant thrombins, with a total of seventy-eight solvent

exposed basic and polar residues substituted with alanine, were used in a two stage clotting assay

with human FV. Seventeen mutants with less than 50% of wild-type (WT) thrombin FV

activation were identified and mapped to anion-binding exosite-I (ABE-I), anion-binding

exosite–II (ABE-II), the Leu45-Asn57 insertion loop and the Na+ binding loop of thrombin. Three

ABE-I mutants (R68A, R70A and Y71A) and the ABE-II mutant R98A had less than 30% WT

activity. The thrombin Na+ binding loop mutants, E229A and R233A, and the Leu45-Asn57

insertion loop mutant, W50A, had a major effect on FV activation with 5%, 15%, 29% of WT

activity respectively. The K52A mutant, which maps to the S´ specificity pocket, had 29% WT

activity. SDS-PAGE analysis of cleavage reactions using the thrombin ABE mutants R68A,

Y71A, and R98A, the Na+ binding loop mutant E229A and the Leu45-Asn57 insertion loop mutant

W50A, showed a requirement for both ABE’s and the Na+ bound form of thrombin for efficient

cleavage at the FV residue Arg709. Several basic residues in both ABE’s have moderate

decreases in FV activation (40-60% WT activity), indicating a role for the positive electrostatic

fields generated by both ABE’s in enhancing complex formation with complementary negative

electrostatic fields generated by FV. The data show that thrombin activation of FV requires an

extensive interaction interface with thrombin. Both ABE-I and ABE-II and the S´ subsite are

required for optimal cleavage and that the Na+ bound form of thrombin is important for its

procoagulant activity.

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INTRODUCTION

Thrombin is a serine protease that interacts with a large number of macromolecular substrates

receptors, cofactors and inhibitors that have both procoagulant and anticoagulant properties.

This functional versatility of thrombin is revealed by its structure (1). Thrombin has the

characteristic serine protease fold but differs in having a deep narrow active site cleft occluded

by the Leu45-Asn57 (60 insertion loop) and Leu144-Gly155 (149 insertion loop) surface loops.

Unique to thrombin are two cationic surface domains termed anion-binding exosite-I (ABE-I)

and –II (ABE-II) important for the binding of ligands to overcome steric hindrance of the

occluded active site cleft. ABE-I is important for the binding of fibrinogen (2-4), fibrin (5),

heparin cofactor II (6, 7), PAR1 (8, 9), thrombomodulin (2, 10-12) and hirudin (13-15). ABE-II

is important for the binding of platelet glycoprotein Ib (16), and the glycosaminoglycan bound

serpins antithrombin III (6), heparin cofactor II (6) and protease nexin I (17, 18).

Both exosites are involved in the binding of coagulation factors V and VIII which is

important for the amplification of the coagulation cascade (19). Studies with the ABE-I specific

inhibitor hirugen (19, 20) has implicated the thrombin ABE-I in cleavage of factor V while

studies using the ABE-II triple mutant thrombin RA (Arg89, Arg93, Arg98→Ala) has implicated

ABE-II (19). The specific proteolytic cleavage of human factor V (FV) by the serine protease

thrombin generates factor Va (FVa) which acts as a cofactor with factor Xa, prothrombin and

Ca2+ ions to form the prothrombinase complex on the surface of anionic phospholipids. This

leads to the amplification of the coagulation pathway at the sites of vascular injury. FV, a single

chain 330,000 kDa cofactor protein, is activated to FVa with the release of the B domain

activation products (E fragment and C1 fragment) by cleavage at the residues Arg709, Arg1018 and

Arg1545 (21). FVa is composed of the 105 kDa heavy chain (A1-A2 domain) and the 74 kDa light

chain (A3-C1-C2 domains) held together by a calcium ion (22, 23). The crystal structure of the

light chain C2 domain implies a Ca2+ independent mode of binding to the phospholipid

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membrane via immersion of hydrophobic residues into the laminar membrane core, with direct

interactions between the basic C2 domain residues and the negatively charged

phosphatidylserine head groups and generalized complementary electrostatic interactions (24).

