Identification of prekallikrein and high-molecular-weight kininogen

Proc. Nati. Acad. Sci. USA Vol. 73, No. 11, pp. 4179-4183, November 1976 Immunology Identification of prekallikrein and high-molecular-weight kinino...
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Proc. Nati. Acad. Sci. USA

Vol. 73, No. 11, pp. 4179-4183, November 1976 Immunology

Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma (Hageman factor/bradykinin generation/contact activation)

ROBERT J. MANDLE*, ROBERT W. COLMANt, AND ALLEN P. KAPLAN** * Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014; and t The Coagulation Unit of the Hematology-Oncology Section, Department of Medicine, University of Pennsylvania, Philadelphia, Pa.

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Communicated by K. Frank Austen, August 19, 1976

ABSTRACT Prekallikrein and high-molecular-weight kininogen were found associated in normal human plasma at a molecular weight of 285,000, as assessed by gel filtration on Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was 115,000. This prekallikrein could be isolated at a molecular weight of 285,000 after plasma deficient in high-molecularweight kininogen was combined with plasma that is congenitally deficient in prekallikrein. Addition of purified 125I-labeled prekallikrein and high-molecular-weight kininogen to the respective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated by incubating prekallikrein with high-molecular weight kininogen. This study demonstrates that prekallikrein and highmolecular-weight kininogen are physically associated in plasma as a noncovalently linked complex and may therefore be adsorbed together during surface activation of Hageman factor. The complex is disrupted when these proteins are isolated by ion exchange chromatography. Activation of Hageman factor upon a negatively charged surface initiates the intrinsic coagulation pathway, the fibrinolytic pathway, and the generation of the vasoactive peptide bradykinin. Recent investigations from several laboratories have shown that the proteins of the kinin-forming pathway, namely, prekallikrein (1, 2) and high-molecular-weight (HMW) kininogen (3-5), are both required for optimal activation and function of Hageman factor. Since prekallikrein and HMW kininogen are intimately associated functionally, it appeared possible that they might be physically associated in plasma. In this paper we demonstrate that prekallikrein and HMW kininogen circulate in plasma as a noncovalently linked complex. Formation of this complex was observed: (a) when prekallikrein-deficient plasma was combined with plasma deficient in HMW kininogen, (b) after prekallikrein-deficient plasma and plasma deficient in HMW kininogen were reconstituted with prekallikrein and HMW kininogen, respectively, and (c) when purified prekallikrein was incubated with HMW kininogen.

MATERIALS AND METHODS Apoferritin and catalase (Calbiochem, San Diego, Calif.), blue dextran, ovalbumin, ribonuclease A, and chymotrypsinogen (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.), chloramine T (Matheson, Coleman and Bell Co., Norwood, Ohio), sodium[125I] iodide (New England Nuclear Co., Boston, Mass.), and human transferrin (Sigma Chemical Co., St. Louis, Mo.) were obtained as indicated. Plasma deficient in Hageman factor and containing 0.38% sodium citrate was obtained from Sera Tec Biologicals, New Brunswick, N.J. Prekallikrein-deficient plasma Abbreviation: HMW kininogen, high-molecular-weight kininogen. f To whom reprint requests should be addressed.

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(Fletcher trait) was a gift from Dr. C. Abildgaard (University of California, Davis, Calif.). Kininogen-deficient plasma was obtained from Ms. Williams and was collected as described (3). Preparation of Plasma Proteins. Fresh plasma used for the isolation of prekallikrein and HMW kininogen was collected in 0.38% sodium citrate. Hexadimethrine bromide (3.6 mg) in 0.1 ml of 0.15 M saline was added for each 10 ml of blood drawn. The tubes were then centrifuged at 900 X g for 20 min at 40 and the plasma was separated with plastic pipettes. Plastic columns and test tubes were used throughout the chromatographic procedures to minimize contact activation of Hageman factor and nonspecific adsorption to glass surfaces. Samples were concentrated by ultrafiltration (Amicon Corp., Lexington, Mass.) through a UM-10 membrane. Hageman Factor Fragments. Prealbumin fragments of Hageman factor were purified by chromatography of plasma on QAE-Sephadex twice, Sephadex G-100, and SP-Sephadex, and elution from alkaline disc gels after electrophoresis as re-

