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A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e , Vol. 1, No. 2Copyright © 1971, Institute for Clinical Science
Theories o f Blood Coagulation Properties and Interactions of Blood Clotting Factors
Introduction
Two main schools of thought dominate the scene of blood coagulation and hemostasis today. Both have their roots in the classic theory of blood coagulation (figure 1) described by Paul Morawitz in 1904.20 In the primitive physiological and biochemical background of that period the formation of a blood clot was envisioned correctly as the result of the conversion of prothrombin to thrombin by the action of tissue thromboplastin, followed by the transformation of a soluble fibrinogen to an insoluble fibrin clot by the action of thrombin, an enzyme derived from an inert precursor, prothrombin. The reaction required calcium ions.
From this simple beginning many elaborate schemes of the mechanism of blood coagulation subsequently were proposed* as newer knowledge of the existence of several other clotting factors evolved starting with the discovery of proaccelerin (factor V ) by Owren.22 After a long period of confusion in regard to both the identity and nomenclature of these clotting activities, some semblance of order was achieved when the International Committee on Nomenclature defined factors and assigned Roman numerals for greater ease of positive identification.45'48 Eventually the many
* Earlier schema of coagulation theory proposed by several investigators may be found in Altman and Dittmer.1
LOUIS A. KAZAL, Ph .D.
Cardeza Foundation for Hematological Research Jefferson M edical College of the
Thomas Jefferson University Philadelphia, PA 19107
clotting factors involved in coagulation began to fall into a more or less definite reaction pattern that permitted some explanation of experimental and clinical observations. By 1964 two theories or hypotheses evolved that are destined to play important roles in shaping the thinking of coagula- tionists in the 1970’s. These are:
1. The Sequential Factor Theory, as set forth by Davie and Ratnoff3 in the United States and by M acFarlane12’13 in Great Britain.
2. The Autocatalytic Prothrombin Derivative Theory of Seegers.30
It remained for the sequential theory to present the first coordinated concept of clotting applicable to the large body of chemical and clinical knowledge about blood coagulation mechanism and disorders. The autocatalytic theory, on the other hand, stresses a challenging complexity at the molecular level for the conversion of prothrombin to thrombin; it places coagulation mechanism in a perspective which has not been acceptable to all coagulation- ists. Conversely, the sequential theory also does not have universal acceptance, although it seems that a majority of coagu- lationists are its proponents. An excellent analysis of current thinking on the subject is presented by Kline.10
Other theories have been proposed which warrant considered attention, for example the Prothrombinogen Theory of Quick25-26
Presented a t the A pplied Seminar on Chem ical H em atology, Novem ber, 1970.
139
140
C L A S S I C T H E O R Y O F B L O O D C O A G U L A T I O N
( P . M o r a w i t z , 1 905 )
KAZAL
T h r o m b o p l a s t i n
+
Ca + +
P r o t h r o m b i n T h r o m b i n
T h r o m b i nF i b r i n o g e n ------------------------------------------------------ ► F i b r i n
F ig u r e 1. Classic theory o f blood coagulation proposed by Paul Morawitz in 1905.
and the more recent inhibitor hypothesis of Mann;16 both approach coagulation problems in an interesting and different manner.
Since the Sequential Factor Theory and the Autocatalytic Prothrombin Derivative Theory are the subject of greater controversy, the main objective of this discussion will be the presentation of the salient and contrasting features of these theories.*
The Nomenclature of Blood Coagulation Factors
The assigned factor numbers, symbols and common names employed in the two theories are presented in table I. The basic difference in the two theories resides in the definition of prothrombin. The sequential theory regards prothrombin as a single precursor substance, a molecule free of factors
°It is not intended in this review to discuss the role of platelets, of fibrinolysis, and of inhibitors, a l l of which obviously influence coagulation mechanism as it relates to hemostasis.
IX, (plasma thromboplastin component),X (Stuart or Stuart-Prower factor) and VII (proconvertin). The derivative theory contends that these activities are hidden in a complex prothrombin molecule as sub-units or autoprothrombins. From this prothrombin complex autoprothrombin I ( factorV II), II, (factor IX ), III (factor X) and C (activated factor X) are derived. All other factors are identical for the two theories; all are proteins except factor IV ( C a + + ) and factor III (tissue factor).
The Sequential Factor Theory
Proposed by British and American co- agulationists, the two clotting mechanisms depicted in figure 2 appeared independently within a month of each other in 1964. The exact sequence of reactions shown is not accepted today; however, the chart serves to illustrate the simplicity of the proposed mechanism.
Davie and Ratnoff3 called their scheme a Waterfall Sequence; M acFarlane12 labeled
THEORIES OF BLOOD COAGULATION 141
Table I
N O M E N C L A T U R E O F C O A G U L A T I O N F A C T O R S
Factor N o . Symbol C o m m o n N a m e *
Sequential Theory Derivative Theory
1 Fibrinogen
11 P ro th ro m b in P ro th ro m b in Complex
111 Tis sue Factor
I V C a + + Io nic C alcium
V A c G Proaccelerin Labile Factor
Accelerator Globulin
V I I S P C A Pro co nve rtin Stable Factor
A u to p ro th ro m b in 1
V I I I A H FA H G
A n tih e m o p h ilic F . A n tih e m o p h ilic
Glo bu lin
Platelet Cofactor
I X P T C PlasmaThromboplastin C om ponent
C h ris tm a s F .
