Role of Hirudin-like Factor Va Heavy Chain Sequences in Prothrombinase Function
2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês
10.1074/jbc.m511419200
ISSN1083-351X
AutoresRaffaella Toso, Rodney M. Camire,
Tópico(s)Vitamin K Research Studies
ResumoProexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659–698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710–1545), FVa699 (des700–1545), FVa692 (des693–1545), FVa678 (des679–1545), and FVa658 (des659–1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an ∼1% clotting activity and reduced affinity for FXa membranes (∼20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa658 exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase. Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659–698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710–1545), FVa699 (des700–1545), FVa692 (des693–1545), FVa678 (des679–1545), and FVa658 (des659–1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an ∼1% clotting activity and reduced affinity for FXa membranes (∼20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa658 exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase. Blood coagulation factor Va (FVa) 2The abbreviations used are: FVa, activated factor V; FV, factor V; FXa, factor Xa; IIa, α-thrombin; BSA, bovine serum albumin; FPR, d-phenylalanyl-l-prolyl-l-arginine; PCPS, small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine; rFV-DT, recombinant partial B-domainless (des811–1491) factor V; PD-FV, plasma-derived factor V; OG488-FXa, factor Xa modified with Oregon Green488; rFVa709, recombinant FV with amino acids 710–1545 deleted; rFVa699, amino acids 700–1545 deleted; rFVa692, amino acids 693–1545 deleted; rFVa678, amino acids 679–1545 deleted; rFVa658, amino acids 659–1545 deleted; MOPS, 4-morpholinepropanesulfonic acid; PACE, paired basic amino acid cleaving enzyme; PT, prothrombin time. 2The abbreviations used are: FVa, activated factor V; FV, factor V; FXa, factor Xa; IIa, α-thrombin; BSA, bovine serum albumin; FPR, d-phenylalanyl-l-prolyl-l-arginine; PCPS, small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine; rFV-DT, recombinant partial B-domainless (des811–1491) factor V; PD-FV, plasma-derived factor V; OG488-FXa, factor Xa modified with Oregon Green488; rFVa709, recombinant FV with amino acids 710–1545 deleted; rFVa699, amino acids 700–1545 deleted; rFVa692, amino acids 693–1545 deleted; rFVa678, amino acids 679–1545 deleted; rFVa658, amino acids 659–1545 deleted; MOPS, 4-morpholinepropanesulfonic acid; PACE, paired basic amino acid cleaving enzyme; PT, prothrombin time. is a heterodimeric protein composed of a heavy chain (residues 1–709; 105 kDa) and a light chain (residues 1546–2196; 74 kDa) which arises from limited proteolysis of the pro-cofactor factor V (FV) (1Mann K.G. Kalafatis M. Blood. 2002; 101: 20-30Crossref PubMed Scopus (176) Google Scholar). Factor Va reversibly associates with the serine protease factor Xa (FXa) on an appropriate membrane surface in the presence of calcium ions to form prothrombinase. This enzyme complex cleaves two peptide bonds in prothrombin, resulting in the generation of α-thrombin (IIa) (2Mann K.G. Nesheim M.E. Church W.R. Haley P.E. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). This reaction is also catalyzed by membrane-bound FXa; however, incorporation of FVa within prothrombinase has a profound effect on the rate of IIa generation. Additionally, prothrombinase has remarkable specificity, as its only known biological substrate is prothrombin (2Mann K.G. Nesheim M.E. Church W.R. Haley P.E. Krishnaswamy S. Blood. 1990; 76: 1-16Crossref PubMed Google Scholar). Although the molecular basis underlying the rate-enhancing effect of FVa remains largely unknown, recent contributions have provided insight into how prothrombinase recognizes its protein substrate (3Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Boskovic D.S. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 5Krishnaswamy S. J. Thromb. Haemost. 