The heavy chain interacts with prothrombin while both chains interact with factor Xa. As part of

the prothrombinase complex, factor Xa has a 300,000 fold increase in catalytic efficiency in

thrombin generation from prothrombin (25). Specific cleavage of FV occurs preferentially first

at Arg709 giving rise to the heavy chain, followed by cleavage at Arg1018 and then by the rate

limiting cleavage at Arg1545 which gives rise to the light chain. Cleavage of both Arg709 and

Arg1545 are important for full cofactor activity of factor Va and the cleavage of Arg1018 enhances

the rate of cleavage of Arg1545 (26).

In this study, we have used 53 mutant thrombins where solvent accessible polar and

charged residues were substituted with alanine in order to fully define thrombin residues

important in the recognition and cleavage of FV. This would give insights into the roles of the

anion binding exosites for specificity towards FV, and residues involved in substrate recognition

and cleavage within the active site cleft.

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EXPERIMENTAL PROCEDURES

Materials- Purified human FV and FVa were purchased from Hematologic Technologies

Inc. Factor V deficient plasma and thromboplastin were purchased from Sigma. The expression

and purification of wild-type (WT) and alanine substituted mutant thrombins from CHO cells

and the mutant R62Q from insect cells, have been described in detail previously (27, 28). The

concentration of active thrombin molecules was determined by titration with D-Phe-Pro-Arg-

chloromethyl ketone (PPACK) using the chromogenic substrate H-D-Val-Leu-Arg-p-nitroanilide

(S-2266, Chromogenix, Sweden). The catalytic activity of the purified recombinant WT and

mutant thrombins towards H-D-Phe-Pip-Arg-p-nitroanilide (S-2238, Chromogenix, Sweden,

fibrinogen, protein C and TAFI has been described (27).

Activation of Factor V by WT and Mutant Thrombins- Cleavage assays were performed

in 50 µl volumes containing 300 nM human FV and 100 pM thrombin in assay buffer (10 mM

Hepes pH 7.4, 150 mM NaCl, 5 mM CaCl2 and 0.1% PEG6000) at 37˚C for 30 minutes then

placed on ice. The concentration of FVa generated in the cleavage assay was determined by a

one stage clotting assay. Cleavage reactions were diluted 1000-fold in assay buffer on ice, 50µl

was then added to 50µl of FV deficient plasma and left at room temperature for 10 minutes. The

clotting assay was started with 100 µl of thromboplastin (containing 5 mM CaCl2, and

equilibrated at room temperature) and the clotting reaction was monitored over time at 600 nm in

a Softmax™ plate reader (Molecular Dynamics) at room temperature until maximum clotting

was achieved (no further change in absorbance at 600 nm). The t1/2 value was recorded as the

time where 50% clot formation occurred. The effective concentration of diluted human factor

Va from the cleavage reaction in the clotting assay was determined from a calibration curve

using various concentrations of purified human FVa vs. the t1/2 value for clotting. For mutants

showing decreased clotting ability, dose response curves were constructed over a range of

thrombin concentrations using the above protocol.

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SDS-PAGE Analysis of Cleavage Reactions- Cleavage reactions containing 300 nM

human FV and 0.5 nM thrombin in assay buffer were incubated from 1 to 120 minutes before

being terminated by the addition of SDS loading buffer and boiling for 5 minutes. Cleavage

products were resolved by electrophoresis on 5%-18% gradient SDS polyacrylamide gels

(Biorad) then stained with biosafe coomassie blue (Biorad). The intensity of coomassie blue

stained bands were determined by direct scanning of stained SDS-PAGE gels using the UMAX

Astra 4000U scanner and Umax VistaScan 3.5.2 software. Scanned images were saved as TIFF

files at a resolution 600 dpi. Pixel densities were calculated for each band from TIFF files

imported into Scion Image 1.62c (http://rsb.info.nih.gov/nih-image/).

Molecular Modeling of Thrombin-The x-ray crystal structure of α-thrombin

(E.C.3.4.21.5) bound with the active site inhibitor PPACK was obtained from the Brookhaven

Protein Data Bank (Brookhaven entry 1PPB). Thrombin is depicted as a space filling (CPK)

model with solvent removed using the RasMol V2.5 software package

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RESULTS

Screening for thrombin mutants defective in Human FV Activation- A site directed

mutagenesis strategy was used to determine residues on thrombin important for the interaction

between thrombin and human FV necessary for activation of FV to FVa. Fifty-three thrombin

mutants (Table 1), where solvent exposed polar and charged residues were mutated to alanine

(27), were used in a two-stage clotting assay using purified human FV to assay FV activation.

