Unique in Vivo Modifications of Coagulation Factor V Produce a Physically and Functionally Distinct Platelet-derived Cofactor (2004)

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from ∼40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2–3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2–3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction (∼10–15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser692 catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr402 with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser692 remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr1543 rather than Arg1545 accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart. Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from ∼40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2–3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2–3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction (∼10–15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser692 catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr402 with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser692 remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr1543 rather than Arg1545 accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart. Factor Va (FVa), 1The abbreviations used are: FVa, factor Va; FV, factor V; APC, activated protein C; PCPS, phosphatidylcholine/phosphatidylserine vesicles (75%:25%); PEG, polyethylene glycol 8000; PGE1, prostaglandin E1; RGDS, Arg-Gly-Asp-Ser peptide; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight.1The abbreviations used are: FVa, factor Va; FV, factor V; APC, activated protein C; PCPS, phosphatidylcholine/phosphatidylserine vesicles (75%:25%); PEG, polyethylene glycol 8000; PGE1, prostaglandin E1; RGDS, Arg-Gly-Asp-Ser peptide; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight. a heterodimeric protein composed of heavy chain (105 kDa) and light chain (74 kDa) subunits, is formed by limited proteolysis of factor V (FV) (1.Jenny R.J. Tracy P.B. Mann K.G. Bloom A.L. Forbes C.D. Thomas D.P. Tuddenham E.G.D. Haemostasis and Thrombosis. Churchill Livingstone, New York1994Google Scholar). In normal hemostasis, FVa functions as a non-enzymatic cofactor of the prothrombinase complex, which consists of a 1:1 stoichiometric and Ca2+-dependent complex of the serine protease factor Xa and FVa, bound to the membrane of appropriately activated platelets, and catalyzes the proteolytic conversion of prothrombin to thrombin (2.Mann K.G. Nesheim M.E. Church W.R. Haley P. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). When incorporated into the prothrombinase complex, the catalytic activity of factor Xa is increased by approximately 5 orders of magnitude, and FVa contributes substantially to this increase (3.Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar). Removal of FVa from the prothrombinase complex results in a 10,000-fold decrease in the rate of thrombin generation (3.Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar), the physiologic effect of which is demonstrated in the bleeding diatheses expressed by FV-deficient individuals (4.Tracy P.B. Giles A.R. Mann K.G. Eide L.L. Hoogendoorn H. Rivard G.E. J. Clin. Investig. 1984; 74: 1221-1228Crossref PubMed Scopus (116) Google Scholar, 5.Chediak J. Ashenhurst J.B. Garlick I. Desser R.K. Blood. 1980; 56: 835-841Crossref PubMed Google Scholar, 6.Onuora C.A. Lindenbaum J. Nossel H.L. Am. J. Med. Sci. 1973; 265: 407-417Crossref PubMed Scopus (17) Google Scholar, 7.Coots M.C. Muhleman A.F. Glueck H.I. Am. J. Hematol. 1978; 4: 193-206Crossref PubMed Scopus (42) Google Scholar, 8.Chiu H.C. Rao A.K. Beckett C. Colman R.W. Blood. 