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• W1980018864 abstract "Thrombin formation results from cleavage of prothrombin following Arg271 and Arg320. Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg320 followed by Arg271 is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (IIWT) contained both cleavage sites; IIQ271 contained a single cleavable site at Arg320; IIQ320 and IIA320 contained a single cleavable site at Arg271; and IIQQ was resistant to cleavage. Cleavage at Arg320 in IIQ271 could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or IIWT, whereas cleavage at Arg271 in either IIQ320 or IIA320 was found to be ∼30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg271 in intact prothrombin resulted from an ∼30-fold reduction in Vmax. Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. IIQ320 was a competitive inhibitor of IIQ271 cleavage, and IIQQ was a competitive inhibitor for each reaction with Ki ≈ Km. The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase. Thrombin formation results from cleavage of prothrombin following Arg271 and Arg320. Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg320 followed by Arg271 is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (IIWT) contained both cleavage sites; IIQ271 contained a single cleavable site at Arg320; IIQ320 and IIA320 contained a single cleavable site at Arg271; and IIQQ was resistant to cleavage. Cleavage at Arg320 in IIQ271 could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or IIWT, whereas cleavage at Arg271 in either IIQ320 or IIA320 was found to be ∼30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg271 in intact prothrombin resulted from an ∼30-fold reduction in Vmax. Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. IIQ320 was a competitive inhibitor of IIQ271 cleavage, and IIQQ was a competitive inhibitor for each reaction with Ki ≈ Km. The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase. The formation of thrombin, a key reaction of the blood coagulation cascade, arises from specific and limited proteolysis of prothrombin (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (447) Google Scholar). Although the serine proteinase, factor Xa, can catalyze this reaction, the rate of thrombin formation is greatly increased following its assembly into prothrombinase through interactions with membranes and factor Va (1Mann K.G. Jenny R.J. Krishnaswamy S. Annu. Rev. Biochem. 1988; 57: 915-956Crossref PubMed Scopus (447) Google Scholar, 2Mann K.G. Trends Biochem. Sci. 1987; 12: 229-233Abstract Full Text PDF Scopus (82) Google Scholar, 3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1609) Google Scholar). Prothrombinase is considered the physiologically relevant catalyst for rapid thrombin formation following the initiation of coagulation (2Mann K.G. Trends Biochem. Sci. 1987; 12: 229-233Abstract Full Text PDF Scopus (82) Google Scholar, 4Mann K.G. Thromb. Haemostasis. 1999; 82: 165-174Crossref PubMed Scopus (414) Google Scholar). Thrombin formation results from cleavage of human prothrombin 1Sequence numbers represent those obtained by consecutive numbering of the 579 residues in mature human prothrombin. 1Sequence numbers represent those obtained by consecutive numbering of the 579 residues in mature human prothrombin. following Arg271 and Arg320 (5Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (95) Google Scholar, 6Degen S.J. MacGillivray R.T. Davie E.W. Biochemistry. 1983; 22: 2087-2097Crossref PubMed Scopus (205) Google Scholar). Initial cleavage at Arg271 followed by cleavage at Arg320 (Scheme I, Reactions 3 and 4) yields thrombin via the formation of prethrombin 2 and fragment 1.2 (P2 plus F1.2) 2Prothrombin fragments are denoted by the following abbreviations: prothrombin, II; fragment 1.2, F1.2; prethrombin 2, P2; meizothrombin, mIIa; and thrombin, IIa. IIa is composed of the thrombin A chain, (IIaA) in disulfide linkage with thrombin B chain, (IIaB). mIIa contains fragment 1.2-thrombin A (F1.2-A) and IIaB, linked by a disulfide bond. 2Prothrombin fragments are denoted by the following abbreviations: prothrombin, II; fragment 1.2, F1.2; prethrombin 2, P2; meizothrombin, mIIa; and thrombin, IIa. IIa is composed of the thrombin A chain, (IIaA) in disulfide linkage with thrombin B chain, (IIaB). mIIa contains fragment 1.2-thrombin A (F1.2-A) and IIaB, linked by a disulfide