Au to p ro th r o m b in 11
X S F S t u a r t F . Prower F .
A u to p ro th ro m b in 111 ( F . X ) C ( F , Xa )
X I P T A PlasmaThromboplastinAn tecedent
X I I H F Hageman F .
X I I I F S F Fib rin Stabilizing F . Fibrinase
° For a complete listing of common names of the earlier discovered, multi-named factors of the sequential theory, see W right.45
his an Enzyme Cascade. Both schemes proposed the same hypothesis, namely, that the formation of fibrin was the result of a series of individual enzymatic reactions in which each factor became activated to an enzyme,
in succession, only to activate the next inert factor to an active enzyme, and so on, until prothrombin is eventually converted to thrombin and fibrinogen to fibrin. Each inert factor (proenzyme) alternately be-
142 KAZAL
S E Q U E N T I A L F A C T O R T H E O R Y
I N T R I N S I C B L O O D C O A G U L A T I O N M E C H A N I S M S 1 9 6 4
W A T E R F A L L S E O U E N C E 1 Davi e a n d Ra tno f f
H ageman F . < X I I ) A c t . Hag ema n F .
1 E N Z Y M E C A S C A D E 1 M a c F a r l a n e
S u r f a c e C o nt a ct
I ’ 'X U — Ï — X 1 1 a
P T A ( X I ) X I a
C a + +C h r i s t m a s F . ( I X ) A c t . C h r i s t m a s F .
C a ++ phosphol ipi d
A n t i h e m o p h i l i c F . ( V I I I ) A c t . A n t i h e m o p hi li c F .
S t u a r t F . ( X ) . A c t . S t u a r t F .
‘ V i l l a
X a
p ho sphol ipi d
P ro a cc e le r in ( V ) A c t . Pr oa c ce le r in V a ?
P r o t h r o m b i n { II ) T h r o m b i n 1 1 .------------ ►
F ib r i n o g e n F i b r i n I
11 a (Th rombi n )
Il a ( F i t > - ¡ n )
F ig u r e 2. Early versions of the sequential factor theory, reconstructed from the Waterfall Sequence Theory by Davie and Ratnoff (1 9 6 4 ) and the Enzyme Cascade Theory by MacFarlane (1964) (w ith the kind permission of the authors).
comes substrate and then enzyme, except for factor XII (Hageman factor) and fibrinogen. Contact with a foreign surface initiates the activation of Hageman factor and subsequent activation occurs in the following order: factors XI, IX, VIII, X, V, and prothrombin. Ionic calcium is not required for the activation of factor XII andXI but it is required in subsequent reactions. Phospholipid, derived from platelets, is necessary for the activation of factorVIII and factor V.
An important aspect of these suggested mechanisms is the concept of biochemical amplification by which the activation of a few molecules of factor XII can lead to the production of increasingly larger amounts of activators through a succession of enzymatic stages, each contributing in larger measure to the ultimate production of a physiologically adequate amount of thrombin for hemostasis.
Intrinsic and Extrinsic CoagulationThe Cascade and Waterfall mechanisms
account only for intrinsic coagulation. It has long been recognized, of course, that the hemostatic mechanism includes more than one route to fibrin formation. Any mechanistic scheme must account for coagulation within the vascular system as well as when the vascular system is injured and exposed to extravascular components; hence, the concept of intrinsic and extrinsic coagulation.
Out of the massive effort of coagulation- minded investigators of all disciplines, e.g., 5000 publications appeared between 1958 and 1964,10 and the efforts of a smaller number of those interested in mechanism, an integrated concept of intrinsic and extrinsic clotting emerged which is shown in figure 3 in a skeletonized form. Intrinsic clotting occurs when contact activated factor XII initiates the factors of the sequence,
THEORIES O F BLOOD COAGULATION 143
GE NE RAL I ZE D SEQUENT I AL FACTOR ME CHANI S M
I n t r i n s i c ------------«- -<--------------E x t r i n s i c----)
SurfaceContact
PLATELET F. 3
•PLASMA FACTORS-
XI
XI
7 . i x ~
F. V I I I F. V I I
F. V— F. 1 V (Ca + + ) —
F. V
F. X F. X
TISSUE ' F. I l l
PROTHROMBI N CONVERTER
PROTHROMBI NASE
PROTHROMBI N V ■THROMBINCa + +
Fl BR I NOGEN■ • F I B R I N
F. X I I I FSF ; F I B R I N A S E
F ig u r e 3 . Generalized sequential factor mechanism for intrinsic and extrinsicblood coagulation.
shown on the left, which eventually produces a prothrombin converter, also called pro- thrombinase and believed to be a complex containing activated factor X. The conversion of prothrombin to thrombin and the transformation of fibrinogen to fibrin follows. Fibrin formation is further controlled by factor XIII, (fibrin stabilizing factor), a thrombin activated enzyme that imparts tensile strength and insolubility to the hemostatic fibrin clot.11
Extrinsic coagulation, which is characteristic of the rapid clotting observed in the prothrombin time determination, requires fewer factors, is initiated by the admixture of blood with a tissue factor, factorIII, and requires the plasmatic factor VII (Proconvertin or Stable Factor).21,43 Factors III, VII, V and X produce activated factor X, the remaining sequence being similar to the intrinsic clotting mechanism.13
Biochemical Properties of Coagulation Factors
A review of biochemical properties of coagulation factors is outside the scope of this presentation. A previous seminar reviews earlier work in this area;9 a more recent one updates this subject.5
Since the intrinsic sequential theory is based on the enzymatic or non-enzymatic nature of certain blood clotting factors, these properties are pertinent to this review. Table II summarizes available information along with information on plasma concentration levels and molecular weights. The factors are arranged in order of their intrinsic sequence, excluding VII and III.
