suspension of platelets, (2) citrated plasma deprived of prothrombin by its

538 J. Physiol. (I953) I22, 538-553 THE INITIAL STAGES OF BLOOD COAGULATION BY ROSEMARY BIGGS, A. S. DOUGLAS AND R. G. MACFARLANE From the Department of Pathology, Radcliffe Infirmary, Oxford (Received 22 June 1953) Previous observations have suggested that blood, some minutes after contact with a foreign surface, suddenly develops a labile but extremely potent thromboplastic activity (Macfarlane & Biggs, 1953; Biggs & Macfarlane, 1953). Investigations of this reaction showed that a mixture of three fractions of normal blood generates a similar activity. These fractions were (1) a suspension of platelets, (2) citrated plasma deprived of prothrombin by its adsorption on A1(OH)3, and (3) serum, or the protein adsorbed from serum by AI(OH)3. After 4 or 5 min incubation of this mixture with CaCl2, samples caused clotting of recalcified citrated normal plasma in 8-1 sec, but did not clot fibrinogen, so that, following conventional definitions, they could be said to contain thromboplastin but not thrombin. If any one of the three components was omitted this thromboplastic activity did not develop (Biggs, Douglas & Macfarlane, 1953). Further experiments were made to identify the factors active in this reaction and contained in the plasma and serum fractions. If the plasma fraction was derived from haemophilic blood, thromboplastin generation was absent or reduced. Generation could be proportionately restored by adding increasing amounts of normal plasma or its fibrinogen fraction to the haemophilic plasma, suggesting that the inactivity of the latter was due to the absence of a normal factor rather than the presence of an inhibitor (Biggs & Douglas, 1953). From this it can be concluded that the factor known as 'anti-haemophilic globulin' is required for thromboplastin generation since this factor is associated with the fibrinogen fraction of normal blood and is deficient in haemophilia (Bendien & van Creveld, 1937; Pohle & Taylor, 1937; Minot & Taylor, 1947). It was also found (Biggs & Douglas, 1953) that if serum from the blood of patients receiving the anticoagulant drug ethyl biscoumacetate (tromexan) was used in the generating mixture thromboplastin formation was similarly reduced and was restored by the addition of normal serum. An effect of ethyl biscoumacetate therapy is to diminish the blood content of factor VII,

INITIAL STAGES OF COAGULATION 539 a clotting factor described by Koller, Loeliger & Duckert (1951) and thought by them to be an accelerator of prothrombrin conversion. Factor VII is present in normal serum and is adsorbed from it by Al(OH)3. In cases of ethyl biscoumacetate therapy it was found that the activity of the serum in thromboplastin generation was proportional to its content of factor VII as measured by an independent method. It is probable therefore that the coagulant activity of this factor has been previously misinterpreted and that it is concerned in thromboplastin generation rather than in the acceleration of the reaction between thromboplastin and prothrombin. Following this tentative identification of three of the components of the thromboplastin-forming system the investigation of a newly recognized haemorrhagic state, Christmas disease, has revealed another probable participant (Biggs, Douglas, Macfarlane, Dacie, Pitney, Merskey & O'Brien, 1952). This state, clinically resembling haemophilia, is due to the deficiency of a hitherto unidentified clotting factor, referred to here as 'Christmas factor'. Christmas factor resembles factor VII in being present in normal serum and adsorbed by Al(OH)3, but its level is not always diminished by ethyl biscoumacetate therapy, nor is there a deficiency of factor VII in Christmas disease. Thromboplastin generation is deficient in the blood of cases of Christmas disease. If the serum of such cases is substituted for normal serum in the thromboplastin-generating mixture, activity does not develop, but appears on addition of normal serum. It must be concluded therefore that Christmas factor is essential for normal thromboplastin generation. The recognition that factor VII takes part in this reaction suggests that factor V, also thought to be an accelerator of prothrombin conversion, might in reality be concerned in thromboplastin formation. Factor V, described by Owren (1947), is probably identical with the labile factor of Quick (1943) and the Ac-globulin (Ware & Seegers, 1948). Since it is present in fresh normal plasma and is not adsorbed by Al(OH)3, it is contained in the plasma fraction of the thromboplastin-generating system we have used. In this investigation experiments have shown that the presence of factor V is necessary for blood thromboplastin formation. Thus the number of factors found to be required for this activity to develop has increased from three to five. Since it is unlikely that five factors react simultaneously to produce thromboplastin an attempt has been made to study the preliminary reactions which probably precede the appearance of active thromboplastin. Since widespread intravascular coagulation is an unusual disaster it is likely that thromboplastin generation does not occur in the normal circulation. Even static blood which is in contact only with vascular endothelium will remain fluid for 24 hr or more (Lister, 1863). Yet the same blood brought into contact with glass or any other 'foreign' surface clots in a few minutes. It is probable that some change induced by surface contact sets in motion a complex

