Activation of coagulation Factor V by calcium-dependent proteinase

Biochimica et Biophysica Acta 929 (1987) 263-270 Elsevier

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BBA 12068

Activation of coagulation Factor V by calcium-dependent proteinase George M. Rodgers a,b, Jingyang Cong c, Darrel E. Goll c and William H. Kane d a Gladstone Foundation Laboratories for Cardiovascular Disease, Cardiovascular Research Institute, San Francisco, CA, h Cancer Research Institute, Department of Medicine, University of California, San Francisco, CA, e Muscle Biology Group, University of Arizona, Tucson, AZ, and a Department of Biochemistry, University of Washington, Seattle, WA (U.S.A.)

(Received 15 January 1987)

Key words: Calcium ion dependence; Proteinase; Blood coagulation; Factor V activation; (Endothelium)

Factor V is a key coagulation cofactor, regulating the rate of Factor Xa-catalyzed prothrombin conversion. Activation of Factor V markedly accelerates coagulation. This study describes a new class of Factor V activators, sulfhydryl proteinases. Of the enzymes studied, calcium-dependent proteinase was the most effective activator. Activation of Factor V by this enzyme was associated with cleavage of 12Sl-labeled Factor V to peptides distinct from those generated by previously described activators. Calcium-dependent proteinase-activated Factor Va peptides with molecular weights of 114 000 and 93 000 bound both to Factor X~ and to cultured endothelial cells. Calcium-dependent proteinase was identified in vascular endothelial cells, a tissue that also synthesizes Factor V. These findings suggest a previously unknown mechanism for cellular regulation of coagulation.

Introduction Factor V is a single-chain glycoprotein (Mr 330000) that circulates in the blood as a procofactor; when activated, Factor V becomes an essential component of the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin [1]. In the prothrombinase complex, activated Factor V (Factor Va) binds to a receptor on membranes and serves as the receptor for Factor Xa, which has proteolytic activity [1]. The assembly of the prothrombinase complex on cellular surfaces serves to localize thrombin

Abbreviations: DFP, diisopropyl fluorophosphate; SDS, sodium dodecyl sulfate; ELISA, enzyme-linked immunosorbent assay. Correspondence: G. Rodgers, Gladstone Foundation Laboratories, Box 40608, San Francisco, CA 94140, U.S.A.

formation and to optimize the rate of prothrombin activation [1]. The prothrombinase reaction was originally studied on the platelet surface [2]. Factor Xa-catalyzed prothrombin activation has since been reported on the surfaces of monocytes and lymphocytes [3], as well as vascular endothelium [4,5]. Factor Va is required for efficient prothrombin activation [1]. When thrombin, considered to be the primary activator of Factor V, activates Factor V, the Factor Va produced is 30-fold more active than the pro-cofactor [6]. Thrombin hydrolysis of Factor V generates three fragments: an aminoterminal heavy chain and a carboxyl-terminal light chain (both function as Factor Va in the presence of calcium) and an activation peptide [6,7]. In other instances, however, Factor V activation does not appear to require thrombin. For example, homocysteine-treated endothelial cells activate Factor V in a manner distinct from that

0167-4889/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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of thrombin [8], and undefined nonthrombin-like cellular proteinases from platelets [9] and monocytes [10] have been reported to activate Factor V. In an attempt to identify cellular activators of Factor V, we selected calcium-dependent proteinase because of its wide distribution among and high activity in a variety of cells [11,12]. Millimolar and micromolar forms of this enzyme exist, differing in their calcium requirements [12-14]. The discovery of Ca2+-dependent proteinase in striated muscle cells originally suggested that this enzyme initiated turnover of myofibrillar proteins [15]; more recently, Ca2+-dependent proteinase has been reported to cleave a variety of coagulation proteins [16-18]. However, the physiologic role of this proteinase is unclear. In this study, we report that Ca2+-dependent proteinase is present in endothelial cells and can activate Factor V. Moreover, other sulfhydryl proteinases were found to be active; together with Ca2+-dependent proteinase, they represent a previously unknown class of Factor V activators. Portions of this work were presented at the 1986 meeting of the American Heart Association, Dallas, TX, and have been published in abstract form [25]. Materials and Methods

