Homocysteine Inhibits Inactivation of Factor Va by Activated Protein C
2001; Elsevier BV; Volume: 276; Issue: 6 Linguagem: Inglês
10.1074/jbc.m004124200
ISSN1083-351X
AutoresAnetta Undas, E.Brady Williams, Saulius Butenas, Thomas Orfeo, Kenneth G. Mann,
Tópico(s)Iron Metabolism and Disorders
ResumoWe report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by α-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min−1 at 0, 0.03, 0.1, 1 mmhomocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm β-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [35S]homocysteine (10–450 μm) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507–709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with β-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s). We report the effect of homocysteine on the inactivation of factor Va by activated protein C (APC) using clotting assays, immunoblotting, and radiolabeling experiments. Homocysteine, cysteine, or homocysteine thiolactone have no effect on factor V activation by α-thrombin. Factor Va derived from homocysteine-treated factor V was inactivated by APC at a reduced rate. The inactivation impairment increased with increasing homocysteine concentration (pseudo first order rate k = 1.2, 0.9, 0.7, 0.4 min−1 at 0, 0.03, 0.1, 1 mmhomocysteine, respectively). Neither cysteine nor homocysteine thiolactone treatment of factor V affected APC inactivation of derived factor Va. Western blot analyses of APC inactivation of homocysteine-modified factor Va are consistent with the results of clotting assays. Factor Va, derived from factor V treated with 1 mm β-mercaptoethanol was inactivated more rapidly than the untreated protein sample. Factor V incubated with [35S]homocysteine (10–450 μm) incorporated label within 5 min, which was found only in those fragments that contained free sulfhydryl groups: the light chain (Cys-1960, Cys-2113), the B region (Cys-1085), and the 26/28-kDa (residues 507–709) APC cleavage products of the heavy chain (Cys-539, Cys-585). Treatment with β-mercaptoethanol removed all radiolabel. Plasma of patients assessed to be hyperhomocysteinemic showed APC resistance in a clot-based assay. Our results indicate that homocysteine rapidly incorporates into factor V and that the prothrombotic tendency in hyperhomocysteinemia may be related to impaired inactivation of factor Va by APC due to homocysteinylation of the cofactor by modification of free cysteine(s). activated protein C β-mercaptoethanol homocysteine N,N,N′,N′-tetramethylethylenediamine 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (sodium salt) 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine phospholipid vesicles polyacrylamide gel electrophoresis Homocysteine is a nonprotein-forming sulfhydryl amino acid derived from the metabolism of methionine. Homocysteine is metabolized in human cells through two pathways of remethylation to methionine and one pathway of transsulfuration to cysteine with cystathionine as an intermediate (1Selhub J. Miller J.W. Am. J. Clin. Nutr. 1991; 55: 131-138Crossref Scopus (467) Google Scholar). 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Res. 1993; 71: 337-359Abstract Full Text PDF PubMed Scopus (254) Google Scholar, 15Welch M.F. Loscalzo J. N. Engl. J. Med. 1998; 338: 1042-1050Crossref PubMed Scopus (1918) Google Scholar, 16Domagala T.B. Undas A. Libura M. Szczeklik A. J. Cardiovasc. Risk. 1998; 5: 239-247Crossref PubMed Scopus (64) Google Scholar, 17D'Angelo A. Selhub J. Blood. 1997; 90: 1-11Crossref PubMed Google Scholar). In vitro studies provide evidence that supraphysiologic homocysteine levels have a direct toxic effect on the endothelium, probably through free radical generation during oxidation of the homocysteine sulfhydryl group (18Loscalzo J. J. Clin. Invest. 1996; 98: 5-7Crossref PubMed Scopus (724) Google Scholar, 19Stamler J.S. Osborne J.A. Jaraki O. Rabbani L.E. Mullins M. Singel D. Loscalzo J. J. Clin. Invest. 1993; 91: 308-318Crossref PubMed Scopus (833) Google Scholar). Homocysteine has also been reported to impair the binding of tissue-type plasminogen activator to its endothelial cell receptor, annexin II (20Hajjer K.A. J. Clin. Invest. 