ported (6). Prekallikrein. Two liters of fresh plasma were dialyzed against 0.003 M phosphate buffer (pH 8.3) and passed over a 20 X 100 cm column of QAE-Sephadex equilibrated with the same buffer. The effluent, containing a mixture of proteins of gamma globulin mobility, was then fractionated by sequential chromatography on SP-Sephadex and Sephadex G-150, as described (7), followed by passage over an immunoadsorbent prepared with antisera to human IgG and ,B2 glycoprotein I. For preparation of the immunoadsorbent, 100 ml of sheep antiserum to human IgG and 132 glycoprotein I was made 45% in ammonium sulfate and stirred at 24° for 1 hr. The mixture was centrifuged at 1200 X g for 90 min at 40, and the precipitate was washed twice in 45% ammonium sulfate. The washed precipitate was dissolved in distilled water, dialyzed against three changes of 10 liters of 0.003 M phosphate buffer-0. 15 M NaCl (pH 7.5) for 24 hr at 40, and coupled to 250 ml of Sepharose 4B by the cyanogen bromide method (8). The Sepharose was poured into a 5 X 20 cm plastic column, washed with one column volume of 1.0% ethanolamine, and then equilibrated in 0.003 M phosphate buffer-0.35 M NaCl (pH 8.0). Samples were dialyzed against this buffer prior to application to the immunoadsorbant. HMW Kininogen. HMW kininogen was isolated by chromatography of normal human plasma on QAE-Sephadex A-50, ammonium sulfate precipitation, fractionation on SP-Sephadex, and Sephadex G-200 gel filtration according to the method of Habal et al. (9). The preparation was quantitated functionally by its ability to selectively correct the coagulation defect in Williams trait plasma as described (3). It was free of any detectable Hageman factor or prekallikrein. The protein content of samples was approximated by ab-

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sorbance at 280 nm with Al%m assumed to equal 10, or was determined by the Lowry method (10); the color reaction was read at an optical density of 700 nm in a Beckman spectrophotometer. Gel filtration on Sephadex G-150 (7), alkaline disc gel electrophoresis (11), and sodium dodecyl sulfate gel electrophoresis (12) were performed as described. Prekallikrein was radioiodinated by the Chloramine T method (13) using sodium [125I]iodide. The iodinated protein was immediately fractionated on a 2 X 100 cm column of Sephadex G-50 equilibrated in 0.003 M phosphate buffer-0.15 M NaCl (pH 8.0). The initial peak of radioactivity was completely separated from the peak of free iodine and contained over 99% trichloroacetic acidprecipitable counts. The 125I-labeled prekallikrein was mixed with a 100-fold excess of nonradiolabeled prekallikrein in order to assess binding to HMW kininogen. Radioactivity was determined in a sodium iodide well scintillation counter (Nuclear Chicago-Searle, Des Plaines, Ill., model 1185) with automatic subtraction of background and an efficiency of 82%. Coagulation Assays. The partial thromboplastin time was measured by the method of Proctor and Rapaport (14). Hageman factor, prekallikrein, and HMW kininogen were determined by a modification of the procedure for determining partial thromboplastin time, with congenitally deficient plasma

(3).

