COAGULATION OF HUMAN · conversion accelerator of Alexander and his co-workers (1949, 1950). Koller and his colleagues (1951, 1952) have re-peated Owren'sexperiments andhave showncon-clusively

62

THE COAGULATION OF HUMAN BLOODBy J. F. ACKROYD, M.B., M.R.C.P.

The Medical Unit, St. Mary's Hospital, London, W.2

The classical theory of blood coagulation ad-vanced by Schmidt (I892) and Morawitz (1905) isrepresented schematically in Fig. i. This theory hasstood the test of time and repeated experimentalinvestigation, and it is on this foundation that allfurther hypotheses must be built.

Conclusive proof that blood coagulation in-volved other factors in addition to those includedin the classical theory was first provided byOwren's (I944, I947) discovery of factor V,although it is clear that Nolf's (I908) work, whichhad largely been overlooked, had anticipatedOwren's discovery by many years. Owren'sfactor V, which was soon to be rediscovered in-dependently by Fantl and Nance (I946) and byWare, Guest and Seegers (I947), stimulated wide-spread interest in the factors concerned in bloodcoagulation with the result that numerous newcoagulation factors, some hypothetical and in-adequately supported by experimental evidence,have been postulated. It is the purpose of thisshort article to describe very briefly the more im-portant properties of the factors included in theclassical theory of blood coagulation; to describethe action pf those newly discovered factors whoseexistence seems adequately established; and to

ProthrombJn LlThromboplastin C

Fibr±n

FIG. i.-The classical theory of blood coagulation.

outline some of the more important observationsthat led to their discovery. Finally an attempt willbe made to show how they, and the older estab-lished factors, interact to cause coagulation of theblood. As many of the coagulation factors havebeen given different names by different workers, alist of some of the more commonly used synonymsis given in Table i.

The Factors Recognized in the ClassicalTheoryTissue ThromboplastinTissue extracts have been known for many years

to hasten the coagulation of blood or plasma. Theyact by accelerating the conversion of prothrombin

TABLE ICOMMONLY USED SYNONYMS FOR COAGULATION FACTORS

FACTOR V (Owren) FACTOR VII (Koller)Thrombogen (Nolf) Stable component (Stefanini)Labile factor (Quick) Co-factor V (Owren)Prothrombin accelerator (Fantl and Nance) Preconvertin-->convertin (Owren)Proaccelerin- >accelerin (Owren) Serum prothrombin conversion accelerator (S.P.C.A.)Plasma Ac. globulin.->Serum Ac. globulin (Seegers) (Alexander and de Vries)

Prothrombin conversion factor (Owen and Bollman)Co-thromboplastin (Mann and Hum)

ANTIHAEMOPHILIc GLOBULIN PLASMA THROMBOPLASTIN COMPONENT (P.T.C.)(Lewis, Tagnon, Davidson, Minot and Taylor) (Aggeler, White, Glendenning, Page, Leake and Bates)

Thromboplastinogen (Quick) Christmas factor (Biggs, Douglas, Macfarlane, Dacie,Thrombocytolysin (Brinkhous) Pitney, Merskey and O'Brien)Plasma thromboplastic factor (Stefanini) Factor IX (Koller)Factor VIII (Koller)

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February 1954 ACKROYD: The Coagulation of Human Blood 63

to thrombin. This activity depends to a con-siderable extent upon the concentration of calciumin the mixture (Biggs and Macfarlane, 1953a).The active principle of the extracts, thrombo-plastin, is particulate, and can be removed bycentrifugation at relatively low speeds (31,000 g.)(Chargaff et al., I944). It can be obtained fromalmost any tissue in the body, brain and lung beingthe most commonly used. Thromboplastin will ac-celerate clotting even when diluted as much asI:100,000. It seems probable, therefore, that itacts as a catalyst. Thromboplastin will not clotfibrinogen solutions or preparations of plasmafrom which the prothrombin has been removed.

Plasma ThromboplastinBlood will clot in glass vessels without admixture

with tissue extracts and must therefore, accordingto the classical theory, contain thromboplastin. Ithas generally been believed that this came fromthe platelets. These, however, contain relativelylittle, but as will be shown later, they are essentialfor normal thromboplastin formation.

Prothrombin and ThrombinThese have been prepared in a highly purified

state by Seegers, McClaughry and Fahey (I950).They are proteins. go per cent. of the pro-thrombin moves electrophoretically with a mobilityof a, globulin. The mobility of thrombin is-slightly less. Purified prothrombinwis convertedinto thrombin when dissolved in 25 per cent.sodium citrate. It therefore seem&;reasonabre toconclude that prothrombin contains all the struc-tural material needed for the formation of thrombin(Seegers et al., 1950). The action of thrombin inconverting fibrinogen to fibrin appears to beenzymatic, for thrombin will convert many timesits weight of fibrinogen. This actiorn is acceleratedby calcium (Biggs and Macfarlane, I953a), and byplatelet extracts (Ware, Fahey and Seegers, 1948).