bond. as intermediates. This cleavage pathway is evident in the action of factor Xa on prothrombin (7Heldebrant C.M. Butkowski R.J. Bajaj S.P. Mann K.G. J. Biol. Chem. 1973; 248: 7149-7163Abstract Full Text PDF PubMed Google Scholar, 8Esmon C.T. Jackson C.M. J. Biol. Chem. 1974; 249: 7782-7790Abstract Full Text PDF PubMed Google Scholar). Cleavage at Arg320 followed by cleavage at Arg271 (Scheme I, Reactions 1 and 2) results in thrombin formation via production of meizothrombin (mIIa) 3The abbreviations used are: mIIa, meizothrombin; PC, l-α-phosphatidylcholine; ATA-FPR-CH2Cl, acetothioacetyl FPR-CH2Cl; DAPA, dansyl-l-arginine N-(3-ethyl-1,5-pentanediyl)amide; FPR-CH2Cl, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone; IIPL, prothrombin purified from human plasma; IIQ271, recombinant prothrombin with Arg at residue 271 replaced with Gln; IIQ320, recombinant prothrombin with Arg at residue 320 replaced with Gln; IIA320, recombinant prothrombin with Arg at residue 320 replaced with Ala; IIQQ, recombinant prothrombin containing Gln at both 271 and 320; IIWT, recombinant wild type human prothrombin; mIIaF, meizothrombin inactivated with ATA-FPR-CH2Cl and modified with 6-(iodoacetamido)fluorescein following thioester hydrolysis; mIIai, meizothrombin inactivated with FPR-CH2Cl; PS, l-α-phosphatidylserine; S2238, H-d-phenylalanyl-l-pipecolyl-l-arginine-p-nitroanilide; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; SELDI/TOF/MS, time-of-flight mass spectrometry using a surface-enhanced laser desorption instrument. 3The abbreviations used are: mIIa, meizothrombin; PC, l-α-phosphatidylcholine; ATA-FPR-CH2Cl, acetothioacetyl FPR-CH2Cl; DAPA, dansyl-l-arginine N-(3-ethyl-1,5-pentanediyl)amide; FPR-CH2Cl, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone; IIPL, prothrombin purified from human plasma; IIQ271, recombinant prothrombin with Arg at residue 271 replaced with Gln; IIQ320, recombinant prothrombin with Arg at residue 320 replaced with Gln; IIA320, recombinant prothrombin with Arg at residue 320 replaced with Ala; IIQQ, recombinant prothrombin containing Gln at both 271 and 320; IIWT, recombinant wild type human prothrombin; mIIaF, meizothrombin inactivated with ATA-FPR-CH2Cl and modified with 6-(iodoacetamido)fluorescein following thioester hydrolysis; mIIai, meizothrombin inactivated with FPR-CH2Cl; PS, l-α-phosphatidylserine; S2238, H-d-phenylalanyl-l-pipecolyl-l-arginine-p-nitroanilide; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; SELDI/TOF/MS, time-of-flight mass spectrometry using a surface-enhanced laser desorption instrument. as an intermediate. Within detection limits, bond cleavage in this order appears to quantitatively account for thrombin formation catalyzed by prothrombinase (9Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 10Krishnaswamy S. Mann K.G. Nesheim M.E. J. Biol. Chem. 1986; 261: 8977-8984Abstract Full Text PDF PubMed Google Scholar, 11Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). Prothrombinase cleaves the substrate in an apparently ordered fashion even though both Arg320 and Arg271 appear accessible to cleavage in prothrombin (9Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar). The kinetic and molecular bases for these observations remain obscure.Fig. 1Cleavage products obtained from prothrombin variants. The indicated variants (2.8 μm) were analyzed by SDS-PAGE with disulfide bond reduction either before (-) or after (+) a 30-min digestion with 3 nm prothrombinase (3 nm Xa, 70 nm Va, and 50 μm PCPS) in the presence of 95 μm DAPA. Bands (∼2.8 μg of protein/lane) were visualized by staining with Coomassie Brilliant Blue R-250. The mobility of various possible prothrombin fragments is shown in the right margin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Kinetic explanations for bond selectivity in prothrombin have been sought from studies using P2 plus F1.2 and mIIa as substrates (9Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 10Krishnaswamy S. Mann K.G. Nesheim M.E. J. Biol. Chem. 1986; 261: 8977-8984Abstract Full Text PDF PubMed Google Scholar, 11Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Nesheim M.E. Mann K.G. J. Biol. Chem. 1983; 258: 5386-5391Abstract Full Text PDF PubMed Google Scholar). The individual bonds in both intermediates are cleaved by prothrombinase (Scheme I, Reactions 2 or 4) with approximately equal catalytic