Assays of Kinin-Forming Proteins. The proteolytic activity of kallikrein was routinely measured by its ability to release bradykinin from heat-inactivated plasma (11). Twenty-five microliters of kallikrein source were incubated with 0.2 ml of substrate for 2 min of 370 and the bradykinin generated was quantitated by bioassay as described (11). Prekallikrein was determined by incubation of 25 ,l of proenzyme source with 25 ,ul of Hageman factor fragments (25 jig/ml) for 5 min at 370 and the kallikrein generated was determined. Unactivated Hageman factor was assayed for its kinin-generating capacity by adding 10 ,l of sample to 200 ,g of plasma deficient in Hageman factor containing 0.9 mg/ml of EDTA. Fifty microliters of a suspension of kaolin in 0.15 M NaCl (10 mg/ml) were added, the mixture was incubated for 2 min at 370C and applied to the bioassay. Incubation of the kaolin suspension with the plasma deficient in Hageman factor generated no detectable bradykinin. Activated Hageman factor was assayed in the same manner, with 0.15 M NaCl in place of the kaolin suspension. Fibrinolytic Assays. Plasminogen, plasmin, and plasminogen proactivator were assayed as described (15). Sephadex G-200 Gel Filtration of Normal and Deficient Plasmas. A 2.6 X 95 cm Pharmacia K26/100 column of Sephadex G-200 was equilibrated with 0.01 M Tris-HOl buffer (pH 7.0) made 0.15 M in NaCl and 10-1 M in EDTA. The column was run at either 40 or 370, the elution rate with upward flow was 10 ml/hr, 4-ml samples were applied, and 3.1-ml fractions were collected. Molecular weight was determined by gel filtration according to the method of Andrews (16). The standards used and their molecular weights by gel filtration were apoferritin (460,000), catalase (195,000), bovine serum albumin (65,000), and chymotrypsinogen (25,000).

RESULTS Four milliliters of normal plasma were fractionated on a 2.6 X 95 cm column of Sephadex G-200 at 40 and the fractions were assayed for prekallikrein, HMW kininogen, and unactivated Hageman factor by their ability to correct the partial thromboplastin time of congenitally deficient plasma. As shown in

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FIG. 1. Sephadex G-200 gel filtration of normal human plasma (upper panel) and plasma deficient in Hageman factor (lower panel). The column fractions were assayed for prekallikrein (A), HMW kininogen (o), and unactivated Hageman factor (0): Protein content was estimated by the Lowry method and the color reaction was read at 700 nm (0).

the upper panel of Fig. 1, the prekallikrein and HMW kininogen eluted in the same fractions, between the first and second protein peaks at an approximate molecular weight of 285,000, while unactivated Hageman factor was eluted between the second and third protein peaks at a molecular weight of 110,000-120,000. When plasma deficient in Hageman factor was fractionated in an identical fashion (lower panel, Fig. 1), prekallikrein and HMW kininogen were again found together at a molecular weight of 285,000. The elution position of prekallikrein and Hageman factor in the normal plasma chromatogram was then confirmed, as assessed by their kiningenerating ability. The location of each protein as assessed by kinin generation was identical to its elution profile as assessed by the coagulation assay, and there was no detectable active kallikrein or activated Hageman factor in these fractions. When plasma deficient in HMW kininogen was fractionated on Sephadex G-200, prekallikrein was found at a molecular weight of 115,000 and overlapped the Hageman factor peak (Fig. 2, upper panel). When prekallikrein-deficient plasma was fractionated on Sephadex G-200 (Fig. 2, center panel), unactivated Hageman factor was found at a molecular weight of 115,000, while the HMW kininogen eluted at a molecular weight of 200,000. When equal volumes of HMW kininogendeficient plasma and prekallikrein-deficient plasma were mixed and 4 ml was fractionated on Sephadex G-200, the prekallikrein was then found with HMW kininogen at a molecular weight of approximately 285,000 (Fig. 2, lower panel). It appeared possible that the association of prekallikrein with HMW kininogen was a cold (4°)-dependent phenomenon; therefore normal plasma was centrifuged at 370, immediately applied to a jacketed Sephadex column, and chromatographed at 370. Prekallikrein was again found in association with HMW kininogen. Chromatography of normal plasma at 240 or use of phosphate rather than Tris buffers did not affect the result. In Fig. 3 is shown an alkaline disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis of the preparation of purified human prekallikrein at 1 mg/ml. A single band is observed in the alkaline disc gel, while a major band with a small minor band below is observed in the sodium dodecyl

immunology: 0.7 0.6 0.5 0.4 0.3 0.2 0.1

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FIG. 2. Sephadex G-200 gel filtration of plasma deficient in HMW kininogen (upper panel) prekallikrein-deficient plasma (middle panel), and a mixture of prekallikrein-deficient plasma and plasma deficient in HMW kininogen (lower panel). The column fractions were assayed for prekallikrein (A), HMW kininogen (o), and unactivated Hageman factor (0).