Fibrinogen and FibrinFibrinogen is a protein with an elongated mole-

cule about 8oo A in length and 40 A in width.Fibrin appears to be formed by the polymerizationof individual fibrinogen molecules. Electronmicroscopic studies suggest that an end to endlinkage of fibrinogen molecules occurs formingfibrils which later aggregate into bundles whichconstitute the individual fibrin strands.

Recently Discovered Coagulation FactorsCoagulation Factors in Plasma and SerumFactor VThe existence of this factor was first established

by Owren's (I944, 1947) brilliant analysis of the

abnormality of blood coagulation in a patient witha hitherto undescribed haemorrhagic disease whichOwren called parahaemophilia. This patient hada history of abnormal bleeding since the age of 31years. The whole blood clotting time was pro-longed, on one occasion having been as long as 70minutes. The prothombin time by Quick's (I935)one-stage method was also prolonged.

In this test the coagulation time of oxalatedplasma is measured after the addition of optimalconcentrations of calcium chloride and brainthromboplastin. On the basis of the classicaltheory, if the plasma contains an adequate amountof fibrinogen, and calcium and thromboplastin arepresent in optimal concentrations, then the co-agulation time should be inversely proportional tothe concentration of prothrombin. The pro-longed coagulation time with this test shouldtherefore have indicated that the prothrombin con-tent of this patient's plasma was reduced. Owrenfound, however, that the prothrombin time could begreatly shortened by the addition of normal plasmafrom which the prothrombin had been removed byadsorption on to aluminium hydroxide, or bySeitz filtration. It seemed clear, therefore, thatthis patient's plasma lacked a substance which ispresent in normal plasma and which is left in theplasma after removal of the prothrombin. Owrencalled this substance factor V. He described amethod for its partial purification and showed thatit was necessary for the rapid conversion of pro-thrombin to thrombin, and that it disappearedrapidly from the blood after clotting. The pro-longed prothrombin time observed with the bloodof his patient was clearly due to the slow and in-complete formation of thrombin resulting from theabsence of factor V from the plasma.

Several other cases of factor V deficiency havenow been described (Frank, Bilhan and Ekren,1950; de Vries, Matoth and Shamir, '95';Stohlman, Harrington and Moloney, I95I; Brinkand Kingsley, I952; Cosgriff and Leifer, 1952;Owren, 1953). In some of these cases there was afamilial incidence of the disease.

Shortly after Owren had announced his dis-covery, Fantl and Nance (1946) and Ware, Guestand Seegers (1947), working independently withpurified preparations of prothrombin, describedexperiments which demonstrated the existence of afactor required for the rapid conversion of pro-thrombin to thrombin. These workers found thatalthough prothrombin prepared by salt precipita-tion rapidly formed thrombin in the presence ofcalcium and thromboplastin, yet prothrombin pre-pared by adsorption on to aluminium hydroxideand subsequent elution, was only converted tothrombin very slowly. The rate of conversioncould be accelerated by the addition of a small

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64 POSTGRADUATE MEDICAL JOURNAL February 1954

quantity of the plasma from which the pro-thrombin had been adsorbed. The factor re-sponsible was partially purified by both groups ofworkers. Fantl and Nance (I948) called the factorprothrombin accelerator, while Ware and Seegers(I948) gave it the name Ac globulin. There seemslittle doubt that these factors are identical withOwren's factor V, and Owren's term will thereforebe used hereafter. The labile factor which dis-appears from oxalated plasma on storage, andwhich was described by Quick in 1943, is probablyalso identical with factor V, although it must bestated that the clotting defect produced by storageis not a clearly defined entity, and that storagealmost certainly alters factors other than factor Vin plasma. Ware, Fahey and Seegers (1948) haveshown that platelets, as well as plasma, havefactor V activity.