54 ROSEMARY BIGGS AND OTHERS reaction culminating in the explosive generation of thromboplastin typical of the normal coagulation process. Attempts have therefore been made to determine which, if any, of the factors or reactions already studied is activated by contact with glass. METHODS The technical methods used in this investigation have been described in detail in previous communications. The thrombin generation test is described by Macfarlane & Biggs (1953) and the thromboplastin generation test by Biggs et at. (1953) and Biggs & Douglas (1953). Factor V and antihaemophilic globulin were prepared from normal plasma treated with Al(OH)3 as described by Biggs & Macfarlane (1953). The antihaemophilic globulin is contained in the fraction precipitated from Al(OH)3-treated plasma by 33% saturation with (NH4)2SO4. Factor V is contained in the fraction precipitated between 33 and 5% saturation with (NH4)2SO4. In the Thrombin generation test either whole blood or recalcified citrated plasma is allowed to clot in glass or silicone-coated tubes, and at intervals the amount of thrombin formed is assessed by transferring samples to fibrinogen solutions, the clotting times of which are recorded. In the Thromboplastin generation test the separated components necessary for thromboplastin formation are incubated collectively with CaCl5, and at intervals the thromboplastin content is assessed by transferring samples to recalcified citrated normal plasma. Modifications of these techniques required by the different experiments will be indicated in the appropriate sections. RESULTS Factor V and antihaemophilic globulin in thromboplastin formation In the thromboplastin generation test platelets, Al(OH)3-treated normal plasma and normal serum are incubated with CaCl2 and at intervals samples are tested for their thromboplastin content. When the Al(OH)3-treated normal plasma used in the thromboplastin generation test is replaced by either factor V or antihaemophilic globulin separately, thromboplastin formation is reduced and delayed. When the two are combined rapid thromboplastin formation is restored (Fig. 1). These findings suggest that both factor V and antihaemophilic globulin are necessary for normal blood thromboplastin formation. The interactions between the thromboplastin components The distribution in the serum and plasma samples used in this investigation of the factors considered necessary for thromboplastin formation is shown in Table 1. It is probable that preliminary reactions involving only two or three of the necessary components precede the appearance of thromboplastin. Evidence for the existence of such serial reactions might be obtained in different ways. For instance, if preliminary reactions occur then the preincubation of suitable combinations should lead to the formation of intermediate products which would form thromboplastin more rapidly when the remaining components are added, than if all five are incubated simultaneously. To determine whether or not any such acceleration occurs, a selection of the five necessary factors may be incubated together with CaCl2, and after 2 or 3 min the remaining reagents may be added and thromboplastin formation