Molecular weights of clotting factors in the non-activated state cover a wide range of values, the prothrombin complex factors VII, X and II having relatively low molec
144 K A Z A L
Table II
P R O P E R T I E S OF B L O O D C O A G U L A T I O N F A C T O R S
Factor Common Name Mol. Wt. Plasma Cone,
mgf 100ml
E n z y m a t i c P r o p e r t i e s of A c t i v a t e d F . M echanism*
Kinetic Esterase 1 n h ' b i t i o n(Clotting) D F P S B T I L B T I Heparin;
X I I Hageman F . 1 0 0 , 0 0 0 1 . 2 + + ? + - + ■ E o r C
X I P T A 2 0 0 , 0 0 0 - + + + - E
IX P T C 1 1 0 , ooo - + - - - + E
V I I I A H F 4 0 0 , 0 0 0 0 . 0 1 2 - - + ! C
V A c G 2 9 0 , 0 0 0 0 . 7 - - C
X Stuart F . 8 6 , 0 0 0 1 . 2 + + + + + E
I I Prothrombin 6 9 , 0 0 0 1 2 . 5 + + + - + + E
1 Fibrinogen 3 4 0 , 0 0 0 4 0 0
X I I I F S F 3 5 0 , 0 0 0 0 . 7 + ,E
V I I Proconvertin 3 5 , 00 0 3 . 0 -
I I I T is sue F. none +? E
* E = Enzymatic, C - Complex formation
ular weights. Of interest is the progressive increase in concentration that parallels the activation sequence. This supports for intrinsic clotting the concept of biochemical amplification, since the plasma seems to provide increasingly greater amounts of most substrates as the terminal stages of clotting are approached.12 Thus, the explosive production of adequate amounts of thrombin from the activation of minute amount of factor XII appears to be an inherent physiologic design of hemostasis.
Evidence for the enzymatic nature of the clotting reactions derives from kinetic studies of clotting using purified factors, from demonstration of esterolytic activity against synthetic substituted amino acid esters such as TAMe and from inhibition by such agents as D FP—diisopropyl fluoro- phosphate, SBTI—soybean trypsin inhibitor, and LBTI—lima bean trypsin inhibitor; these properties also are summarized in table II along with observations on the effect of heparin. The heparin column illustrates where this anticoagulant exerts its action against the clotting activity of
factors. Observations on esterase activity and on inhibition by trypsin inhibitors support the proteolytic nature of activated factors. Activated prothrombin (throm bin) also has peptidase activity. Many investigations have contributed to the basic knowledge of this aspect of coagulation, references to which may be found in Williams.42
It has not been possible to clearly demonstrate the activation of factors VIII and V to an enzymic state. Evidence from gel filtration studies, however, indicates that these factors form complexes which have enzymatic properties. Factors VIII, factor IXa, phospholipid and calcium ions form such a complex;2’8 the lipid for this reaction is supplied by platelets. Factor V also forms a coagulant active complex with factor Xa, phospholipid and calcium; this complex is believed to be prothrombin convertor or prothrombinase.23 Possible complex formation between factors XIa and XII has been suggested.7
The enzymatic nature of extrinsic factors (factors III and V II) are not as clearly de
THEORIES O F BLOOD COAGULATION 145
In the intrinsic system exposure of blood to a “foreign” surface produces activation of Hageman factor (factor X II), followed by activation of plasma thromboplastin antecedent (factor XI) and plasma thromboplastin component or Christmas Factor (factor IX). Activated factor IX then complexes with AHF (factor V III) in the presence of ionic calcium, most likely on the surface of negatively charged phospholipids, such as phosphatidyl-ethanola- mine,-serine, and -choline, or mixtures of these, which are derived from platelets as platelet factor 3. I t is this complex containing the active factor IX enzyme that converts Stuart Factor to factor Xa. Thrombin at low concentrations is. known to increase the reactivity of factor VIII in this complex; at high concentrations, it destroys it. Its activating role in physiologic clotting is not clear, however.
F ig u r e 4 . Sequential factor mechanism of blood coagulation reactions leading to fibrin formation.
lineated b u t there is some evidence in its favor inasmuch as the protein moiety of thromboplastic lipoprotein can activate factor X enzymatically in the presence of factor VII.44
Sequential Mechanism of Blood Coagulation
An acceptable scheme of reactions for the sequential factor theory, which is consistent with present clinical and biochemical evidence, is shown in figure 4. The reactions in this coagulation scheme are arranged to illustrate intrinsic clotting from the left and extrinsic from the right. In the center are those reactions that are common to both systems. The enzymatic activation of a factor is indicated by the lower case letter, a; complex formation, by the areas boxed with dash lines.