Factor VIIThat there is another factor in addition to factor

V which is responsible for accelerating the con-version of prothrombin to thrombin was firstsuggested by Owen and Bollman (1948). Theywere investigating Schofield's (I924) observationthat spoiled sweet clover disease in cattle (whichis due to the presence of dicoumarol in the fodder)is improved by the injection of normal serum.This condition was believed to be due solely to areduction in plasma prothrombin which was un-likely to have been significantly increased by theadministration of serum which normally containslittle or no prothrombin. These authors thereforeinvestigated the blood of dogs fed on, or injectedintravenously with, dicoumarol. They found thatthe plasma prothrombin, as estimated by the one-stage method, was greatly reduced, but that the ad-dition of normal prothrombin free serum caused aconsiderable rise in the apparent prothrombinlevel as indicated by this test. They concludedfrom this and other experiments that normal serumcontains a factor necessary for prothrombin con-version. This factor is unlikely to have beenfactor V because factor V disappears rapidly fromserum (Owren, 1947) and also because factor V isnot significantly reduced in the plasma of patientsreceiving dicoumarol (Owren, I95ob). The factordescribed by Owen and Bollman appears, there-fore, to be a further prothrombin conversion factor.

In 1949, de Vries, Alexander and Goldsteinobserved that oxalated serum, in the presence ofcalcium and thromboplastin, accelerates the co-agulation of oxalated plasma. This occurred if theoxalated plasma was diluted with plasma whichhad been adsorbed with barium sulphate to re-move most of its prothrombin whilst leaving anexcess of factor V. They therefore considered thatthe serum contained a new coagulation factor

which they have partially purified (Alexander.Goldstein and Landwehr, 1950), and which theycall serum prothrombin conversion accelerator(S.P.C.A.). They showed that dicoumarol causesa reduction in both plasma prothrombin and theserum prothrombin conversion accelerator (Alex-ander, de Vries and Goldstein, 1949).

In 1950 Owren (195oa) showed that thrombinwas formed when human serum was added toSeitz filtered ox plasma in the presence of calciumand thromboplastin. He thought at first that theprothrombin must be in the serum but latershowed that it was in the ox plasma which had beeninadequately filtered (Owren, 195oa; 195I). Thisremaining prothrombin was not converted tothrombin by calcium, thromboplastin and factor Valone. Owren therefore concluded that the serummust contain a further factor necessary for pro-thrombin conversion, and that this factor had beenadsorbed from the plasma by Seitz filtration. Itis also adsorbed by aluminium hydroxide (Biggs,Douglas and Macfarlane, 1953a). Owren calledthis factor Convertin. It seems almost certain thatit is identical with the factor described by Owenand Bollman (1948) and the serum prothrombinconversion accelerator of Alexander and his co-workers (1949, 1950).

Koller and his colleagues (1951, 1952) have re-peated Owren's experiments and have shown con-clusively that this factor accelerates the conversionof prothrombin to thrombin. They call it factorVII, factor VI being the name given by Owr rnto a hypothetical substance derived from factor V.The importance of this factor, which will here-

after be referred to as factor VII, was finally estab-lished by Alexander and his co-workers (I95I)who described a patient with a haemorrhagic syn-drome apparently due to a congenital deficiency ofthe factor. The disease was characterized by anormal whole blood clotting time and a prolongedone-stage prothrombin time which was correctedin vitro by the addition of crudely purified factorVII and temporarily, in vivo, by the intravenousadministration of human serum. The factor Vcontent of the patient's plasma was shown to benormal. Similar cases have recently been reportedby Beaumont and Bernard (I953) and by Jiirgens(I953); and Owren (I953) has described a familyof which several members were affected by thiscondition.

Further studies have shown that deficiency ofthis factor is the main abnormality caused by tro-mexan therapy (Douglas, I953). It is also reducedin advanced liver disease and in the newborn (deNicola, 1953).

Factor XThe existence of this factor has recently been

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postulated by Koller (I953) who found that iffactor VII was prepared in a sufficiently pure stateit lost its power completely to correct the coagula-tion defect of the blood of patients receiving tro-mexan. He suggested that crude factor VII con-tained two factors: factor VII and a new factorwhich he called factor X, factors VIII and IXbeing antihaemophilic globulin and P.T.C. (Christ-mas factor) respectively. Koller found that factorX, like factor VII, is present in high concentrationin serum, and is reduced in advanced liver disease,in the newborn, and in the blood of patients re-ceiving dicoumarol and tromexan. He believesthat he has seen -a case of congenital factor Xdeficiency.

Antihaemophilic GlobulinHaemophilia is inherited as a sex-linked re-

cessive character, although sporadic cases, result-ing from spontaneous mutation, frequently occur.The blood characteristically shows a prolongedclotting time although this is not an essentialfeature of the disease. The one-stage prothrombintime is normal and there is an impaired conversionof prothrombin to thrombin during coagulation.