Seventeen thrombin mutants with approximately 50% or less FV activation compared to WT

thrombin were identified and mapped to ABE-I, ABE-II, the Leu45-Asn57 insertion loop and the

Na+ binding loop (Fig. 1, Table 2). ABE-I had 6 residues (K21A, K65A, H66A, R70A, R73A,

K77A) with less than 50% WT activity. Two mutant thrombins showed less than 15% WT

activity (R68A, Y71A) (Fig. 1, Table 2). The double mutant K106A/K107A showed only 24%

of the WT type activity, indicating the effects of the single mutations (K106A = 62% and K107A

= 52%) were additive for the double mutant. Interestingly, substitution of Arg62 with glutamine

gave a greater decrease in clotting activity (23% WT activity) compared to the alanine

substituted form of Arg62 (65% WT activity). ABE-II showed one mutant (R98A) with greatly

reduced FV activation (27% WT activity) while two triple mutants (with a total of 6 residues

substituted) showed less than 50% WT activation (R89A/R93A/E94A and

R245A/K248A/Q251A). Both the ABE-I and ABE-II mutants have normal kcat/Km values toward

the chromogenic substrate S-2238 suggesting the reduced activity is due to impaired binding to

the exosites rather than an effect on catalysis (Table 1). The residues Glu229, Arg233 and Asp234

form part of a surface exposed Na+ binding loop which modulates the activity of thrombin (29).

Mutation of each residue resulted in 5%, 15% and 32% of WT activity respectively.

Interestingly, the E229A mutant shows a 13-fold reduction in the kcat/Km for S-2238 (Km = 36.3

µM, kcat= 33.2 s-1) compared to WT thrombin (Km = 4.7 µM, kcat= 55.9 s-1) while the remaining

two mutants have normal catalysis towards S-2238, suggesting that substitution of Glu229 affects

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substrate binding within the active site cleft. Two residues, situated on the Leu45-Asn57 loop, also

have reduced FV activation. Trp50 forms part of a hydrophobic “YPPW lid” which restricts the

specificity of thrombin by occluding the active site, and Lys52 protrudes and contributes to the

specificity of the S´ subsite (30, 31). Alanine substitution at these residues showed FV activation

of 29%. The mutant thrombin W50A has a 5-fold reduction in kcat/Km for S-2238 reflected by a

6-fold increase in Km, while K52A has normal catalysis towards S-2238. Mutation of Trp50, like

Glu229, appears to alter the active site cleft affecting substrate binding.

Dose Response Curves for Mutants with Diminished Human FV Activation- For mutants

with a marked decreased in FV activation (less than 20% WT activity), dose response curves

were constructed since these responses in the screening assay may not be in the linear range for

cleavage (Fig. 2). The mutants R68A and R233A had 12.5% and 15.4% WT activity in the

initial screen, each requiring 6-fold more thrombin to generate the same amount of human FVa

generated by cleavage with 100 pM WT thrombin. The 6-fold more thrombin required for

cleavage by the mutants corresponded well with the initial screen of the clotting assays.

Consistent with this, the 5.2% WT activity of E229A in the initial screen also corresponded well

in requiring 12-fold more thrombin to generate the same amount of FVa obtained by 50 pM WT

thrombin. The mutant W50A required 4-fold more thrombin to generate the same amount of

FVa compared to 100 pM WT thrombin. Of all the mutants tested, Y71A appears to have had the

greatest effect on cleavage of FV. Even at a concentration of 600 pM, only 25 nmoles of FVa

were produced in 30 minutes by Y71A, which is equivalent to a 30-fold decrease in FV

activation by Y71A as compared to WT thrombin.

SDS-PAGE analysis of cleavage reactions- SDS-PAGE analysis of cleavage reactions on

FV by representative thrombin mutants of the major interaction interfaces (ABE-I, ABE-II, the

Na+ binding loop and the Leu45-Asn57 loop) was employed to assess their role in the differential

cleavage of the three thrombin cleavage sites Arg709, Arg1018 and Arg1545 (Fig. 3A and 3B).

