Activation of Factor IX by the reaction product of tissue factor and

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1 Proc. Natl. Acad. Sci. USA Vol. 74, No. 12, pp , December 1977 Biochemistry Activation of Factor IX by the reaction product of tissue factor and Factor VII: Additional pathway for initiating blood coagulation (bypass of activated Factor XI/assay for activated Factor IX) BJARNE 0STERUD AND SAMUEL I. RAPAPORT Department of Medicine, University of California, San Diego, California and the San Diego Veterans Administration Hospital, San Diego, California Communicated by Helen M. Banney, August 19, 1977 ABSTRACT A study was carried out on mechanisms, independent of activated Factor XI, capable of activating Factor IX. The reaction product of tissue factor and Factor VII functioned as a potent Factor IX activator in the assay system used. Activated Factor IX itself activated Factor X; thrombin failed to activate Factor IX. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis confirmed that the reaction product of tissue factor and Factor VII activated Factor IX, with replacement of the band corresponding to native factor IX [molecular weight (Mi) 55,000] by bands corresponding to the heavy chain (Mr 27,000) and light chain (Mr 17,000) of activated Factor IX. When either Factor VII or calcium ions were left out of incubation mixtures, the band of native Factor IX persisted unchanged. Contact of blood with tissue factor represents a second mechanism, bypassing activated Factor XI, for the activation of Factor IX during hemostasis. It. may help to explain the discrepancy between the mild bleeding of hereditary Factor XI deficiency and the severe bleeding of hereditary Factor IX deficiency. When blood clots in glass without added materials (intrinsic clotting), the negative charge of the surface initiates reactions involving Factor XII, prekallikrein, high molecular weight kininogen, and Factor XI that generate activated Factor XI (Factor XIa). Factor XIa then activates Factor IX. Indirect evidence suggests that the activity arising during extrinsic clotting-the reactions initiated when tissue factor is added to blood-may also activate Factor IX (1-3). Moreover, it has been reported that purified activated Factor X (Factor Xa) can activate purified Factor IX in a feedback reaction (4). In the studies reported herein, we used purified human Factor IX and a new assay for activated Factor IX (Factor IXa) to examine activation of Factor IX. These studies establish that the reaction product of tissue factor and Factor VII functions as a potent activator of Factor IX. MATERIALS AND METHODS Tissue factor (5) and cephalin (6) were prepared from human brain tissue. Purified bovine Factor VIII (80 units/ml) was generously supplied by James Brown. One unit of clotting activity was defined as the activity present in 1 ml of a normal plasma pool. A Factor IX antiserum was raised as described elsewhere (7). Factor IX was purified by a modification of the method of 0sterud and Flengsrud (8) in which a heparin-agarose affinity column (9) replaced the Factor X antibody-column. Preparations gave comparable values (5-20 units/ml) for clotting activity measured in a partial thromboplastin time assay (3) and antigen concentration measured by electroimmunoassay (10). Specific activity was 200 units/mg of protein, and Factor X1a cleaved up to 95% of the protein in the preparations. Factor IXa was prepared by incubation of 500 Ml of purified Factor IX (12 units/ml), 25 Al of Factor XIa (24 units/ml), 50 Al of 50 mm CaCI2, and 1 ml of distilled water. Then, 25 Al of 0.15 M sodium citrate was added. The Factor IXa was separated by DEAE-cellulose chromatography and stored at -70 with added bovine albumin (0.5 mg/ml). Factor XIa,was prepared by adsorption of plasma with Celite (11), elution with 10% NaCl, chromatography on a heparinagarose column, and elution in 0.05 M sodium acetate (ph 5.5) containing 0.5 M NaCl. Bovine thrombin (Parke-Davis) was absorbed (12) and then purified further by affinity chromatography on a benzamidine-sepharose column (13). Factor VII was prepared from human plasma as for the initial steps of the purification of Factor IX. Factor VII was eluted with prothrombin from the preparative polyacrylamide gel electrophoresis, separated from prothrombin by gel filtration on a Sephadex G-100 column (14), and concentrated by chromatography on DEAE-cellulose. Preparations contained units of Factor VII activity per ml, were devoid of prothrombin, Factor IX, or Factor X activity, gave two weak bands on sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis, and contained no protein measurable by the Lowry et al. method (15). Factor X was obtained as the initial fractions from the heparin-agarose column used to prepare Factor IX. These fractions contained units of Factor X activity per ml (specific activity, 57 units/mg of protein) and gave two bands on Na- DodSO4/polyacrylamide gel electrophoresis, one of which migrated in the position of albumin. After reduction with 2- mercaptoethanol, the nonalbumin band was replaced by bands corresponding to the heavy chain band and light chain band of Factor X. Factor Xa was prepared by incubating Factor X, CaCl2, and a fraction from whole Russell's viper venom (RVV) that activates Factor X (16), and then stopping the reaction with 0.15 M sodium citrate. The resulting Factor Xa was separated from the RVV fraction by preparative polyacrylamide gel electrophoresis (8). Amidolytic Assays for Factor Xa and Factor X. Factor Xa was assayed by measuring the initial rate of hydrolysis of Bz- Ile-Glx-Gly-Arg-p-nitroanilide (S-2222, Kabi, Sweden). Fifty microliters of S-2222 (1 mg/ml) and 600 Al of Tris-buffered saline (TBS) were warmed at 370 in a cuvette. Then, 100 Atl of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C solely to indicate this fact Abbreviations: Factor IXa or IXa, activated Factor IX; Factor XIa or XIa, activated Factor XI; Factor Xa or Xa, activated Factor X; Na- DodSO4, sodium dodecyl sulfate; RVV, Russell's viper venom; TBS, Tris-buffered saline; Mr, molecular weight.