INITIAL STAGES OF COAGULATION 541 studied. In these experiments, antihaemophilic globulin, factor V and platelets were prepared from normal plasma. The serum from a patient treated with ethyl biscoumacetate was used as a source of the Christmas factor and serum - -o 2. I- 1 9 8 7 6 5 4 3 2 1 2 3 4 5 Incubation time (min) Fig. 1. Curves demonstrating thromboplastin formation in mixtures containing factor V, antihaemophilic globulin and a mixture of the two. O, represents the thromboplastic activity developed in a mixture of equal parts of antihaemophilic globulin, -85% NaCl, platelets, normal serum and M/4-CaCl2; x x, factor V, -85% NaCl, platelets, normal serum and CaCl2;, antihaemophilic globulin, factor V, platelets, normal serum and CaCl2. In this and subsequent illustrations the thromboplastic activity was measured by removing -1 ml. samples at intervals and adding these to.1 ml. amounts of citrated normal plasma which were recalcified with -1 ml. of M/4-CaCl2 and observing the clotting times. The clotting times were interpreted in terms of thromboplastin concentrations using a dilution curve (Biggs et al. 1953). The results are the average figures derived from twelve independent experiments. TABLE 1. Type of sample Normal plasma Normal Al(OH)3-treated plasma Normal serum Haemophilic plasma Haemophilic Al(OH),-treated plasma Haemophilic serum Plasma in Christmas disease Christmas disease plasma Al(OH),- treated Serum in Christmas disease Plasma in ethyl biscoumacetate therapy Plasma in ethyl biscoumacetate therapy Al(OH)3-treated Serum in ethyl biscoumacetate therapy PH. CXXII. Thromboplastin components present in a variety of blood samples Platelets Antihaemophilic globulin + Christmas factor + Factor V + ± Factor VII + + + + _ + _ + + + + + + + +- + + _ + + + 35

542 ROSEMARY BIGGS AND OTHERS from a patient with Christmas disease was used as a source of factor VII (see Table 1). Since there is no clue as to the nature of any of the possible preliminary reactions there is no alternative but to test all of the possible combinations of factors taken two, three or four at a time. To illustrate the general plan of these experiments one will be described in detail. Thromboplastin formation was followed in a mixture containing 3 ml. of preparations of each of the five necessary components and CaCJ4. The progress of thromboplastin formation is shown in Fig. 2. In another test, 3 ml. of each of factor V, platelets, 1 8.1 6. E24 2 1 2 3 4 5 6 Incubation time (min) Fig. 2. Curves demonstrating blood thromboplastin generation, when all of the necessary thromboplastin components are mixed at the same time ( O); when antihaemophilic globulin, platelets and CaCl2 are first incubated at 37 C for 3 min and the mixture completed to contain all of the necessary components ( x x ); and when platelets, factor V, the Christmas factor and CaCl2 are incubated at 37 C for 3 min and the mixture completed to contain all of the thromboplastin components ( ). the Christmas factor and CaCl2 were incubated for 3 min at 37 C. After this time 3 ml. of each of antihaemophilic globulin and factor VII were added, and thromboplastin formation studied as before. When the results of this pre-incubation experiment are compared with those of the test in which all five reagents were present from the start it will be seen that pre-incubation led to accelerated thromboplastin formation (Fig. 2). From these results it is to be presumed that some reaction involving platelets, factor V and the Christmas factor precedes the formation of active thromboplastin. In another experiment platelets, anti-haemophilic globulin and CaCI2 were first incubated, and then factors V and VII and the Christmas factor were added (Fig. 2). In this experiment there was no acceleration of the final speed of thromboplastin formation, and it must be concluded that platelets and antihaemophilic globulin do not react together.

INITIAL STAGES OF COAGULATION 543 In this way all of the twenty-five possible groups of factors taken two, three or four at a time were pre-incubated with CaCl2 and the mixtures completed to contain the five necessary factors. Pre-incubation of one of the ten combinations of factors taken two at a time led to acceleration of thromboplastin formation. Pre-incubation of four of the ten possible combinations of factors taken three at a time led to acceleration and pre-incubation of four of the possible five combinations of factors taken four at a time produced acceleration on addition of the fifth. The combinations of factors which led TABLE 2. The combinations of factors which, when incubated together, lead to accelerated thromboplastin formation on completion of the mixture to contain all of the components required for thromboplastin formation. + indicates that the substance was present in the initial mixture. - indicates that the substance was omitted. Antihaemophilic Christmas Factor Factor Expt. CaCI2 Platelets globulin factor V VII 1 + + _ + + _ 2 + + + + + _ 3 + + - + + + 4 + - + + _ 5 + - + + + _ 6 + + + + 7 + - + + - + 8 + + + + - + 9 + - + + + + to acceleration of the final reaction are shown in Table 2. These results can be divided into two groups. In Expts. 1-3 the pre-incubated mixtures all contained platelets, Christmas factor and factor V. In Expts. 2 and 4-9 all of the pre-incubated mixtures contained the antihaemophilic globulin and the Christmas factor. These results suggest that at least two preliminary reactions may be involved in thromboplastin formation, in one platelets, factor V and the Christmas factor react, and in the other antihaemophilic globulin and the Christmas factor are involved. Factor VII does not apparently take part in any appreciably time-consuming preliminary reaction. Glass and siltcone surfaces and the generation of thrombin The generation of thrombin in normal whole blood. The five components used in these tests react together to form thromboplastin in the presence of CaCI2. In the circulating blood this reaction does not, apparently, occur, and it must be presumed that one or more of the components are inactive until the blood is shed. The effect of glass contact on the generation of thromboplastin is studied in an attempt to determine which, if any, of the factors is changed by such contact. These experiments must necessarily be carried out with whole blood or plasma, and the evidence on individual factors must be inferred. The problem has therefore been approached from several points of view in the hope that the summation of evidence may be more reliable than that from one source alone. When untreated normal blood is transferred to 35-2