146 KAZAL
In the extrinsic system, factor Xa is formed by the action of a complex of factor III and factor V II which appears to have enzymatic properties. Russell’s viper venom, which also has enzymatic properties of its own, functions in the same manner as the factor III + VII complex. I t has been valuable in exploring the activation of Stuart Factor, since it is capable of substituting for the action of factor III and factor VII.
W hen clotting reactions have been started in either the intrinsic or the extrinsic system, they converge unto a common pathway through the activation of Stuart Factor. Once factor Xa is formed, it complexes w ith factor V (proaccelerin or AcG-accelerator globulin). This combination also occurs on the surface of phospholipid; the phospholipid is derived from either the platelets or factor III (tissue factor), depending on the system that is functioning. Actually, factor Xa alone will convert prothombin to thrombin slowly, as is evident from the work of Milstone19 with thrombokinase and Seegers36 with autoprothrombin C, both of these agents
being identical to factor Xa, bu t the presence of factor V and calcium greatly accelerates the conversion of prothrombin. Gel filtration studies provide the evidence for complex formation. Thrombin can increase the reactivity of factor V or destroy it a t low and high concentrations respectively, and again one can only speculate as to physiologic significance. Perhaps this is part of the autocatalytic nature of the clotting mechanism. Various reviews of coagulation mechanism have been published.4-5'10,13,18,32,42
In the final stages thrombin first transforms fibrinogen to a loose fibrin polymer, fibrin-S, which is soluble in 5 M urea, and other agents. This intermediate stage is short-lived in the presence of thrombin activated F.S.F. (factor X III) which produces an insoluble hemostatic fibrin, fibrin-I.11
The Autocatalytic Prothrombin Derivative Theory
The Autocatalytic Prothrombin Derivative Theory35-36 has its origin in observations, made as early as 1949, that purified
A U T O C A T A L Y T I C A C T I V A T I O N O F P R O T H R O M B I N I N C I T R A T E S O L U T I O N
S o y b e a n T r y p s i n I n h i b i t o r 3 , 4 , 4 ' T r i a m i n Ö d i p h e n y l s u l f o n e
2 5 % C i t r a t eP r o t h r o m b i n T h r o m b i n
A u t o p r o t h r o m b i n CF ig u r e 5. Basic reaction in the conversion of prothrombin to thrombin in citrate
solution (Seegers 1969).
THEORIES O F BLOOD COAGULATION 147
bovine prothrombin slowly generated the enzyme, thrombin, in a 25 percent solution of sodium citrate (figure 5). I t was demonstrated that thrombin itself accelerated the conversion of prothrombin, accounting for the autocatalysis that is characteristic of blood clotting. Autoprothrombin C, an enzyme derived from prothrombin, however, was the substance primarily responsible for the conversion of prothrombin. Both enzymes were inhibited by soybean trypsin inhibitor (SBTI) and by 3, 4, 41 triamino- diphenylsulfone.
The observation that a physicochemically well-characterized protein, like prothrombin, can yield one or more subunit protein components, i.e. derivatives, with enzymatic activities has been well documented.34 Consequently, prothrombin occupies a central position in the analysis of the blood coagulation mechanism that is quite different from that of the sequential factor scheme. It is defined as a molecular complex responsible for the production of the many observed autoprothrombin derivatives.
The purified bovine prothrombin34 is a
homogeneous protein according to many physicochemical criteria bu t it is electro- phoretically and chromatographically heterogeneous. The subunits obtained differ in N-terminal amino acids, molecular weights, amino acid composition, and other physicochemical properties. They originate from the prothrombin complex by biological activation with different clotting factors, alone or in combination or by biochemical manipulation, for example by chromatography. Each derivative behaves differently in its capacity to generate thrombin in the two- stage assay procedure in the presence of certain clotting factors.
The central substance, prothrombin, is prepared by procedures involving adsorption from plasma on inorganic precipitates, salt precipitation, isoelectric precipitation at pH 4.6, and ion exchange chromatography. It is equally well-characterized by physicochemical procedures.34 Some of the properties of the prothrombin complex and the auto-derivatives are presented.34
According to the Derivative Theory35 whose principal reactions are shown in
S E E G E R S A U T O P R O T H R OMB I N D E R I V A T I V E ME C HA N I S M
F ig u r e 6 . The autoprothrombin theory o f blood coagulation. The prefix “auto,” suggested by Kline (1 9 6 5 ), represents a convenient abbreviated terminology for the term, auto- prothrombin. (Modified from Seegers, 1969).
148 KAZAL
figure 6, the chemistry of blood coagulation follows three basic reactions:37
1. The formation of autoprothrombin C2. The formation of thrombin3. The formation of fibrin
The one precursor molecule, a prothrombin complex, can be made to generate several auto-derivatives and thrombin; two of these, auto C and thrombin are key enzymes in the theory. All other factor-activities become accessories for the conversion of the prothrombin complex to thrombin. The transformation of fibrinogen to fibrin follows standard concepts.
The principal autoprothrombins derived from the prothrombin complex are:
1. Auto III, having the properties of factor X, without enzymatic activity
2. Prethrombin, a larger subunit also without enzymatic activity
3. Auto II, capable of correcting factor IX deficient plasma
4. Auto Ic, probably having factor VII activity
5. Derivatives with capacity to inhibit the conversion and
6. Auto C, now recognized as activated factor X or the thrombokinase of Mil- stone. It is the primary enzyme.
The physiologic splitting of the prothrombin complex into auto III and prethrombin unfortunately is not so well defined but the existence of the two components in the test tube is clear. Auto III is the subunit that responds to the action of accessory factors as follows:
1. An approximation of the extrinsic system is seen in the effect of tissue factorIII, cothromboplastin (factor V II) and calcium ions which transform auto III to auto C. Peptides are released in this re action.