It has been known for many years that theclotting time could be reduced to normal by theaddition of thromboplastin or of small quantities ofnormal plasma (Weil, I906o; Addis, i9 ii;Govaerts and Gratia, I93I). These findingsstrongly suggest that haemophilia is caused bydeficient formation of thromboplastin, and thatthis results from the absence of some factor presentin normal plasma. This factor appears to beutilised during coagulation and is almost absentfrom normal serum. It is not adsorbed byaluminium hydroxide (Biggs et al., I952) or bariumsulphate (Rosenthal et al., 1953). It occurs mainlyin Cohn's plasma fraction I, which also containsthe fibrinogen (Cohn, I946). It will be referred tobelow as the antihaemophilic globulin. FactorV (Owren, 1947) and factor VII (Owren, I95oa)are present in normal concentration in haemo-philia.

Plasma Thromboplastin Component (P. T.C.) or'Christmas Factor

Pavlovsky, in 1947, observed the surprising factthat occasionally a patient is found suffering fromwhat is apparently 'classical haemophilia, whoseblood or plasma has the power to reduce theclotting time of the blood of another haemophilicpatient.Three years later Koller and his colleagues

(I950) reported the results of their investigationson a patient who seems to have been a furtherexample of the condition described by Pavlovsky.Since then further cases have been reported by

Aggeler and his co-workers (1952), Schulman andSmith (I95z), Biggs, Macfarlane and Dacie andtheir colleagues (Biggs et al., 1952), Poole (1953),Lewis and Ferguson (1953) and by Cramer,Matter and Loeliger (i953).The clinical picture in all these cases was in-

distinguishable from that of haemophilia. Thecondition is inherited as a sex-linked recessivecharacter, although sporadic cases occur. As inhaemophilia, the coagulation time is usually pro-longed. The one-stage prothrombin time isnormal, although the conversion of prothrombinto thrombin during coagulation is reduced. Thecondition is not due to, a deficiency of anti-haemophilic globulin, for the plasma of thesepatients reduces the clotting time of haemophilicplasma. The clotting time of the plasma of thesepatients is reduced by normal and by haemophilicplasma, but not by antihaemophilic globulin (Biggset al., 1952).

Investigation of the factor concerned shows that,unlike anti-haemophilic globulin, it is present inlarge amounts in normal serum. It is removedfrom normal serum or plasma by adsorption on toaluminium hydroxide, or by Seitz filtration (Biggsand Macfarlane, 1953a), or by adsorption on tobarium sulphate (Rosenthal et al., 1953). In theserespects it resembles factor VII. That it is dis-tinct from factor VII is shown by the followingobservations: (i) Its deficiency does not causeprolongation of the one-stage prothrombin time.(2) Serum from patients deficient in the factorcorrects the coagulation defect of the plasma ofpatients receiving tromexan. (3) The coagulationdefect in patients deficient in the factor is correctedby some serum samples from patients receivingtromexan (Biggs and Macfarlane, 1953a).As the one-stage prothrombin time of the plasma

of patients deficient in this factor is normal, itappears that brain thromboplastin rectifies thedefect, and therefore that this factor, like anti-haemophilic globulin, must be concerned in theformation of thromboplastin. The factor has beenpartially purified by Aggeler and his colleagues(1952), who have called it the plasma thrombo-plastin component (P.T.C.). Macfarlane andDacie and their co-workers (Biggs et al., I952)have called it the Christmas factor after the firstpatient in whom they found it to be deficient.Both terms will be used when the factor is referredto below.

Plasma Thromboplastin Antecedent (P.T.A.)Rosenthal, Dreskin and Rosenthal (I953) have

recently reported a family of which three membershad a mild haemorrhagic disease which resembledhaemophilia except for the fact that two of thoseaffected were females. The clotting time was pro-

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longed and there was an abnormal utilization ofprothrombin. The one-stage prothrombin timewas normal. The abnormality was corrected by theaddition of blood from patients with either haemo-philia or with P.T.C. deficiency (Christmasdisease). It appears therefore that this conditionmay be due to deficiency of a third factor whichthese authors have called plasma thromboplastinantecedent (P.T.A.). According to Rosenthal andhis colleagues, the three conditions can readily bedistinguished as follows: The clotting defect inhaemophilia is corrected by normal plasma ad-sorbed with barium sulphate, but not by normalserum; P.T.C. deficiency is corrected by normalserum, but not by barium sulphate adsorbedplasma; and P.T.A. deficiency is corrected by bothnormal serum and normal plasma which has beenadsorbed with barium sulphate.