Trizma base, bromelain, ficin, papain, cathepsin C, pyroglutamate aminopeptidase, bovine serum albumin, cyanogen bromide-activated Sepharose, diisopropylfluorophosphate (DFP) and plasma deficient in Factor V were obtained from Sigma Chemical Co. (St. Louis, MO). Leupeptin was provided by Vega Biotechnologies (Tucson, AZ). Sodium [125I]iodide was obtained from Amersham (Clearbrook, IL). Iodogen was supplied by Pierce Chemical Co. (Rockford, IL). Adult bovine aortic endothelial and human umbilical vein endothelial cells were cultured as described [4,81. Factor V and Factor X were purified from human plasma as described [4,6]. Factor V was iodinated with no loss of coagulant activity [6]. Protein concentrations were assayed using the technique of Bradford [19]. Factor X was activated as described [4], then inactivated with DFP prior to coupling to Sepharose [20]. The method of

Suzuki et al. [20] was used to identify 125I-Factor Va peptides that bound to a DFP-inactivated Factor X a-Sepharose column. A purified millimolar form of Ca2+-dependent proteinase prepared from chicken breast muscle (Wolfe, F.H., Sathe, S.K., Goll, D.E., Kleese, W.C., Edmunds, T. and Duperret, S.M., unpublished data) was used in these studies. Briefly, chicken breast muscle tissue was disrupted in 20 mM Tris-acetate, 5 mM ethylenediaminetetraacetate (EDTA) (pH 7.5) containing 0.1% fl-mercaptoethanol and centrifuged at 17 800 × g for 20 min. The supernatant was further purified by O-(diethylaminoethyl) (DEAE) cellulose chromatography using a linear 0-500 mM KC1 gradient. Millimolar Ca2+-dependent proteinase activity was eluted in fractions containing 180-350 mM KC1. Calcium-dependent proteinase activity was further purified by hydrophobic chromatography using a phenyl-Sepharose column equilibrated with 20 mM Tris-acetate, 1 mM EDTA (pH 7.5) containing 0.1% fl-mercaptoethanol, 150 mM KC1 and 1 mM NaN 3. Calcium-dependent proteinase activity was eluted with a 150-0 mM KC1 gradient, followed by flushing the column with 1 mM EDTA and 0.1% fl-mercaptoethanol. Additional purification was achieved with successive chromatography steps using a Sephacryl S-300 column and a DEAE-TSK-fine column. This purification method routinely resulted in a Ca2+-dependent proteinase preparation of approx. 85% purity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In typical experiments, 125I-Factor V (300 n g / ml) in Tris-buffered saline (20 mM Tris, 150 mM NaC1) containing 5 mM CaC12 (pH 7.4) was incubated at 21°C with purified Ca2+-dependent proteinase. Factor V activity was monitored by a clotting assay [8]; cleavage of ~25I-Factor V was followed by reduced SDS-gel electrophoresis and autoradiography. An enzyme-linked immunosorbent assay (ELISA) was used to quantitate Ca2+-dependent proteinase levels in lysates of adult bovine aortic endothelial and human umbilical vein endothelial cells. Washed cells were lysed in Tris-buffered saline and freeze-thawed; Ca2+-dependent proteinase levels were determined using monoclonal antibodies specific for this proteinase. In this

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maximal activity occurring at 15 min. When similar experiments with the Ca2+-dependent proteinase were performed at 37°C, activation of Factor V was reduced by approx. 50%, presumably reflecting rapid autolysis of this enzyme at this temperature [12]. Omission of Factor V resulted in only about 0.6% coagulant activity, indicating that CaZ+-dependent proteinase cleavage of Factor V, and not the enzyme alone, accounted for the increased coagulant activity. Incubation of Factor V in the buffer without enzyme resulted in basal Factor V activity that did not change over the period of study. Fig. 2 compares cleavage of 125I-Factor V by Ca2+-dependent proteinase and thrombin. Calcium-dependent proteinase cleaved a25I-Factor V into at least nine peptides; hydrolysis was inhibited by leupeptin (100 ~tg/ml, 1 rain and 4 min). Diisopropylfluorophosphate (0.2 raM) did not inhibit a CaZ+-dependent proteinase-induced cleavage of 125I-Factor V, but this reagent did inhibit a thrombin-induced cleavage of azsI-Factor V (data not shown). A comparison of the apparent molecular weights of the Factor V, cleavage peptides generated by CaZ+-dependent proteinase and thrombin (Fig. 2) indicates that the former proteinase produces distinct peptides. When Factor V was activated with CaZ+-depen dent proteinase (2.5 ~tg/ml) and then treated with thrombin (0.5 U/ml), Factor V coagulant activity rapidly reached the same maximal values as those generated by thrombin alone (a 20-fold increase).