1985; 65: 810-818Crossref PubMed Google Scholar) Factor V circulates in two pools in whole blood. The majority (75–80%) is found in the plasma as an inactive, single chain procofactor of 330 kDa, whereas the remainder (20–25%) is found in the α-granules of platelets (9.Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar), where it is stored in a partially cleaved and activated state (9.Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar, 10.Osterud B. Rapaport S.I. Lavine K.K. Blood. 1977; 49: 819-834Crossref PubMed Google Scholar). The unique activation state of platelet-derived FV/Va and its proximity to the procoagulant surface of platelets make it an excellent source of cofactor activity for assembly and function of the prothrombinase complex at a site of vascular injury. Its paramount importance in the maintenance of normal hemostasis, when compared with that of plasma-derived FVa, has been demonstrated in several clinical studies. Individuals expressing substantially decreased levels of FV in their platelets exhibit a life-threatening bleeding diathesis despite their expression of relatively normal levels of plasma-derived FV (4.Tracy P.B. Giles A.R. Mann K.G. Eide L.L. Hoogendoorn H. Rivard G.E. J. Clin. Investig. 1984; 74: 1221-1228Crossref PubMed Scopus (116) Google Scholar). An individual with an antibody that neutralized greater than 99% of the plasma-derived FV pool endured surgical challenge as the antibody was without effect on platelet-derived FV/Va, indicating that the platelet stores of FV/Va were sufficient to maintain normal hemostasis (11.Nesheim M.E. Nichols W.L. Cole T.L. Houston J.G. Schenk R.B. Mann K.G. Bowie E.J. J. Clin. Investig. 1986; 77: 405-415Crossref PubMed Scopus (132) Google Scholar). This latter observation is consistent with the ability of platelet transfusions to correct bleeding episodes in individuals who have expressed acquired FV inhibitors for several months (5.Chediak J. Ashenhurst J.B. Garlick I. Desser R.K. Blood. 1980; 56: 835-841Crossref PubMed Google Scholar). Even though platelet-derived FV originates from the plasma pool of FV (12.Camire R.M. Pollak E.S. Kaushansky K. Tracy P.B. Blood. 1998; 92: 3035-3041Crossref PubMed Google Scholar, 13.Gould W.R. Simioni P. Kalafatis M. Luni S. Tracy P.B. Blood. 2001; 98 (Abstr. 2938): 703Google Scholar, 14.Bouchard B.A. Meisler N.T. Bomard J. Williams J.L. Long M.W. Tracy P.B. Blood. 2000; 96 (Abstr. 2688): 625Crossref PubMed Google Scholar, 15.Bouchard B.A. Meisler N.T. Tracy P.B. Blood. 2001; 98 (Abstr. 2157): 516Google Scholar), several studies have demonstrated distinct differences in the physical nature and proteolytic susceptibility of platelet- and plasma-derived forms of FV. Previous studies (16.Viskup R.W. Tracy P.B. Mann K.G. Blood. 1987; 69: 1188-1195Crossref PubMed Google Scholar, 17.Monkovic D.D. Tracy P.B. J. Biol. Chem. 1990; 265: 17132-17140Abstract Full Text PDF PubMed Google Scholar) have shown that platelet-derived FV/Va is composed of several peptides, some of which are distinct and have molecular weights unlike any intermediate species observed during the activation of the plasma-derived molecule. Whereas plasma-derived FV exists as a single chain procofactor expressing little if any cofactor activity (3.Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar), the platelet-derived FV/Va molecule, which is stored in the α-granule, has undergone partial proteolysis and expresses substantial cofactor activity upon platelet secretion (17.Monkovic D.D. Tracy P.B. J. Biol. Chem. 1990; 265: 17132-17140Abstract Full Text PDF PubMed Google Scholar, 18.Vicic W.J. Lages B. Weiss H.J. Blood. 1980; 56: 448-455Crossref PubMed Google Scholar, 19.Kane W.H. Mruk J.S. Majerus P.W. J. Clin. Investig. 