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Cleavage of FV at Arg709 by WT thrombin occurred within 1 minute with the appearance of the

heavy chain (VaH). The 75 kDa band appearing within 2-5 minutes represents both the light

chain (VaL) and the E fragment (generated by cleavage at Arg709 and Arg1018), which are difficult

to resolve under these conditions. Within 5 minutes the majority of the 330 kDa FV was

converted into VaH and other intermediate protein species. Within 30 minutes there was almost

complete conversion to the VaL and VaH species. For the mutant thrombins W50A, R68A and

R98A, the VaH appeared to be delayed by 1-2 minutes compared to WT thrombin, with the

intact protein persisting for 10 minutes, suggesting that cleavage at Arg709 is delayed. Generation

of the VaH protein species in cleavage reactions for the mutants Y71A and E229A was further

delayed, requiring 5-10 minutes before detection of the heavy chain. The appearance of the VaH

chain by specific cleavage at Arg709 between WT and mutant thrombins were assessed by

running cleavage products from 1 minute and 5 minute reactions on the same gel. This was

performed in order to avoid making comparisons between several gels showing differing degrees

of staining. The amount of VaH generated was quantified by scanning stained gels directly and

the mean pixel density for each band was determined and the results are presented as a

percentage of the VaH band intensity generated by cleavage with WT thrombin (Fig. 4). The

effect of the Y71A and E229A substitutions on cleavage at Arg709 were most prominent with

almost undetectable levels of VaH at 1 minute compared to WT thrombin (0.2 ± 0.5 % and 2.4 ±

2.4% band intensity, respectively). After 5 minutes, the Y71A and E229A mutants generated

only 5.1 ± 1.1% and 24.9 ± 3.0 % of the VaH compared to WT thrombin. The effects of the

R68A, W50A and R98A substitutions were less severe, showing 26.0 ± 2.8%, 34.8 ± 2.1% and

43.8 ± 2.7% of the VaH band intensity respective to WT thrombin within 1 minute. Cleavage

reactions with the mutant thrombin W50A showed persistence of the E-C1-VaL protein species

over the entire time course, suggesting impairment of cleavage at Arg1018. To confirm this,

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cleavage reactions of WT and W50A were run on the same gel (Fig. 3C), which clearly shows

the persistence of the E-C1-VaL fragment over 120 minutes.

The appearance of the Va-L species for the cleavage reactions by all the mutant

thrombins was difficult to interpret since cleavages at Arg709 and Arg1018 generate the E fragment

which co-migrates with the VaL chain. Also, cleavage at Arg1018 enhances cleavage at Arg1545

(32). Hence, it is difficult to assess the effect of the substitutions on cleavage at Arg1545.

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DISCUSSION

Using a total of 53 mutant thrombins, which have a total of 78 alanine substitutions, we were

able to map the interaction interface between thrombin and FV using a two-stage clotting assay.

This revealed an extensive binding surface which utilizes ABE-I, ABE-II, the Na+ binding loop

and the Leu45-Asn57 insertion loop (Fig. 5).

The ABE-I of thrombin is a surface patch with a high density of surface exposed basic

amino acids. This region extends away from the active site cleft and is essential for the binding

of fibrinogen (2-4), heparin cofactor II (5,6), PAR1 (8,9), thrombomodulin (2, 10-12) and the

inhibitor hirudin (13-15). Binding to ABE-I is important to overcome steric hindrance to the

occluded active site. Basic residues in ABE-I have a dual role in binding its many ligands.

Studies with PAR1 (33) and hirudin (34) show that the ABE-I residues contribute to a positive

electrostatic field which extends into the solvent. This is important for enhancing the rate of

complex formation with complementary electrostatic fields generated by its ligands (electrostatic

steering). Secondly, basic residues are used for direct interactions in complex formation (ionic

tethering) although which residue involved differs between the various ligands (33, 34). The

ABE-I also has a number of hydrophobic residues important for hydrophobic interactions with

PAR1 (9), thrombomodulin (35) and hirudin (14, 15). Alanine substitution of the ABE-I

residues Arg68, His66, Arg70 and Tyr71 significantly reduced FV activation and was due to

impaired binding within ABE-I rather than a direct effect on catalysis. The mutations either

affect direct interactions with FV or indirectly by affecting local protein structure. Both Arg68

and Arg70 have been shown to form direct interactions with ligands in the crystal structure of

thrombin bound to either a peptide based on the N-terminal domain of the PAR1 receptor (Arg68)