sulfate gel. The minor band may represent a contaminating protein or may be prekallikrein that has been digested during purification. Functional assessment of this prekallikrein dem-

onstrated that it contained no detectable kallikrein, Hageman factor, kininogen, factor XI, or plasminogen. It did, however, contain plasminogen proactivator activity. The molecular weight of the highly purified human prekallikrein as assessed by sodium dodecyl sulfate gel electrophoresis was 104,000, a value which was similar to the molecular weight of prekallikrein in the plasma deficient in HMW kininogen. These results suggested that plasma prekallikrein is normally complexed with HMW kininogen and that this complex is independent of the presence of unactivated Hageman factor. We next attempted to reconstitute the prekallikrein-deficient plasma and plasma deficient in HMW kininogen with the respective proteins isolated from normal plasma to determine whether binding would be observed. Fifty microliters

! w~~~~~~w FIG. 3. Alkaline disc gel electrophoresis (top) and sodium dodecyl sulfate gel electrophoresis (bottom) of 30 jg of purified human prekallikrein.

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FIG. 4. Sephadex G-200 gel filtration of 12-I-labeled prekallikrein added to plasma deficient in HMW kininogen (upper panel), Sephadex G-200 gel filtration of 125I-labeled prekallikrein plus nonradiolabeled prekallikrein added to prekallikrein-deficient plasma (center panel), and Sephadex G-200 gel filtration of 125I-labeled prekallikrein and HMW kininogen added to plasma deficient in HMW kininogen. The prekallikrein coagulant activity is shown by the shaded area.

of 125I-labeled prekallikrein were mixed with 1 ml of plasma deficient in HMW kininogen and fractionated on Sephadex G-200. 25I-labeled prekallikrein eluted as a major peak of radioactivity at a molecular weight of 115,000, which corresponded to the peak of prekallikrein procoagulant activity (Fig. 4, upper panel). The 125I-labeled prekallikrein was next mixed with an excess of nonradiolabeled prekallikrein. The mixture was added to 2 ml of prekallikrein-deficient plasma and incubated at 24° for 10 min, and the plasma was then fractionated on Sephadex G-200. Two peaks of radioactivity were obtained; the first peak was eluted at a molecular weight of 285,000, and the second peak at a molecular weight of 115,000 (Fig. 4, center panel). When the column was assayed for prekallikrein by a coagulation assay, a peak of prekallikrein activity was found at 285,000 and activity could be detected through the 100,000 molecular weight region. One milliliter of plasma deficient in HMW kininogen was next reconstituted with HMW kininogen such that plasma levels were achieved. Fifty microliters of 125I-labeled prekallikrein were added to the plasma; the mixture was incubated at 240 for 10 min and fractionated on Sephadex G-200 (Fig. 4, lower panel). Again, peaks of '25I-labeled prekallikrein were observed at molecular weight 285,000 and 115,000, while the prekallikrein in the plasma deficient in HMW kininogen that was previously found at molecular weight 115,000 (Fig. 4, upper panel) was now identified at a molecular weight of 285,000. Finally the mixture of 125I-labeled prekallikrein and nonradiolabeled prekallikrein was incubated with HMW kininogen for 10 min at 240 and then fractionated on Sephadex G-200. As

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shown in Fig. 5, '25I-labeled prekallikrein was found complexed with the HMW kininogen and a second peak of radioactivity was also found at molecular weight 115,000. When the column fractions were assayed for prekallikrein functionally, virtually all of the activity was found at a molecular weight of 285,000. HMW kininogen was found functionally from tube 70 to 90; however, a trough separating complexed and uncomplexed kininogen was not clearly distinguished in the presence of excess kininogen.