2. An intrinsic clotting type of activation is proposed for the conversion of auto III
to auto G by platelet cofactor (factorV III), platelet factor 3, and calcium ions.
3. Auto C itself will catalyze this conversion; it provides with thrombin the autocatalysis that is characteristic of blood coagulation.
4. Not indicated in the figure is effect of 25 percent citrate solution which releases auto III from the complex leaving the prethrombin subunit available for further reaction, and which also converts auto III to auto C.
Autoprothrombin C, like factor Xa or thrombokinase19 will convert prothrombin to thrombin at a slow rate. Auto C also slowly activates prethrombin; however, AcG (factor V ), platelet factor 3, and calcium ions accelerate the reaction maximally.
This scheme of reactions represents the core of the Derivative Theory. A more elaborate total scheme has been published embodying concepts of platelet alterations, fibrinolysis and inhibitor functions.35-38
The Expanded Classical Theory of Quick
A third theory of blood coagulation, presented by Quick,27' 30 is also based on the classical concepts of Morawitz.20 Quick’s theory places greater emphasis on physiologic and clinical observations for an explanation of the interaction of clotting factors. The current mechanism (figure 7) suggested by Quick25 invokes a four-step reaction sequence which accounts not only for all recognized clotting factors b u t in addition postulates the existence of pro- thrombinogen, a precursor of prothrombin, and of erythrocytin, a phospholipid derived from platelets by the action of a contact factor. Erythrocytin, produced in step 1, is necessary for the formation of a plasma thromboplastin derived from the interaction of factor VIII and factor IX in step 2. In step 3 the interaction of thromboplastin with calcium, factor V, factor VII and
THEORIES OF BLOOD COAGULATION 149
Step 1Activated Contact Factor -)- Platelets- ►Erythrocytin
Factor IXStep 2
Factor VIII Activated Factor IX + Erythrocytin------ > Plasma Thromboplastin
Step 3 Calcium Factor VFactor VII -)- Plasma Thromboplastin—Factor X Prothrombin
» Thrombin
Prothrombinogen
Step 4Fibrinogen -)- Thrombin- > Fibrin
F ig u r e 7 . Expanded classical coagulation theory of Quick (M odified from Quick, 1 9 6 6 ) .
factor X converts prothrombin to thrombin, which transforms fibrinogen to fibrin in step 4. The first-formed thrombin catalyzes the activation of contact factor to speed-up the reactions, bu t initially contact factor is activated by any foreign surface, e.g., glass. Autocatalysis by thrombin is controlled by its adsorption to fibrin.
According to Quick prothrombin exists in two forms: 1) an inactive prothrombinogen which may be a combination of active prothrombin and an inhibitor and 2) an active prothrombin that participates in clotting reaction. A substance in plasma, the prothrombin time-fixing agent (PTFA ),29 maintains an active level of prothrombin; a hereditary deficiency of this factor has been observed.31 The one-stage prothrombin time measures only active prothrombin; the two-stage method measures both inactive and active prothrombin. The two forms of prothrombins have been observed in human bu t not in rabbit or dog bloods. The hum an infant has all of its prothrombin in the active-form while in the adult
about 75 percent is in the inactive or prothrombinogen form.28
The most potent form of erythrocytin is found in red blood cells: this erythrocytin acts directly on step 2 and does not require activation by contact factor, as does the platelet precursor type of erythrocytin. The release of erythrocytin as a result of red cell lysis may be an important factor in disseminated intravascular coagulation.
Another aspect of this theory indicates that tissue thromboplastin which is not present in blood acts directly on step 3 of the mechanism, by-passing the action of erythrocytin and plasma thromboplastin formation and accounting for the rapid clotting observed with the one-stage prothrombin time test.
An analysis of the three-clotting theories described thus far has been presented.25
The Inhibitor Theory of Mann
Mann15-16 recently proposed a simplified concept of coagulation. His hypothesis emphasizes the controlling role of inhibi
150 KAZAL
tors, stresses the importance of lipid throm- boplastic surface, and attempts to simplify the clotting process by visualizing a local concentration of clotting factors on a lipid surface as reacting components for the conversion of prothrombin to thrombin. The clotting factors are grouped into two reacting components. One is the hemophilic factor (A H F) whose coagulation active state, represented by “a”, is repressed by combination with an inhibitor (i-AHFa); the other is the vitamin K dependent group of factors (K D F) whose coagulation active state, “a”, also is inhibited by bound inhibitor (i-K DFa). Inhibitor is defined as any substance(s) which prevents the active molecule from functioning. Calcium is an essential factor. The lipid surface is provided by lipid thromboplastic substances (phospholipid, lipoprotein) derived from disintegrating platelets or from tissue thromboplastin, which are not “available” in normal circulating blood.