(9) or the C-terminal tail of hirudin (Arg68 and Arg70) (14, 15). Hence, it is possible that these

residues may also be in direct interactions with FV. Likewise, the residue Tyr71 is important for

hydrophobic interactions with the C-terminal tail of hirudin (14, 15) and the thrombomodulin 4-

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5-6 EGF-like domain (35). The substitution of residue His66 has a large effect on FV activation,

however this residue does not make any direct interactions from the published crystal structures

of thrombin bound with various ligands. Further, the H66A mutation has a large effect on the

thrombomodulin dependent activation of TAFI and protein C (27) however, the crystal structure

of thrombin bound with the 4-5-6 EGF-like domains of thrombomodulin showed this residue

making no direct major interactions (35). Together with the observation of normal catalysis

towards S-2238, it would suggest the H66A substitution has a local effect on the binding in

ABE-I. Interestingly, the effect of substitution of Arg62 with glutamine is more severe than that

seen with alanine. The effect of the R62Q mutation is similar to that of the dysthrombin Quick I

(R62C), where the cysteine substitution appears to distort the 60-70 autolysis loop affecting the

binding of hirudin (36), fibrinogen (37) heparin cofactor II (38) within ABE-I. Hence, it appears

that alanine substitution does not affect loop structure to the same degree as glutamine or

cysteine substitutions. The role of ABE-I in the recognition and cleavage at the thrombin

cleavage sites within FV was difficult to determine due to the complexity of cleavage patterns on

SDS-PAGE. What is clear from cleavage studies using the ABE-I mutants of Arg68 and Tyr71 is

the importance of ABE-I in the recognition and cleavage of FV at Arg709. These studies suggest

a major interaction with a hirudin-like domain N-terminal to the Arg709 cleavage site (21) and are

consistent with equilibrium binding studies by Bock and colleagues (39).

The ABE-II, which is located on the opposite side of the molecule, is involved in the

binding of glycosaminoglycan bound serpins antithrombin III (6), protease nexin I (17, 18),

heparin cofactor II (6, 7, 28), bovine FV and recombinant human FVIII (19), as well as the

binding of prothrombin fragment F2 (40) and platelet membrane glycoprotein Ib (16). The

mutant thrombins R98A and R89/R93/E94A have an effect on the activation of FV (27-40% WT

activity), suggesting an important role for the interaction of ABE-II with human FV. The

importance of these three residues has been shown by Esmon and Lollar (19) using the triple

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thrombin mutant R89A/R93A/R98A which had greatly reduced ability to activate bovine FV.

The residues Arg89 and Arg98 lie in close proximity (4Å), while Arg93 is over 12Å away from

these residues (Fig. 5). Mutation of these residues impairs heparin binding and heparin-ATIII

accelerated inhibition of thrombin (41) and chondroitin sulphate enhanced affinity of

thrombomodulin to ABE-II (41). The R98A substitution has a major effect on cleavage of Arg709

on FV, and compares well to cleavage studies of bovine FV using the thrombin triple mutant RA

(R89A/R93A/R98A), suggesting a role of ABE-II for cleavage of human FV at Arg709 (19).

It is less clear if both ABE-I and ABE-II are important for cleavage at Arg1018 and Arg1545.

Studies by Thorelli and colleagues (26, 32) show that cleavage at Arg1018 facilitates cleavage at

Arg1545. Therefore, the late appearance of the VaL could be a direct consequence of poor

cleavage of Arg1018 delaying the appearance of the C1-VaL species and subsequent cleavage at

Arg1545 or alternatively, the mutations may directly affect recognition and cleavage at the

individual cleavage sites. The effect of the ABE-I and ABE–II mutations on cleavage of Arg1018

and Arg1545 was difficult to determine due to co-migration of the E-fragment and VaL on SDS-

PAGE.