1982; 70: 1092-1100Crossref PubMed Scopus (26) Google Scholar). Furthermore, platelet-derived FV/Va is a unique substrate for several proteases when compared with its plasma counterpart. Kinetic analyses have demonstrated that plasma-derived FV is the preferred substrate for thrombin, whereas platelet-derived FV/Va is activated by factor Xa 50–100 times more effectively than by thrombin (17.Monkovic D.D. Tracy P.B. J. Biol. Chem. 1990; 265: 17132-17140Abstract Full Text PDF PubMed Google Scholar). Plasmin-catalyzed cleavage of platelet-derived FVa bound to activated platelets results in a 4-fold increase in cofactor activity that is sustained for as long as 4 h, whereas plasmin-catalyzed cleavage of plasma-derived FVa bound to activated platelets results in a rapid loss of cofactor activity (20.Conlon S.J. Camire R.M. Kalafatis M. Tracy P.B. Thromb. Haemostasis. 1997; 77 (Abstr. 2507): 616Crossref PubMed Scopus (8) Google Scholar). An intriguing observation was made in work published by Rand et al. (21.Rand M.D. Kalafatis M. Mann K.G. Blood. 1994; 83: 2180-2190Crossref PubMed Google Scholar) who demonstrated that the heavy chain of platelet-derived FV/Va is totally resistant to phosphorylation by a platelet-associated kinase, under circumstances where the plasma-derived molecule is readily phosphorylated. Finally, several studies have demonstrated that platelet-derived FVa is more resistant to activated protein C (APC)-catalyzed inactivation when compared with plasma-derived FVa (22.Camire R.M. Kalafatis M. Cushman M. Tracy R.P. Mann K.G. Tracy P.B. J. Biol. Chem. 1995; 270: 20794-20800Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 23.Camire R.M. Kalafatis M. Simioni P. Girolami A. Tracy P.B. Blood. 1998; 91: 2818-2829Crossref PubMed Google Scholar). This apparent insensitivity to APC results from a reduced rate of cleavage at Arg506 and is preferentially displayed when platelet-derived FVa is associated with the activated platelet membrane (22.Camire R.M. Kalafatis M. Cushman M. Tracy R.P. Mann K.G. Tracy P.B. J. Biol. Chem. 1995; 270: 20794-20800Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Thus, platelet-derived FV/Va appears to be a unique cofactor when compared with its plasma counterpart. All of the studies detailed above were done with platelet-derived FV/Va present in platelet releasates prepared with a variety of agonists. To date, functional studies with purified, platelet-derived FV/Va have been precluded by an inability to isolate the platelet-derived cofactor pool in its native, functional state. Therefore, this work outlines for the first time a protocol to purify platelet-derived FV/Va in order to define its physical and functional characteristics allowing the discovery of molecular characteristics unique to platelet-derived FV/Va. Reagents—Prostaglandin E1 (PGE1), phenylmethylsulfonyl fluoride, benzamidine, leupeptin, soybean trypsin inhibitor, heparin (from bovine lung), Arg-Gly-Asp-Ser (RGDS) peptide, and α-cyanohydroxycinnamic acid were purchased from Sigma. ATP, [γ-32P]ATP, and crystallized bovine serum albumin were purchased from ICN (Aurora, OH). Casein kinase II was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Phospholipid vesicles composed of 75% (w/w) PC and 25% (w/w) PS (PCPS) were prepared as described previously (24.Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson R.D. Biochemistry. 1977; 16: 2806-2810Crossref PubMed Scopus (727) Google Scholar), and the concentration of the vesicles was determined by phosphorous assay as described (25.Gomori G. J. Lab. Clin. Med. 1942; 27: 955-960Google Scholar). Mouse anti-human FV monoclonal antibodies αHFVaLC9 (26.Foster W.B. Tucker M.M. Katzmann J.A. Miller R.S. Nesheim M.E. Mann K.G. Blood. 1983; 61: 1060-1067Crossref PubMed Google Scholar), αHFVaHC17 (27.van't Veer C. Golden N.J. Kalafatis M. Mann K.G. J. Biol. Chem. 1997; 272: 7983-7994Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and burro anti-human FV polyclonal antibody (9.Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar) were provided by the Antibody Core Facility, Department of Biochemistry, University of Vermont (Burlington, VT). A rabbit antiphosphoserine polyclonal antibody was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Sequencing grade trypsin was purchased from Promega (Madison, WI). ZipTipc18 microcolums were purchased from Millipore (Billerica, MA). Preparation of Coagulation Proteins—All proteins were of human origin and purified from fresh frozen plasma unless otherwise noted. Factor X and prothrombin were isolated as described (28.Bajaj S.P. Rapaport S.I. Prodanos C. Prep. Biochem. 1981; 11: 397-412Crossref PubMed Scopus (155) Google Scholar, 29.Jesty J. Nemerson Y. Methods Enzymol. 1976; 45: 95-107Crossref PubMed Scopus (75) Google Scholar). Plasma-derived FV was isolated by immunoaffinity chromatography as described (30.Katzmann J.A. Nesheim M.E. Hibbard L.S. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 162-166Crossref PubMed Scopus (145) Google Scholar, 31.Nesheim M.E. Katzmann J.A. Tracy P.B. Mann K.G. Methods Enzymol. 1981; 80: 249-274Crossref PubMed Scopus (96) Google Scholar). Thrombin was prepared by activation of prothrombin with Taipan snake venom by the method of Owen and Jackson (32.Owen W.G. Jackson C.M. Thromb. Res. 1973; 3: 705-714Abstract Full Text PDF Scopus (79) Google Scholar). Human APC was purchased from Haematologic Technologies Inc. (Essex Junction, VT). All proteins used were >95% pure as judged by SDS-PAGE before and after disulfide bond reduction according to the method of Laemmli (33.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). Molecular weights and extinction coefficients (E280nm1%) of the various proteins used were taken as follows: prothrombin, 72,000, 13.8 (34.Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (178) Google Scholar); thrombin, 37,000, 17.4 (34.Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (178) Google Scholar, 35.Fenton II, J.W. Landis B.H. Walz D.A. Finlyason J.S. Lundblad R.L. Fenton II, J.W. Mann K.G. Chemistry and Biology of Thrombin. Ann Arbor Science Publishers Inc., Ann Arbor, MI1977Google Scholar); FV, 330,000, 9.6 (9.Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar, 31.Nesheim M.E. Katzmann J.A. Tracy P.B. Mann K.G. Methods Enzymol. 1981; 80: 249-274Crossref PubMed Scopus (96) Google Scholar); APC, 56,200, 14.5 (36.Kisiel W. J. Clin. Investig. 1979; 64: 761-769Crossref PubMed Scopus (336) Google Scholar); and factor Xa, 46,000, 11.6 (28.Bajaj S.P. Rapaport S.I. Prodanos C. Prep. Biochem. 1981; 11: 397-412Crossref PubMed Scopus (155) Google Scholar). Preparation of Human Washed Platelet Suspensions—Platelets required for functional studies were obtained from non-medicated, consenting adults whose blood (24 ml) was drawn into a 30-ml syringe containing 4 ml of ACD (0.022 m citrate, 0.014 m dextrose, final concentrations). Platelet-rich plasma was obtained from whole blood as described previously (23.Camire R.M. Kalafatis M. Simioni P. Girolami A. Tracy P.B. Blood. 1998; 91: 2818-2829Crossref PubMed Google Scholar), and platelets were washed according to the procedure of Mustard et al. (37.Mustard J.F. Perry D.W. Ardlie N.G. Packham M.A. Br. J. Haematol. 1972; 22: 193-204Crossref PubMed Scopus (650) Google Scholar), with modifications (22.Camire R.M. Kalafatis M. Cushman M. Tracy R.P. Mann K.G. Tracy P.B. J. Biol. Chem. 1995; 270: 20794-20800Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Platelets were brought to a final concentration of 2 × 109 platelets/ml in 0.5 mm HEPES-Tyrode's buffer containing 0.35% bovine albumin (HTA; 5 mm HEPES, 0.14 m NaCl, 2.7 mm KCl, 12 mm NaHCO3, 0.42 mm NaH2PO4, 1 mm MgCl2, 2 mm CaCl2, 5 mm dextrose, pH 7.35) and maintained at 37 °C. For the isolation of platelet-derived FV/Va, human platelets were obtained from either outdated platelet-pheresis packs or single unit donor packs (10–12 days post-draw) purchased from Haematologic Technologies Inc. (Essex Junction, VT). Platelets from ∼50 to 100 units of plasma were pooled and washed in the presence of PGE1 (5 μm) by centrifugation according to the procedure of Mustard et al. (37.Mustard J.F. Perry D.W. Ardlie N.G. Packham M.A. Br. J. Haematol. 