2005; 3: 54-67Crossref PubMed Scopus (128) Google Scholar). These studies support a model in which binding specificity is determined by two resolvable steps involving an interaction at an exosite followed by active site docking. These exosites have yet to be fully defined, but there is some evidence that at least part of the enzyme exosite lies on the catalytic domain of FXa (6Buddai S.K. Toulokhonova L. Bergum P.W. Vlasuk G.P. Krishnaswamy S. J. Biol. 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J. Biol. Chem. 2004; 279: 20786-20793Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). A series of observations suggest that the proexosite I region, the precursor site to IIa exosite I (19Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), contributes either directly or indirectly to this substrate binding site. For example, reagents that block proexosite I such as hirugen and bothrojaracin inhibit macromolecular substrate cleavage by prothrombinase or the FXa-FVa complex (3Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 21Monteiro R.Q. Zingali R.B. Thromb. Haemost. 2002; 87: 288-293Crossref PubMed Scopus (27) Google Scholar). Additionally, proexosite I prethrombin-1 mutants and a naturally occurring prothrombin variant (Arg67 to His, chymotrypsin numbering system (22Bode W. Mayr I. Bauman Y. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 8: 3467-3475Crossref PubMed Scopus (821) Google Scholar)) are poor substrates for prothrombinase and FXa-FVa (23Chen L. Yang L. Rezaie A.R. J. Biol. Chem. 2003; 278: 27564-27569Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 24Akhavan S. De Cristofaro R. Peyvandi F. Lavoretano S. Landolfi R. Mannucci P.M. Blood. 2002; 100: 1347-1353Crossref PubMed Scopus (28) Google Scholar). Interestingly, these probes or mutations reduced the rate of IIa generation only in the presence of the cofactor, suggesting a link between FVa and proexosite I. Consistent with this, IIa exosite I mutants and exosite I probes inhibit FV activation (25Esmon C.T. Lollar P. J. Biol. Chem. 1996; 271: 13882-13887Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 26Dharmawardana K.R. Bock P.E. Biochemistry. 1998; 37: 13143-13152Crossref PubMed Scopus (49) Google Scholar, 27Myles T. Yun T.H. Hall S.W. Leung L.L. J. Biol. Chem. 2001; 276: 25143-25149Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Additionally, detailed studies by Bock and co-workers have provided compelling evidence that the FVa heavy chain binds to exosite I of IIa (26Dharmawardana K.R. Bock P.E. Biochemistry. 1998; 37: 13143-13152Crossref PubMed Scopus (49) Google Scholar, 28Dharmawardana K.R. Olson S.T. Bock P.E. J. Biol. Chem. 1999; 274: 18635-18643Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Taken together, these data are consistent with the idea that proexosite I plays an important role in macromolecular substrate recognition by facilitating productive interactions between FVa and prothrombin. Based on these findings it is reasonable to hypothesize that the corresponding binding site on FVa for proexosite I is found within the hirudin-like C-terminal region of the FVa heavy chain (659–698; Fig. 1). Evidence to support this derives from recent experiments employing synthetic FVa peptides. One of these peptides (DYDYQ; 695–699, see Fig. 1), like hirugen, was found to bind specifically to IIa and inhibit prothrombin activation, suggesting this region of FVa provides a substrate binding site within prothrombinase (29Kalafatis M. Beck D.O. Mann K.G. J. Biol. Chem. 2004; 278: 33550-33561Abstract Full Text Full Text PDF Scopus (44) Google Scholar, 30Beck D.O. Bukys M.A. Singh L.S. Szabo K. Kalafatis M. J. Biol. Chem. 2004; 279: 3084-3095Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). In support of this, changing DYDY to KFKF (FVa2K2F) significantly reduced FVa cofactor activity within prothrombinase (30Beck D.O. Bukys M.A. Singh L.S. Szabo K. Kalafatis M. J. Biol. Chem. 2004; 279: 3084-3095Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, it is unlikely that these data provide a complete explanation since proteolysis within the