1993; 91: 2873-2879Crossref PubMed Scopus (303) Google Scholar), to induce tissue factor expression (21Fryer R.H. Wilson B.D. Gubler D.B. Fitzgerald L.A. Rodgers G.M. Arterioscler. Thromb. 1993; 13 (1233): 1327Crossref PubMed Google Scholar) and to suppress heparan sulfate expression (22Nishinaga M. Ozawa T. Shimada K. J. Clin. Invest. 1993; 92: 1381-1386Crossref PubMed Scopus (251) Google Scholar). Several studies suggest that homocysteine may interfere with the protein C pathway (23Rodgers G.M. Kane W.H. J. Clin. Invest. 1986; 77: 1909-1916Crossref PubMed Scopus (385) Google Scholar, 24Rodgers G.M Conn M.T. Blood. 1990; 75: 895-901Crossref PubMed Google Scholar, 25Lentz S.R. Sadler J.E. J. Clin. Invest. 1991; 88: 1906-1914Crossref PubMed Scopus (453) Google Scholar, 26Hayashi T. Honda G. Suzuki K. Blood. 1992; 79: 2930-2936Crossref PubMed Google Scholar). The most important function of this pathway appears to be the inactivation of factor Va, the essential cofactor for the prothrombinase complex which converts prothrombin to thrombin. Human factor V (M r = 330,000) is activated by α-thrombin cleavages at Arg-709, Arg-1018, and Arg-1545 (27Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 28Jenny 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). The product, factor Va, is composed of a heavy chain, residues 1–709 (M r = 105,000), derived from the NH2terminus of factor V (A1-A2 domains) and a noncovalently associated light chain, residues 1546–2196 (M r = 74,000), derived from the COOH terminus (A3-C1-C2 domains) (27Nesheim M.E. Mann K.G. J. Biol. Chem. 1979; 254: 1326-1334Abstract Full Text PDF PubMed Google Scholar, 28Jenny 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 proteolytically inactivated by activated protein C (APC)1 by three cleavages of the heavy chain at Arg-506, Arg-306, and Arg-679 (29Kalafatis M. Mann K.G. J. Biol. Chem. 1993; 268: 27246-27257Abstract Full Text PDF PubMed Google Scholar, 30Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar). The cleavage of factor Va at Arg-506 is enhanced in the presence of a phospholipid surface, whereas cleavage at Arg-306 is lipid-dependent (30Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar). After cleavage at Arg-306, the A2 domain (residues 307–709) dissociates as two fragments (residues 307–506 and 507–709) from the factor Va molecule, leading to the complete inactivation of factor Va (31Mann K.G. Hockin M.F. Begin K.J. Kalafatis M. J. Biol. Chem. 1997; 272: 20678-20683Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 32Hockin M.F. Cawthern K.M. Kalafatis M. Mann K.G. Biochemistry. 1999; 38: 6918-6934Crossref PubMed Scopus (33) Google Scholar). The regulation of factor Va is a key process for maintaining hemostasis, as evidenced by two congenital thrombotic disorders,i.e. the G1691A mutation (Arg-506 to Gln) in factor V Leiden and protein C deficiency (33Bertina R.M. Koeleman B.P.C. Koster T. Rosendaal F.R. Dirven R.J. de Ronde H. van der Velden P.A. Reitsma P.H. Nature. 1994; 369: 64-67Crossref PubMed Scopus (3823) Google Scholar, 34Lane D.A. Mannucci P.M. Bauer K.A. Bertina R.M. Bochkov N.P. Boulyienko V. Chandy M. Dahlback B. Ginter E.K. Miletich J.P. Rosendaal F.R. Seligsohn U. Thromb. Haemostasis. 1996; 76: 651-670Crossref PubMed Scopus (637) Google Scholar). There is also evidence for impaired factor Va inactivation by APC in the absence of the factor V Leiden mutation, which is associated with increased risk of venous thrombosis (35de Visser M.C. Rosendaal F.R. Bertina R.M. Blood. 1999; 93: 1271-1276Crossref PubMed Google Scholar). The present study was undertaken to evaluate the ability of homocysteine at concentrations of 1 mm or less to modulate activation of human factor V and inactivation of human factor Va. We examined the effect of homocysteine on these processes using clotting assays and Western blot analyses along with radiolabeling of factor V by [35S]homocysteine. Our data suggest a potential contributor to the prothrombotic tendency observed in hyperhomocysteinemia. dl-Homocysteine, l-homocystine,dl-homocysteine thiolactone, and l-cysteine as well as HEPES were purchased from Sigma. 