2 Biochemistry: Osterud and Rapaport the test material was added and the rate of increase of absorbance at 405 nm was measured with a recording spectrophotometer. Factor X was assayed after conversion to Factor Xa with RVV. The test sample, 200Wul, was incubated with 20 Al of RVV diluted 1:15,000 in TBS and 20,ul of 25 mm CaC12 for 5 min at 370. Then, 10Al of 0.3 M EDTA was added and the mixture was chilled in ice water. A 100-Al subsample was assayed, without delay, for Factor Xa activity as described above. Initial AA was converted to Factor X or Factor Xa activity units/ml from standard curves made with dilutions of a normal pooled plasma Ȧssay for Factor IX.. A three-step assay was designed. In the first step, materials being tested for their ability to activate Factor IX were incubated at 370 with purified Factor IX. In the second step, a subsample from the first mixture was incubated with a mixture of thrombin-activated Factor VIII, cephalin, calcium, and Factor X. After 3 min at 370, reactions were stopped by adding EDTA and chilling in ice water. In the third step, a subsample of the second incubation mixture was assayed for Factor Xa activity as described above. By adjusting concentrations and dilutions of the first incubation mixture, one could utilize this assay to test the Factor IX-activating ability of materials which in themselves activate Factor X directly (e.g., a mixture of tissue factor, Factor VII and calcium). Details are described in Results. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis (10% gels) in the presence of NaDodSO4 was performed by the technique of Weber et al. (17). Gels were stained with Coomassie blue. RESULTS Validation of Factor IX, Assay Results as a Measure of the Ability of a Test Material to Activate Factor IX. In a negative control experiment, native Factor IX (6.5 units/ml) was incubated with TBS and Ca2+, and subsamples over time were added to the second mixture of the Factor IXa assay. No chromogenic activity was formed (i.e., the assay was insensitive to native Factor IX). In a positive control experiment, native Factor IX (6.5 units/ml) was incubated with Factor XIa (0.24 unit/ml) and Ca2+. Subsamples over time from this first incubation mixture supported the formation of increasing amounts of Factor Xa in the second incubation mixture (Fig. 1). The assay results clearly reflected the known capability of Factor XIa to activate Factor IX. A dilution curve was then prepared by using dilutions in TBS of a subsample from a first incubation mixture that had incubated at 370 for 10 min. The final concentration of Factor X in the second incubation mixture was 0.82 unit/ml and the final concentration of Factor IXa varied between and 0.08 units/ml. (Details of both incubation mixtures were as described in the legend to Fig. 1 except that 25 Ml of the diluted test sample was added to the second incubation mixture.) An arithmetic plot of units of Factor Xa activity generated (y) against Factor IXa concentration (x) yielded a straight line described by the equation: y = x. Because bovine Factor Xa has been reported to activate bovine Factor IX in the presence of lipid and Ga2+ (4), the possibility existed of a feedback activation in our assay that would overestimate the ability of a test material to activate Factor IX. Conceivably, generation of a small amount of Factor IXa in the first incubation mixture-through supporting an initial generation