544 ROSEMARY BIGGS AND OTHERS a glass tube there is, prior to coagulation, a latent period lasting 3-4 min during which no thrombin can be detected (Macfarlane & Biggs, 1953). This latent period represents the duration of the reactions required for the formation of thromboplastin from its precursors. After this delay phase, rapid thrombin formation immediately follows the formation of a powerful thromboplastin. In this test thrombin formation is an indicator of thromboplastin formation provided that prothrombin is present in normal amounts. If instead of using glass tubes the blood is run into silicone-coated tubes the delay phase is increased usually by 5-1 min. This observation suggests that glass alters some factor or factors in the blood which are necessary for thromboplastin formation while the silicone surface is relatively inactive. If this supposition is true then the addition to normal blood collected into siliconetreated tubes of appropriate blood derivatives already exposed to glass should accelerate thrombin formation and, as it were, by-pass the usual effects of glass contact. To test this hypothesis 2 ml. of whole normal blood was collected into a silicone-treated tube containing -2 ml. of the derivative to be tested. Thrombin formation was then followed and the results compared with a similar test in which the supposedly active derivative was replaced with 85 % NaCl. Using this technique serum samples from a normal subject, a haemophilic subject, a patient under treatment with ethyl biscoumacetate and from a patient with Christmas disease were tested (Fig. 3). From the results of these tests it is clear that the serum from the normal, the haemophilic, and the patient treated with ethyl biscoumacetate, all contain some factor which can accelerate thromboplastin formation in blood which has not been in contact with glass. The serum from the patient with Christmas disease was inactive. Interpreting these results from Table 1 it appears that when the Christmas factor is present the serum is active in substituting for glass contact, but when it is absent there is no activity. The presence or absence of factor VII does not influence the result. That factor VII is not influenced by contact is supported by the observation that when the blood of patients treated with ethyl biscoumacetate is placed in a glass tube there is no unusual delay in thrombin formation and therefore also presumably no delay in thromboplastin generation. This finding suggests that the blood of these patients does not lack any substance normally activated by contact (Fig. 4). In a similar manner, platelet suspensions and brain emulsion were tested in their ability to accelerate thrombin formation in normal blood collected into silicone-treated tubes (Fig. 3). Untreated brain emulsions eliminate the whole of the delay phase. Suspensions of washed platelets prepared from normal plasma are as active as the serum samples. When normal serum and platelets are present together the mixture has a greater effect than that of either component separately (Fig. 3).

INITIAL STAGES OF COAGULATION 545 Freshly collected citrated plasma does not accelerate thrombin formation in whole normal blood in silicone-coated tubes, but if the plasma is stored in contact with glass some activity develops (Fig. 5), possibly as a result of a change in the Christmas factor. 11 - VW._._E E 1 9 8 7 6 5 4 3 v 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Incubation time (min) Fig. 3. Curves demonstrating thrombin formation from whole normal blood placed in silicone tubes to which various reagents were added in the proportion of 1 part of; reagent to 1 parts of blood. x x, saline added;, serum from a patient with Christmas disease added; A A, platelets added; A A, platelets and normal serum added;, brain extract added. The discontinuous lines represent additions of normal serum, haemophilic serum and serum from a patient treated with ethyl biscoumacetate. The thrombin concentrations in this and subsequent diagrams were deduced from a dilution curve using the observed clotting times. By the method used a clotting time of 12 sec represents 1 units/ml. of thrombin in terms of a specific preparation of thrombin topical (Parke Davis). 9 8 'A E 6 5 4 3 1 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Incubation time (min) Fig. 4. Curves demonstrating thrombin formation in whole normal blood collected into glass tubes ( ), and in the whole blood of patients treated with ethyl biscoumacetate ( x -x ). The results are the average of seven similar experiments.

