Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 958-961, February 1979 Medical Sciences Interactions among Hageman factor, plasma prekallikrein, high molecular weight kininogen, and plasma thromboplastin antecedent (Factor XII/Fletcher factor/Fitzgerald factor/Factor XI) OSCAR D. RATNOFF AND HIDEHIKO SAITO Department of Medicine, The School of Medicine, Case Western Reserve University, Cleveland, Ohio; and University Hospitals of Cleveland, Cleveland, Ohio 44106 Contributed by Oscar D. Ratnoff, December 1, 1978

ABSTRACT To investigate the earliest steps of the intrinsic M sodium citrate, and bovine PTA-deficient plasma was from clotting pathway, Hageman factor (Factor XII) was exposed to blood containing 1/9th vol of 0.13 M sodium citrate buffer (pH Sephadex gels to which ellagic acid had been adsorbed; Hage- 5.0). A standard pool of normal adult plasmas (14) was said to man factor was then separated from the gels and studied in the 1 and PTA. fluid phase. Sephadex-ellagic acid-exposed Hageman factor, contain unit/ml each of HF, PK, HMWK, whether purified or in plasma, activated plasma thromboplastin Plasmas simultaneously deficient in PTA and other factors antecedent, but only when high molecular weight kininogen were prepared by incubating Fletcher or Fitzgerald trait was present. In the absence of plasma prekallikrein, maximal plasmas for 1 hr at 370C with specific immunoglobulins. Plas- activation of plasma thromboplastin antecedent was slightly mas simultaneously deficient in PK and other factors were delayed in plasma, a delay not observed with similarly treated prepared from Fletcher trait plasma. Absorption with specific purified Hageman factor. Thus, high molecular weight kini- immunoglobulins removed more than 99% of PTA and PK, and nogen was needed for expression of Hageman factor's clot- more than 98% of HMWK and plasminogen; immunoabsorp- promoting properties and plasma prekallikrein played a minor role in the interaction of ellagic acid-treated Hageman factor tion did not bring about activation of HF. The mixtures were and plasma thromboplastin antecedent. adsorbed with 1/50th vol of Cy-alumina gel (Calbiochem) for 10 min at room temperature to remove vitamin K-dependent Plasma exposed to negatively charged agents clots through clotting factors, centrifuged at 2000 X g for 10 min to sediment reactions of the intrinsic pathway of thrombin formation. The the Cy-alumina gel and insoluble antigen-antibody precipitates, first step in this pathway is activation of Hageman factor (HF, and stored at -70'C until used. Factor XII). Indirect evidence implies that negatively charged Plasmas simultaneously deficient in HF and other clotting substances bring about a conformational change in HF needed factors were prepared by immunoabsorption of Fletcher or for its clot-promoting activity (1-3). Recent studies suggest that Fitzgerald trait plasmas on columns of insoluble immuno- activation of human (4) and bovine (5) HF also involves its globulin against HF covalently bonded to cyanogen bromide- proteolytic scission within an internal disulfide loop. Once ac- activated agarose (Pharmacia) (15) and elution with barbital/ tivated, HF activates plasma thromboplastin antecedent (PTA, saline buffer (0.025 M sodium barbital in 0.125 M sodium Factor XI). The roles of plasma prekallikrein (PK, Fletcher chloride, pH 7.5). (Unless otherwise noted, the term "buffer" factor) and high molecular weight kininogen (HMWK, Fitz- refers to this buffer.) The contents of tubes containing protein gerald, Williams, Flaujeac, or Reid factor) in the intrinsic were pooled, 1/50th vol of 0.5 M sodium citrate buffer (pH 5.0) pathway are not fully elucidated. This pathway is impaired in was added, and the solution was concentrated to its original plasmas deficient in either PK (6) or HMWK (7). In purified volume by ultrafiltration through an Amicon PM-10 membrane systems, however, while activation of PTA requires the presence (Amicon Corporation, Lexington, MA) at 4VC. Simultaneous of HMWK (8-11), PK is not a necessary reactant (12, 13). One deficiency of HF, PK, and plasminogen was produced by in- explanation for the difference between plasma and purified