of Factor Xa in the second incubation mixture-could trigger a cycle in the second incubation mixture in which Factor E- 0.5 Proc. Natl. Acad. Sci. USA 74 (1977) Xto/ 0.4 I L Mixture 1 incubation time, min FIG. 1. Generation of Factor Xa induced by subsamples over time from an incubation mixture of Factor IX and Factor XI. contrasted with failure of generation of Factor Xa by subsamples over time from an incubation mixture of Factor IX and Factor Xa. Three series of first incubation mixtures were prepared. One series contained 10 ul of Factor IX (6.5 units/ml), 10 Al of Factor XIa (0.24 unit/ml), 5 Al of cephalin, and 3jul of 50 mm CaC12 (-). The second series contained 10 gi of Factor IX (6.5 units/ml), 10 MI of Factor X. (2.3 units/ml), 5 jd of cephalin, and 3 Al of 50mM CaCi2 (a). The third series contained 10 Al of TBS, 10,ul of Factor Xa (2.3 units/ml), 5 Al of cephalin, and 3 1l of 50 mm CaCi2 (0). At different incubation times, a 10-Ml subsample was removed and added to a second incubation mixture containing 50 AI of native Factor X (2.7 units/ml), 50 Al of bovine Factor VIII (4 units/ml), 10,l of thrombin (0.1 unit/ml), 20AI of cephalin, and 20 AI of 50 mm CaCI2. It stood for 3 min in ice water before the addition of the subsample from the first incubation mixture. Then, the complete second mixture was incubated for 3 min at 370, the reaction was stopped by addition of 10 Ai of 0.3 M Na2EDTA, and the mixture again was chilled in ice water. Immediately thereafter, 100 Mi of the second incubation mixture was added to 600 AI of TBS and 50 Ml of S-2222 (1 mg/ml) at 370 in the cuvette of the spectrophotometer. Xa, lipid, and Ca2+ caused further formation of Factor IXa and, in turn, further formation of Factor Xa. Therefore, an experiment was carried out to evaluate the ability of human Factor Xa (final concentration, 0.83 unit/ml), cephalin (lipid source), and Ca2+ to activate Factor IX in the first incubation mixture of the assay. Two types of first incubation mixture were prepared: both contained Factor Xa, lipid, and Ca2+. One also contained Factor IX (6.5 units/ml) and the other contained TBS (control). At varying incubation times, subsamples from these incubation mixtures were added to the second incubation mixture and, after 3 min, the Factor Xa activity of the second incubation mixture was measured. As shown in Fig. 1, the amount of Factor Xa activity in the second incubation mixture was independent of the presence or absence of Factor IX in the first incubation mixture. It reflected only the carry-over of minimal Factor Xa activity from the first incubation mixture. Similar negative results were obtained when a commercial cephalin (Sigma) or frozen-and-thawed platelets (18) were used as the source of phospholipid. An experiment was also carried out to examine whether Factor IXa itself can activate Factor IX in the presence of phospholipid and Ca2+. Because the Factor IXa was prepared by activating native Factor IX with Factor XIa, a Factor XI antibody was added to some incubation mixtures to minimize error due to failure of ion exchange chromatography to remove Factor XIa completely. As shown in Table 1, the Factor IXa preparation failed to activate notable amounts of native Factor IX in incubation mixtures containing the antibody. This experiment was repeated with the addition of thrombin-activated Factor VIII to the first incubation mixture and again yielded negative results.