1,2-Dioleoyl-sn-glycero-3-[phospho-l-serine] (sodium salt) (PS) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (PC) were purchased from Avanti (Alabaster, AL). Acrylamide, bisacrylamide, TEMED, ammonium persulfate, and nitrocellulose were purchased from Bio-Rad. Pooled normal plasma (number of donors >30) was purchased from George King Bio-Medical (Overland Park, KS). Factor V was purified from human plasma by immunoaffinity chromatography as previously described (36Katzmann 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). Factor V concentrations were estimated spectrophotometrically. Human APC and α-thrombin were gifts from Hematologic Technologies Inc. (Essex Junction, VT). Hirudin was obtained from Genentech (South San Francisco, CA). Phospholipid vesicles (PCPS) composed of 75% PC and 25% PS were prepared according to Barenholz et al. (37Barenholz Y. Gibbes D. Litman B.J. Goll J. Thompson T.E. Carlson F.D. Biochemistry. 1977; 16: 2806-2810Crossref PubMed Scopus (729) Google Scholar), with the concentrations determined by phosphorous assay. Monoclonal antibodies against the heavy chain of human factor Va (αFVaHC 17) were from the Biochemistry Antibody Core Laboratory (University of Vermont). Goat anti-mouse IgG peroxidase was purchased from Southern Biotech (Birmingham, AL). Molecular weight standards were purchased from Life Technologies, Inc. The chemiluminescent substrate, luminol, was purchased from PerkinElmer Life Sciences as was the fluorography reagent, Entensify.l-[35S]Methionine andl-[35S]cysteine were purchased from Amersham Pharmacia Biotech. Factor V was dialyzed against 20 mm HEPES, 0.15 m NaCl, 5 mm CaCl2, pH 7.4 (HBS-Ca2+) with or without the addition of homocysteine or other thiols (solubilized in 0.15 m HCl) at varying concentrations at 4 °C for 2 h followed by dialysis against HBS-Ca2+ for 2 h. Homocystine was solubilized in 0.15 m HCl. Factor V was activated by treatment with 1 nm α-thrombin for 10–13 min at 37 °C. α-Thrombin was inhibited by the addition of hirudin at a 4–5-fold molar excess. APC at various concentrations (see legends to figures) was added to factor Va in the presence or absence of phospholipids. At selected time intervals, samples were removed and diluted into HBS-Ca2+ for assay. Factor Va activity was immediately measured in a clotting assay at 37 °C using factor V-deficient plasma (38Nesheim M.E. Katzmann J.A. Tracy P.B. Mann K.G. Lorand L. Methods in Enzymology, Proteolytic Enzymes Part C. Academic Press, Inc., New York1981: 249-285Google Scholar). In a typical assay, 50 μl of factor V or Va diluted in HBS-Ca2+ was added to an equal volume of factor V-deficient human plasma followed by the addition of 100 μl of PT (prothrombin time) reagent (Simplastin, Organon Teknika, Durham, NC). All experiments were repeated three times on separate days with comparable results. Time course data for the APC inactivation of factor Va were fit to a single exponential equation using software IGOR Pro Version 3.1, WaveMetrics Inc. (Lake Oswego, OR). Initial slopes from the fit curves were used to calculate the pseudo first order rate constants. Pseudo first order rate constants are all normalized to constant APC concentration (2.5 nm). Aliquots from the activation/inactivation experiments were withdrawn and quenched in 2% β-mercaptoethanol, 2% SDS for electrophoretic analysis. Samples were separated on a 5 to 15% linear gradient gel (SDS-PAGE) under reducing conditions according to Laemmli (39Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207227) Google Scholar). Transfer from the gel to nitrocellulose (Bio-Rad) was performed as previously described (40Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar). Immunoreactive fragments were detected using the monoclonal antibody αFVaHC 17, which recognizes an epitope located between residues 307 and 506 in the heavy chain of factor Va (41Kalafatis M. Bertina R.M. Rand M.D. Mann K.G. J. Biol. Chem. 1995; 270: 4053-4057Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Blots were developed using the Renaissance chemiluminescent reagent (Dupont). Film was developed in a Kodak X-Omat on Reflection film (PerkinElmer Life Sciences). Densitometry of immunoblots was performed on a Hewlett-Packard ScanJet 4C/T equipped with a transparency adapter for backlighting the x-ray film (Hewlett-Packard, Palo