546 ROSEMARY BIGGS AND OTHERS These results, taken together, suggest that during the delay phase of normal coagulation some change occurs involving the Christmas factor and platelets, this change being brought about by contact of plasma with glass. The generation of thrombin in citrated plasma. In a second method of approach to this problem, citrated plasma samples from patients with various clotting abnormalities were prepared in silicone-coated tubes. In normal platelet-containing plasma all of the necessary thromboplastin constituents are present, but blood from patients with haemophilia lacks antihaemophilic 9 8 7 E 3 62-1 11 31 2 I 1 2 3 4 5 6 7 8 9 1 11 12 13 14 Incubation time (min) Fig. 5. Thrombin formation in 2 ml. of normal blood collected into silicone-coated tubes. The tubes contained (1)-2 ml.of saline (x - x ); (2)-2 ml.of fresh normal citrated plasma (-); (3) -2 ml. of plasma stored for 24 hr ( O). globulin and blood from patients with Christmas disease lacks the Christmas factor. Using slowly or rapidly centrifuged samples from normal subjects and patients with haemophilia or Christmas disease, one or two of the five components for thromboplastin formation can be excluded from the system, and the remaining three or four factors can be exposed to glass contact in various combinations and the generation of thrombin can be followed in specimens stored in silicone in parallel with those exposed to glass. Experiments carried out in this way are shown in Fig. 6 and Table 3. To illustrate the general plan of these experiments a test on haemophilic plasma (Fig. 6B1) will be described in detail. Blood was collected from a haemophilic subject into a silicone-coated container and centrifuged at 1 rev/min for 5 min. The platelet-containing plasma was separated and divided into two parts. One part was stored in a silicone-coated tube and the other was placed in a small glass bottle containing 3 glass beads and rotated slowly on an inclined turn-table for 2 hr. At the end of this time 1 ml. of the plasma stored in silicone tubes was placed in a silicone-coated tube and '1 ml. of an antihaemophilic globulin preparation was added and the mixture

INITIAL STAGES OF COAGULATION 547 incubated at 37 C. In this experiment AM(OH)3-treated normal plasma was used as a source of antihaemophilic globulin. 1 ml. of M/4-CaCl2 was added to the mixture and the generation of thrombin was followed by removing -1 ml. samples at intervals and adding them to fibrinogen. The glasscontacted specimen was tested in the same way. The two test samples differed only in their exposure to glass. The antihaemophilic globulin was added to the specimens because in its absence no thromboplastin formation can occur. 8 6 4 2 Slowly centrifuged Rapidly centrifuged I E _ o z C c E F- 8 6 4 2 B. E I 8 6 4 2 Cl i a ai C2-2'< 2 4 6 8 1 12 2 4 Incubation time (min) 6 8 1 12 E u C1 r_ e Fig. 6. Thrombin formation in slowly and rapidly centrifuged plasma samples from norma subjects A1 and A2, haemophilic patients B1 and B2, and patients with Christmas disease C1 and C2. The plasma was either stored in silicone tubes or exposed to glass contact for 2 hr., specimens exposed to glass; x- x, specimens stored in silicone. In each experiment any thromboplastin-generating component known to be lacking was added. Thus in the curves of diagram B1 the results of experiments on slowly centrifuged haemophilic plasma are shown and in this experiment antihaemophilic globulin was added before testing. The same amount of the same preparation of antihaemophilic globulin was added in each of the two tests so that from the point of view of contact with glass the effect on the antihaemophilic globulin is eliminated in this experiment. The effect of contact, which is very large in this example, must have operated on the factors contained in the haemophilic plasma and, as Table 1 shows, these are factors V and VII, the Christmas factor and platelets.