Plasma ThromboplastinOne of the main problems in understanding

blood coagulation on the basis of the classicaltheory is the origin of thromboplastin. As bloodtaken by vein puncture, without contaminationwith tissue juices, clots readily when placed in aglass vessel, it is clear that blood contains all thefactors necessary for coagulation. The plateletshave usually been assumed to be the origin of thethromboplastin under these conditions, but plate-lets, in fact, contain very little (Ware, Fahey andSeegers, 1948). The addition of platelets to re-calcified oxalated plasma does not reduce itsclotting time to less than about 6o to 8o seconds(Biggs and Macfarlane, 1953a), whereas with brainthromboplastin it is easily possible to reduce theclotting time to about 12 seconds. Is this weakthromboplastic action sufficient to promote normalcoagulation or is there some other source ofthromboplastin in normal plasma?The most convincing demonstration that plasma

contains a potent thromboplastin has been pro-vided by the thromboplastin generation test ofBiggs and Douglas (I953). In this test plasma isadsorbed with aluminium hydroxide which re-moves prothrombin, factor VII and P.T.C.(Christmas factor) and, as shown by Koller (I953),probably also factor X, but leaves antihaemophilicglobulin and factor V in the plasma which, inaddition, probably contains plasma thrombo-plastin antecedent (P.T.A.), the factor recentlydescribed by Rosenthal and his colleagues (1953).The deficiencies. in the factors other than pro-thrombin, removed by adsorption with aluminiumhydroxide, are made good by the addition of serumfreed of both prothrombin and thrombin. Plate-lets are then added to the mixture. Finally calciumchloride is added and the thromboplastic activitythat develops in the mixture is estimated at

120

110

100

90

808 70 .

A 60 .

50\

V 40 '

30-

20

10

10 20 3040 50 60 70 80 90 100 110 120 140T6nbmoplestin %

FIG. 2.-Thromboplastin dilution curve. The curveshows the relation between clotting time andthromboplastin concentration.

Reproduced from Biggs and Douglas, Y7. Clin. Path., 1953, 6,23, by courtesy of the authors and publishers.

intervals by observing the coagulation time ofsamples of normal oxalated plasma to which cal-cium chloride and aliquot quantities of the above-mentioned mixture are added. As the mixturecontains no significant amount of prothrombin,the coagulation of the normal plasma will be duealmost entirely to the thromboplastin developedin the mixture, and the coagulation times will beinversely proportional to the concentration ofthromboplastin. Under these conditions thrombo-plastin forms gradually over a period of 4 to 5minutes, but when fully developed has a potencyat least as great as that of a potent brain extract.By observing the coagulation times with differentdilutions of this thromboplastin, a thromboplastindilution curve can be drawn (Fig. 2) relatingclotting time to thromboplastin concentration.The diagram (Fig. 3) shows the thromboplastinconcentrations generated in 40 different tests.After five minutes, when the thromboplastingenerated in the mixtures was maximal, the meanclotting time of normal plasma to which themixture was added was less than io seconds. Thisis as short, if not shorter, than that obtained withthe most potent tissue extracts. It is clear, there-fore, that normal plasma contains all the factorsrequired for producing a potent thromboplastin.Moreover, using purified reagents, Biggs, Douglasand Macfarlane (I953b) have shown that all thefollowing components: platelts, antihaemophilicglobulin, P.T.C. (Christmas factor), factor V andfactor VII, are required, and that if any one ofthese factors is missing, abnormal thromboplastinformation will result. The reagents used in theseinvestigations almost certainly contained the re-

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February 1954 ACKROYD: The Coagulation of Human Blood 67140.-----

120-

_100

- I0-80. Io g0E

60 I

40 I

20 -

Incubation time in minutes

FIG. 3.-The normal range of the thromboplastingeneration test. The central curve represents theaverage of 40 different tests. The shaded areashows the range of variation obtained in 38 of 40observations in which the adsorbed plasma wasobtained from different normal subjects, but serumand platelets were from one subject. The extremelimits show the range of variation when all thereagents are varied simultaneously.

Reproduced from Biggs and Douglas, J. Clin. Path., 19S3, 6,23, by courtesy of the authors and publishers.

cently described factors X and plasma thrombo-plastin antecedent (P.T.A.), and it seems probablethat these are also necessary for thromboplastinformation.

Coagulation Factors in the PlateletsThe thromboplastin generation test has shown

that platelets are necessary for the formation ofplasma thromboplastin. Three coagulation factorshave been extracted from platelets:

Platelet factor I. This was first described byWare, Fahey and Seegers in 1948. It is found,together with platelet factor II, in watery extractsof platelets. These two factors have since beenseparated by van Creveld and Paulssen (I95I).Platelet factor I has an activity resembling that offactor V of normal plasma, but unlike factor V itis particulate and can be made to sediment bycentrifuging at about 32,000 g.

Platelet factor II. This accelerates the clottingof fibrinogen by thrombin.

Platelet factor III. This is insoluble in water.When suspended in plasma it antagonizes theaction of heparin and has thromboplastic activity.This factor was first described by van Creveld andPaulssen (I95I, 1952).