Alto, CA) and analyzed using NIH Image 1.6 (Bethesda, MD). Factor V was dialyzed against HBS-Ca2+ containing 1 mmβ-mercaptoethanol (BME) or 2 mm homocysteine (HCY) for 2 h at 4 °C and then for 2 h at 4 °C against HBS-Ca2+. Dialysis was performed in filled, capped bottles in buffer that had been boiled in a reflux apparatus and purged extensively with nitrogen. The resulting stocks, FVBME (2.2 μm) and FVHCY (2.0 μm) were used immediately. FVHCY could be flash-frozen in methanol/dry ice and successfully recovered, whereas FVBMEproved sensitive to freezing, showing a decrease in APC sensitivity after thawing. Mixtures of FVBME and FVHCY with a total factor V concentration of 400 nm in which the amount of FVHCY varied from 0 to 100% were constructed by appropriate dilution in HBS-Ca2+, and their relative susceptibility to APC proteolysis assessed by a modification of the method of Dahlback (42Dahlback B. Carlsson M. Svensson P.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1004-1008Crossref PubMed Scopus (2011) Google Scholar). Five μl of each factor V mixture was added to 95 μl of factor V-deficient (immunodepleted) plasma followed by the addition of 100 μl of aPTT (activated partial thromboplastin time) reagent (Organon Teknika). This reformulated plasma ([factor V] = 20 nm) was incubated for 5 min at 37 °C. One hundred μl of 3 nm APC in HBS-30 mmCa2+ or 100 μl of HBS-30 mm Ca2+was then added, the mixture rocked at 37 °C, and clotting time was recorded. Clotting times were plotted against the percentage of FVBME present. Best-fit lines were determined by linear regression analysis. The slopes of these lines from 3 separate sets of FVBME/FVHCY mixtures varied by less than 10%. To assay plasma, 100 μl of patient or normal plasma was combined directly with 100 μl of aPTT reagent and then processed as above. Plasma samples from individuals with elevated homocysteine levels were a generous gift from Dr. Andrzej Szczeklik, Jagiellonian University School of Medicine, Cracow, Poland. Plasmas were obtained from four asymptomatic men with genetically determined hyperhomocysteinemia stemming from a mutation in the 5,10-methylenetetrahydrofolate reductase gene (C677T). The presence of factor V Leiden was excluded on the basis of polymerase chain reaction analysis. Blood was drawn after a 16-h overnight fast, and plasma homocysteine concentration was determined according to Mansoor et al. (43Mansoor M.A. Svardal A.M. Ueland P. Anal. Biochem. 1992; 200: 218-229Crossref PubMed Scopus (494) Google Scholar). l-[35S]Methionine (201 mCi/mmol) was converted tol-[35S]homocysteine thiolactone and subsequently to l-[35S]homocysteine using the method of Baernstein (44Baernstein H.D. J. Biol. Chem. 1934; 106: 451-456Abstract Full Text PDF Google Scholar). The product was identical with authentic homocysteine by thin layer and paper chromatography and contained a small amount (<10%) of homocystine. Reported concentrations ofl-[35S]homocysteine were determined by liquid scintillation counting. Radiolabeled l-homocysteine was added to factor V that had been dialyzed against HBS-Ca2+at room temperature for 2 h. Subsequently, factor V was activated with α-thrombin, and then, upon addition of hirudin and PCPS vesicles, factor Va was inactivated by APC. At designated time points, aliquots were removed from the mixture and subjected to SDS-PAGE. Protein bands were visualized by staining the gel with Coomassie Blue and destaining by diffusion. Gels were then soaked in the fluorography reagent as per the manufacturer's instructions and dried, and radiolabeled protein was detected using Fuji Medical x-ray film (Fuji Medical Systems USA, Inc., Stamford CT). Densitometry was performed on the Coomassie-stained gel before drying and on the resulting autoradiographs as described for immunoblots. Details are given in the legends to figures. Kinetic analyses of [35S]homocysteine modification of factor V were performed in HBS-Ca2+ at room temperature in which times of treatment were varied at a fixed [35S]homocysteine concentration, or concentrations of [35S]homocysteine were varied and the reaction allowed to proceed for a fixed time. Reactions were quenched with iodoacetic acid (Sigma) that