efficiency (9Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 10Krishnaswamy S. Mann K.G. Nesheim M.E. J. Biol. Chem. 1986; 261: 8977-8984Abstract Full Text PDF PubMed Google Scholar, 11Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Consequently, an explanation for ordered bond cleavage by prothrombinase requires that Arg271 and Arg320 in intact prothrombin are cleaved with different catalytic efficiencies. Therefore, formal consideration of the reactions of prothrombin activation requires a distinction to be made between cleavage at Arg271 before and after Arg320 cleavage (Arg271and Arg271*, Scheme I) or at Arg320 before and after Arg271 cleavage (Arg320 and Arg320*, Scheme I). As previous measurements have established that recognition and cleavage at Arg320 are independent of prior cleavage at Arg271 (11Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), apparently ordered bond cleavage by prothrombinase can only result if cleavage at Arg271 is slow in comparison to cleavage at Arg271* (11Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar, 12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Despite general agreement with this logical construct, comparable kinetic constants have been reported for the action of prothrombinase on each of the four individual cleavage reactions assessed using recombinant derivatives of prothrombin (12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Kinetic discrepancies have also led to the suggestion that a significant or large fraction of thrombin is produced by channeling without intermediate release (14Boskovic D.S. Bajzar L.S. Nesheim M.E. J. Biol. Chem. 2001; 276: 28686-28693Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 15Weinreb G.E. Mukhopadhyay K. Majumder R. Lentz B.R. J. Biol. Chem. 2003; 278: 5679-5684Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 16Tans G. Janssen-Claessen T. Hemker H.C. Zwaal R.F.A. Rosing J. J. Biol. Chem. 1991; 266: 21864-21873Abstract Full Text PDF PubMed Google Scholar). Studies with recombinant human prothrombin derivatives have yielded the novel suggestion that the two bonds in the substrate are recognized and cleaved by kinetically distinct and slowly interconverting conformers of prothrombinase (12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Yet binding studies indicate that all possible substrates and product bind competitively through exosite interactions to prothrombinase with affinities that are independent of the active site of the enzyme (17Boskovic D.S. Troxler T. Krishnaswamy S. J. Biol. Chem. 2004; 279: 20786-20793Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). These contradictory findings point to difficulties in providing a valid explanation for the action of prothrombinase on prothrombin by using kinetic models such as Scheme I. They are also inconsistent with models implicating a major role for exosite binding in substrate recognition (18Betz A. Krishnaswamy S. J. Biol. Chem. 1998; 273: 10709-10718Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Boskovic D.S. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 20Krishnaswamy S. Betz A. Biochemistry. 1997; 36: 12080-12086Crossref PubMed Scopus (86) Google Scholar, 21Orcutt S.J. Pietropaolo C. Krishnaswamy S. J. Biol. Chem. 2002; 277: 46191-46196Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We have studied the action of human prothrombinase on a series of recombinant derivatives of human prothrombin to investigate these contradictory ideas. We present a model that adequately explains the pathway for prothrombin cleavage on the basis of the kinetic constants for the four possible enzyme-catalyzed reactions illustrated in Scheme I. Our findings are inconsistent with the previous proposal (12Brufatto N. Nesheim M.E. J. Biol. Chem. 2003; 278: 6755-6764Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and instead suggest equivalent exosite binding interactions between all four possible substrate species and prothrombinase. We propose that ordered cleavage of the two bonds is driven by interactions between a single form of prothrombinase and two distinct conformations of substrate generated in the pathway for cleavage. Materials—Hen egg l-α-phosphatidylcholine (PC) and porcine brain l-α-phosphatidylserine (PS) were from Avanti Polar Lipids (Alabaster, AL); 6-(iodoacetamido)fluorescein was from Molecular Probes (Eugene, OR). The proteinase inhibitors d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (FPR-CH2Cl, Calbiochem), p-amidinophenylmethane-sulfonyl fluoride (Sigma), and dansyl-l-arginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA, Hematologic Technologies, Essex Junction, VT) were obtained from the sources indicated. The acetothioacetyl derivative of FPR-CH2Cl (ATA-FPR-CH2Cl) was prepared by the method of Bock (22Bock P.E. Methods Enzymol. 