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• W2019908848 abstract "Blood coagulation factor V circulates as a procofactor with little or no procoagulant activity. It is activated to factor Va by thrombin following proteolytic removal of a large central B-domain. Although this reaction is well studied, the mechanism by which bond cleavage and B-domain release facilitate the transition to the active cofactor state has not been defined. Here we show that deletion or substitution of specific B-domain sequences drives the expression of procoagulant function without the need for proteolytic processing. Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids that is highly basic and well conserved across the vertebrate lineage. Our findings demonstrate that discrete sequences in the B-domain serve to stabilize the inactive procofactor state, with proteolysis primarily functioning to remove these inhibitory constraints. These unexpected results provide new insight into the mechanism of factor V activation. Blood coagulation factor V circulates as a procofactor with little or no procoagulant activity. It is activated to factor Va by thrombin following proteolytic removal of a large central B-domain. Although this reaction is well studied, the mechanism by which bond cleavage and B-domain release facilitate the transition to the active cofactor state has not been defined. Here we show that deletion or substitution of specific B-domain sequences drives the expression of procoagulant function without the need for proteolytic processing. Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids that is highly basic and well conserved across the vertebrate lineage. Our findings demonstrate that discrete sequences in the B-domain serve to stabilize the inactive procofactor state, with proteolysis primarily functioning to remove these inhibitory constraints. These unexpected results provide new insight into the mechanism of factor V activation. Many proteins involved in hemostasis circulate in blood in a quiescent state and only express activity following limited proteolysis. One of these proteins, factor V (FV), 2The abbreviations used are: FV, factor V; FVIII, factor VIII; FVa, activated factor V; FX, factor X; FXa, factor Xa; S2238, H-d-phenylalanyl-l-pipecolyl-l-arginyl-ρ-nitroanilide; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide; PCPS, small unilamellar phospholipid vesicles composed of 75% (w/w) phosphatidylcholine and 25% (w/w) phosphatidylserine; PD-FV, plasma-derived factor V; rFVa, recombinant FVa; OG488-FXa, factor Xa modified with Oregon Green488. is synthesized as a large multi-domain (A1-A2-B-A3-C1-C2) protein sharing homology with factor VIII (FVIII), except in the B-domain (1Mann K.G. Kalafatis M. Blood. 2002; 101: 20-30Crossref PubMed Scopus (177) Google Scholar, 2Kane W.H. Colman R.W. Marder V.J. Clowes A.W. George J.N. Goldhaber S.Z. Hemostasis and Thrombosis. 2006: 177-192Google Scholar). Factor V circulates as a procofactor and has minimal procoagulant activity (3Nesheim M.E. Taswell J.B. Mann K.G. J. Biol. Chem. 1979; 254: 10952-10962Abstract Full Text PDF PubMed Google Scholar, 4Foster W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar). Thrombin catalyzes the conversion of FV to factor Va (FVa) following three cleavages (Arg709, Arg1018, and Arg1545), thereby releasing the heavily glycosylated B-domain that spans amino acids 710–1545 (5Jenny R.J. Pittman D.D. Toole J.J. Kriz R.W. Aldape R.A. Hewick R.M. Kaufman R.J. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4846-4850Crossref PubMed Scopus (343) Google Scholar). Factor Va is a heterodimer consisting of a noncovalently associated heavy and light chain and functions as a cofactor within the prothrombinase complex. Because FV cannot function in prothrombinase, the proteolytic conversion of FV to FVa must result in structural changes necessary for function. Most approaches aimed at understanding FV activation are principally based on correlating proteolysis within the B-domain with the expression of procoagulant activity (4Foster W.B. Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 13970-13977Abstract Full Text PDF PubMed Google Scholar, 6Esmon C.T. J. Biol. Chem. 1979; 254: 964-973Abstract Full Text PDF PubMed Google Scholar, 7Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 8Suzuki K. Dahlbaöck B. Stenflo J. J. Biol. Chem. 1982; 257: 6556-6564Abstract Full Text PDF PubMed Google Scholar, 9Monkovic D.D. Tracy P.B. Biochemistry. 