548 ROSEMARY BIGGS AND OTHERS Similar experiments carried out with other samples give other combinations of factors in which the effects of glass contact may be studied. In experiments with rapidly centrifuged plasma 1 ml. of a concentrated platelet preparation was added before testing, and in experiments with plasma from patients with Christmas disease -1 ml. of normal serum or serum from a patient treated with ethyl biscoumacetate was added just before testing. The results of these tests are shown in Fig. 6. In Table 3 the factors contrasted by the design of each experiment are indicated. Type of plasma Normal TABLE 3. Rapid (Fig. 6A2) Haemophilic Slow (Fig. 6B1) The constitution of plasma samples used in the experiment illustrated in Fig. 6 Factors present in the original Preparaticon added specimen* just befor( e testing Platelets, anti- Nil haemophilic globulin, Christmas factor, factor V and factor VII Antihaemophilic Platelets Platelets Speed of centrifuging Slow (Fig. 6A1) Rapid (Fig. 6B2) globulin, Christmas factor, factor V and factor VII Platelets, Christmas factor, factor V and factor VII Christmas factor, factor V and factor VII Al(OH)3-ttreated normal plasma Platelets and Al(OH)3--treated normal Iplasma Factors present in the added preparationt Nil Antihaemophilic globulin and factor V Platelets, antihaemophilic globulin and factor V Christmas factor or Christmas factor + factor VII Plasma from Slow (Fig. 6C1) Platelets, antihaemophilic patient treated Serum from a a patient with Christmas factor and factor VII biscoumacetate globulin, factor V with ethyl deficiency or normal serum Rapid (Fig. 6C2) Antihaemophilic Platelets and Platelets and globulin, factor V serum from a Christmas factor, and factor VII patient treated or platelets, with ethyl Christmas factor biscoumacetate and factor VII or normal serum * The experiment shows the effect of glass contact on these factors. t The effect of glass contact on these factors is eliminated from the experiment. Reference to Table 3 and Fig. 6 shows that contact with glass had no significant effect in one experiment (Fig. 6C2). In this experiment the effect of contact with glass on factors V and VII and the antihaemophilic globulin were tested. It must be concluded that these factors are not altered by contact with glass. The maximum effect of glass contact occurred in the experiments illustrated in Fig. 6 A1 and B1. In these two experiments both platelets and Christmas factor were exposed to glass contact. In the remaining experiments either platelets or the Christmas factor were involved, and the effect of contact was intermediate in type between the experiments in which both

INITIAL STAGES OF COAGULATION 549 factors were involved or neither were present. The findings are summarized in Fig. 7 by averaging the results of experiments in which both of the relevant factors were exposed to contact and comparing the resulting curve with the average results of exposing to glass either the Christmas factor or platelets or neither of these factors. These experiments show that both platelets and the Christmas factor are influenced by contact with glass, and that the maximum effect is achieved when both factors are present in large amounts. Factors V and VII and the antihaemophilic globulin are not, apparently, altered by contact. 8 7-.t Ste t6 IM 6- E.3-2 1 2 3 4 5 6 7 8 9 1 11 12 Incubation time (min) Fig. 7. Curves demonstrating the average thrombin formation in various types of plasma sample., both the Christmas factor and platelets were exposed to glass contact; * - the Christmas factor was exposed to contact but the platelets were not exposed; V V platelets were exposed to glass but the Christmas factor was not exposed; x x, neither the Christmas factor nor the platelets were exposed to glass. Glass contact and the generation of thromboplastin. In the third method of approach to this problem the effect of contact with glass on the activity of the Christmas factor in thromboplastin generation was studied. Rapidly centrifuged haemophilic plasma diluted 1/1 with -85 % NaCl was used as a source of Christmas factor and factor VII in the thromboplastin generation test. Al(OH)3-treated normal plasma was used as a source of antihaemophilic globulin, and factor V and platelets were prepared from normal whole plasma. To the mixture of antihaemophilic globulin, factor V and platelets was added either a dilution of rapidly centrifuged haemophilic plasma which had been kept in silicone-coated tubes, or one that had been exposed to glass. From Fig. 8 it is clear that contact with glass facilitates thromboplastin formation. Since previous evidence has shown that factor VII is not influenced by contact with glass the increased activity is probably due to a change in the Christmas factor.