DISCUSSION Colman et al. (3) and Wuepper et al. (4) have recently reported patients who possessed unique abnormalities of the Hageman-factor-dependent pathways. In each instance, the patient was found to be deficient in kininogen and the factor that corrected each of the functional abnormalities was identified as a high-molecular-weight form of human kininogen. Two other patients with the identical functional abnormalities have been reported (5, 17), and they have also been found to lack HMW kininogen (5). Patients with Fletcher trait are deficient in prekallikrein (1) and possess a diminished rate of surface'dependent coagulation and fibrinolysis which has been shown to reflect a diminished rate of formation of activated Hageman factor (2). Although kallikrein can be shown to directly cleave Hageman factor in the fluid phase (18, 19), kallikrein does not activate Hageman factor in human plasma unless a suitable surface is present (20). Recent reports by Liu et al. (21), Meier et al. (22), Webster et al. (23), Kaplan et al. (24), and Griffen and Cochrane (25) have shown that HMW kininogen augments the function of activated Hageman factor and HMW kininogen is a cofactor required for kallikrein to activate surface-bound Hageman factor. Thus, it appeared that prekallikrein and HMW kininogen act together to yield a normal rate of Hageman factor activation and HMW kininogen is further necessary for optimal function of the activated Hageman factor. We have now shown that prekallikrein and HMW kininogen normally circulate in plasma as a complex and they may therefore be adsorbed by negatively charged surfaces together. Unactivated Hageman factor was not found associated with this complex. Liu et al. (21) have presented evidence that the effect of HMW kininogen upon.the function of activated Hageman factor is to enhance

the activity of the active site, and a stoichiometric relationship between activated Hageman factor and HMW kininogen has been observed (22, 24, 25). In addition, it is also possible that HMW kininogen enhances the interaction of Hageman factor with prekallikrein by sterically positioning the prekallikrein to facilitate its cleavage. Nagasawa and Nakayasu (26) have previously reported that human plasma prekallikrein was found at a molecular weight of approximately 300,000 and first suggested that it must circulate complexed to some other protein. We find a complex of molecular weight 285,000 containing prekallikrein and HMW kininogen. However, the molecular weight of the complex (285,000) was less than that predicted for the sum of the reactants (315,000). It is possible that the apparent molecular weight of the complex or the reactants by gel filtration is different from its true molecular weight. Habal et al. (27) have reported a molecular weight of 210,000 for their purified HMW kininogen, which is similar to the value we find for HMW kininogen in prekallikrein-deficient plasma or partially purified HMW kininogen (200,000). However, they report a molecular weight of only 110,000 for purified HMW kininogen in guanidine Sepharose 4B. Thus, HMW kininogen may contain two noncovalently bound subunits of equal size or its molecular weight, as determined by Sephadex G-200 gel filtration, is considerably greater than its true value. We were able to demonstrate binding of prekallikrein to HMW kininogen by mixing prekallikrein-deficient plasma and plasma deficient in HMW kininogen or by reconstituting these deficient plasmas with purified components. We did not obtain complete binding of '25I-labeled prekallikrein in the presence of an excess of HMW kininogen although most of the nonradiolabeled prekallikrein added was complexed, as assessed by a functional assay. This may reflect the presence of denatured prekallikrein in the radiolabeled material and/or the presence of a nonbinding contaminant having the same molecular weight. The complex of prekallikrein and HMW kininogen is dissociated when plasma is fractionated by ion exchange chromatography, suggesting that the binding is in part attributable to a charge interaction between the alkaline prekallikrein [isoelectric point of 8.75 (7)] with the very acidic HMW kininogen [isoelectric point 4.3 (9)]. Divalent cations do not appear to be required for binding since 10-3 M EDTA did not alter the chromatographic patterns of the plasma.

Immunology: Mandle et A Prekallikrein and HMW kininogen are intimately associated functionally since they react in sequence to liberate the vasoactive peptide bradykinin and react together upon surfaces to enhance the activation and function of Hageman factor. Patients that are deficient in prekallikrein have normal levels of HMW kininogen. However two bf the patients with HMW kininogen deficiency have diminished levels of circulating prekallikrein (3, 17). This may reflect a shortened half-life of prekallikrein when not associated with HMW kininogen or the genes coding for the synthesis of these proteins may be linked. We have not ruled out the possibility that other proteins may also participate in the interaction between prekallikrein and HMW kininogen, although molecular weight considerations suggest that this is unlikely. Plasma proteins other than prekallikrein may also complex with HMW kininogen, as suggested by the observation that in one of three gel filtrations of prekallikrein-deficient plasma, a second HMW kininogen peak at a molecular weight of approximately 400,000 was observed; this will require further investigation. 1. Wuepper, K. D. (1973) J. Exp. Med. 138, 1345-1355. 2. Weiss, A. S., Gallin, J. I. & Kaplan, A. P. (1974) J. Clin. Invest.