1990; 29: 1118-1128Crossref PubMed Scopus (211) Google Scholar, 10Nesheim M.E. Foster W.B. Hewick R. Mann K.G. J. Biol. Chem. 1984; 259: 3187-3196Abstract Full Text PDF PubMed Google Scholar, 11Camire R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar, 12Marquette K.A. Pittman D.D. Kaufman R.J. Blood. 1995; 86: 3026-3034Crossref PubMed Google Scholar, 13Keller F.G. Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. 1995; 34: 4118-4124Crossref PubMed Scopus (56) Google Scholar, 14Thorelli E. Kaufman R.J. Dahlbaöck B. Eur. J. Biochem. 1997; 247: 12-20Crossref PubMed Scopus (64) Google Scholar, 15Steen M. Dahlbaöck B. J. Biol. Chem. 2002; 277: 38424-38430Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 16Kalafatis M. Beck D.O. Mann K.G. J. Biol. Chem. 2004; 278: 33550-33561Abstract Full Text Full Text PDF Scopus (44) Google Scholar, 17Kane W.H. Majerus P.W. J. Biol. Chem. 1981; 256: 1002-1007Abstract Full Text PDF PubMed Google Scholar). These studies support the idea that bond cleavage at Arg1545 is required for the expression of full cofactor activity. However, how proteolysis directly contributes to the development of FVa cofactor activity is not well defined. An alternative way of looking at this problem is to evaluate how FV is preserved as an inactive procofactor. One possibility is that binding sites on the heavy and/or light chain that are important to cofactor function are in a conformational state that precludes factor Xa (FXa)/prothrombin binding. Proteolysis could then drive cofactor activation in a manner analogous to the activation strategy used by the chymotrypsin-like serine proteases, protease activated receptors, and fibrinogen (18Khan A.M. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (384) Google Scholar, 19Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2145) Google Scholar, 20Doolittle R.F. J. Thromb. Haemostasis. 2003; 1: 1559-1565Crossref PubMed Scopus (43) Google Scholar). In this mechanism, bond cleavage is required to unmask new sequences, which then act to facilitate the necessary conformational change for activation (18Khan A.M. James M.N.G. Protein Sci. 1998; 7: 815-836Crossref PubMed Scopus (384) Google Scholar). A second possibility is that B-domain sequences serve an inhibitory function by rendering binding sites on the heavy and/or light chain inaccessible to FXa or prothrombin. Proteolysis would then promote dissociation of inhibitory B-domain sequences effecting activation. Aspartic and cysteine proteases use this approach to control the inactivity of the zymogen (21Khan A.R. Khazanovich-Bernstein N. Bergmann E.M. James M.N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10968-10975Crossref PubMed Scopus (50) Google Scholar). A signature feature of this strategy is the ability to constitutively activate the protein by removing inhibitory sequences in the absence of proteolysis. Evidence that FV may use this strategy comes from the expression of a FV derivative in which a large segment of the B-domain was deleted (FVdes811–1491 or FV-810; see Scheme 1). This variant was shown to have significant procoagulant activity (13Keller F.G. Ortel T.L. Quinn-Allen M.A. Kane W.H. Biochemistry. 1995; 34: 4118-4124Crossref PubMed Scopus (56) Google Scholar, 22Kane W.H. Devore-Carter D. Ortel T.L. Biochemistry. 1990; 29: 6762-6768Crossref PubMed Scopus (45) Google Scholar), and more recent work indicates that FV-810 binds FXa membranes with high affinity and functions in an equivalent way to FVa in the absence of intentional proteolysis (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These observations suggested that the B-domain must somehow interfere with binding interactions that govern the function of FVa. In the current study we used a series of activity and direct binding measurements to characterize a panel of progressively finer B-domain-truncated variants to explore this idea further and define regions within the B-domain that may be involved in stabilizing the FV procofactor state. Materials—The peptidyl substrate H-d-phenylalanyl-l-pipecolyl-l-arginyl-ρ-nitroanilide (S2238) was from Diapharma Group, Inc. (West Chester, OH). Benzamidine, bovine serum albumin, and 4-amidinophenylmethanesulfonyl fluoride hydrochloride were purchased from Sigma. Dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was from Hematologic Technologies (Essex Junction, VT). All of the tissue culture reagents were from Invitrogen except insulin-transferrin-sodium selenite, which was from Roche Applied Science. Small unilamellar phospholipid vesicles (PCPS) composed of 75% (w/w) hen egg l-α-phosphatidlycholine and 25% (w/w) porcine brain l-α-phosphatidylserine (Avanti Polar Lipids, Alabaster, AL) were