had been recrystallized from heptane. Aliquots from reacting mixtures of factor V and [35S]homocysteine were combined with an equal volume of 100 mm iodoacetic acid, 0.5 m Tris, pH 8.7, before the addition of SDS-PAGE sample buffer. The radiolabeled factor V was resolved by electrophoresis on 4–12% linear gradient SDS-polyacrylamide gels under nonreducing conditions, and the gels were processed as described above. Densitometric data from resulting autoradiographs were normalized to correct for variations in protein loading and then converted to the percentage of maximum values using the highest densitometric value within a data set. These transformed data were then fit to a double exponential equation using software IGOR Pro Version 3.1, WaveMetrics Inc. There was no significant alteration in α-thrombin activation of factor V after dialysis against 1 mm homocysteine in HBS-Ca2+for 2 h. Homocystine, homocysteine thiolactone, and cysteine incubated with factor V under the same conditions did not affect activation of factor V by α-thrombin (data not shown). Preincubation of factor V with 1 mmhomocysteine for 2 h inhibited the APC anticoagulant effect toward factor Va (Fig. 1). Factor V was treated with homocysteine at 1 mm, 100 μm, or 30 μm at 4 °C for 2 h, dialyzed against HBS-Ca2+ for an additional 2 h, and then activated by α-thrombin. A progressive decrease in the initial rates of APC inactivation of factor Va, generated from the homocysteine-treated factor V, is associated with increasing homocysteine concentrations (Fig. 1). The rate constant for the APC-catalyzed inactivation of factor Va, generated from factor V treated with 1 mmhomocysteine, is about one-third the rate constant for control experiments (Fig. 1). To test whether the interference was related to a decrease in APC activity after incubation with homocysteine, APC was preincubated with homocysteine at 1 mm for 30, 60, and 120 min at 4 °C. No differences were observed in the rate of inactivation of 100 nm factor Va by 2.5 nm APC whether treated with homocysteine or untreated (data not shown). Contrary to previous reports (23Rodgers G.M. Kane W.H. J. Clin. Invest. 1986; 77: 1909-1916Crossref PubMed Scopus (385) Google Scholar, 25Lentz S.R. Sadler J.E. J. Clin. Invest. 1991; 88: 1906-1914Crossref PubMed Scopus (453) Google Scholar), we did not observe a direct effect of homocysteine on APC activity per se. The cleavage at Arg-306 is phospholipid-dependent, whereas the cleavage at Arg-506 is enhanced in the presence of phospholipids (30Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar). In the absence of phospholipids, the rate of APC inactivation of factor Va derived from factor V treated with 1 mm homocysteine (above) was significantly delayed compared with controls (Fig.2). The ∼3-fold decrease in the rate constant induced by homocysteine is similar to that observed in the presence of phospholipids. Since the lipid-independent inactivation proceeds only through cleavage at Arg-506, the significant impairment of APC inactivation of factor Va without phospholipids indicates that homocysteine must slow cleavage at Arg-506. Factor V (100 nm) was treated with either 1 mmhomocysteine, 1 mm homocystine, or HBS-Ca2+ at 4 °C for 2 h. After activation by α-thrombin (see "Methods"), the factor Va preparations were cleaved by 2.5 nm APC at 37 °C, and the time course of each reaction was followed by Western blot analysis using the monoclonal antibody αFVaHC 17, which recognizes an epitope in the fragment 307–506 (36Katzmann 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), the ultimate APC cleavage product of factor Va heavy chain. Data for this experiment over 60 min are presented in Fig.3. Lanes 1–6 correspond to the control; lanes 13–18 correspond to the homocystine-treated factor V. The samples from treatment with homocysteine are presented in lanes 7–12. In both the control and in the homocystine-treated samples, the 60-min reaction sample (lane 6 and lane 18) show almost complete cleavage of the heavy chain and the appearance of fragments corresponding to the final cleavage product of factor Va. In contrast, some heavy chain remains intact in the homocysteine-treated samples at 60 min (lane 12). The