3 5262 Biochemistry: 0sterud and Rapaport Table 1. Present in incubation mixture 1 Failure of Factor IXa to activate Factor IX Factor Xa (units/ml) formed in incubation mixture 2 on addition of: XI 10-min 25-min IXa IX antibody subsample subsample _ Incubation mixture 1 contained 10 Al of IXa (6.7 units/ml), 10 Al of IX (6.5 units/ml) or TBS, 10 Ml of a 1:200 dilution in TBS of a Factor XI antiserum or TBS, 5 Al of cephalin, and 3 Al of 50 mm CaCl2. After 10 and 25 min of incubation at 370, 2-Ml subsamples were added to incubation mixture 2 (see legend to Fig. 1). Activation of Factor IX by the Reaction Product of Tissue Factor and Factor VII. In repeated experiments, incubation mixtures of tissue factor, Factor VII, Factor IX, and Ca2+ consistently generated Factor IXa activity. The data from one experiment, with its accompanying controls, are summarized in Table 2. When all reactants were present in the incubation mixture 1, potent Factor IXa activity was formed, as indicated by full activation of Factor X in mixture 2. When Factor IX was left out of mixture 1, only about 10% of the Factor X of mixture 2 was converted to Factor Xa, presumably reflecting the direct action of the reaction product of tissue factor and Factor VII on Factor X. Neither Factor VII nor tissue factor, acting alone, could generate Factor IXa activity in mixture 1; consequently, Factor Xa activity failed to form in mixture 2. Ca2+ was required to generate Factor IXa activity [more factor Xa activity (0.29 unit/ml) was formed in this control experiment than in the control experiment without Factor IX (0.09 unit/ml), presumably because of carry-over of tissue factor, Factor VII, and Factor IX into mixture 2, which contained Ca2+I. The activity generated when all reactants were present in mixture 1 failed to generate Factor Xa in the absence of Factor VIII in incubation mixture 2. Finally, no Factor X was present as a Table 2. Evidence that mixtures of tissue factor, Factor VII, Factor IX, and Ca2+ generate Factor IXa Factor Xa Present in Present in (units/ml) incubation incubation formed in mixture 1 mixture 2 incubation IX VII TF* Ca2+ VIII X mixture Incubation mixture 1 contained 10 Ml of IX (15.4 units/ml), 10 Ml of VII (0.20 unit/ml), 5 Ml of tissue factor, and 3 Ml of 50 mm CaCl2. When a reactant was left out, it was replaced with an equal volume of TBS. After 10 min at 370,150,l of incubation mixture 2 was added to incubation mixture 1. When VIII or X was left out of incubation mixture 2, it was replaced with 50 Ml of TBS. After 3 min of further incubation, the reaction was stopped with EDTA and a subsample was removed for Xa assay. Details are given in the legend to Fig. 1. * TF, tissue factor. E. '. 0.4 E0. C x~0.4 X u0 Proc. Natl. Acad. Sci. USA 74 (1977) - I I I I 7 j~~~~~~~ Mixture 1 incubation time, min FIG. 2. Evidence for a time-consuming reaction between the product of the interaction of tissue factor and Factor VII and Factor IX in the generation of Factor IXa. The first incubation contained 10 gl of Factor VII (0.20 unit/ml), 5,ul of thromboplastin, 10 Ml of Factor IX (6.5 units/ml), and 3 Ml of 50 mm CaCl2. In one experiment, all reagents were added together (0). In a second experiment, Factor VII, thromboplastin, and CaCl2 were incubated together for 6 min before addition of Factor IX (@). In a third control experiment, 10 MAl of TBS was substituted for Factor IX (A). Times on the abscissa represent incubation times of the completed mixtures. At the times shown, 10 Ml from incubation mixture 1 was added to a second incubation mixture. Further details are as described in the legend to Fig. 1. contaminant of the reactants of the incubation mixture 1; this last set of conditions also confirms the specificity of the chromogenic substrate for Factor Xa. Subsamples of a first incubation mixture containing tissue factor, Factor VII, Factor IX, and Ca2+ developed increasing ability over time to support the generation of Factor Xa in the second incubation mixture (Fig. 2). Because tissue factor and Factor VII are known to interact in a time-consuming reaction (19), this experiment was repeated with preliminary incubation of tissue factor, Factor VII, and Ca2+ for 6 min before the addition of Factor IX. A similar time-consuming reaction was demonstrable under these conditions. The effect of a Factor IX antiserum on the activity formed in the first incubation mixture was evaluated. When tissue factor, Factor VII, Factor IX, and Ca2+ were incubated together for 10 min and then a Factor IX antiserum was added, the mixture lost its ability to support the generation of notable amounts of Factor Xa (0.67 unit/ml without antibody; 0.06 unit/ml with antibody). Finally, an experiment was