cubation of immunoglobulin against plasminogen with Fletcher HF thus obscuring trait plasma for 1 hr at 370C before filtration through insoluble systems is that is altered during purification, anti-HF. Alternatively, plasma was depleted of more than 98% a need for PK. of plasminogen by filtration through lysine-agarose (16). The We have reexamined the early steps in the intrinsic pathway treated plasmas, containing no detectable HF, were stored at to determine whether activation of HF in plasma requires PK -70°C in silicone-coated polyethylene containers until used. or HMWK. Plasma containing HF, or purified HF, acquired A crude immunoglobulin fraction was separated from normal clot-promoting properties upon exposure to ellagic acid ad- rabbit serum or from monospecific rabbit antisera to human sorbed to Sephadex gels. Expression of these properties required HF (17), PTA (18), kallikrein (19), HMWK (13), and plas- the presence of HMWK but not PK. minogen (Behring, Sommerville, NJ) (20). The volume of an- tiserum needed was determined by incubating dilutions of MATERIALS AND METHODS immunoglobulin in buffer with equal volumes of pooled plasma Normal human plasma and plasma from individuals with for 1 hr at 37°C and measuring the residual titer of the specific Hageman trait, Fitzgerald trait (HMWK deficiency), and PTA factor. deficiency were separated from venous blood containing 1/50th Ellagic acid, synthesized by James D. Crum (21), was dis- vol of 0.5 M sodium citrate buffer (pH 5.0) (14). Fletcher trait solved at 0.1 mM in 0.05 M glycine buffer (pH 10.0), adjusted (PK deficiency) and Christmas disease (Factor IX deficiency) to pH 7.5 with 1 M sodium acetate buffer (pH 4.8), and filtered plasmas were prepared from blood containing 1/9th vol of 0.13 through Whatman no. 1 paper, leaving a faint yellow-green residue. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: HF, Hageman factor; PTA, plasma thromboplastin vertisement" in accordance with 18 U. S. C. §1734 solely to indicate antecedent; PK, plasma prekallikrein; HMWK, high molecular weight this fact. kininogen; CT, clotting time; PTT, partial thromboplastin time. 958 Downloaded by guest on September 28, 2021 Medical Sciences: Ratnoff and Saito Proc. Natl. Acad. Sci. USA 76 (1979) 959

Sephadex-ellagic acid was prepared by swelling Sephadex Table 1. Effect of Sephadex-ellagic acid upon the coagulant G-10, G-15, G-25, G-100, G-150 or G-200 (Pharmacia) in'A3 vol properties of HF in plasma of buffer for 1 hr. The supernatant was decanted and the pro- Addition to Clotting activity cess was repeated twice, with 0.02% sodium azide added to the PTA-deficient Centrolex Kaolin/Centrolex third wash. The swollen Sephadex was stored at 40C until used. plasma Sec Units/ml Sec Units/ml Ten milliliters settled volume of Sephadex was mixed with 30 ml of 0.1 mM ellagic acid, stirred for several min, and allowed Sephadex-ellagic acid 80.6 0.48 77.9 0.57 to settle, and the supernatant was decanted. Approximately 25% Sephadex-buffer >300 <0.01 63.5 > 1.0 of the ellagic acid was adsorbed to the Sephadex, as estimated Two milliliters of Sephadex G-10-ellagic acid or Sephadex-buffer, by the clot-promoting properties of the supernatant after cen- washed 15 times, was mixed with 2.0 ml of human PTA-deficient trifugation at 2000 X g for 10 min, relative to fresh solutions plasma, then diluted to 4 ml with-buffer and centrifuged, and a sample of ellagic acid, using the partial thromboplastin time (PTT) of of the supernatant was tested on HF-deficient substrate plasma normal plasma as an indicator. The Sephadex-ellagic acid was without further dilution for activated HF (Centrolex added) or total supernatant HF (kaolin/Centrolex added), -incubating the final mixture 8 min washed 6-15 times with 100 ml of buffer, with the before recalcification. Clotting activity was compared to that ofpooled discarded each time after the gel had settled; the last buffer normal plasma that was diluted serially with buffer and assayed for contained 0.02% sodium azide. Sephadex-ellagic acid retained total HF without exposure to Sephadex-ellagic acid or Sephadex- its clot-promoting properties for at least a month at 4VC. The