Several residues in both ABE-I and ABE-II have reduced FV activation ranging from

52% (K107A) to 65% (R62A). However the magnitude in reduction suggests that these residues

are unlikely to participate in major interactions. Mutagenesis studies support the hypothesis that

ABE-I basic residues, not involved in direct interactions with hirudin (33) or PAR1 (34),

contribute small but collectively important contributions to the localized positive electrostatic

field generated by ABE-I. Hence, it is likely that both ABE-I and ABE-II positive electrostatic

fields are important in enhancing complex formation through the interaction of complementary

electrostatic fields with FV (42, 43). Both Arg709 and Arg1545 have sequences N-terminal to the

cleavage site with a high density of acidic residues, which if exposed to the solvent, could

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contribute to a negative electrostatic potential allowing rate enhancement by electrostatic

steering with complementary electrostatic fields of thrombin ABE-I and -II.

The residues Glu229 and Arg233 make ion pair interactions with residues Lys236 and Asp146

respectively, important for maintaining the structural integrity of the Na+ binding loop (44, 45).

Mutation of either residue leads to a conformational change of the Na+ binding loop, favoring the

anticoagulant form of thrombin (46). Consistent with this, E229A and R233A showed markedly

reduced FV activation, suggesting that efficient recognition and cleavage of FV requires the Na+

form of thrombin. The thrombin substitution W50A appears to have an effect on recognition and

cleavage at Arg709, but the persistence of the E-C1-Val species shows that recognition and

cleavage at Arg1018 is also impaired. Trp50 is spatially well separated (17Å) from the Na+ binding

site, however mutation of Trp50 may have an indirect effect on the Na+ binding loop.

Substitution of Trp50 with serine appears to affect Na+ binding and favors the anticoagulant form

of thrombin with enhanced protein C specificity (45). Likewise, it is possible that alanine

substitution of Trp50 could favor the anticoagulant form of thrombin with reduced ability to

activate FV.

Specificity of the thrombin S1´ substrate binding site is restricted to small polar P1´

residues, due in part to occlusion by the Lys52 side chain (1). Alanine substitution of this residue

resulted in a decrease in FV activation suggesting a role of this residue, and the S1´ subsite for

defining the specificity of thrombin towards FV. Alanine substitution of this residue has also

been shown to be important in defining the specificity of thrombin towards antithrombin III (31)

and fibrinogen (12, 47).

In summary, thrombin activation of human FV requires an extensive interaction interface

involving ABE-I, ABE-II, the Leu45-Asn57 loop and the Na+ binding form of thrombin. It will be

interesting to analyze and compare the structural requirements for the activation of human factor

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VIII, which is homologous to human FV, and important for the assembly of the intrinsic tenase

enzyme complex.

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FOOTNOTES

*This work was supported by National Institutes of Health Grants R01 HL57530 and the Cheong

Har Family Foundation.

1Abbreviations used: WT, wild-type; FV, factor V; ABE, anion-binding exosite; PEG,

polyethylene glycol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;

PAR 1, protease activated receptor 1; VaH, factor Va heavy chain and VaL, factor Va light

chain, serpin; serine protease inhibitor.

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FIGURES

Fig. 1: Human FV activation by thrombin mutants. The ability of purified WT and mutant

thrombins to activate human FV was determined using a two-stage clotting assay as described in

“Experimental Procedures”. The effect of alanine substitutions on FV activation is reported

relative to 100% WT activity. The error bars represent the standard deviation of at least two

separate independent experiments (carried out in duplicate).

Fig. 2: Dose dependence of thrombin cleavage of human FV. FV (390 nM) was incubated

with several concentrations of WT and mutant thrombins ranging from 50 to 600 pM at 37˚C for

30 minutes. The concentration of human FVa generated after the 30 minute cleavage reaction

was determined using a two-stage clotting assay as outlined in “Experimental Procedures”.

Panel A shows the dose dependence curves for WT thrombin (squares) and the ABE-I mutants

R68A (circles) and Y71A (triangles). Panel B sows the dose dependence curves for WT

thrombin (squares) and the Na+ binding loop mutants E229A (circles), R233A (triangles) and the

Leu45-Asn57 loop mutant W50A (diamonds).

Fig. 3: SDS-PAGE analysis of cleavage reactions for ABE-I, ABE-II, Na+ binding and

Leu45-Asn57 loop mutants. Cleavage reactions containing 390 nM human FV and 0.5 nM

thrombin were performed 37˚C at several time points ranging from 1 to 120 minutes. Cleavage

products were resolved by SDS-PAGE on a 5-18% gradient gel. Panel A shows the expected

sizes of cleavage products. Panel B shows gels for WT thrombin, W50A, R68A, Y71A, R98A

and E229A. Panel C shows time course experiments for the cleavage of WT and W50A

thrombins over 0, 1, 5, 10 and 60 minutes.