ELISA method, plates were coated with known amounts of purified Ca2+-dependent proteinase or cell lysates. Monoclonal antibodies to the enzyme were added, and quantitation was performed using a horseradish peroxidase detection system and an ELISA plate reader. To identify the 125I-Factor Va peptides that bind to cultured endothelial cells, adult bovine aortic endothelial cells were washed three times with serum-free medium containing 1% bovine serum albumin; 1 ml of that medium was then added to the cells. Factor Va activated by Ca 2+dependent proteinase was then added to the cells. After incubation, the cells were washed with serum-free medium, then processed for reduced SDS-gel electrophoresis and autoradiography [8]. Results

Fig. 1 compares activation of Factor V by Ca2+-dependent proteinase or thrombin. Calciumdependent proteinase (8/xg/ml) activated Factor V 14-fold within 1 min; over the next 15 min, Factor V coagulant activity slowly decreased, but remained above basal levels. When tested at a concentration of 1 /~g/ml, the CaZ+-dependent proteinase-activated Factor V 4-fold after 8 min. The minimal Ca2+-dependent proteinase concentration found to activate Factor V was 200 ng/ml; this resulted in a 25-50% increase in coagulant activity (data not shown). Thrombin (1 U / m l ) activated Factor V approx. 20-fold, with

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Fig. 1. Comparison of Factor V activation by calcium-dependent proteinase (CDP) and thrombin. ]251-Factor V (300 ng/ml) in Tris-buffered saline containing 5 mM CaCI 2 (pH 7.4) at 21"C was incubated either with purified Ca2+-dependent proteinase (2 or 8 /~g/ml) or with thrombin (1 U / m l , 21 ° C). Factor V coagulant activity was measured at intervals. In this assay, 100% activity is equivalent to the Factor V activity present in 1 ml of pooled normal plasma.

266

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Fig 2. Cleavage of 125I-Factor V by Ca2+-dependent proteinase (CDP) and thrombin. Aliquots of Ca2+-dependent proteinase or thrombin incubations described in Fig. 1 were processed at intervals for reduced sodium dodecyl sulfate gel electrophoresis (5%). Autoradiograms of the dried gels are shown. Leupeptin (100 /~g/ml) was included in the incubation mixture in certain experiments (1-min and 4-min incubation periods). A comparison of the 1251-Factor V~, peptides generated by thrombin (1 U/ml, 5 min, 21 o C) is shown in the far right lane.

This contrasted with a 4-fold increase in coagulant activity generated by Factor V incubated with Ca2+-dependent proteinase alone. Analysis of the Factor V~ peptides in these studies revealed that thrombin treatment of Ca 2+-dependent proteinase-activated Factor V converted the complex Ca2+-dependent proteinase cleavage pattern to an activation profile very similar to that seen with thrombin-activated Factor V (Fig. 3, left side, lane A). The ability of several sulfhydryl proteinases to activate Factor V was compared with that of Ca 2 ~-dependent proteinase and thrombin. Bromelain, ficin and papain activated Factor V 4- to 6-fold (compared with a 14-fold and 20-fold increase in activity for Ca2+-dependent proteinase and thrombin, respectively), whereas cathepsin C and pyroglutamate aminopeptidase, at concentrations from 0.1 to 10 /~g/ml, did not enhance the coagulant activity of Factor V. Calcium-dependent proteinase was the most potent sulfhydryl enzyme activator of Factor V, being at least 7-fold

more active than other sulfhydryl proteinases on a molar basis. 1251-Factor Va cleavage peptides generated by bromelain, ficin and papain were very similar to each other, but different from those generated by Ca2+-dependent proteinase. The most prominent t251-Factor V~ peptides generated by bromelain, ficin and papain had molecular weights of 114000 and 93000. In initial experiments, the question of whether vascular endothelial cells exhibit Ca2+-dependent proteinase activity was addressed using an ELISA technique to identify the Ca2+-dependent proteinase in endothelial cell lysates. Both the millimolar and micromolar forms of this proteinase were identified by this technique; confluent adult bovine aortic endothelial and human umbilical vein endothelial cells contained 9-11 ng of micromolar Ca2+-dependent proteinase per 106 cells. To determine which of the Ca2+-dependent proteinase-induced Factor V~ peptides were important in coagulation, Factor Va species generated by thrombin or by Ca2+-dependent pro-

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Fig. 3. Binding of 12SI-Factor Va peptides to a diisopropylfluorophosphate-inactivated Factor Xa-Sepharose affinity column. 12SI-Factor V was activated by thrombin (1 U/ml, 5 min) or by Ca2+-dependent proteinase (4 ~g/ml, 5 rain). The column was equilibrated in 50 mM Tris-HC1, 50 mM NH4C1 and 5 mM CaC12 (pH 7.5). Elution of bound material was achieved with 50 mM Tris-HC1, 500 mM NH4C1 and 100 mM CaC12 (pH 7.5) [20]. Results from (A) the incubation mixture before chromatography, (B) the flow-through, and (C) the eluted material are shown after reduced SDS-gel electrophoresis (7.5%) and autoradiography. Equivalent cpm were added to each lane.