prepared and characterized as described (24Higgins D.L. Mann K.G. J. Biol. Chem. 1983; 258: 6503-6508Abstract Full Text PDF PubMed Google Scholar, 25Gomori G. J. Lab. Clin. Med. 1942; 27: 955-960Google Scholar). Oregon Green488 maleimide and succinimidyl acetothioacetate were from Amersham Biosciences. Simplastin Excel was from BioMerieux (Durham, NC), and FV-deficient plasma was from George King Bio-medical Inc. (Overland Park, KS). Unless otherwise noted, all of the 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 (26Buddai 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, 27Baugh R. Krishnaswamy S. J. Biol. Chem. 1996; 271: 16126-16134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 28Katzmann J.A. Nesheim M.E. Hibbard L.S. Mann K.G. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 162-166Crossref PubMed Scopus (147) Google Scholar). Thrombin, prethrombin-2, and FXa were prepared and purified by established procedures (29Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (179) Google Scholar, 30Camire R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 31Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar). Human plasma derived FV (PD-FV) and FV-810 were proteolytically processed with thrombin to generate PD-FVa and rFVa as described (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Kalafatis M. Krishnaswamy S. Rand M.D. Mann K.G. Methods Enzymol. 1993; 222: 224-236Crossref PubMed Scopus (35) Google Scholar). Oregon Green488-human FXa (OG488-FXa) was prepared as described (26Buddai 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). Molecular weights and extinction coefficients (E0.1%280 nm) of the various proteins used were: prothrombin, 72,000 and 1.47 (29Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (179) Google Scholar); prethrombin-2, 37,500 and 1.95 (29Mann K.G. Methods Enzymol. 1976; 45: 123-156Crossref PubMed Scopus (179) Google Scholar); thrombin, 37,500 and 1.94 (33Lundblad 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 (34DiScipio 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 (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); rFVa, 175,000 and 1.78 (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar); and FV-810, 216,000 and 1.54 (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), respectively. The calculated molecular weights and extinction coefficients for the rFV derivatives were: FV-902, 229,000 and 1.55; FV-956, 232,000 and 1.59; FV-1033, 239,000 and 1.52; FV-1053, 241,000 and 1.50; FV-1106, 258,000 and 1.49; and FV-1152, 264,000 and 1.46, respectively. The molecular weights and extinction coefficients of FVB8–131, FVB8–104, and FVB8–46 were assumed to be the same as FV-1033. Construction of Recombinant FV Derivatives—The construction of FV-810 (see Scheme 1) has been previously described (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Specific oligonucleotides used to generate FV-902 were as follows: primer A, 5′-GAAGAGGTGGGAATACTT-3′ corresponds to the cDNA sequence encoding for amino acid residues 319 to 325; primer B, 5′-CTCAATGTAATCTGTATCACTAGGAGGGTC-3′ in which the first 15 bases encode for residues 1496–1492 and the last 15 bases encode for residues 902–898; primer C, 5′-GACCCTCCTAGTGATACAGATTACATTGAG-3′, in which the first 15 bases encode for residues 898–902 and the last 15 bases encode for residues 1492–1496; and primer D, 5′-TCTGTCCATGATAAGAAATGG-3′ corresponds to the FV cDNA sequence encoding for residues 1877–1871. The resulting DNA fragment was TOPO cloned (Invitrogen), then digested with Bsu36I and SnaBI, gel-purified, and subcloned into pED-FV digested with the same enzymes (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). To ensure the absence of polymerase-induced errors, the entire modified cDNA was sequenced. The remaining constructs outlined in Scheme 1 were prepared in the same way, except primers B and C were appropriately changed. Expression and Purification of rFV Derivatives—Plasmids encoding each of the FV constructs were transfected into baby hamster kidney cells, and stable clones were established as described (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 35Toso R. Camire R.M. J. Biol. Chem. 2006; 281: 8773-8779Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Protein expression levels varied from 0.5 to 4 μg/106cells/24 h. Each of the FV derivatives was purified essentially as described (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) with a final yield of ∼0.5–2 mg of protein/liter of conditioned medium. Protein purity was assessed by SDS-PAGE under reducing conditions followed by staining with Coomassie Brilliant Blue R-250. FV-specific Prothrombin Time-based Clotting Assay—Factor V/Va (200 nm) derivatives were prepared in assay buffer. For experiments in which pretreatment with thrombin was intended, the samples (200 nm) were incubated at 37 °C for 10 min with 2 nm thrombin, followed by the addition of 3 nm hirudin. The samples were then diluted to less than 1 nm in assay buffer with 0.1% bovine serum albumin, and specific clotting activity using FV-deficient plasma was assayed as described (11Camire R.M. Kalafatis M. Tracy P.B. Biochemistry. 