53,622-633. 3. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F., Seavey, M., Guimaraes, J. A., Pierce, J. V. & Kaplan, A. P. (1975)

J. Clin. Invest. 56,1650-1662. 4. Wuepper, K. D., Miller, K. R. & LaCombe, M. J. (1975) J. Clin. Invest. 56, 1663-1672. 5. Donaldson, V. H., Glueck, H. I., Miller, M. A., Movat, H. Z. & Habal, F. (1976) J. Lab. Clin. Med. 87,327-337. 6. Kaplan, A. P., Spragg, J. S. & Austen, K. F. (1971) in Second International Symposium, Biochemistry of the Acute Allergic Reactions, eds. Austen, K. F. & Becker, E. L. (Blackwell Scientific Publications, Ltd., Oxford, England), pp. 279-278. 7. Kaplan, A. P., Kay, A. B. & Austen, K. F. (1972) J. Exp. Med. 135, 81-97. 8. Axen, R., Dorath, J. & Ernback, S. (1967) Nature 214, 13021330.

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9. Habal, F. M., Movat, H. Z. & Burrowes, C. E. (1974) Biochem. Pharmacol. 23, 2291-2303. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. C. & Randall, R. J.

(1951) J. Biol. Chem. 193,265-275. 11. Kaplan, A. P. & Austen, K. F. (1970) J. Immunol. 105, 802811. 12. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 13. Greenwood, F. C., Hunter, W. M. & Glover, J. S. (1963) Biochem. J. 89, 114-123. 14. Proctor, R. R. & Rapaport, S. I. (1961) Am. J. CGn. Pathol. 35, 212-219. 15. Kaplan, A. P. & Austen, K. F. (1972) J. Exp. Med. 136, 13781393. 16. Andrews, P. (1965) Biochem. J. 96,595-606. 17. Saito, H., Ratnoff, 0. D., Waldmann, R. & Abraham, J. P. (1975) J. Clin. Invest. 55,1082-1084. 18. Cochrane, C. G., Revak, S. D. & Wuepper, K. D. (1973) J. Exp. Med. 138, 1564-1583. 19. Bagdasarian, A., Lahiri, B. & Colman, R. W. (1973) J. Biol. Chem. 248,7742-7747. 20. Kaplan; A. P., Meier, H. L., Yecies, Y. D. & Heck, L. W. (1976) in Fogarty International Center Proceedings, The Kallikrein System in Health and Disease, eds. Pisano, J. J. & Austen, K. F. (U.S. Government Printing Office, Washington, D.C.), No. 27, in press. 21. Liu, C. Y., Bagdasarian, A., Meier, H. L., Scott, C. F., Pierce, J. V., Kaplan, A. P. & Colman, R. W. (1976) Fed. Proc. 35, 692

(abstr.). 22. Meier, H. L., Webster, M. E., Liu, C. Y., Colman, R. W. & Kaplan, A. P. (1976) Fed. Proc. 35, 692 (abstr.). 23. Webster, M. E., Guimaraes, J. A., Kaplan, A. P., Colman, R. W. & Pierce, J. V. (1976) Adv. Exp. Med. Biol. 70, 285-299. 24. Kaplan, A. P., Meier, H. L. & Mandle, R., Jr. (1976) Seminars in Thrombosis and Haemostasis 3,1-26. 25. Griffin, J. H. & Cochrane, C. G. (1976) Proc. Natl. Aced. Sci. USA

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26. Nagasawa, S. & Nakayasu, T. (1973) J. Biochem. 74, 401-403. 27. Habal, F. M., Underdown, B. J. & Movat, H. Z. (1975) Biochem. Pharmacol. 24, 1241-1243.

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