C-terminal FVa heavy chain region by a variety of enzymes only modestly reduces cofactor function in certain assay systems (29Kalafatis M. Beck D.O. Mann K.G. J. Biol. Chem. 2004; 278: 33550-33561Abstract Full Text Full Text PDF Scopus (44) Google Scholar, 31Bakker H.M. Tans G. Thomassen M.C. Yukelson L.Y. Ebberink R. Hemker H.C. Rosing J. J. Biol. Chem. 1994; 269: 20662-20667Abstract Full Text PDF PubMed Google Scholar, 32Egan J.O. Kalafatis M. Mann K.G. Protein Sci. 1997; 6: 2016-2027Crossref PubMed Scopus (44) Google Scholar, 33Camire R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar, 34Safa O. Morrissey J.H. Esmon C.T. Esmon N.L. Biochemistry. 1999; 38: 1829-1837Crossref PubMed Scopus (14) Google Scholar). To evaluate the importance of this region of FVa to prothrombinase function in more detail, we have employed a novel strategy to express recombinant FVa derivatives with specific truncations within the C terminus of the FVa heavy chain and used a series of physical and kinetic measurements to assess the contribution of this region to substrate binding within prothrombinase. Materials—Bovine serum albumin (BSA) and 5,5′-dithiobis(2-nitrobenzoic acid) were purchased from Sigma. Hippuryl-d-phenylalanyl-l-pipecolyl-l-arginyl-p-nitroanilide (S2238) was purchased from Diapharma Group, Inc. (West Chester, OH), and its concentration was verified (in water) using E342 = 8270 m-1 cm-1 (35Lottenberg R. Jackson C.M. Biochim. Biophys. Acta. 1983; 742: 558-564Crossref PubMed Scopus (133) Google Scholar). Oregon Green488 maleimide and succinimidyl acetothioacetate were from Molecular Probes (Eugene, OR). d-Phenylalanyl-l-prolyl-l-arginine (FPR) chloromethyl ketone was from Calbiochem. SulfoLink coupling gel was purchased from Pierce. Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide was purchased from Hematologic Technologies (Essex Junction, VT). All tissue culture reagents were from Invitrogen except insulin-transferrin-sodium selenite, which was from Roche Applied Science. l-α-Phosphatidylserine (brain, sodium salt) and l-α-phosphatidylcholine (egg yolk) were purchased from Avanti Polar Lipids (Alabaster, AL). Small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine (PCPS) were prepared as described previously (36Higgins D.L. Mann K.G. J. Biol. Chem. 1983; 258: 6503-6508Abstract Full Text PDF PubMed Google Scholar). The concentration of the phospholipid vesicles was determined by phosphorous assay (37Gomori G. J. Lab. Clin. Med. 1942; 27: 955-960Google Scholar). Unless otherwise noted, all functional assays were performed at 25 °C in 20 mm Hepes, 0.15 m NaCl, 2 mm CaCl2, 0.1% polyethylene glycol 8000, pH 7.5 (assay buffer). Proteins—Human prothrombin, FX, and FV were isolated from plasma as described previously (6Buddai S.K. Toulokhonova L. Bergum P.W. Vlasuk G.P. Krishnaswamy S. J. Biol. Chem. 2002; 277: 26689-26698Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 38Baugh R. Krishnaswamy S. J. Biol. Chem. 1996; 271: 16126-16134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 39Katzmann 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). Prethrombin-1, prethrombin-2, and IIa were prepared from human prothrombin and purified using established procedures (40Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (178) Google Scholar). A recombinant B-domainless form of FV (rFV-DT) used to prepare rFVa was expressed and purified as previously described (41Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Human plasma derived FV (PD-FV) and rFV-DT were proteolytically processed with IIa to generate PD-FVa and rFVa, and both were subsequently purified on a Poros HS/20 (0.46 × 10 cm) column as described (41Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 42Kalafatis M. Krishnaswamy S. Rand M.D. Mann K.G. Methods Enzymol. 