1998; 37: 11896-11906Crossref PubMed Scopus (41) Google Scholar). The data are presented as the mean values ± S.D. Fluorescence Intensity Measurements—Samples (2.5 ml) in assay buffer were maintained at 25 °C in 1 × 1-cm2 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 as described (26Buddai 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, 36Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (74) 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 (30Camire R.M. J. Biol. Chem. 2002; 277: 37863-37870Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Krishnaswamy S. Walker R.K. Biochemistry. 1997; 36: 3319-3330Crossref PubMed Scopus (38) Google Scholar). Progress curves of prethrombin-2 activation were performed using the following reaction conditions: PCPS (20 μm), DAPA (3 μm), and prethrombin-2 (1.4 μm) were incubated with the various cofactors (3.0 nm) in assay buffer, and the reaction was initiated with FXa (3.0 nm). When prothrombin was used as protein substrate, the following reaction conditions were used: PCPS (20 μm), DAPA (3 μm), and prothrombin (1.4 μm) were incubated with the various cofactors (0.1 nm), and the reaction was initiated with FXa (1.0 nm). At various time points, aliquots of the reaction mixture were quenched, and thrombin generation was determined by using the chromogenic substrate S2238. Data Analysis—The binding data were analyzed according to the referenced equations by nonlinear least squares regression analysis using the Marquardt algorithm (38Bevington P.R. Robinson K.D. Data Reduction and Error Analysis for the Physical Sciences. 1992; (McGraw-Hill, New York)Google Scholar). The quality of each fit was assessed by the criteria described (39Straume M. Johnson M.L. Methods Enzymol. 1992; 210: 87-105Crossref PubMed Scopus (111) Google Scholar). 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 (40Krishnaswamy S. J. Biol. Chem. 1990; 265: 3708-3718Abstract Full Text PDF PubMed Google Scholar). Reported estimates of error for binding data represent ± 2 S.D. Expression of FV Derivatives—We have previously shown that FV-810 is functionally equivalent to FVa (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). We hypothesized that reintroducing portions of the B-domain would yield a molecule with procofactor-like properties. Because the C-terminal half of the B-domain (i.e. residues 1019–1545) varies considerably in length or is absent among different vertebrates (41Davidson C.J. Tuddenham E.G. McVey J.H. J. Thromb. Haemostasis. 2003; 1: 1487-1494Crossref PubMed Scopus (132) Google Scholar, 42Jiang Y. Doolittle R.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7527-7532Crossref PubMed Scopus (119) Google Scholar, 43Vos H.L. J. Thromb. Haemostasis. 2005; 3: P0041Google Scholar), we chose to extend the length of FV-810 by ∼50–100-amino acid increments from the N-terminal side (Scheme 1). SDS-PAGE analyses indicated that each of the proteins migrated with the expected mobility (Fig. 1). Plasma-derived FV, PD-FVa, and rFVa used as controls are included on the gel. Following treatment with thrombin, the proteins migrated in the expected way on the gel yielding FVa. Additional control experiments revealed no significant difference in the rate of proteolysis of the variants compared with FV when using thrombin or FXa (data not shown). Characterization of FV Variants—Initial experiments focused on the clotting activity of the B-domain variants prior to and following treatment with thrombin (Table 1). In this assay system, PD-FV has a low specific activity, which increases ∼10-fold following thrombin activation. This specific activity is comparable with the levels observed for PD-FVa, rFVa, and FV-810. Extending the B-domain length by adding amino acids 811–902 to FV-810 (resulting in FV-902) did not significantly change the specific clotting activity. A further increase in B-domain length to 378 amino acids (FV-1033) decreased the specific activity to levels seen for the procofactor, PD-FV. Extension beyond 378 amino