55 ROSEMARY BIGGS AND OTHERS a3. E - 2 1 1 2 3 4 5 6 Incubation time (min) Fig. 8. Curves demonstrating thromboplastin formation in mixtures containing the Christmas factor supplied by rapidly centrifuged haemophilic plasma. a, the haemophilic plasma was exposed to glass before use. x x, the haemophilic plasma was not exposed to contact. DISCUSSION The purpose of the foregoing experiments is to gain further information on the mechanism by which thromboplastin is formed in normal blood. They seem to show that at least five recognizable factors are concerned, these being the platelets, antihaemophilic globulin, factor V, factor VII and the Christmas factor. Calcium is also required. The implication of a number of reactants in the production of a final (thromboplastic) activity suggests some system of preliminary interaction between pairs of factors and between their products, rather than the simultaneous interaction of all five factors and calcium. By studying the activity of thromboplastin-generating mixtures made up of different pre-incubated combinations of factors, it was found that the Christmas factor appears to undergo preliminary reactions with the factor V and platelets, and with antihaemophilic globulin. Since contact with a foreign surface seems to be the initial stimulus to natural coagulation it is probable that some reaction set in motion by surface contact is the first link in a chain of events leading to the appearance of thromboplastin. By a second series of experiments in which various factors or combinations of factors previously exposed to glass were added to a thromboplastin-generating system which had not been so exposed, evidence was obtained suggesting that the Christmas factor and platelets are activated by contact. In blood which has not been exposed to a foreign surface, therefore, Christmas factor and platelets are

INITIAL STAGES OF COAGULATION presumably inactive. From these two sources of evidence it appears that the initial stage of coagulation may be the activation of Christmas factor and its interaction with the platelets. There is evidence to link factor V with the reactions involving the platelets. Factor VII is necessary for the final formation of thromboplastin and calcium is required apparently at all stages. There is no direct information at present on the possible nature of these suggested reactions. It has been shown (Douglas & Biggs, 1953) that both factor V and antihaemophilic globulin are consumed in the process of forming thromboplastin, a finding which agrees with previous observations by Graham, Penick & Brinkhous (1951) and Alexander, Goldstein & Landwehr (1951) on the consumption of these factors during normal coagulation. Factor VII and Christmas factor, on the contrary, are not consumed, being thus found in the serum after thromboplastin formation is complete. It is probable, therefore, that factor V and antihaemophilic globulin are converted into other substances (perhaps into thromboplastin itself) and may be substrates for enzyme action, whereas factor VII and Christmas factor may be catalysts, and, in fact, enzymes. Several hypothetical schemes of interaction can be constructed to fit the experimental evidence so far obtained. Since this is neither conclusive nor complete, little would be gained by discussing at length such hypotheses which, in the present stage of the investigation, are useful only in indicating further experiments. Two examples, however, are briefly presented. On the literal interpretation of the observations described it would appear that the Christmas factor, following activation by surface contact, takes part in separate and simultaneous reactions with the platelets and factor V and with antihaemophilic globulin. The products of these two reactions might then be expected to interact to form thromboplastin. There is some evidence suggesting that factor VII operates at a late stage of the mechanism. The scheme below fulfils these observations. This scheme has the disadvantage that it postulates a dual role for the Christmas factor, fails to explain apparent Contact with glass Contact with glass 551 Platelets+ Factor V Antihaemophilic globulin Christmas factor and < CaCI2 Product I Product II Factor VII r ocaci2 n Thromboplastin *