1993; 222: 224-236Crossref PubMed Scopus (33) Google Scholar). Oregon Green488-human FXa (OG488-FXa) was prepared and characterized as described (6Buddai S.K. Toulokhonova L. Bergum P.W. Vlasuk G.P. Krishnaswamy S. J. Biol. Chem. 2002; 277: 26689-26698Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Protein concentrations were determined using the following molecular weights and extinction coefficients (E0.1%280nm): prothrombin, 72,000 and 1.47 (40Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (178) Google Scholar); prethrombin-1 49,900 and 1.78 (40Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (178) Google Scholar); prethrombin-2, 37,500 and 1.95 (40Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (178) Google Scholar); IIa, 37,500 and 1.94 (43Lundblad R.L. Kingdon H.S. Mann K.G. Methods Enzymol. 1976; 45: 156-176Crossref PubMed Scopus (233) Google Scholar); FXa, 45,300 and 1.16 (44Di Scipio R.G. Hermodson M.A. Yates S.G. Davie E.W. Biochemistry. 1977; 16: 698-706Crossref PubMed Scopus (415) Google Scholar); PD-FVa, 173,000 and 1.78; rFVa, 175,000 and 1.78 (41Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The calculated molecular weights for the rFVa derivatives were taken as: rFVa709, 175,000; rFVa699, 174,060; rFVa692, 173,190; rFVa678, 171,478; rFVa658, 171,165; an extinction coefficient of 1.78 was used for each derivative. N-terminal sequence analysis was performed in the laboratory of Dr. Alex Kurosky and Steven Smith at the University of Texas Medical Branch at Galveston. Construction of rFVa Derivatives—The human FV cDNA was subcloned into the pED expression plasmid as previously described (41Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Splicing by overlap extension was used to generate a series of FV derivatives in which the entire B-domain and portions of the C terminus of the heavy chain were deleted. To facilitate intracellular proteolytic processing, a DNA sequence encoding a PACE/furin recognition site was inserted between the new C terminus on the heavy chain and the light chain. The following five constructs were prepared (RKRRKR was inserted before amino acid 1546 for all constructs, see Fig. 1): rFVa709 (des710–1545), rFVa699 (des700–1545), rFVa692 (des693–1545), rFVa678 (des679–1545), and rFVa658 (des659–1545). Specific oligonucleotides used for rFVa709 were as follows: primer A, 5′-GAAGAGGTGGGAATACTT-3′ corresponds to the cDNA sequence coding for amino acid residues 319–325; primer B, 5′-GCGCTTTCTACGCTTTCTCCTAATTCCTAATGCTGC-3′ in which the first 18 bases correspond to cDNA sequence coding for a PACE/furin recognition sequence (RKRRKR), and the last 18 bases correspond to cDNA sequence coding for residues 709–704; primer C, 5′-AGAAAGCGTAGAAAGCGCAGCAACAATGGAAACAGAAGA-3′ in which the first 18 bases correspond to cDNA sequence coding for RKRRKR and the last 21 bases correspond to cDNA sequence coding for residues 1546–1552; primer D, 5′-TCTGTCCATGATAAGAAATGG-3′, which corresponds to the FV cDNA sequence coding for residues 1877–1871. The resulting DNA fragment was digested with Bsu36I and SnaBI, gel-purified, and subcloned into pED-FV digested with the same enzymes. To ensure the absence of polymerase-induced errors, the entire modified cDNA was sequenced. The remaining constructs were prepared in the same way, except primer B was appropriately changed. Expression and Purification of rFVa Derivatives—Plasmids encoding each of the rFVa constructs were transfected into baby hamster kidney cells, and stable clones were established essentially as described (41Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Protein expression levels varied from 1 to 4 μg/106cells/24 h. Each of the rFVa derivatives was expanded into triple flasks, and approximately 1 liter of conditioned media was harvested daily for 4 days and immediately loaded onto a SP-Sepharose (Amersham Biosciences) column equilibrated in 20 mm Hepes, 0.18 m NaCl, 5 mm CaCl2, pH 7.4. The column was washed with the same buffer and then eluted with 0.65 m NaCl. Fractions containing FVa activity were pooled and concentrated by ultrafiltration (Millipore), and the buffer was exchanged to reduce the NaCl concentration. The protein was then loaded onto a Poros HS/20 (0.46 × 10 cm) column equilibrated with 20 mm Hepes, 0.15 m NaCl, 5 mm CaCl2, pH 7.4. Bound protein was