When blood clots, the reactions involve 1) the adsorption of AHF, i-AHFa, KDF, i-KDF, and Ca to the lipid surface receptor sites provided by the platelets or tissue thromboplastin, whichever becomes involved in the coagulation process, and 2) a
concomitant shift of inhibitor to species of AHF or KDF clotting factors which do not carry bound inhibitor and which, therefore, are free to accept the inhibitor molecule. The inhibitor-shift releases active forms of AHF (A H Fa) and KDF (K D Fa) on the surface of the lipid on which prothrombin now is converted to thrombin in the presence of calcium ions.
The diagram in Figure 8 attempts to portray the dynamic aspect of activation. Figure 9 reproduces the original diagram of Mann10 which depicts the active factors in the normal state and the various combinations of inhibitor and clotting factors which result in factor deficiency states.
Discussion
The derivative theory is notably at odds with the sequential factor theory, not so much in the clotting factors that each requires for an explanation of blood coagulation, bu t primarily in the nature of the plasmatic existence of certain clotting factors in blood. In the beginning the two theories had little in common from the viewpoint of mechanism, and in principle, remain so today; however, in recent times it has become obvious that certain aspects
PLASMA INACTIVE STATE ACTIVE STATE
AHF AHF-------
i-AHFa i-A H Fa—
KDF Ca++----------->-
i-KDFa i-K D Fa— ■
KDF-------
Prothrom bin
F i g u r e 8 .
INHIBITOR
SH IFTS
l ip i ds u r fa c e
P R 0 T H R 0 M B IN
TTHROMBIN
l-A H F-4
AHFa—
Ca'H—
KDFa—
l - K D F -
H p ids u r fa c e
Conversion of prothrombin according to concepts based on the inhibitor theory of Mann (1 9 7 0 ).
THEORIES OF BLOOD COAGULATION 151
of each theory meet on common ground in so far as some of the prothrombin factors in one are recognized as the same factors in the other (table I) . Furthermore, both theories recognize the following as individual plasmatic clotting factors: Ca++, factor VIII, factor V, fibrinogen and factor XIII. Each holds to the essentiality of platelet factor 3 and tissue factor III. Evidence, pro and con, is strong for both theories and it is difficult to choose camps on biochemical grounds. Most clinical observations have been explained by sequential theory, but their interpretation by derivative theory is just as plausible.
The major difference between the two theories really resides in those clotting
activities whose synthesis is controlled by vitamin K, i.e., factors VII, IX, X, and prothrombin. The Sequential Theory regards these as individual entities and the prothrombin as a single molecular precursor of thrombin. The inference is made that the “prothrombin complex” may be contaminated with other factors; however, it is claimed by proponents of Derivative Theory that such activities are not detectable in the complex bu t only in the activated prothrombin subunits of the molecule. The Derivative Theory holds that these factor activities are part of a larger parent molecular complex whose subunits are held together by unidentified bonds;39 furthermore, this complex is believed to
Normal
Factor JX deficiency
Normal Inhibitor shift and
formation of active complex
FactorSIII deficiency
Factor ¥ deficiency Factor SE deficiency
FactorXdeficiency i at unusual site, blocks reactivity to tissue as well as lipid thromboplastin
a = a c tiv e s ite of A H F o r K D F , so m etim es lack in g in otherw ise sim ilar m olecu les ,
i = inh ib itorIn h ib itor can sh ift to recep tor s ite n o t b ea ring i. R ecep tor sites m a y va ry .In an y of th e ab n orm alities illu stra ted , th e a c tiv e com plex A I I F a K D F a c a n n o t form . W h ile o n ly 4 m olecu les are illu stra ted there w ou ld a c tu a lly be m a n y m o lecu les per particle of throm b op lastin , so w ith m ix ed p o p u la tio n s of a n y tw o ty p es, recep tor site s for i w ou ld bo availab le so th a t A H F a K D F a cou ld form .
(1970) (w ithF i g u r e 9. Inhibitor Theory of Mann. Reproduced from Mann kind permission of the author).
the
152 KAZAL
exist in plasma as a single complex precursor with hidden active sites that are released when accessory factors activate it and release coagulant active subunits. Inherited coagulopathies in the group of prothrombin factors are viewed by sequential theorists as structural or chemical abnormalities imposed on individual molecules while in the derivative theory they are considered as abnormalities at the active site level for one or more of the inherent biological activities of the single molecule.
Several non-chromatographed preparations of prothrombin have shown multiple factor activities.17’41 A highly purified prothrombin complex possessing multi-factor activity has been obtained as a crystalline barium glycoprotein from bovine plasma.40 Factors VII, IX and X activities were present in the complex to the extent of 20 percent of the total protein; interestingly, factorIX activity could not be separated from factor II activity by chromatographic procedures and changes in molecular weight of the factors VII and X were believed to take place during activation. The observations indicate that the prothrombin complex is a t least a family of very closely related proteins which undergo proenzyme- enzyme transformation and which have similar molecular properties bu t separate coagulation activities. The physicochemical properties of the barium-free complex were very similar to those of Seegers’ prothrombin. This does not necessarily equate the “family of prothrombin proteins”40 with the “parent prothrombin molecule”;39 however, it does bring coagulationists closer to a clearer understanding of prothrombin on the molecular level, providing some insight on how such differing original concepts in the two theories could arise.