1993; 222: 478-503Crossref PubMed Scopus (31) Google Scholar) and quality controlled as described. The peptidyl substrate, H-d-phenylalanyl-l-pipecolyl-l-arginine-p-nitroanilide (S2238) was from Chromogenix (West Chester, OH). Reagents for recombinant DNA manipulation were from Invitrogen as were cell culture media and most media supplements. Fetal bovine serum and G418 were obtained from Cellgro (Herndon, VA). Small unilamellar phospholipid vesicles composed of 25% PS and 75% PC (PCPS) were prepared and characterized as described (11Walker R.K. Krishnaswamy S. J. Biol. Chem. 1994; 269: 27441-27450Abstract Full Text PDF PubMed Google Scholar). For initial velocity studies, large unilamellar phospholipid vesicles of the same composition (PCPSLUV) were prepared by repeated extrusion (25 times) through 400-nm polycarbonate filters (Liposofast, Avestin, Ottawa, Canada) and clarification by centrifugation (90,000 × g, 45 min). Quasi-elastic light scattering (Nicomp 380, Nicomp, Santa Barbara, CA) of a typical PCPSLUV preparation yielded a monodisperse Gaussian distribution centered at d = 299 ± 72 nm. Human plasma used for the isolation of proteins was a generous gift of the Plasmapheresis Unit of the Hospital of the University of Pennsylvania. All kinetic measurements were conducted in 20 mm Hepes, 0.15 m NaCl, 2 mm Ca2+, 0.1% (w/v) PEG-8000, pH 7.5 (Assay Buffer), at 25 °C. Recombinant Proteins—Prothrombin variants were cloned into pcDNA 3.1(+) by using the Gateway Cloning System (Invitrogen) (23Landy A. Annu. Rev. Biochem. 1989; 58: 913-949Crossref PubMed Google Scholar). PCR amplification of the cDNA encoding human prothrombin (6Degen S.J. MacGillivray R.T. Davie E.W. Biochemistry. 1983; 22: 2087-2097Crossref PubMed Scopus (205) Google Scholar), followed by unidirectional cloning into the TOPO-adapted pENTR entry vector, yielded a cassette containing a Kozak sequence, translation start site, signal, propeptide, and mature protein sequence followed by a 3′-untranslated sequence (97 bases) extending to the polyadenylation site. This cassette was used as a template for further mutagenesis using the QuickChange mutagenesis kit (Stratagene). Mutagenic primers were used to introduce a codon for Gln in place of Arg271 for the expression of IIQ271, Gln in place of Arg320 for the expression of IIQ320,or Ala in place of Arg320 for preparation of IIA320. Mutagenesis using the cDNA encoding IIQ271 as template was used to generate an expression cassette encoding a prothrombin variant containing codons for Gln in place of both Arg271 and Arg320 (IIQQ). The integrity of each construct was established by DNA sequencing. Each variant cassette was subjected to λ phage integrase-mediated recombination into an adapted pCDNA 3.1(+) destination vector. Final expression constructs were sequenced again before transfection. HEK 293 cells in Opti-MEM (Invitrogen) were transfected by treating with 7.5 μg of plasmid DNA and 30 μl of LipofectAMINE 2000 (Invitrogen)/3 × 105 cells for 4–5 h followed by the addition of Dulbecco's modified Eagle's medium/F-12 containing 15 mm Hepes, 5% (v/v) fetal bovine serum, and 1 mm l-glutamine. Stable cell lines, generated by selection in media containing G418 (0.5 mg/ml), were screened for protein production using an immunoassay (Enzyme Research Laboratories, South Bend, IN) and by a functional assay for thrombin produced following the addition of prothrombinase or ecarin. Typical production levels ranged from 4 to 7 μg/ml prothrombin/24 h in a confluent T-25 flask with 10 ml of serum-free medium. Stable cell lines were expanded into cell factories (Nunclon, Nunc), and large scale protein production was conducted in Dulbecco's modified Eagle's medium/F-12 media without phenol red containing 15 mm Hepes, 5 μg/ml insulin/transferrin/sodium selenite supplement (Invitrogen), 1 mm l-glutamine, and 10 μg/ml reduced vitamin K (Abbott). Conditioned media, harvested daily, were