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.

154 KAZAL

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.