buffer. Clotting activity is expressed both as CT (in sec) and in units 15th wash contained less than 0.1 sM ellagic acid, measured of HF/ml in comparison to the standard. The PTA-deficient plasma by the PTT after centrifugation. Sephadex-buffer was prepared had a titer of 0.78 unit of HF/ml. in the same way except for substitution of buffer for ellagic acid. observed when the treated PTA-deficient plasma was incubated Purified HF (22) varied in specific activity from 63 to 83 with HF-deficient plasma for 8 min before recalcification units/mg of protein. Partially purified PK contained 1.25 (Table 1). The same result was obtained with bovine PTA- coagulant units of PK per ml (3.2 units/mg of protein) and no deficient plasma and also when the supernatant, diluted plasma detectable contamination with other clotting factors; the bulk was recentrifuged at 30,000 X g for 15 min to ensure that no of extraneous protein was IgG (13). Sephadex-ellagic acid was transferred to the substrate plasma. Bovine serum albumin (crystallized, Pentex, Miles) was Sephadex G-25-, G-100-, G-150-, and G-200-ellagic acid were dissolved at a concentration of 1% in buffer. successively less effective, while Sephadex-buffer, without el- Clotting factors in plasma were titrated by a modified ka- lagic acid, was not clot-promoting. No superiority for either olin-activated PTT. The assays detected as little as 0.001 Sephadex G-10 or G-15 was observed. unit/ml of HF, PTA, or PK, and 0.01 unit/ml of HMWK. When human PTA-deficient plasma was mixed with HF was "activated" by stirring plasma containing HF with Sephadex-ellagic acid and separated from the gel by centrifu- 10 vol of settled Sephadex-ellagic acid in polystyrene tubes at gation, the titer of HF in the supernatant plasma was often room temperature 2 min. After standing 5 min, 9 vol buffer substantially less than in the original plasma; essentially all was were added, diluting the plasma approximately 1:20. The activated. The gel no longer activated HF after two-successive mixture was restirred for 2 min and allowed to settle for 1 min. additions of human PTA-deficient plasma. At the same time, The supernatant, diluted plasma was centrifuged in a poly- the titer of HF in the supernatant plasma increased as if no propylene tube at 30,000 X g for 15 min and tested for clot- further sites for binding or activating HF were available. promoting activity by incubating 0.1-ml samples in 10 X 75 mm The action of Sephadex-ellagic acid did not seem to reflect polystyrene tubes with 0.1 ml of 0.1% crude soybean phos- elution of ellagic acid during its exposure to plasma. Human phatides (Centrolex "O," the gift of Central Soya Company, PTA-deficient plasma was mixed with Sephadex G-10-ellagic Chicago) in 0.15 M sodium chloride for 2 min at 37°C. acid. After centrifugation, the supernatant plasma 'was incu- Thereafter, 0.1 ml of HF-deficient plasma was added and in- bated at 60°C for 60 min. Such plasma did not promote clotting cubation was continued for periods up to 32 min. Prewarmed of HF-deficient plasma. Its clot-promoting properties for 0.025 M calcium chloride (0.1 ml) was added and the clotting normal plasma were comparable to those of 0.3 ,uM ellagic acid, time (CT) was measured at 37°C by continual tilting. In control as tested by its effect upon the PTT of normal plasma. The experiments, plasma was mixed with Sephadex-buffer. The heated plasma did not inhibit the action of additional ellagic total available HF in the supernatant, diluted plasmas exposed acid. Similar results were obtained when HF-deficient plasma to Sephadex-ellagic acid or Sephadex-buffer was estimated by was exposed to Sephadex G-10-ellagic acid and tested in the substituting 0.1 ml of kaolin/Centrolex (10 mg/ml of 0.1% same way without heating. Further, addition of ellagic acid at Centrolex) for Centrolex. The titer of HF was compared to that a final concentration of 10,uM to human PTA-deficient plasma of pooled normal plasma that had been diluted serially in buffer that had been mixed with Sephadex-buffer had only trivial and tested for total available HF. clot-promoting activity, as tested upon a substrate of HF-de- Purified HF was "activated" by substituting it for plasma ficient plasma. Thus, the clot-promoting action of Sephadex- in the method described in the preceding paragraph, usually ellagic acid-treated plasma was not due to elution of sufficient preceded by addition of an equal volume of 1% bovine serum free ellagic acid to influence the assays. Whether some ellagic albumin. acid was bound to HF under these conditions is not known. The clot-promoting activity of PTA-deficient plasma exposed RESULTS to Sephadex-ellagic acid seemed attributable to HF. No such properties generated when HF-deficient plasma was mixed Effect of Sephadex-ellagic acid upon the coagulant with Sephadex-ellagic acid and tested upon plasmas deficient properties of HF in plasma in HF, PTA, or Christmas factor. Moreover, PTA-deficient Human PTA-deficient plasma was mixed with Sephadex G-10- plasma, similarly treated, did not shorten the CT of plasmas or G-15-ellagic acid, diluted with buffer, and separated from deficient in PTA or Christmas factor. Thus, clot-promoting the gel by centrifugation. Such plasma shortened the prolonged activity depended upon the presence of HF in plasma, and was modified PTT of HF-deficient plasma; the minimal CT was recognized only if the substrate contained PTA. Clotting factors Downloaded by guest on September 28, 2021 960 Medical Sciences: Ratnoff and Saito Proc. Natl. Acad. Sci. USA 76 (1979) acting subsequent to participation of PTA in the intrinsic pathway did not appear to have been activated in the absence >300. of PTA. I PTA-deficient plasma contains normal amounts of HF, PK, 240J and HMWK. Activity attributable to HF, comparable to that co in PTA-deficient plasma, was demonstrated in PK- or ' 160 HMWK-deficient plasmas treated with Sephadex-ellagic acid, as though these factors were not needed to alter the behavior of HF in the initial mixture. In the experiments described thus far, plasma that had been 80120 exposed to Sephadex-ellagic acid was incubated with HF- 60 deficient plasma for 8 min before the PTT was measured. 1 2 4 8 16 32 HF-deficient plasma contains both PK and HMWK. PK-defi- Preliminary incubation time, min cient plasma, depleted of PTA, HMWK, and vitamin K-de- FIG. 2. Evolution of coagulant activity in purified HF mixed with pendent clotting factors and exposed to Sephadex G-10-ellagic Sephadex-ellagic acid. Purified HF (0.2 ml) [2.5 units (30 ,gg of pro- acid, corrected the clotting defect of plasmas deficient in HF tein) per ml] was substituted for plasma in the protocol described for alone or deficient in HF and PK, but not that of plasma defi- Fig. 1. Symbols are as for Fig. 1. cient in HF and HMWK (Fig. 1). Similar results were obtained when HMWK-deficient plasma was depleted of PTA and PK then separated from the gel. HF thus treated gradually short- before exposure to Sephadex-ellagic acid. In repeated experi- ened the clotting time of plasma deficient in HF alone or in HF ments, the CT of the substrate plasma, a reflection of the rate and PK, but was without clot-promoting activity when the of activation of PTA, reached its nadir more slowly in substrates substrate was deficient in HF and HMWK (Fig. 2). The yield deficient in HF and PK compared to plasma deficient in HF of activated HF was consistently low, about 10% of the HF alone; the ultimate clot-promoting activity generated was often applied; almost all was in the activated form. greater. In experiments not illustrated, addition of purified PK Effect of depletion of plasminogen on the coagulant (1 clotting unit/ml) to a substrate deficient in HF and PK cor- properties of HF rected this minor abnormality. Thus, the clot-promoting activity evolving in mixtures of When HF is incubated with plasmin, a 30,000 molecular weight Sephadex-ellagic acid and plasmas that contained HF was ex- fragment is separated that readily activates PK but is only pressed only when HMWK was present. Whether HMWK in- weakly procoagulant (23). Addition of immunoglobulin against duced a further change in HF or was needed for activated HF plasminogen to normal plasma did not impede generation of to act upon PTA was not clarified. The presence of PK in the clot-promoting activity by Sephadex-ellagic