To continue the comparison of the two theories, in the Sequential Theory coagulation begins with the activation of Hageman factor (factor X II); in contrast, the Deriva
tive Theory views factor XII as a cofactor for platelet activation, and platelets as the focus for incipient clotting. I t is noteworthy that intravenously administered factor XII is reported not to produce disseminated intravascular clotting,14 that Hageman deficiency is not accompanied by serious bleeding problems,24 and that myocardial infarction and thrombotic events have occurred in Hageman deficiency.6,33 Such observations cast doubt upon the role of factor XII in initiating blood coagulation and indicate that as much remains unknown and unexplained in this regard in the Sequential Theory as in the Derivative Theory.
Both theories have been critically analyzed10’ 25 and the analysis need not be extended further here.
The Prothrombinogen Theory of blood coagulation proposed25 is based essentially on a sequential factor hypothesis. I t incorporates some not-to-be-ignored observations especially at the clinical level. Chief among these is the explanation of the observation that the one-stage normal prothrombin time of 12 seconds is the same in newborn and adult plasma while the concentrations of total prothrombin in newborn plasma is considerably less than that of the adult. Closely modelled to Morawitz’s theory,20 Quick’s investigations disclosed the existence of stable and labile factors in plasma28 which were subsequently related to factor VII and V, respectively. The concept of a prothrombinogen was presented to explain the observations in the newborn.
The inhibitor hypothesis of Mann attempts to simplify coagulation by reducing the number of reactants. It envisions only two reactants, one related to factor VIII and the other to the vitamin K dependent factors and factor V. This obviously would simplify the interpretation of kinetics of coagulation; however, the predication of inhibitor-bound and inhibitor-free species
THEORIES OF BLOOD COAGULATION 153
of the same molecules, and the shift of inhibitor from one molecule to another is largely a hypothetical mechanism, although the requirement for a lipid surface in coagulation is clearly well-documented and actually is part of the Sequential Theory. The Inhibitor Theory seeks to explain rapid reaction mechanism, congenital defects, and several physiologic observations r slating to in vivo activity of clotting factors. The literature abounds in observations on blood coagulation not in harmony with or seemingly having no bearing to existing theories and Mann s hypothesis attempts to correlate these observations into a working hypothesis.16
It is obvious that there are many unsettled problems in the field of blood coagulation mechanism and perhaps it will take many years of research before a single theory will emerge. A better understanding of the nature of the existence of clotting factors in their unactivated state in plasma is essential, and this is a problem beset by the difficulties of being able to recognize clotting factors only in their activated state and mainly through the measurement of a clotting time. Until such knowledge is enlarged and until all clotting factors, existing and predicated, are obtained in an absolute state of purity, it will be difficult to unravel the present confusion in regard to mechanism.
References1. A l t m a n , P. L. a n d D i t t m e r , D . S.: Blood
and Other Body Fluids, pp. 231-236, Federation of American Society of Experimental Biology, W ashington DC, 1961.
2. B a r t o n , P. G.: Sequence theories of blood coagulation re-evaluated w ith reference to lipid protein interactions. Nature 215: 1508-9,1967.
3. D a v ie , E. W . a n d R a t n o f f , O. D .: Waterfall sequence for intrinsic blood clotting. Science 1 4 5 :1310-12,1964.
4. E s n o u f , M . P.: Biochemical aspects o f blood coagulation. Proc. Roy. Soc. (B iol.) 173: 269-75, 1969.
5. E s n o u f , M . P. a n d M a c F a r l a n e , R. G.:
Enzymology and the blood clotting m echanism. Advances Enzym. 3 0 :255-315, 1968.
6. G l u e c k , H. I., R o e h l l , W ., J r .: Myocardial infarction in a patient w ith a Hageman (factor X II) defect. Ann. Int. Med. 64: 390-6, 1966.
7. H a n n e n , C., M o r s e l t , G ., S c h o e n m a k e r s , J.: Contact activation of Hageman factor and the interaction of Hageman factor and plasma thrombo-plastin antecedent. Thrombos. Diathes. Haemorrh. 1 7 :307-20, 1967.
8. H o u g ie , C., D e n s o n , K. W. E., a n d B ig g s , R.: A study of the reaction product of factor VIII and factor IX by gel filtration. Thrombos. Diathes. Haemorrh. 1 8 :211-22, 1967.
9. K a z a l , L. A.: Coagulation Proteins. Serum Proteins and the Dysproteinemias, pp. 261- 288, F . W . Sunderman and F. W . Sunderman, Jr., eds., Lippincott, Philadelphia, 1964.
10. K l i n e , D . L.: Blood coagulation: Reactions leading to prothrombin activation. Ann. Rev. Physiol. 27: 285-306, 1965.
11. L o r a n d , L.: Physiologic crosslinking of fibrin. Thrombos. Diathes. Haemorrh. Suppl. 34: 75-102, 1970.
12. M c F a r l a n e , R. G .: An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier. Nature 202: 498- 99, 1964.
13. Ibid., The blood clotting mechanism. The development of a theory of blood coagulation. Proc. Roy Soc. Biol. 1 7 3 :261-8, 1969.
14. M a m m e n , E. F. a n d G r a m m e n s , G . L.: The purification and some properties o f bovine Hageman factor. Thrombos. Diathes. Haemorrh. 18: 306-7, 1967.