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Fig. 4: Comparison of human FV heavy chain release by WT and mutant Thrombins. The

intensity of coomassie blue stained FV heavy chain bands (VaH) generated from cleavage

reactions at 1 and 5 minutes for WT and thrombin mutants (W50A, R68A, Y71A, R98A, and

E229A) were determined by direct scanning of stained SDS-PAGE gels as described in the

“Experimental Procedures”. The band intensity for each mutant is presented on the bar graph as

the percent VaH band intensity with respect to WT thrombin.

Fig. 5: Space filling model of thrombin residues with decreased Human FV activation.

Thrombin is depicted as a space filling model using the RasMol V2.5 software package showing

residues, when substituted with alanine, with less than 50% WT human FV activation. Mutants

with ≤15% WT activity are depicted in a darker color. Residues of interest are labeled using the

single amino acid code. The top panel shows the classic front view of thrombin (31) with the

active site cleft running horizontally left to right, with the Leu45-Asn57 insertion loop on the top

of the cleft occluding the active site Ser205 (red). The active site is shown with the bound active

site inhibitor PPACK as a brown stick model. The Leu45-Asn57 insertion loop residues are

colored magenta and the Na+ binding loop residues are green. ABE-I runs to the right of the

active site cleft and ABE-II on the opposite side of the molecule. The middle and bottom panels

show the same molecule but rotated 90˚ to the left and right respectively to show ABE-I and

ABE-II. ABE-I residues are blue while ABE-II residues are depicted in yellow.

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Table IThrombin nomenclature and S-2238 amidolytic activities of purified thrombin mutants

Mutationa Chymotrypsinogennumberingb

S-2238 hydrolysiskcat/Km (s-1µM-1)

Wild-type 11.9 ± 1.3S4aA/E6aA/D8aA 1E/1C/1A 13.1 ± 1.5

K17aA/K18aA/S19a 9/10/11 10.1 ± 2.4K23aA 14A 12.0 ± 1.9R26aA 14D 14.3 ± 2.3E27aA 14E 10.7 ± 4.0

E30aA/D34aA 14H/14L 12.5 ± 0.6E3A/D6A 18/21 11.7 ± 3.0

R20A 35 11.1 ± 2.6K21A 36 13.3 ± 1.2

S22A/Q24A/E25A 37/38/39 13.9 ± 2.5S22A 37 11.7 ± 0.8Q24A 38 12.7 ± 0.1E25A 39 13.7 ± 2.7D35A 49 14.1 ± 2.2R36A 50 12.9 ± 3.2W50A 60D 2.6 ± 0.4D51A 60E 14.0 ± 1.6K52A 60F 14.5 ± 2.5

N57A/D58A 62/63 11.4 ± 3.2R62A 67 12.5 ± 2.8K65A 70 13.2 ± 2.0H66A 71 11.5 ± 2.9R68A 73 12.0 ± 2.2T69A 74 13.6 ± 0.1R70A 75 12.0 + 1.0Y71A 76 10.6 ± 2.2R73A 77A 14.0 ± 1.1N74A 78 13.5 ± 2.2K77A 81 14.4 ± 2.8

E82A/K83A 86/87 15.0 ± 2.1R89A/R93A/E94A 93/97/97A 8.9 ± 2.1

R98A 101 13.9 ± 2.7K106A/K107A 109/110 12.8 ± 0.7

K106A 109 14.2 ± 2.9K107A 110 12.6 ± 1.0D113A 116 10.3 ± 3.1Y114A 117 10.0 ± 1.7

D122A/R123A/E124 125/126/127 8.5 ± 2.7S128A/Q131A 129B/131 12.5 ± 1.8

K145A/T147A/W148 145/147/148 8.1 ± 0.5T149A/N151A 149/149B 11.0 ± 3.6

K154A 149E 11.4 ± 4.4S158A 153 10.1 ± 4.8

E169A/K174A/D175 164/169/170 8.1 ± 3.2R178A/R180A/D183 173/175/178 10.6 ± 2.9

D193A/K196A 186A/186D 10.2 ± 2.6N216A/N217A 204B/205 12.6 ± 0.8

E229A 217 0.9 ± 0.3R233A 221 8.3 ± 1.7D234A 222 7.4 ± 1.8

R245A/K248A/Q251 233/236/239 11.2 ± 1.0R245A 233 12.7 ± 4.9K248A 236 10.6 ± 3.4Q251A 239 11.4 ± 0.9

aNumbering of thrombin is based on human α−thrombin. The letter “a” denotes the A-chain, theremaining residues are the B-chain. The numbering system is compared to the crystallographicconvention with respect to bovine chymotrpsinogenb. Table 1 has been reported previously (27).