acids (FV-1053, FV-1106, and FV-1152) was without functional consequence, because each of these variants had a procofactor-like specific activity. Factor V-956 had an intermediate specific activity (Table 1).TABLE 1Characterization of B-domain-truncated FV variantsCofactor speciesSpecific activityaThe mean values ± S.D. are presented from three determinations. The data are representative of two to three similar experimentsSpecific activityaThe mean values ± S.D. are presented from three determinations. The data are representative of two to three similar experiments,bActivated with thrombin prior to the addition to the clotting assayKdcReported errors represent ± 2 S.D. The data are representative of two to three similar experimentsncReported errors represent ± 2 S.D. The data are representative of two to three similar experimentsunits/nmolunits/nmolnmmol/molPD-FVa235 ± 40NAdNA, not applicable0.50 ± 0.081.2 ± 0.04rFVa215 ± 26NA0.90 ± 0.131.0 ± 0.03FV-810220 ± 17213 ± 6.50.96 ± 0.171.1 ± 0.05FV-902210 ± 9.0206 ± 230.81 ± 0.131.1 ± 0.04FV-95674.1 ± 6.7187 ± 6.15.2 ± 1.00.98 ± 0.09FV-103317.5 ± 1.9222 ± 33-e-, we were not able to accurately determine a value-FV-105325.0 ± 1.1210 ± 11--FV-110623.6 ± 1.3206 ± 37--FV-115224.9 ± 3.2165 ± 16--PD-FV19.4 ± 2.5172 ± 20--a The mean values ± S.D. are presented from three determinations. The data are representative of two to three similar experimentsb Activated with thrombin prior to the addition to the clotting assayc Reported errors represent ± 2 S.D. The data are representative of two to three similar experimentsd NA, not applicablee -, we were not able to accurately determine a value Open table in a new tab To verify the results of the clotting assay, a purified component assay was employed. Progress curves of the conversion of prethrombin-2 to thrombin using equimolar concentrations of (pro)cofactor (3 nm) and FXa (3 nm) yielded results that were consistent with the clotting assay. Thrombin increased linearly over time when using PD-FVa, rFVa, FV-810, and FV-902, and the initial rates of thrombin generation were within a factor of two (Table 2 and Fig. 2). Once again, FV-956 had reduced activity compared with FVa. In contrast, PD-FV, FV-1152, FV-1106, FV-1053, and FV-1033 had very little activity (Table 2 and Fig. 2). Furthermore, the progress curves were characterized by an obvious lag in thrombin generation, which is expected when the product further activates the procofactor (Fig. 2). Similar results were obtained when using prothrombin as the macromolecular substrate (data not shown). In both the clotting and purified component assays, pretreatment of each of these rFV derivatives with thrombin resulted in full activation (Tables 1 and 2).TABLE 2Initial rates of prethrombin-2 activationCofactor speciesInitial velocityaThe data are representative of two to three similar experimentsInitial velocityaThe data are representative of two to three similar experiments,bActivated with thrombin prior to the addition in the prothrombinase assaynm IIa/minnm IIa/minPD-FVa23NAdNA, not applicablerFVa23NAFV-8103522FV-9023030FV-9561518FV-1033-c-, a lag was observed; thus, we were not able to determine a value26FV-1052-26FV-1106-22FV-1152-24PD-FV-20a The data are representative of two to three similar experimentsb Activated with thrombin prior to the addition in the prothrombinase assayc -, a lag was observed; thus, we were not able to determine a valued NA, not applicable Open table in a new tab Binding of (Pro)Cofactors to FXa Membranes—Based on the functional measurements, the B-domain-truncated FV derivatives could be grouped into two categories: cofactor-like (FV-810, FV-902, and FV-956) and procofactor-like (FV-1033, FV-1053, FV-1106, and FV-1152). Because FV binds poorly to active site-blocked FXa (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), we evaluated the ability of each variant to bind FXa membranes. Equilibrium fluorescence measurements were used to establish binding parameters describing the interaction between the cofactor and membrane-bound FXa. Using a fixed concentration of OG488-FXa, subsequent titration with incremental additions of rFVa, FV-810, and FV-902 (Fig. 3) yielded a saturable increase in fluorescence intensity with comparable dissociation constants (Kd) and stoichiometries (n) (Table 1). Recombinant FV-956 bound with a 5–10-fold reduced affinity, which possibly accounts for the decreased cofactor activity observed in the functional measurements (Table 1), because both of these assays use limiting concentrations of cofactor. In contrast, no significant increase in