performed to test whether the tissue factor-factor VII activation of Factor IX contributed significantly to the total amount of Factor Xa generated when, simultaneously, the tissue factor-factor VII interaction was also directly activating Factor X. Incubation mixtures were made up containing Factor X at a final concentration of 0.48 unit/ml (0.95,tg of protein) and either control TBS buffer or Factor IX at a final concentration of 0.84 unit/ml (0.5,ug of protein). The other reactants were as described in Table 3. Under these experimental conditions, about twice as much Factor Xa was generated within 7 min when tissue factor-factor VII acted upon both Factor IX and Factor X as when tissue factor-factor VII acted upon Factor X alone (Table 3). Electrophoretic Evidence that the Reaction Product of Tissue Factor and Factor VII Activates Factor IX. Na- DodSO4/polyacrylamide gel electrophoresis of an incubation mixture containing all reactants-tissue factor, Factor VII, Factor IX, and a2+--demonstrated replacement of the protein band of native Factor IX [molecular weight (Mr) 55,000] by a protein band corresponding to the heavy chain of Factor IXa (Mr 27,000) and a very weak protein band corresponding to the

4 Biochemistry: Osterud and Rapaport Table 3. Evidence that tissue factor-factor VII activation of both Factors IX and X yields more Factor Xa activity than tissue factor-factor VII activation of Factor X alone Factor X. Present in incubation mixture (units/ml) VII TF X IX VIII formed Fifty microliters of VIII (4 units/ml) or TBS was incubated with 10 A1 of thrombin (0.1 unit/ml) for 3 min at 40 and then 20 Al ofx (2.7 units/ml), 10 Al of IX (10 units/ml) or TBS, 3Al of tissue factor (TF), 5 Al of VII (0.15 unit/ml), 10 Al of cephalin, and 10 Ml of 50 mm CaCi2 were added. The whole incubation mixture was incubated for 7 min at 37. Then, 10 Ml of 0.3 M Na2EDTA was added to stop the reaction and Xa activity was measured as described in the legend to Fig. 1. light chain of Factor IXa (Mr 17,000). Gel C of Fig. 3 illustrates this; the weak light chain band, visible on direct inspection, cannot be seen in the reproduction. In contrast, electrophoresis of incubation mixtures in which either Ca2+ (gel A) or Factor VII (gel B) was left out demonstrated an unchanged band of native Factor IX and no evidence of the characteristic heavy chain protein band of Factor IXa. DISCUSSION The above results establish that the reaction product of tissue factor, Factor VII, and Ca2+ can activate purified human Factor IX. This was shown in two ways: first, by demonstrating that an incubation mixture of tissue factor, Factor VII, Factor IX, and Ca2+ generated Factor IXa clotting activity; and second, by demonstrating on NaDodSO4/polyacrylamide gel electrophoresis of such a mixture that the band of native Factor IX was replaced by the heavy and light chain bands of Factor IXa. The reported feedback activation of Factor IX by Factor Xa (4) would complicate interpretation of oar data. As shown in Fig. 1, human Factor Xa, cephalin, and CA2+ could not activate human Factor IX under our conditions. These experiments ruled out possible error due to generation of Factor IXa by Factor Xa in the second incubation mixture. They also established that activation of Factor IX by Factor Xa does not play a significant role in human blood coagulation. It was also crucial to determine whether Factor IXa itself could activate Factor IX. If so, then a material producing only a minimal initial conversion of Factor IX to Factor IXa in the first incubation mixture could be interpreted as a potent activator of Factor IX. However, when Factor IXa reagent was used as the test material in the first incubation mixture, it made no significant difference whether the mixture contained a large amount of native Factor IX or control buffer (Table 1). The modest amount of Factor Xa generated in the second incubation mixture under both conditions reflected only carry-over of the Factor IXa reagent into the second mixture. Thus, Factor IXa could not activate Factor IX, and the assay reflected only the capability of the test material of a first incubation mixture to activate Factor IX. With the assay validated, it was possible to conclude, unequivocally, that mixtures of tissue factor and Factor VII in the presence of Ca2+ can function as a potent Factor IX activator (Table 2; Fig. 2). In contrast, mixtures containing either tissue factor without Factor VII or Factor VII without tissue factor were inert. The product of the tissue factor-factor VII reaction interacted with Factor IX in a time-consuming reaction (Fig. Proc. Natl. Acad. Sci. USA 74 (1977) 5263 * _rn 55,000 A B C 27,OO0 FIG. 3. NaDodSO4/polyacrylamide gel electrophoresis