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Table IIMutant thrombins with less than 50% human factor V activation

Mutant Thrombina Location on Thrombin FV Activation

(%WT Activity)

K21A ABE-I 38.1 ± 3.5R62Q ABE-I 22.2 ± 5.0K65A ABE-I 46.2 ± 4.6H66A ABE-I 30.5 ± 0.3R68A ABE-I 12.5 ± 1.9R70A ABE-I 26.9 ± 3.6Y71A ABE-I 6.2 ± 4.1R73A ABE-I 37.8 ± 0.8

K106A/K107A ABE-I 23.8 ± 5.0R89A/R93A/E94A ABE-II 8.9 ± 2.1

R98A ABE-II 13.9 ± 2.7R245A/K248A/Q251A ABE-II 11.2 ± 1.0

W50A Leu45-Asn57 Loop 28.6 ± 9.1K52A Leu45-Asn57 Loop 28.8 ± 4.0E229A Na+ Loop 5.2 ± 5.0R233A Na+ Loop 15.4 ± 3.7D234A Na+ Loop 31.9 ± 16.4

aNumbering based on human α−thrombin.

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0 10 20 30 40 50 60 70 80 90

100

110

120

130

140

150

160

170

180

% WT Human FV Activation

S4a/E6a/D8a

K17a/K18a/S19a

K23a

R26a

E27a

E30a/D34a

E3/D6

R20

K21

S22

Q24

E25

D35

R36

W50

D51

K52

N57/D58

R62A

R62Q

K65

H66

R68

T69

R70

Y71

R73

N74

K77

E82/K83

R89/R93/E94

R98

K106

K107

K106/107

D113

Y114

D122/R123/E124

S128/Q131

K145/T147/W148

T149/N151

K154

S158

E169/K174/D175

R178/R180/D183

D193/K196

N216/N217

E229

R233

D234

R245/K248/Q251

R245

K248

Q251

Throm

bin Mutants

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0 25 50 75

100

125

150

175

200

225

250

275

HFVa (nM)/30 min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

thrombin (pM

)

A

0 25 50 75

100

125

150

175

200

225

250

275

HFVa (nM)/30 min

0

50

100

150

200

250

300

350

400

450

500

550

600

650

thrombin (pM

)

B

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W50AWT0 1 2 5 10 30 60

B

A

A1 A2 B Domain A3 C1 C2

709 1018 1545

Heavy Chain E Fragment C1 Fragment Light Chain (VaH) (VaL)

105 kDa 71 kDa 150 kDa 74 kDa

120 (mins)

R68A0 1 2 5 10 30 60 120 (mins)

HFV

VaH

R98A0 1 2 5 10 30 60 120 (mins)

E229A

Y71A

0 1 2 5 10 30 60 120 (mins)

0 1 2 5 10 30 60 120 (mins)

0 1 2 5 10 30 60 120 (mins)

E-C1VaL

VaL/E

C1VaL

CHFV

C1VaL

VaH

E-C1VaL

VaL/E

0 5 30 60 120 5 30 60 120 (mins)WT W50A

Arg Arg Arg

78 kDa120 kDa

209 kDa

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0 10 20 30 40 50 60 70 80 90

100

110

% VaH Band Intensity

WT

W50A (1 min)

W50A (5 min)

R68A (1 min)

R68A (5 min)

Y71A (1 min)

Y71A (5 min)

R98A (1 min)

R98A (5 min)

E229A (1 min)

E229A (5 min)

Throm

bin Mutants

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Timothy Myles, Thomas H. Yun, Scott W. Hall and Lawrence L.K. Leungfactor V activation

An extensive interaction interface between thrombin and factor V is required for

published online April 18, 2001J. Biol. Chem. 

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