acid. Similar results substrate had a minor influence upon the rate of generation of were observed when plasma containing HF (but depleted of clot-promoting properties, suggesting its role in the activation PTA, PK, HMWK, and plasminogen) was tested upon a sub- of PTA in plasma. strate containing PTA and HMWK (but deficient in HF, PK, and plasminogen). Relatively little total HF was eluted under Effect of Sephadex-ellagic acid upon the coagulant these conditions from Sephadex-ellagic acid; essentially all was properties of purified HF procoagulant, and maximal activation of the substrate plasma Like HF in plasma, purified HF preparations acquired clot- was observed within 16 min. Similar results were obtained when when mixed with acid and purified HF that had been incubated with anti-plasminogen promoting activity Sephadex-ellagic and mixed with Sephadex-ellagic acid was tested on substrates of plasma deficient in HF or HF, PK, and plasminogen (plas- minogen neutralized by specific antiserum or by filtration through lysine-agarose). >3001 , I These several experiments do not support the view that 2404 plasminogen is essential for activation of HF by Sephadex-el- lagic acid. u 160- 0) DISCUSSION 120- The present study provides a fresh approach to study of the initial steps of the intrinsic pathway. In normal plasma, exposure 80- of HF to activators such as glass, kaolin, or ellagic acid brings about activation of PTA. Two additional agents, PK and 60 HMWK, functionally deficient in individuals with Fletcher trait 2 4 8 16 and Fitzgerald trait (Williams trait, Flaujeac trait, Reid trait) Preliminary incubation time, min respectively, are implicated in the activation of PTA. The FIG. 1. Evolution of coagulant activity in depleted plasmas mixed coagulant defect in Fletcher trait is corrected by purified PK with Sephadex-ellagic acid. Two milliliters of Sephadex G-10-ellagic (24, 25) or kallikrein (22) and in Fitzgerald trait, by HMWK acid, washed 15 times, was mixed with 0.44 ml ofPK-deficient plasma, absorbed with rabbit immunoglobulins against HMWK and PTA and (7, 26-28). The prolonged activated PTT shortens when PK- adsorbed with C-y-alumina gel (equivalent to 0.2 ml of PK-deficient deficient plasma is exposed for a brief time to glass (6), a phe- plasma), diluted to 4 ml with buffer, and centrifuged. Samples of nomenon not observed in Fitzgerald trait plasma (7). Thus, in supernatant (0.1 ml) were mixed without further dilution with 0.1 ml plasma, HMWK, but not PK, seems required for activation of of Centrolex and 0.1 ml of substrate plasma and incubated at 370C PTA by HF. for 1-32 min before addition of 0.1 ml 0.025 M calcium chloride, and Whether PK and HMWK are needed to convert HF to an the CT was measured at 370C. The substrate plasmas were deficient In in HF alone (0), HF and prekallikrein (0), or HF and HMWK enzymatically active state is disputed (4, 9-11, 13, 29). pu- (A). rified systems, HMWK must be present for activation of PTA Downloaded by guest on September 28, 2021 Medical Sciences: Ratnoff and Saito Proc. Natl. Acad. Sci. USA 76 (1979) 961

by HF and kaolin (8) or ellagic acid (13). HMWK apparently 3. Fair, B. D., Saito, H., Ratnoff, 0. D. & Rippon, W. B. (1977) Proc. acts stoichiometrically (8, 9), but whether to "adtivate&'MIr Soc. Exp. Biol. Med. 155, 199-202. to allow enzymatically active HF to act upon PTA is unclear. 4. Revak, S. D., Cochrane, C. G. & Griffin, J. H. (1977) J. Clin. Despite contrary evidence (4, 11), PK is not required for acti- Invest. 59, 1167-1175. PTA in 5. Fujikawa, K., Kurachi, K. & Davie, E. W. (1977) Biochemistry vation of purified systems (12, 13). Enzymatically active 16, 4182-4188. fragments of HF, separated by tryptic digestion, activate PTA 6. Hathaway, W. E., Belhasen, L. P. & Hathaway, H. S. (1965) weakly in the absence of PK or HMWK (13). This observation Blood 26,521-532. does not illuminate the site of action of PK or HMWK. A further 7. Saito, H., Ratnoff, 0. D., Waldmann, R. & Abraham, J. P. (1975) uncertainty is whether in plasma, as opposed to purified sys- J. Clin. Invest. 55, 1082-1089. tems, PK is necessary for the activation or action of HF. 8. Schiffman, S. & Lee, P. (1975) J. Clin. Invest. 