15. M a n n , F. D .: A simpler view of the coagulation of blood. Am er. J. Clin. Path. 37: 263-7,1962.
16. Ibid., Simplification of the concept o f coagulation by revival of the inhibitor theory. Thrombos. Diathes. Haemorrh. 23: 12-18,1970.
17. M a r c in ia k , E. a n d Se e g e r s , W . H.: Prethrombin as a new sub-unit of prothrombin. Nature 209: 621-622, 1966.
18. M il s t o n e , J. H.: Thrombokinase as prime activator of prothrombin: Historical perspectives and present status. Fed. Proc. 23: 742- 48, 1964.
19. M i l s t o n e , J. H., O l i a n o f f , N., a n d M i l - s t o n e , V. K.: Activities associated with thrombokinase derived from bovine plasma. Proc. Soc. Exptl. Biol. Med. 1 1 9 :804-7,1965.
20. M o r a w it z , P.: D ie Chemie der Blutgerin- nung. Ergebn. Physiol. 4 : 307-422, 1904.
21. N e m e r s o n , Y.: The reaction betw een bovine brain tissue factor and factors VII and X. Biochemistry 5: 601-8, 1966.
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22. O w b e n , P. A.: The coagulation of blood: Investigations on a new clotting factor. Acta Med. Scand. Suppl. 194: 1-327, 1947.
23. P a p a h a d j o p o u l o s , D . a n d H a n a h a n , D . J.: Observations on the interactions o f phospholipids and certain clotting factors in prothrombin activator formation. Biochim. Biophys. Acta 90: 436-9, 1964.
24. P r e n t i c e , C. R . M., a n d R a t n o f f , O. D.: Genetic disorders o f blood coagulation. Seminars Hemat. 4: 93-125, 1967.
25. Q u i c k , A. J.: Current blood clotting schemes. Thrombos. Diathes. Haemorrh. 16: 318-330, 1966.
26. Ibid., Hemorrhagic Diseases and Thrombosis, 2nd ed., Lea & Febiger. Philadelphia,1966.
27. Ibid., Influence of erythrocytes on the coagulation of blood. Amer. J. Med. Sci. 239: 51-60, 1960.
28. Ibid., On the constitution of prothrombin. Amer. J. Physiol. 140: 212-20, 1943.
29. Ibid., The determinant of the prothrombin time in normal human plasma. Thrombos. Diathes. Haemorrh. 2 : 226-35, 1958.
30. Ibid., The diagnosis of common hereditary hemorrhagic diseases. Ann. Intern. Med. 55: 201-9, 1961.
31. Q u i c k , A. J. a n d H u s s e y , C. V.: Hereditary thrombopathic thrombocytopenia. Amer. J. Med. Sci. 245: 643, 1963.
32. R a t n o f f , O. D.: The biology and pathology of the initial stages o f blood coagulation. Progr. Hemat. 5: 204-245, 1966.
33. R a t n o f f , O. D ., B u s s e , J. R . , a n d S h o e n , R . P.: The demise o f John Hageman. N ew Eng. J. Med. 279:760-1 , 1968.
34. S e e c e r s , W . H.: Blood Clotting Enzymology, Academic Press, N ew York, 1967.
35. Ibid., Blood clotting mechanisms: Threebasic reactions. Ann. Rev. Physiol. 31: 269-94,1969.
36. Ibid., Enzyme theory of blood clotting. Fed. Proc. 23.- 749-756, 1964.
37. S e e g e r s , W . H., M c C o y , L., a n d M a r c i n i a k ,E.: Blood clotting enzymology. Three basic reactions. Clin. Chem. 14: 97-115, 1968.
38. S e e g e r , W . H., M c C o y , L., M a r c i n i a k , E., a n d M u r a n o , G.: Theory of blood coagulation: Applications in disseminated intravascu- lar coagulation. Thrombos. Diathes. H aemorrh. Suppl. 36: 239-268, 1969.
39. S e e g e r s , W . H., M u r a n o , G., a n d M c C o y , L.: Structural changes in prothrombin during activation: A theory. Thrombos. Diathes. Haemorrh. 2 3 : 26-36, 1970.
40. T i s h k o f f , G. H., W i l l i a m s , L. C., a n d B r o w n , D. M.: Preparation of highly purified prothrombin complex; Crystallization: b iological activity and molecular properties. J. Biol. Chem. 243:4151-67 , 1968.
41. v o n Voss, D.: Das verhalten von prothrombin, faktor VII, IX und X bei der säulen- chromatographie an anionen—austauschem. Hoppe-Seyler Z. Physiol. Chem. 3 4 8 :1172-78,1967.
42. W i l l i a m s , W . J.: Recent concepts of the clotting mechanism. Seminars Hemat. 5: 32- 44, 1968.
43. I bid., T he activity of lung microsomes in blood coagulation. J. Biol. Chem. 239: 933-42,1966.
44. W i l l i a m s , W . J., a n d N o r r i s , D. G.: Purification of a bovine plasma protein (factor V II) which is required for the activity o f lung microsomes in blood coagulation. J. Biol. Chem. 2 4 1 :1847-56, 1966.
45. W r i g h t , I. S.: Concerning the function and nomenclature of blood clotting factors, with a preliminary report o f the profile of blood clotting factors in young males. Ann. Int. Med. 51:841-850 , 1959.
46. Ibid., The nomenclature of blood clotting factors. J.A.M.A. 180: 733-735, 1962.