So far no attempt appears to have been made todiscover which of these factors is utilized in thegeneration of plasma thromboplastin. The pro-perties of factors I and III suggest that these mustbe required, but it is not known whether thesealone are sufficient or whether the platelets havestill other properties which are also involved.

In thrombocytopenic purpura, presumably be-cause there are insufficient platelets to permitnormal thromboplastin formation, prothrombinconversion is incomplete. Apart from this, theassociation of a haemorrhagic disease with anabnormality of the platelets has been reported bytwo groups of workers. van Creveld and Paulssen(I95I, 1952) have described two cases of a haemo-rrhagic disorder which they attributed to a partialor complete absence of factor III from the platelets,and Biggs and Douglas (953), although they madeno attempt to assay the platelets for their contentof the different platelet factors, were able to showthat the platelets in a case of thromboasthenia(Glanzmann) were grossly deficient in their powerto form normal plasma thromboplastin when in-cubated with normal serum and aluminiumhydroxide adsorbed plasma.

The Initiation of Blood CoagulationIf blood is taken from a vein into a silicone lined

syringe and is transferred to a silicone lined tube,coagulation may not take place for several hours.If the platelets from such a preparation are re-moved by centrifugation, the plasma may not cloteven if it is transferred to a glass container (Patton,Ware and Seegers, 1948). It seems clear, there-fore, that both platelets and contact with a suitablesurface are necessary, and it may be surmised thatcontact precipitates platelet disintegration and soinitiates the formation of plasma thromboplastin.

In haemophilia, in which the plasma is d2ficientin antihaemophilic globulin, the blood may notclot for an hour or more even when it is in contactwith glass. Moreover, the platelets in haemo-philic blood disintegrate much less rapidly duringcoagulation than do normal platelets in normalblood (Howell and Cekada, 1926). As plat 1atsfrom haemophilic blood, when suspended innormal plasma, in glass vesszls, disrupt normallyduring coagulation (Patek and Stetson, I936), itwould seem that the platelets in haemophilia areprobably normal, and that antihaemophilic globu-lin may be concerned with the normal disruptionof platelets when these come into contact with asuitable surface.

Further evidence of the importance of contact isprovided by the observation that the capacity ofserum to accelerate prothrombin conversion is re-duced if coagulation is delayed by the use ofsilicone lined apparatus (Alexander, de Vries and

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Goldstein, 1949; Biggs and Macfarlane, 1953a).This observation was originally interpreted asindicating that factor VII must exist in plasma asa relatively inactive precursor which was activatedby contact, for at this time factor VII was the onlyconversion factor known which existed in equalconcentration in serum and plasma (the concentra-tions of both factor V (Owren, i947) and anti-haemophilic globulin (Graham et al., I951) fallrapidly after coagulation). Subsequently it wasrrealized that P.T.C. (Christmas factor) and prob-ably the recently postulated factors X and plasmathromboplastin antecedent (P.T.A.) also exist inhigh concentration in serum. More recent workhas indicated that contact probably has no effecton factor VII (Biggs and Macfarlane, I953b), andthat it activates one or more of the other factorsfound in serum.

Naturally Occurring Inhibitors ofCoagulationAntithrombinNormal blood, in addition to its complex

mechanisms for producing thrombin, also containspowerful mechanisms for neutralizing it. It hasbeen calculated, from the results of experiments onthe addition of purified thrombin to plasma, thatX ml. of normal plasma is capable of neutralizingrapidly the amount of thrombin that would berequired to clot 2,000 ml. of blood in I5 seconds(Biggs and Macfarlane, 1953a).Thrombin inactivation depends upon two

mechanisms:I. Adsorption on to fibrinogen. This is the

cause of the immediate inactivation of thrombin.The thrombin is not destroyed and can be re-covered after lysis of the fibrin (Klein and Seegers,1950).

2. Neutralization by a constituent of normalplasma, antithrombin, which, except with lowconcentrations of thrombin, appears to reactstoichiometrically with thrombin rendering itinert (Klein and Seegers, 1950).

HeparinHeparin is a powerful anticoagulant, and

although it is not present in the blood in measur-able quantity, it can be extracted from most of thetissues of the body, particularly the liver andlungs. There is evidence that it arises from themast cells. These cells are commonly foundalong the capillaries suggesting perhaps that theymay have some action in preventing local capillarythromboses.

Heparin is inactive in the absence of thealbumin fraction of the plasma which contains asubstance essential for its action (Quick, 1938). Itseems probable that this substance is antithrombin(Lyttleton, 1950).