treated with 5 mm benzamidine and stored at -20 °C. Conditioned media (10 liters) were thawed, pooled, and applied at room temperature to a 4.8 × 6 cm column of Q-Sepharose (Amersham Biosciences) equilibrated with 20 mm Hepes, 1 mm benzamidine, pH 7.5. After washing with 20 mm Hepes, pH 7.5, bound protein was eluted with 20 mm Hepes, 0.6 m NaCl, pH 7.5. Fractions containing protein were pooled, cooled to 4 °C, and treated with 11 mm Na3citrate followed by the addition of 1 m BaCl2 over 15 min to a final concentration of 74.1 mm. The precipitate was collected by centrifugation, dissolved with 0.2 m EDTA, pH 8.0, dialyzed overnight against 20 mm Hepes, 1 mm EDTA, 1 mm benzamidine, pH 7.5, and applied to a HQ POROS column (10 × 100 mm) (Applied Biosystems) equilibrated in 20 mm Hepes, pH 7.5. Bound protein was eluted with a gradient of increasing NaCl (0–1.0 m, 15 ml/min, 100 min) in the same buffer. Fractions containing prothrombin were pooled, dialyzed against 1 mm NaPi, pH 6.8, applied to a ceramic hydroxyapatite matrix, CHT5-I (10 × 64 mm) (Bio-Rad) equilibrated in the same buffer, and eluted with a gradient of increasing NaPi, pH 6.8 (1–500 mm, 3.0 ml/min, 17 min). Under- or un-carboxylated prothrombin elutes early in this gradient separated from fully carboxylated material that elutes at higher ionic strength. 4S. J. Orcutt and S. Krishnaswamy, unpublished observations. Fractions were pooled to minimize contamination with under-carboxylated prothrombin, precipitated with solid (NH4)2SO4 (80% saturation), collected by centrifugation (56,000 × g, 30 min), dissolved in 50% (v/v) glycerol, and stored at -20 °C. Final yields of all prothrombin derivatives were typically ∼2 mg per liter of conditioned media. Plasma Proteins—Procedures for the purification of factor X, factor V, and prothrombin from plasmapheresis plasma have been described (21Orcutt S.J. Pietropaolo C. Krishnaswamy S. J. Biol. Chem. 2002; 277: 46191-46196Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 24Baugh R.J. Krishnaswamy S. J. Biol. Chem. 1996; 271: 16126-16134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Human factors Xa and Va were prepared and characterized as described previously (9Krishnaswamy S. Church W.R. Nesheim M.E. Mann K.G. J. Biol. Chem. 1987; 262: 3291-3299Abstract Full Text PDF PubMed Google Scholar, 25Nesheim M.E. Katzmann J.A. Tracy P.B. Mann K.G. Methods Enzymol. 1981; 80: 249-274Crossref PubMed Scopus (96) Google Scholar, 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). Kinetic titration of Xa preparations with p-nitrophenol-p′-guanidinobenzoate (27Chase Jr., T. Shaw E. Methods Enzymol. 1967; 19: 20-27Crossref Scopus (522) Google Scholar) yielded 0.96–1.22 mol of active sites/mol of factor Xa. Further quality control of factor Va preparations was performed by fluorescence binding measurements assessing its ability to assemble into prothrombinase 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). Typical results from this approach yielded Kd = 1.74 ± 0.45 nm for the assembly of prothrombinase and n = 1.37 ± 0.05 mol of Va bound/mol of Xa at saturation. Proteolytic derivatives of human prothrombin (IIPL), fragment 1.2 (F1.2), prethrombin 2 (P2), and thrombin were purified and characterized by established procedures (5Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (95) Google Scholar). Ecarin was purified from the venom of Echis carinatus pyramidum (Latoxan, Valence, France) (19Boskovic D.S. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Modifications to procedures developed with bovine prothrombin were employed to prepare and purify human mIIa covalently inactivated with FPR-CH2Cl (mIIai) and mIIa inactivated with ATA-FPR-CH2Cl and labeled with 6-(iodoacetamido)fluorescein (mIIaF) (17Boskovic D.S. Troxler T. Krishnaswamy S. J. Biol. Chem. 2004; 279: 20786-20793Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 19Boskovic D.S. Krishnaswamy S. J. Biol. Chem. 2000; 275: 38561-38570Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Following purification, both mIIai and mIIaF were dialyzed into 20 mm Hepes, 0.15 m NaCl, pH 7.5, concentrated by ultrafiltration, and stored at -20 °C. Protein concentrations were determined using the following extinction coefficients (E280 mg-1·cm2) and molecular weights: human Xa, 1.16, 45,300 (28Di Scipio R.G. Hermodson M.A. Davie E.W. Biochemistry. 