552 ROSEMARY BIGGS AND OTHERS activation of two factors by contact and proposes two intermediate products. An alternative and in some ways more satisfactory scheme is set out as follows: Precursor + platelets Antihaemophilic globulin Product I + Factor V Contact Christmas factor Thromboplastin J- Factor VII This scheme accounts for the action of contact on both the Christmas factor and platelets; it suggests that antihaemophilic globulin and the Christmas factor react together, which would account for the similar clotting defects caused by deficiency of these factors; it places factors V and VII together which is reasonable since it is these two factors which are required for the activity of brain thromboplastin, and it proposes only one hypothetical intermediate product. On the other hand, this scheme does not account for the apparent association between factor V and platelets. It is clear that further information must be obtained before the complicated mechanism of thromboplastin formation can be elucidated. The approach to the problem is beset with technical difficulties, and caution must therefore be exercised in the interpretation of experimental results. Restraint is also necessary in the formulation of hypotheses since it must be borne in mind that such hypotheses may soon be rendered obsolete by new observations and that other factors, at present undiscovered, may be concerned in the reaction. SUMMARY 1. Five factors, platelets, anti-haemophilic globulin, the Christmas factor and factors V and VII, together with CaCl2, are necessary for blood thromboplastin formation. 2. From study of the activity of thromboplastin-generating mixtures made up of different pre-incubated combinations of factors it is suggested that preliminary reactions involving only two or three of the essential components precedes the formation of active thromboplastin. The exact nature of these reactions has not been determined. 3. It has been shown that exposure to glass accelerates thrombin formation if the Christmas factor or platelets are present. The greatest effect was demonstrated when both factors were present in large amounts. Factor V, factor VII and anti-haemophilic globulin are, apparently, not affected by contact with glass.

INITIAL STAGES OF COAGULATION 553 We should like to thank Miss G. Richards for technical assistance and Dr A. H. T. Robb-Smith for reading the manuscript and making valuable suggestions. One of us (A. S. D.) wishes to thank the Medical Research Council for a Fellowship enabling him to take part in this work. REFERENCES ALEXANDER, B., GOLDSTEIN, R. & LANDWEEX, G. (1951). The labile factor of prothrombin conversion: its consumption under normal and pathological conditions affecting blood coagulation. J. clin. Invest. 3, 252-262. BENDIEN, W. M. & VAN CREVELD, S. (1937). Investigations on haemophilia. Amer. J. di8. Child. 54, 713-725. BIaGs, R. & DOUGLAS, A. S. (1953). The thromboplastin generation test. J. clin. Path. 6, 23-29. BIaGS, R., DouGLAS, A. S. & MAcFARLANE, R. G. (1953). The formation of thromboplastin in human blood. J. Phy8iol. 119, 89-11. BIGGs, R., DoluaLs, A. S., MAcFARLANE, R. G., DACIE, J. V., PITNEY, W. R., MERSKEY, C. & O'BziEN, J. R. (1952). Christmas disease. A condition previously mistaken for haemophilia. Brit. med. J. ii, 1378-1388. BIaaS, R. & MACFARLANE, R. G. (1953). Human Blood Coagulation and its Disorders. Oxford: Blackwell's Scientific Publications. DOUGLAS, A. S. & BIGGS, R. (1953). The consumption of some components involved in physiological coagulation. Glaagow med. J. GRAHAM, J. B., PE1NrIK, G. D. & BRmKHOUS, K. M. (1951). Utilization of the antihaemophilic factor during clotting of canine blood and plasma. Amer. J. Phy8iol. 164, 71-715. KOLLER, F., LOELIGER, A. & DUCKERT, F. (1951). Experiments on a new clotting factor (factor VII). Acta haemat. 6, 1-18. LISTER, J. (1863). On the coagulation of the blood. Proc. Roy. Soc. 12, 58-6O1. MACFARLANE, R. G. & BIGGs, R. (1953). A thrombin generation test. J. clin. Path. 6, 1-8. MNOT, S. R. & TAYLOR, F. H. L. (1947). Haemophilia. The clinical use of antihaemophilic globulin. Ann. intern. Med. 26, 363-367. OWBEN, P. A. (1947). The coagulation of blood. Acta med. acand. Suppl. 194. POHLE, F. J. & TAYLOR, F. H. L. (1937). The coagulation defect in haemophilia. The effect in haemophilia of intramuscular administration of a globulin substance derived from normal human plasma. J. clin. Invest. 16, 741-747. QuicK, A. J. (1943). On the constitution of prothrombin. Amer. J. Phy8iol. 14, 212-22. WARE, A. G. & SEEGERS, W. H. (1948). Plasma accelerator globulin: partial purification, quantitative determination and properties. J. biol. Chem. 172, 699-75.