data show that homocysteine inhibits cleavage of factor Va heavy chain by APC and that the reaction requires the reduced form of the amino acid. This effect on inactivation is homocysteine-specific, since for factor V preincubated with 1 mml-cysteine for 2 h, the time course of the inactivation of factor Va by APC was identical to those in control and homocystine experiments (data not shown). Human plasma contains a wide range of the sulfhydryl compounds, including cysteine, cysteinylglycine, homocysteine, and glutathione, which are in the reduced (<5%) or oxidized form or are protein-bound (43Mansoor M.A. Svardal A.M. Ueland P. Anal. Biochem. 1992; 200: 218-229Crossref PubMed Scopus (494) Google Scholar). Total plasma concentration of cysteine, the most abundant aminothiol, is around 250 μm, with levels of reduced cysteine amounting to 10 μm (43Mansoor M.A. Svardal A.M. Ueland P. Anal. Biochem. 1992; 200: 218-229Crossref PubMed Scopus (494) Google Scholar). Moreover, it is known that homocysteine binds to numerous plasma proteins, mainly albumin (plasma concentration 0.5–0.7 mm) at an equimolar ratio. To determine whether the addition of homocysteine to normal human plasma would alter the APC sensitivity of factor V subsequently purified from such plasma, human plasma was incubated with homocysteine (10 mm) at 4 °C for 2 h, and then factor V was purified, as described under "Methods." Homocysteine added to plasma effectively retarded the inactivation of subsequently purified and α-thrombin-activated factor V by APC in the presence of PCPS vesicles (Fig. 4). The ∼3-fold decrease in the rate constant is the same as observed when purified factor V was treated with 1 mm homocysteine (Fig. 1). Slower inactivation of factor Va, derived from factor V purified from plasma treated with homocysteine, was also observed in the absence of phospholipids (data not shown). To determine whether homocysteine can be incorporated into human factor V, factor V was incubated with 450 μm [35S]homocysteine at room temperature for 2 h. It was then activated by α-thrombin, and the resulting factor Va was treated with 6 nm APC in the presence of phospholipids. At selected time points aliquots were removed and subjected to analysis by SDS-PAGE. Coomassie Blue staining of the gel with nonreduced samples (Fig.5 A) displays [35S]homocysteine-treated factor V (lane 1), the derived factor Va (lane 2), and the time course of APC degradation of factor Va (lanes 3–8). Degradation of the heavy chain of factor Va, derived from [35S]homocysteine-treated factor V, is not complete at 180 min (lane 8). In contrast, APC degradation of factor Va, derived from untreated factor V, is complete under these conditions in 60 min (30Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar). The APC cleavage of factor Va, derived from [35S]homocysteine-treated factor V, yielded products ofM r = 45,000 (), M r= 30,000 (), and M r = 28/26,000 (). Bands corresponding to the light chain of factor Va are stable throughout the 180-min incubation with APC. This pattern is identical to that seen with untreated factor V (30Kalafatis M. Rand M.D. Mann K.G. J. Biol. Chem. 1994; 269: 31869-31880Abstract Full Text PDF PubMed Google Scholar). Comparing the Coomassie Blue-stained (Fig. 5 A) gel with the corresponding autoradiograph (Fig. 5 B) revealed that only the protein fragments that contain free sulfhydryl groups (45Xue J. Kalafatis M. Silveira J.R. Kung C. Mann K.G. Biochemistry. 1994; 33: 13109-13116Crossref PubMed Scopus (30) Google Scholar, 46Xue J. Kalafatis M. Silveira J.R. Kung C. Mann K.G. Biochemistry. 1993; 32: 5917-5923Crossref PubMed Scopus (23) Google Scholar) (intact factor V (lane 1), the heavy and light chains of factor Va (lane 2), the B region (lanes 2–8), and the doublet of M r = 28/26,000 (lanes 3–8)) incorporate [35S]homocysteine. Consistent with the pattern observed by Coomassie Blue staining, the intensity of the radiolabeled heavy chain band decreased with time, whereas the intensity of the light chain bands remained unchanged. The fragments ofM r = 45,000 () and 30,000 (), which do not contain any free cysteines, are not visualized on the autor
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