fluorescence intensity was observed when using PD-FV or FV-1033 (Fig. 3). Similar results were obtained with FV-1053, FV-1106, and FV-1152 (data not shown). Treatment of PD-FV, FV-1033, FV-1053, FV-1106, and FV-1152 with thrombin followed by assessment of direct binding yielded dissociation constants consistent with FVa (data not shown), indicating that once activated each of the variants can assemble within prothrombinase. Consistent with our previous findings (23Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), control experiments indicated that each of the FV variants was not cleaved during the binding measurements (data not shown; see Fig. 5B for FV-810 and FV-1033).FIGURE 5Direct binding measurements with FV-1033 derivatives. A, reaction mixtures containing 10 nm OG488-FXa and 50 μm PCPS in assay buffer were titrated with increasing concentrations of PD-FVa (▪), FVB8–131 (×), FVB8–104 (▪), FVB8–46 (□), and FV-1033 (▾). The data for FV-1033 are provided as a comparison and are the same as those in Fig. 3. Fluorescence (F) intensity was measured at 25 °C. The lines are drawn following analysis to independent, noninteracting sites, and the fitted values (Kd and n) are given in Tables 1 and 3. The data are representative of two to three similar experiments. B, SDS-PAGE analysis. Purified proteins (4 μg/lane) prior to the start of the experiment (Start) and taken from the cuvette at the end of the experiment (End) were subjected to SDS-PAGE. Lane 1, FV-810; lane 2, FVB8–131; lane 3, FVB8–104; lane 4, FVB8–46; lane 5, FV-1033. FV-1033 was run on a separate gel. The apparent molecular weights of the standards are indicated on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Contribution of Specific B-domain Sequences to Preserving the Procofactor State—The findings suggest that either specific sequences (residues 902–1033) or a specific length (∼378 residues) of the B-domain somehow plays a role in the mechanism by which FV is maintained as a procofactor. To discriminate between these possibilities, we constructed additional FV variants that retained a B-domain length of 378 amino acids but that had portions of residues 900–1030 exchanged with FVIII B-domain sequences that share no homology with sequences within the FV B-domain. Using the procofactor-like FV-1033 as a scaffold, three derivatives were prepared with B-domain lengths of 378 amino acids: FVB8–131, FVB8–104, and FVB8–46, representing 131, 104, and 46 amino acids from the FV B-domain exchanged with FVIII B-domain (Fig. 4). We chose regions of the FVIII B-domain that did not contain known thrombin or intracellular furin cleavage sites (44Vehar G. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblat F. Oppermann H. Keck R. Wood W. Harkins R. Tuddenham E.G.D. Lawn R. Capon D. Nature. 1984; 312: 337-342Crossref PubMed Scopus (659) Google Scholar). As expected, SDS-PAGE revealed that FVB8–131, FVB8–104, and FVB8–46 have apparent molecular weights equivalent to FV-1033 (Fig. 4). Following incubation with thrombin, each of the derivatives was processed to yield the expected heavy and light chains, and the rate of proteolysis was comparable with FV (data not shown). By using either a purified component assay (data not shown) or a one-stage prothrombin time-based clotting assay or by monitoring direct binding to FXa membranes, we found that each of these variants had properties most consistent with the cofactor-like form (Table 3 and Fig. 5A). Control experiments revealed that the variants were not cleaved during the course of the binding measurements (Fig. 5B). This indicates that proteolysis of the variants is not a requirement for high affinity binding to FXa membranes. When the variants were incubated with thrombin, their specific activities increased only ∼1.5-fold compared with an activation quotient of ∼12 with FV-1033. The activity and direct binding measurements indicate that these derivatives were not completely identical to FVa, because they had ∼65% of full cofactor activity. This is likely due to the finding that the variants bound with a slightly reduced affinity to FXa membranes (∼2–4-fold). In addition" @default.
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• W2019908848 date "2007-05-01" @default.
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• W2019908848 title "Inhibitory Sequences within the B-domain Stabilize Circulating Factor V in an Inactive State" @default.
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• W2019908848 doi "https://doi.org/10.1074/jbc.m701315200" @default.
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