56, 1082-1092. In the present experiments, HF was activated in plasma to 9. Griffin, J. H. & Cochrane, C. G. (1976) Proc. Natl. Acad. Sci. USA avoid its alteration during purification. We took advantage of 73,2554-2558. the adsorption of ellagic acid to Sephadex gels (21). Plasmas 10. Webster, M. E., Guimaraes, J. A., Kaplan, A. P., Colman, R. W. & Pierce, J. V. (1976) Adv. Exp. Med. Biol. 70, 285-299. containing HF and exposed to Sephadex-ellagic acid gels cor- 11. Meier, H. L., Pierce, J. V., Colman, R. W. & Kaplan, A. P. (1977) rected the clotting defect of HF-deficient plasma. The alter- J. Clin. Invest. 60, 18-31. ation in HF brought about by Sephadex-ellagic acid occurred 12. Schiffman, S., Pecci, R. & Lee, P. (1977) Thromb. Res. 10, in plasmas lacking PTA, PK, or HMWK, but its recognition 319-323. required the presence of HMWK, but not PK, in the HF-defi- 13. Saito, H. (1977) J. Clin. Invest. 60,584-594. cient substrate plasma, as had been reported for purified sys- 14. Zimmerman, T. S., Ratnoff, 0. D. & Powell, A. E. (1971) J. Clin. tems (13). Generation of clot-promoting properties in the sub- Invest. 50, 244-254. strate was slower in the absence of PK, as noted by Schiffman 15. Cuatrecasas, P., Wilchek, M. & Anfinsen, C. B. (1968) Proc. Nati. Lee PK Acad. Sci. USA 61, 636-643. and (8), a delay corrected by addition of to the substrate. 16. Deutsch, D. G. & Mertz, E. T. (1970) Science 170, 1095-1096. Thus, our earlier studies on the role of HMWK and PK in the 17. Saito, H., Ratnoff, 0. D., Donaldson, V. H., Harvey, G. T. & activation of PTA in purified systems were not vitiated by un- Pensky, J. (1974) J. Lab. CGun. Med. 84, 62-73. suspected changes in HF during purification. 18. Saito, H. & Goldsmith, G. H. (1977) Blood 50,377-385. These experiments are consistent with the view that HMWK 19. Saito, H. & Ratnoff, 0. D. (1974) Nature (London) 248,597- is needed for expression of the coagulant properties of activated 598. HF rather than for activation of HF. In additional studies (30), 20. Ratnoff, 0. D. (1972) J. Lab. Clin. Med. 80, 704-710. evidence in support of this hypothesis is provided by demon- 21. Ratnoff, 0. D. & Crum, J. D. (1964) J. Lab. Clin. Med. 63, stration that purified HF, activated by Sephadex-ellagic acid 359-377. 22. Saito, H., Ratnoff, 0. D. & Donaldson, V. H. (1974) Circ. Res. in the apparent absence of other agents, had amidolytic prop- 31,641-651. erties. This activation was not related to detectable scission of 23. Kaplan, A. P. & Austen, K. F. (1971) J. Exp. Med. 133, 696- HF, as though the conformational changes induced by its ex- 712. posure to negatively charged agents were sufficient to elicit its 24. Weiss, A. S., Gallin, J. I. & Kaplan, A. P. (1974) J. Clin. Invest. enzymatic properties. 53,622-633. 25. Wuepper, K. D. (1972) in Inflammation: Mechanisms and These studies were ably assisted by Ms. Laura Stith. Dr. Robert Controls, eds. Lepow, I. H. & Ward, P. A. (Academic, New Waldmann kindly provided Fitzgerald trait plasma, and Dr. Gary York), pp. 93-117. Kociba, bovine PTA-deficient plasma. These studies were supported 26. Wuepper, K. D., Miller, D. R. & Lacombe, M. J. (1975) J. Clin. in part by Grants HL 01661 and HL 15195 from the National Heart, Invest. 56, 1663-1672. Lung and Blood Institute, The National Institutes of Health, U.S. Public 27. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F., Health Service, and in part by grants from the American Heart Asso- Skevey, M., Guimaraes, S. A., Pierce, J. V. & Kaplan, A. P. (1975) ciation and its Northeast Ohio Affiliate. O.D.R. is a Career Investigator J. Clin. Invest. 56, 1650-1689. of the American Heart Association. 28. Donaldson, V. H., Glueck, H. L., Miller, M. A., Movat, H. A. & Habal, F. M. (1976) J. Lab. Clin. Med. 87,327-337. 1. Donaldson, V. H. & Ratnoff, 0. D. (1965) Science 150, 754- 29. Chan, J. Y. C., Habal, F. M., Burrowes, C. E. & Movat, H. Z. 756. (1976) Thromb. Res. 9,423-433. 2. McMillin, C. R., Saito, H., Ratnoff, 0. D. & Walton, A. G. (1974) 30. Ratnoff, 0. D. & Saito, H. (1979) Proc. Natl. Acad. Sci. USA 76, J. Clin. Invest. 54, 1312-1322. in press. Downloaded by guest on September 28, 2021