Heparin appears to have two main actions:i. It increases the adsorption of thrombin by

fibrin (Klein and Seegers, I950).2. In association with antithrombin it combines

with thrombin, greatly increasing the rate ofneutralization of thrombin by antithrombin, butapparently not increasing the amount of thrombinneutralized (Biggs and Macfarlane, I953a).According to Biggs and Macfarlane (1953a),

when heparin is added to whole blood it delaysor prevents the conversion of prothrombin tothrombin. They consider that this effect isprobably due to the rapid neutralization of throm-bin, this neutralization preventing the auto-catalytic effect of thrombin on the development ofplasma thromboplastic activity (see below).

AntithromboplastinThis substance has been postulated by Tocantins

but its existence has not yet been finally proved.

Clot RetractionWhen blood clots, the clot occupies a volume

equal to that of the blood from which it wasformed. After a variable period of minutes, theexact time depending mainly on the temperature,the clot begins to retract, extruding serum and asmall number of red cells. Clot retraction de-pends upon the presence in the blood of anadequate number of normal platelets. If theplatelets are removed from plasma, either bycentrifugation (Arthus and Chapiro, I908; LeSourd and Pagniez, I9iI) or by lysis (Tocantins,1934; Ackroyd, I949a, I949b), clot retraction isabolished. Tocantins (I934) and Budtz-Olsen(095i) have produced a considerable amount ofevidence to show that platelets cause clot retractionby settling on the fibrin threads and later fusinginto small masses, so drawing the fibrin strandstogether. It is not clear what part clot retractionplays in the haemostatic mechanism.

FibrinolysisNormal human blood contains enzymes which,

under certain circumstances, become activated anddigest fibrin, and sometimes also fibrinogen. Theterminology used below is that of Biggs andMacfarlane (1953a).PlasminThis enzyme is found in the globulin fraction

of normal human plasma. Its activity is inhibitedby the albumin fraction which has therefore beenconsidered to contain a specific inhibitor ' anti-plasmin.' Plasmin normally exists in the blood inthe form of an inactive precursor ' plasminogen.'It can be activated by separation from the albuminfraction of plasma or by the addition to plasma of

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chloroform or bacterial filtrates, the most com-monly used being streptokinase which is obtainedfrom certain strains of p-haemolytic streptococci.Plasmin causes lysis of fibrin and also destroysfibrinogen.

FibrinolysinHuman and animal tissues contain an activator

'fibrinokinase' which activates a fibrinolysinwhich causes lysis of both fibrin and fibrinogen.This enzyme is also found in the globulin fractionof the plasma, but there are reasons for believingthat it is not identical with plasmin (Astrup andPermin, 1948).

Fibrinolysis Post Mortem and in VivoPost mortem blood in cases of sudden death is

often found to be fluid, the fibrin in the post-mortem clots having been liquefied by a fibrino-lytic enzyme which attacks only fibrin and notfibrinogen (Mole, 1948).

Fibrinolytic activity is induced in vivo in manby acute anxiety, severe exerciso or by injectionsof adrenalin (Macfarlane and Biggs, 1946; Biggs,Macfarlane and Pilling, I947). The enzyme con-cerned differs from plasmin and fibrinolysin in thatit does not digest fibrinogen (Bidwell and Macfar-lane, 1951). It closely resembles the lysin foundpost mortem in cases of sudden death. Nothing isknown of the mode of activation of either of theseenzymes.

The Interaction of the Factors Concerned inBlood CoagulationThe Formation of ThromboplastinThe first step in the initiation of blood coagula-

tion under normal circumstances is the formationof thromboplastin. Where there is much con-tamination with tissue juices these supply thrombo-plastic activity, although there is evidence that thetissues supply a thromboplastin which is biologic-ally incomplete, and that its activity is increased bythe action of factors V and VII (Biggs, Douglasand Macfarlane, I953b).

In the absence of tissue thromboplastin, bloodcoagulation depends upon the production of in-trinsic plasma thromboplastin, the development ofwhich appears to be dependent upon contact witha water-wettable surface. This seems to be neces-sary for the disintegration of platelets. Plateletdisintegration is probably accelerated by anti-haemophilic globulin.

Contact is apparently also necessary for theactivation of one or more of the factors, other thanfactor VII, found in high concentration in serum(plasma thromboplastin component (Christmasfactor) and probably the recently postulated factorsplasma thromboplastin antecedent and factor X).

These ' serum ' factors then react with anti-haemophilic globulin and the products of plateletdisintegration (probably platelet factors I and III)and with factors V and VII, to produce a highlyactive thromboplastin. This develops slowly atfirst and does not appear in maximal concentrationfor 4 to 5 minutes. The mode of its formation isentirely obscure.