of the activation of Factor IX by the reaction product of tissue factor and Factor VII in the presence of Ca2+. Gels are shown from three incubation mixtures. Each mixture contained 150 Ml of Factor IX (6.5 units/ml). The incubation mixture of gel C also contained 20 JAI of factor VII (0.20 unit/ml), 20 Ml of tissue factor (from which soluble protein had been removed by centrifugation at 20,000 X g and resuspension of the pellet in TBS for a total of five times), and 20 Ml of 50 mm CaCI2. The incubation mixture of gel A contained 20 Ml of TBS in place of the CaCI2. The incubation mixture of gel B contained 20 Al of TBS in place of Factor VII. After 30-min incubation at 370, 10 Ml of 0.3 M EDTA was added to each mixture, which was then centrifuged at 20,000 X g for 10 min. The supernatants were subjected to electrophoresis. 2). As expected of Factor IXa, the final product of the interaction of tissue factor, Factor VII, Factor IX, and Ca2+ required Factor VIII to generate Factor Xa in a second incubation mixture (Table 2) and was inhibited by a Factor IX antiserum. The experiments with NaDodSO4/polyacrylamide gel electrophoresis, illustrated in Fig. 3, provided further strong direct data that Factor IXa forms in incubation mixtures of tissue factor, Factor VII, Factor IX, and Ca2+. When all components were present, electrophoresis revealed that a band corresponding to the position of native Factor IX was replaced by bands corresponding to the positions of the heavy and light chains of Factor IXa (20). However, when incubation mixtures lacked either Ca2+ or Factor VII, the band corresponding to native Factor IX persisted, and no bands appeared corresponding to the heavy and light chains of Factor IXa. Thus, both activity assays and electrophoresis yielded direct evidence that the reaction product of tissue factor and Factor VII in the presence of Ca2+ activates Factor IX. Thus, the interaction of the reaction product of tissue factor and Factor VII with Factor IX represents an alternate pathway, independent of Factor XIa, for initiating "intrinsic" clotting. Activation of Factor IX by this mechanism can be as rapid and complete as activation of Factor IX by Factor XIa (compare Figs. 1 and 2). Moreover, the tissue factor-factor VII activation of Factor IX can increase significantly the generation of Factor Xa under experimental conditions in which the tissue factor- Factor VII reaction product is simultaneously directly activating Factor X (Table 3). Thus, activation by the reaction product of tissue factor and Factor VII could well play a meaningful role in the activation of Factor IX during hemostasis. It could partly explain the striking difference between the mild bleeding of

5 5264 Biochemistry: 0sterud and Rapaport patients with hereditary Factor XI deficiency and the severe bleeding of patients with hereditary Factor IX deficiency. We thank Mrs. K. K. Lavine and Mr. S. Brown for their technical assistance. This work was supported by U.S. Public Health Service Program Project Grants HL and -02 from the National Heart, Lung, and Blood Institute, National Institutes of Health. 1. Biggs, R. & Nossel, H. L. (1961) Thromb. Diath. Haemorrh. 6, Josso, F. & Prou-Wartelle, 0. (1965) Thromb. Diath. Haemorrh. Suppl. 17, Rapaport, S. I., Hjort, P. F., Patch, M. J. & Jeremic, M. (1966) Scand. J. Haematol. 3, Kalousek, F., Koningsberg, W. & Nemerson, Y. (1975) FEBS Lett.. 50, Owren, P. A. (1949) Scand. J. Clin. Lab. Invest. 1, Bell, W. N. & Alton, H. G. (1954) Nature 174, Kasper, C. K., 0sterud, B., Minami, J. Y., Shonick, W. & Rapaport, S. I. (1977) Blood 50, sterud, B. & Flengsrud, R. (1975) Biochem. J. 145, Fujikawa, K., Thompson, A. R., Legaz, M. E., Meyer, R. G. & Davie, E. W. (1973) Biochemistry 12, Proc. Nati. Acad. Sci. USA 74 (1977) 10. 0rstavik, K. H., 0sterud, B., Prydz, H. & Berg, K. (1975) Thromb. Res. 7, Nossel, H. L. (1964) The Contact Phase of Blood Coagulation (Blackwell Scientific Publications, Oxford). 12. Rapaport, S. I., Schiffman, S., Patch, M. J. & Ames, S. B. (1963) Blood 21, Thompson, A. R. & Davie, E. W. (1971) Biochim. Biophys. Acta 250, sterud, B. & Schiffman, S. (1972) Thromb. Diath. Haemorrh. 28, Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, Schiffman, S. I., Rapaport, S. I. & Theodor, I. (1968) Biochemistry 8, Weber, K., Pringle, J. R. & Osborn, M. (1972) in Methods in Enzymology, eds., Hirsh, C. H. W. & Timasheff, S. N. (Academic Press, New York), Vol. XXVI, Part C, pp sterud, B., Lavine, K. K. & Rapaport, S. I. (1977) Blood 49, sterud, B., Otnaess, A-B., Bjorklid, E. & Prydz, H. (1972) Biochemistry 11, Fujikawa, K., Legaz, M. E., Kato, H. & Davie, E. W. (1976) Biochemistry 13,

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