1972; 22: 193-204Crossref PubMed Scopus (650) Google Scholar) with minor modifications (22.Camire R.M. Kalafatis M. Cushman M. Tracy R.P. Mann K.G. Tracy P.B. J. Biol. Chem. 1995; 270: 20794-20800Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Platelets were centrifuged at 1,500 × g in 250-ml conical polypropylene tubes for 15 min at ambient temperature. The supernatant was decanted, and the platelets were resuspended in a total of 1 liter of modified Tyrode's buffer (MTB; 0.14 m NaCl, 2.7 mm KCl, 12 mm NaHCO3, 0.42 mm NaH2PO4, 1 mm MgCl2, 2 mm CaCl2, 5 mm dextrose and 5 μm PGE1, pH 7.35) with the addition of 5 units/ml heparin. Red blood cells and those platelets that were not readily resuspended were discarded, and the suspended platelets were centrifuged at 1,500 × g. Platelets were washed two additional times in MTB buffer, and the final platelet pellet was resuspended in 20 mm Tris, 150 mm NaCl, pH 7.4, containing 5 mm CaCl2(TBS/Ca2+). The final platelet suspension was counted on a model Z1 Coulter Counter (Coulter Corp., Miami, FL). Isolation of Platelet-derived FV/Va—Platelet-derived FV/Va was isolated by a modification of the procedure for the purification of plasma-derived FV (30.Katzmann J.A. Nesheim M.E. Hibbard L.S. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 162-166Crossref PubMed Scopus (145) Google Scholar). Washed platelets were diluted to a final concentration 4 × 109 platelets/ml in TBS/Ca2+ containing 5 μm PGE1 yielding ∼250–350 ml of platelet suspension. Subsequently, the protease inhibitors benzamidine (10 mm), phenylmethylsulfonyl fluoride (0.5 mm), leupeptin (0.5 mm), and soybean trypsin inhibitor (20 μg/ml) were added to prevent protein cleavage. Platelets were lysed with the addition of Triton X-100 (1% v/v) for 5 min at 25 °C with occasional mixing yielding ∼560 units of FV. The platelet lysate was then incubated at 0 °C for 90 min to generate a cryoprecipitate, which was removed by centrifugation at 16,000 × g for 40 min at 4 °C with greater than 98% recovery of platelet-derived FV/Va. An anti-human FV monoclonal antibody (30.Katzmann J.A. Nesheim M.E. Hibbard L.S. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 162-166Crossref PubMed Scopus (145) Google Scholar) coupled to Sepharose CL-4B resin (50 ml resin; 3–4 mg antibody/ml resin) was added to the supernatant, and the suspension was slowly stirred at 4 °C for 2 h to adsorb the FV onto the resin. The resin was then transferred to a sintered glass funnel, and the supernatant was removed by vacuum. The resin was then slurried with ambient temperature TBS/Ca2+ containing 1 mm benzamidine (TBS/Ca2+/Benz), transferred to a 2.5 × 20 cm chromatography column, and washed at ambient temperature with TBS/Ca2+/Benz until the corrected A280 (A280C) was below 0.01. This was followed by an identical wash with TBS/Ca2+/Benz containing 0.35 m NaCl until an A280C < 0.01 was reached in the flow through. The bound FV was subsequently eluted with TBS/Ca2+/Benz containing 5.0 m NaCl at a flow rate of ∼0.2 ml/min. Collected fractions (5 ml) were assayed for FV cofactor activity using a prothrombin time-based clotting system employing FV-deficient plasma. Fractions containing detectable FV cofactor activity were pooled and generally exhibited nearly 10% of the starting FV/Va present in the platelet lysate. Buffer exchange of the pool into TBS/Ca2+ was accomplished at 4 °C using an Amicon ultrafiltration cell with a YM30 