eluted with a gradient of increasing NaCl (0.15–1.0 m). The final yield was ∼0.5–2.0 mg of rFVa/liter of conditioned media, and the purified protein was stored at -80 °C. Protein purity was assessed by SDS-PAGE using pre-cast 4–12% gradient gels (Invitrogen) under reducing conditions using the MOPS buffer system followed by staining with Coomassie Brilliant Blue R-250. FV-specific PT-based Clotting Assay—Factor Va (200 nm) derivatives were prepared in assay buffer. Samples were then diluted to less than 1 nm in assay buffer with 0.1% albumin and specific clotting activity using FV-deficient plasma (George King Bio-medical Inc., Overland Park, KS) was performed as described (33Camire R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar). Fluorescence Intensity Measurements—Samples (2.5 ml) in assay buffer were maintained at 25 °C in 1 × 1-cm2 stirred quartz cuvettes, and steady state fluorescence intensity was measured using λex = 480 and λem = 520 nm with a long pass filter (KV500, Schott, Duryea, PA) in the emission beam. Measurements, including controls, were performed essentially as described (3Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 6Buddai S.K. Toulokhonova L. Bergum P.W. Vlasuk G.P. Krishnaswamy S. J. Biol. Chem. 2002; 277: 26689-26698Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Kinetics of Protein Substrate Cleavage—Steady state initial velocities of macromolecular substrate cleavage by prothrombinase were determined discontinuously at 25 °C as described (17Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar, 45Camire R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The kinetic parameters of prothrombinase-catalyzed prothrombin, prethrombin-2/fragment 1.2, or prethrombin-1 activation (Km and Vmax) were determined in assay buffer by measuring the initial rate of IIa formation at increasing concentrations of macromolecular substrate. Assay mixtures contained PCPS (20 or 50 μm), FVa (20 nm), and various concentrations of prothrombin (0–1.5 μm), prethrombin-2/fragment 1.2 (0–1.5 μm; 1.5 molar excess of fragment 1.2), or prethrombin-1 (0–12 μm). The reaction was initiated with 0.1 nm FXa for prothrombin and prethrombin-2/fragment 1.2 or 0.5 nm FXa for prethrombin-1. Initial rates of prothrombin activation in the absence of membranes were determined essentially as described (20Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16435-16442Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Reaction mixtures in assay buffer contained FVa (5 nm; 200 nm for rFVa658) and various concentrations of prothrombin (0–15 μm). The reaction was initiated with 10 nm FXa, and the rate of thrombin generation was measured as described (17Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar, 45Camire R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Chromatography of FVa on IIa-agarose—Nα-[(acetylthio)acetyl]-FPR-chloromethyl ketone was prepared and characterized as previously reported (46Bock P.E. Biochemistry. 1988; 27: 6633-6639Crossref PubMed Scopus (56) Google Scholar, 47Bock P.E. J. Biol. Chem. 1992; 267: 14974-14981Abstract Full Text PDF PubMed Google Scholar). Nα-[(acetylthio)acetyl]-FPR-IIa and Nα-[(acetylthio)-acetyl]-FPR-IIa coupled to agarose were prepared using previously established procedures (26Dharmawardana K.R. Bock P.E. Biochemistry. 1998; 37: 13143-13152Crossref PubMed Scopus (49) Google Scholar). The IIa-agarose matrix contained 3.7 mg of IIa coupled/ml of gel. Free binding sites were blocked with BSA. The BSA-agarose matrix used as the control for nonspecific binding contained 10 mg of BSA coupled/ml of gel. Columns were equilibrated in 50 mm Hepes, 110 mm NaCl, 5 mm CaCl2, 0.1% polyethylene glycol 8000, pH 7.4. Factor Va (∼200 μg) was loaded onto each column at a flow rate of 8 ml/h at 22 °C and washed with the same buffer. Protein was eluted with 50 mm Hepes, 2 m NaCl, 5 mm CaCl2, 0.1% polyethylene glycol 8000, pH 7.4. Fractions (0.5 ml) were collected, and the absorbance at 280 nm and clotting activity were measured. Data Analysis—Data were analyzed according to the referenced equations by nonlinear least squares regression analysis using the Marquardt algorithm (48Bevington P.R. Robinson K.D. Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill Inc., New York1992Google Scholar). The qualities of the fits were assessed by the criteria described (49Straume M. Johnson M.L. Methods Enzymol. 1992; 210: 87-105Crossref PubMed Scopus (110) Google Scholar). Fitted parameters are reported ±95% confidence limits. Dissociation constants (Kd) and stoichiometries (n) for the interaction between FXa and membrane-bound FVa were obtained from the dependence of the fluorescence intensity on the concentrations of cofactor (50Krishnaswamy S. J. Biol. Chem. 1990; 265: 3708-3718Abstract Full Text PDF PubMed Google Scholar). Initial velocity measurements of prothrombin cleavage by prothrombinase were analyzed by fitting the data to the Henri-Michaelis-Menten equation (51Segal I.H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady State Enzyme Systems. John Wiley & Sons, Inc., New York1975Google Scholar), to yield fitted values for Km and Vmax. Construction of rFVa Variants—To directly assess the contribution of hirudin-like FVa sequences (659–698) to cofactor function, the entire B-domain and specific heavy chain sequences were removed, and a PACE-furin cleavage site was introduced between these deleted sequences and the light chain generating rFVa658 (des659–1545), rFVa678 (des679–1545), rFVa692 (des693–1545), rFVa699 (des700–1545), and rFVa709 (des710–1545; see Fig. 1). Recombinant FVa709 served as a control as it should be equivalent to PD-FVa and rFVa. This system takes advantage of the endogenous intracellular proteolytic processing machinery allowing for internally cleaved two-chain FVa variants to be secreted into the cell culture media. We used this strategy to overcome potential problems in the activation of the deletion variants by IIa or RVV-V (factor V, cleaving protease isolated from Russell's viper venom) because of the elimination of possible enzyme binding sites contributed by heavy chain and B-domain sequences. Characterization of rFVa Variants—Western blot analysis of conditioned media derived from stable cell lines revealed that each variant was intracellularly processed and secreted in the two chain form (data not shown). These derivatives were subsequently purified to homogeneity by ion exchange chromatography. SDS-PAGE analyses (Fig. 2) indicated that the proteins migrated according to the expected molecular weight. N-terminal sequence analysis of the light chains for each derivative showed that the PACE/furin cleavage site was completely removed, as the N terminus was found to be SNNGNRRN (Table 1). Because it has been previously shown that baby hamster kidney cells process C-terminal basic amino acids from the light chain of protein C (52Foster D.C. Sprecher C.A. Holly R.D. Gambee J.E. Walker K.M. Kumar A.A. Biochemistry. 1990; 29: 347-354Crossref PubMed Scopus (42) Google Scholar) and rFVa709 appears functionally equivalent to plasma-derived and rFVa (see Table 1), we speculate that the RKRRKR sequence is removed form the C terminus of the heavy chain, most likely by an Arg/Lys carboxypeptidase (53Rouille Y. Duguay S.J. Lund K. Furuta M. Gong Q. Lipkind G. Oliva Jr., A.A. Chan S.J. Steiner D.F. Front. Neuroendocrinol. 1995; 16: 322-361Crossref PubMed Scopus (314) Google Scholar, 54Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 55Rockwell N.C. Krysan D.J. Komiyama T. Fuller R.S. Chem. Rev. 2002; 102: 4525-4548Crossref PubMed Scopus (169) Google Scholar).TABLE 1Functional and physical characterization of FVa variantsCofactor speciesSpecific activity ± S.D.aSpecific clotting activity was determined by a FV-specific PT-based clotting assay as described under “Experimental Procedures.” The data represent the average of three measurements, and the error represents the S.D.Initial velocitybInitial steady state rates were determined in assay buffer using the indicated cofactors (0.1 nm) p
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