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Fig. 4. Binding of Ca2+-dependent proteinase-generated 125IFactor Va peptides to cultured adult bovine aortic endothelial cells. 125I-Factor V was prepared and activated with C a 2 + - d e -

teinase were bound to a Factor X a affinity column. As shown in Fig. 3, thrombin-activated Factor V~ peptides with molecular weights of 120 000, 78000 and 73 000 bound to the column and were eluted by high-salt buffer (lane C). These data are similar to those reported by Suzuki et al. [20]. Also shown in Fig. 3 are CaZ+-dependent proteinasegenerated Factor Va peptides that bound to Factor X a. These peptides had molecular weights of 114000,93000 and 82000. The M r 82000 peptide was only weakly detected in the initial incubation mixture (lane A) and was presumably concentrated by this column. These data suggest that pendent proteinase as described in Fig. 1. Aliquots of Ca2+-dependent proteinase-activated Factor V~ were added to washed endothelial cells and were incubated for 30, 60 or 90 min. After each interval, the cells were washed then processed for reduced SDS-gel electrophoresis (7.5%) and autoradiography. The figure depicts the autoradiogram: 1251-Factor V (left lane); Ca2+-dependent proteinase-Factor V~ (center lane); and cellbound, Ca:+-dependent proteinase-activated Factor V~ (three right lanes) after 30, 60 or 90 min of incubation.

268 three peptides may be active in the Factor Va generated by Ca2+-dependent proteinase activation, To determine whether Ca2+-dependent proteinase Factor Va peptides can bind to cultured endothelial cells, a25I-Factor V was incubated with Ca2+-dependent proteinase as described in Fig. 1, then added to washed adult bovine aortic endothelial cells. Analysis of the cell-bound Factor V.d is shown in Fig. 4. The prominent peptides associated with endothelial cells had molecular weights of 114000 and 93000, values similar to those of the most prominent Factor Va peptides that bound to the Factor X~-Sepharose column. Co-incubation of excess unlabeled CaZ+-dependent proteinase-generated Factor Va prevented binding of the labeled peptides to endothelial cells (data not shown). These data suggest that the coagulantly active Factor Va peptides generated by Ca2+-de pendent proteinase may associate with the surface of vascular endothelial cells to modulate coagulation. Discussion

This report demonstrates that Ca2+-dependent proteinase (and other sulfhydryl proteinases) can activate Factor V. Previously described activators of Factor V include serine proteinases, such as thrombin [6] and Factor X~ [21], as well as a variety of cellular proteinases [8-10]. Calcium-dependent proteinase has been reported to cleave fibrinogen [16] and von Willebrand factor [17], but the functional consequence of this cleavage is uncertain. Although Ca2+-dependent proteinase is found in numerous tissues [11,12], there have been no reports of its presence in vascular endothelium. Calcium-dependent proteinase isolated from different tissues has been found to have similar properties [12-14]. Consequently, in these studies we used a millimolar form of the Ca2+-dependent proteinase purified from chicken muscle because this form was available in large quantity. The observation that Ca2+-dependent proteinase and other sulfhydryl proteinases generate similar Factor V~ peptides (114000 and 93000) is consistent with the significant amino acid homology around the active sites of Ca2+-dependent proteinase and these other enzymes [22]. It is interesting to note