1977; 16: 5253-5260Crossref PubMed Scopus (115) Google Scholar); human Va 1.78, 173,000 (29Toso R. Camire R.M. J. Biol. Chem. 2004; 279: 21643-21650Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), F1.2, 1.12, 34,800; P2, 1.94, 37,500 (5Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (95) Google Scholar); IIa, 1.94, 37,500 (30Lundblad R.L. Kingdon H.S. Mann K.G. Methods Enzymol. 1976; 45: 156-176Crossref PubMed Scopus (232) Google Scholar); mIIa derivatives, 1.42, 72,000, IIPL, and all recombinant prothrombin variants, 1.42, 72,000 (5Mann K.G. Elion J. Butkowski R.J. Downing M. Nesheim M.E. Methods Enzymol. 1981; 80: 286-302Crossref PubMed Scopus (95) Google Scholar). All prothrombin derivatives were exchanged into Assay Buffer either by dialysis or by centrifugal gel filtration before use. Characterization of Prothrombin Variants—N-terminal sequence analysis of prothrombin species was performed by automated Edman degradation at the Emory University Microchemical Facility. Intact prothrombin species and their cleavage products were characterized by sequencing bands excised following SDS-PAGE and electroblotting as described (24Baugh R.J. Krishnaswamy S. J. Biol. Chem. 1996; 271: 16126-16134Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Analysis of 4-carboxyglutamic acid content was performed by base hydrolysis and quantitative determination of Gla and Asx separated by high pressure liquid chromatography and detected following post-column derivatization (31Price P.A. Methods Enzymol. 2002; 91: 13-17Crossref Scopus (37) Google Scholar, 32Camire R.M. Larson P.J. Stafford D.W. High K.A. Biochemistry. 2000; 39: 14322-14329Crossref PubMed Scopus (60) Google Scholar). Molecular weights were determined by mass spectrometry using SELDI/TOF/MS (Ciphergen, Fremont, CA). Prolonged Digestion of Prothrombin Variants—Reaction mixtures in Assay Buffer contained 2.8 μm of each prothrombin variant with no additions (-) or 50 μm PCPS, 95 μm DAPA, 70 nm Va, and 3 nm Xa (+). Samples (20 μl), withdrawn following a 30-min incubation at 25 °C, were mixed with 15 μl of SDS quenching buffer to achieve final concentrations of 62.5 mm Tris, 25 mm EDTA, 62 mm dithiothreitol, 1% (w/v) SDS, 10% (v/v) glycerol, 0.01% (w/v) bromphenol blue, pH 6.8, and heated at 90 °C for 2.5 min. SDS-PAGE of quenched samples (25 μl, ∼2.8 μg of protein) was performed using 4–12% NOVEX BisTris gradient gels run with MES buffer (Invitrogen). Bands were visualized following staining with 0.25% (w/v) Coomassie Brilliant Blue R-250 in 45% (v/v) MeOH, 10% (v/v) AcOH, and destaining with 18% (v/v) MeOH, 9% (v/v) AcOH. Kinetics of Bond Cleavage in Prothrombin Variants—Reaction mixtures containing 5.0 μm prothrombin variant, 50 μm PCPS, 20 μm DAPA, and 50 nm Va in Assay Buffer and maintained at 25 °C were initiated by the addition of 1 nm Xa. Samples (15 μl) withdrawn at the indicated times were quenched by mixing with an equal volume of 125 mm Tris, 20% (v/v) glycerol, 2% (w/v) SDS, 0.02% (w/v) bromphenol blue, 50 mm EDTA, pH 6.8. Samples were treated with 62 mm dithiothreitol, heated at 90 °C for 2.5 min, and subjected to electrophoresis (3.6 μg of protein/lane) using 10% NOVEX Tris-glycine gels (Invitrogen). Protein bands visualized by staining with Coomassie Brilliant Blue R-250 and destaining were imaged in transmitted light using an EDAS290 digital camera system (Eastman Kodak). Kinetics of Bond Cleavage in Meizothrombin Variants—Each prothrombin variant (5.8 μm, 3 ml) was cleaved with ecarin (7 μg/ml) for 18 min at 25 °C in 20 mm Hepes, 0.15 m NaCl, 0.1% PEG 8000, pH 7.5, containing 50 μm FPR-CH2Cl to yield mIIai. Reaction mixtures were quenched with 10 mm EDTA, diluted with 3 ml of 20 mm Hepes, pH 7.5, maintained on ice, and treated with two sequential additions of 50 μm p-amidinophenylmethanesulfonyl fluoride. Ecarin was rapidly removed by application of each reaction mixture to a column (0.25 ml) of S-Sepharose (Amersham Biosciences) equilibrated in" @default.
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• W1980018864 date "2004-12-01" @default.
• W1980018864 modified "2023-09-30" @default.
• W1980018864 title "Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin" @default.
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• W1980018864 doi "https://doi.org/10.1074/jbc.m410866200" @default.
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