,Conversion of Prothrombin to ThrombinThromboplastin is essential for the conversion of

THROMBIN UNITS

0 2 4 6 S 10 12 14 16TIUE IN MINUTES

FIG. 4.-Thrombin generation in normal whole bloodCurve i: Whole blood alone. Curve 2: Blood towhich a small amount of thrombin was added at thebeginning of the test.

Reproduced from Macfarlane, Lecture 13 in ' Lectures on theScientific Basis of Medicine, 195I-52,' by courtesy of the authorand publishers.

THROMBOPLASTINCONC.

70

60

650140

30.

2 4 6 8 10 12 14 16TIME IN MINUTES

FIG. S.-The formation of thromboplastin from plate-lets, aluminium hydroxide adsorbed plasma, normalserum and calcium chloride in the presence (x-x)and absence (o-o) of thrombin.

Reproduced from Biggs and Macfarlane, 'Human Blood Co-agulatiOn,' 1953, by courtesy of the authors and publishers

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CONTACTwith water-wettable surface andpossibly with fibrin generated

during coagulation

Platelets

Products of platelet Anti- P.T.C. ? Plasma thrombo-disintegration (probably haemophilic Factor V Factor VII (Christma3 plastin ante- FFactor X Calcium ThrombinPlatelet factors I &III) globulin factor) cedent (P.T.A.)

Calcium Plasma thromboplasti Prothrosmbin

factorI

pFibrinol tc |H F eparin l

enzymes / Antithrombin

Lyed Adscorbed | nacltLvatedfirn \ t / thrombin | thrombin

Fi1FIG. 6.-The factors concerned in normal blood coagulation.

prothrombin to thrombin during coagulation. Itappears to act as a catalyst in this reaction.Thrombin formation can readily be studied byaspirating aliquot quantities of blood during co-agulation, and transferring them to solutions offibrinogen. The thrombin content will be in-versely proportional to the clotting time of thefibrinogen. Fig. 4 curve i shows thrombin pro-duction during the clotting of normal whole blood.There is a lag period of about 3 minutes before anythrombin appears. It then rapidly increasas inamount for a further 3 minutes and then rapidlydisappears, the disappearance being due to theaction of antithrombin. That thrombin canaccelerate its own production is shown by theobservation (Fig. 4 curve 2) that if a very smallamount of thrombin is added to the blood as soonas it is taken, thrombin begins to appear afterabout i minute although its subsequent rate ofproduction is not increased. It seems probablethat this autocatalytic property of thrombin is dueto its capacity to accelerate the production ofthromboplastin, an activity that can readily bedemonstrated by means of the thromboplastingeneration test (see Fig. 5). Quick (i95i) andStefanini (I95i) believe that thrombin acceleratesits own formation by causing platelet disruption,so increasing thromboplastin production. Therate at which thrombin produces detectablechanges in the platelets is relatively slow and the

quantity of thrombin required to produce thesechanges is very much greater than that found byBiggs, Douglas and Macfarlane (1953a) to be re-quired to accelerate thromboplastin generation. Itseems doubtful therefore whether the action ofthrombin on the platelets is an important cause ofthe autocatalytic action of thrombin.

The Conversion of Fibrinogen to FibrinFibrin begins to appear as soon as thrombin is

detectable in the blood. The action of thrombinin converting fibrinogen to fibrin is probablyenzymatic. Although not essential, both calciumand the products of platelet disintegration (plateletfactor II) appear to enhance the activity of throm-bin. It has been suggested (Tocantins, I952;Owren, I952) that fibrin may increase thrombo-plastin production by forming an extensive water-wettable surface. Fibrin on the other handadsorbs large quantities of thrombin, thus actingas a potent antithrombin.

In conclusion, it will be seen that blood coagula-tion involves several complex mechanisms. Theseare summarized in Fig. 6. Some of these mechan-isms tend to increase clotting and others to hold itin check. The factors which determine the re-sultant of these opposing forces are ill understood,and it is clear that many of the views expressedhere may have to be modified as further discoveriesare made in this rapidly advancing field.

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February I954 ACKROYD: The Coagulation of Human Blood 71

The author is indebted to Dr. Rosemary Biggs andDr. R. G. Macfarlane for their criticisms of the manu-script and to the Medical Research Council for a grantfor expenses.

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Copies of Title Page and Index for Vol. 29 of The Postgraduate MedicalJournal are now available on request. See page I04 for binding particulars.

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Documents

COAGULATION OF HUMAN · conversion accelerator of Alexander and his co-workers (1949, 1950). Koller and his colleagues (1951, 1952) have re-peated Owren'sexperiments andhave showncon-clusively