that Ca 2+-dependent p r o t e i n a s e - F a c t o r V~ peptides and thrombin-Factor V~ peptides that bind to Factor Xa-Sepharose are of similar apparent molecular weight. However, given the complexity of the Factor V cleavage generated by Ca 2+-dependent proteinase, we cannot definitively identify the active species. Future studies are needed to determine whether the peptides that result from the CaZ+-dependent proteinase cleavage of Factor V are analogous to the thrombin cleavage products representing the heavy and light chains of Factor Va, and whether these are the peptides that bind to Factor X a. A comparison of the ability of the Ca 2 +-dependent proteinase and thrombin to activate Factor V indicates that thrombin is the more potent activator. This conclusion is based on an analysis using the following molecular weights and concentrations of reactants: Factor V, M r 330000, 300 n g / m l ; Ca2 +-dependent proteinase, Mr 120 000, 1 /xg/ml; and thrombin, M r 36000, 1 U / m l (spec. act., 3000 U / m g ) . From these data, the following molar ratios are obtained: Ca2+-dependent proteinase/Factor V = 8 nM : 0.9 nM (approx. 9 : 1); t h r o m b i n / F a c t o r V = 9 n M : 0 . 9 nM (approx. 10: 1). At these equimolar concentrations of activator to substrate, Ca2+-dependent proteinase increased Factor V coagulant activity 4-fold, whereas thrombin activation resulted in a 20-fold enhancement in coagulant activity. Thus, on a molar basis, thrombin activated Factor V approx. 5-fold more efficiently than Ca2+-dependent proteinase. The existence of a vascular activator of Factor V and the ability of Ca2+-dependent proteinaseactivated Factor Vd peptides to bind to Factor X~, as well as to endothelium indicate that Ca2+-de pendent proteinase may regulate coagulant events on the vascular surface. The observations in this report suggest a potential thrombin-independent mechanism for prothrombin activation following vascular injury. Perturbed endothelial cells express tissue factor activity [23], resulting in activation of Factor X. The presence of a Factor V activator (such as Ca2+-dependent proteinase) in perturbed endothelium would efficiently promote Factor X,-catalyzed prothrombin activation in the absence of thrombin. With regard to the identity of the Factor V activator present in homocysteine-

269

treated endothelial cells, preliminary data suggest that the activator may not be CaZ+-dependent proteinase or another sulfhydryl proteinase. This tentative conclusion is based on the Factor Va peptides generated by homocysteine-treated cells [8]; the peptides produced by CaZ+-dependent proteinase and other sulfhydryl enzymes are distinct from those reported in the homocysteine study. However, it is conceivable that the endothelial cell Ca2+-dependent proteinase-Factor Va activation peptides might be different from those generated by the muscle enzyme. Isolation and characterization of the endothelial cell activator will resolve this issue. It is of interest that Ca2+-dependent proteinase-activated Factor Va peptides can be further activated by thrombin to generate maximal coagulant activity. This suggests a mechanism for cellular activation of Factor V in which perturbed endothelium would express Ca2+-dependent proteinase activity to activate the cofactor initially. The resultant thrombin would feed back and further activate Factor V. The fact that thrombin can further cleave CaZ+-dependent proteinase-Factor Va peptides to peptides similar to those generated by thrombin alone supports this hypothesis. In a previous study, Ca2+-dependent proteinase has been described as a cytoplasmic enzyme [12]. Therefore, for this proteinase to activate surfacebound Factor V on platelets or vascular endothelial cells,, one would have to postulate that the enzyme had access to membrane-bound ligands. Perhaps perturbation of cells would allow such proteolysis to occur. There is precedence for this hypothesis: McGowan et al. [24] have reported surface proteolytic activity on stimulated platelets that is characteristic of Ca2+-dependent proteinase. Identification of Ca2+-dependent proteinase and other sulfhydryl proteinases as activators of Factor V indicates that thrombin may not be the only physiologic activator of this pro-cofactor. The ubiquitous nature of Ca2+-dependent proteinase, including its presence in platelets [11] and vascular endothelium, and its expression after cellular stimulation [24] suggest that this proteinase (and perhaps other sulfhydryl proteinases) may contribute to activating coagulation.

Acknowledgements The authors thank Regina Lim, William Kleese, Donna Sloan and Kevin Kiene for technical assistance, and Dr. David Phillips and Dr. Marc Shuman for comments and criticism. Purified human c~-thrombin was provided by Dr. John Fenton (New York State Department of Health, Albany, NY). This research was supported by a Clinical Investigator Award No. 1 K08 HL01031 (GMR) from the National Heart, Lung and Blood Institute and by Grants HL 33005 (GMR) and HL 20984 (DEG) from the National Institutes of Health, as well as a grant from the Muscular Dystrophy Association (DEG). This work was completed during the tenure of a Clinician-Scientist award, with funds contributed in part by the Washington affiliate of the American Heart Association and by E.R. Squibb and Sons (WHK). Kate Sholly and Michele Prator are acknowledged for manuscript typing, A1 Averbach and Sally Gullatt Seehafer for editorial assistance and James X. Warger and Norma Jean Gargasz for illustration and photography.

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