filter that had been soaked for 30 min in TBS/Ca2+ containing 1% PEG-8000 to reduce FV binding. The pool was then concentrated down to a of followed by the addition of 2 mm and stored at °C. The concentration of platelet-derived FV/Va was determined by of the light chain by using the were by of the activated platelet-derived cofactor to a of plasma-derived FVa. the purified platelet-derived FV/Va exhibited a yield and a specific cofactor activity of with greater than SDS-PAGE and Western of and Platelet-derived platelet-derived FV/Va (0.5 μm) in 20 mm HEPES, 150 mm NaCl, 5 mm CaCl2, pH containing PEG-8000 was with thrombin (20 at 37 °C for 15 min to activation in the presence of from the FV a of purified plasma-derived FV was thrombin-activated for sufficient to The activated FVa was then with to the (50 were diluted with of mm 10% 10% pH at 100 °C for 2 and to SDS-PAGE on by the method of Laemmli (33.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). were either by the method of et al. D. Biochem. 1981; PubMed Scopus Google Scholar), or protein was transferred to as described by et al. H. J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: PubMed Scopus Google Scholar) and by blotting using either anti-human FV monoclonal antibodies αHFVaLC9 (26.Foster W.B. Tucker M.M. Katzmann J.A. Miller R.S. Nesheim M.E. Mann K.G. Blood. 1983; 61: 1060-1067Crossref PubMed Google Scholar), αHFVaHC17 (26.Foster W.B. Tucker M.M. Katzmann J.A. Miller R.S. Nesheim M.E. Mann K.G. Blood. 1983; 61: 1060-1067Crossref PubMed Google Scholar), or a burro anti-human FV polyclonal antibody (9.Tracy P.B. Eide L.L. Bowie E.J. Mann K.G. Blood. 1982; 60: 59-63Crossref PubMed Google Scholar), as described previously (12.Camire R.M. Pollak E.S. Kaushansky K. Tracy P.B. Blood. 1998; 92: 3035-3041Crossref PubMed Google Scholar). of Platelet-derived of the of prothrombin activation were used to define the apparent the of platelet- or plasma-derived FVa with of the prothrombinase complex in an assay of purified containing factor Xa and of FVa in assay buffer (20 mm HEPES, m NaCl, 5 mm CaCl2, pH were incubated for 5 min followed by the addition of prothrombin μm were removed and diluted with of buffer (20 mm HEPES, m NaCl, mm pH to the The thrombin concentration in was determined using the substrate Inc., by to a thrombin The rate of thrombin generation in the various prothrombin activation was by of the data obtained from the removed were determined by to described previously S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar, R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; PubMed Scopus Google Scholar). of of Platelet- and Plasma-derived trypsin and of FVa peptides, FVa heavy chain and light chain were isolated by from a SDS-PAGE (33.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar) and with sequencing grade trypsin as described D.A. PubMed Scopus Google Scholar). containing either FVa heavy chain or light chain peptides were into and washed times with 25 mm pH for 15 The were with for 5 min and under by for 25 A of trypsin trypsin in m pH was added to the and for h at 37 °C. To the FVa peptides from the the was removed to a and with of acid followed by at ambient temperature for The supernatant was added to the with an additional of the and subsequently incubated with the an additional 30 The were then pooled and under by for MALDI-TOF analyses were prepared using a as the and on a in α-cyanohydroxycinnamic acid as MALDI-TOF of FVa were obtained on a mass with a laser and a The were obtained in with All were using a mixture of and chain obtained were into the of the University of San Francisco, for to the of human FV. modifications present on the peptides were identified by of to modifications using the of the system from the of of was obtained by of into the of the of and