Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain.

Bakker, Harry M.; Tans, Guido; Thomassen, M. C. L. G. D.; Yukel'son, L. Ya.; Ebberink, R.H.M.; Hemker, H. Cœnraad; Rosing, Jan

doi:10.1016/s0021-9258(17)32044-6

Cited by 45 publications

(36 citation statements)

References 32 publications

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“…In spite of limiting plasma concentrations, factors V and VIII are activated very rapidly during the early stages of blood coagulation. Various cleavages have been reported to occur during activation, but only three of the proposed sites appear critical for triggering full activity in each cofactor (Toole et al, 1986;Pittman & Kaufman, 1988;Krishnan et al, 1991;Guinto et al, 1992;Bakker et al, 1994;Keller et al, 1995;Regan & Fay, 1995). In bovine factor V, the cleavage site within the B domain has the same optimal P 2 -P 2 ′ sequence as the thrombin receptor (SPR-SFH) and no repressor residues.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

et al. 1996

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The importance of substrate residues P2' and P3' on thrombin catalysis has been investigated by comparing the hydrolysis of a series of fluorescence-quenched substrates. Each consisted of a 10-residue peptide, carrying a 2-aminobenzoyl (Abz) group at the N-terminus, and a penultimate 2,4-dinitrophenyl (Dnp) derivatized lysine. Cleavage of such a peptide relieves the intramolecularly-quenched fluorescence, allowing determination of the kinetic parameters. The nature of the P2' residue was found to have a major influence on the rate of cleavage: the Kcat/Km value for the hydrolysis of the Arg-Ser bond in Abz-Val-Gly-Pro-Arg-Ser-Phe-Leu-Leu-Lys(Dnp)-Asp-OH was nearly 3 orders of magnitude higher than that for the hydrolysis of the same substrate with aspartate instead of phenylalanine at the P2' position. Comparatively, the P3' side chain was less important: the kcat/Km value for the substrate with the least effective residue (aspartate) was only 33 times lower than that of the substrate with the most favorable amino acid (lysine). The role of thrombin residues Arg35, Lys36, Glu39 and Lys60f in the putative P2' and P3' binding sites was also examined. Replacement of Lys60f by glutamine improved the rate of cleavage for peptides with P2' lysine or leucine. Compared with thrombin, mutants E39K and E39Q hydrolyzed faster substrates with an acidic residue in P2' or P3', but slightly slower those with a lysine at either position. Mutations R35Q and K36Q only improved the hydrolysis of substrates with an acidic P2' residue. Overall, thrombin prefers bulky hydrophobic side chains in subsite S2' and positively charged residues in S3', whereas acidic residues are markedly antagonistic to both subsites.

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Section: Discussionmentioning

confidence: 99%

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

et al. 1996

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“…The Carboxy-Terminal Region of the Factor Va IIa HeaVy Chain Is Essential for Expression of Maximum Cofactor ActiVity. Using a protease purified from the venom of Naja naja oxiana, which cleaves factor Va IIa at Asp 683 , generating FVa NO (101 kDa heavy chain and 74/71 kDa light chain), Bakker et al were able to demonstrate using limiting concentrations of factor Xa or prothrombin, that this novel cofactor has a 10-fold and a 4-fold reduced affinity for those proteins, respectively (28). Although factors Va IIa/CG and Va IIa/HNE have similar subunit compositions relative to factor Va NO , we did not observe any significant defect in factor Xa binding or interactions with prothrombin.…”

Section: T H Imentioning

confidence: 99%

“…For example, our laboratory has demonstrated that human factor Xa activates human plasma factor V in a membrane-and Ca 2+ -dependent manner following cleavage at or near Arg 709 and at Arg 1018 (23). Processing by other proteases includes an activator from Russell's viper venom (RVV-V) (24), calpain (25), plasmin (26), platelet proteases (22,27), Naja naja oxiana venom (28), venoms of snakes belonging to the genus Elapidae (29), and meizothrombin (30) as well as elastase (31)(32)(33)(34) and cathepsin G (32,33).…”

mentioning

confidence: 99%

Proteolysis of Factor V by Cathepsin G and Elastase Indicates That Cleavage at Arg¹⁵⁴⁵ Optimizes Cofactor Function by Facilitating Factor Xa Binding

1998

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The single-chain procofactor factor V is cleaved by thrombin (FVaIIa) at Arg709, Arg1018, and Arg1545 and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/HNE. The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa. Significant differences in cofactor function were observed between the two assay systems. Whereas FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa bi...

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“…The K d value of the hfVa–hPro interaction is 1 μM. , hfVa provides binding sites for proexosite 1 and the Gla domain of hPro, explaining one of the possible mechanisms by which the cofactor functions to increase enzyme efficiency. , It has been proven that several acidic amino acids at the very end of the heavy chain of hfVa are vital for hfVa cofactor activity − and participate in the interaction of hPro with prothrombinase. It has been proposed that amino acids from the region 680–709 are required for proper interaction and catalysis of hPro by hfXa within prothrombinase. ,, Further, we have hypothesized that these acidic residues may be involved in the direct binding of hfVa with hPro and/or thrombin through positively charged amino acids.…”

Section: Introductionmentioning

confidence: 99%

Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function

Hirbawi

Kalafatis

2017

ACS Omega

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Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg701 of hfVa for human prothrombin (hPro) activation by prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by prothrombinase. The difference between the two cofactor molecules was also found within the Asn700–Arg701 dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp–Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution 700NR701→DE. rhfVNR→DE together with the wild-type molecule (rhfVWT) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the Kd of rhfVaNR→DE for human fXa as well as the kcat and Km of prothrombinase made with rhfVaNR→DE for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVaWT. Remarkably, sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of hPro activation time courses demonstrated that the rate of cleavage of hPro by prothrombinase reconstituted with rhfVaNR→DE was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVaWT. These unanticipated results provide significant insights on the role of the carboxyl-terminal end of the heavy chain of hfVa for hPro cleavage and activation by prothrombinase and show that residues 700NR701 regulate at least in part the enzyme–substrate/product interaction during fibrin clot formation.

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Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain.

Cited by 45 publications

References 32 publications

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Proteolysis of Factor V by Cathepsin G and Elastase Indicates That Cleavage at Arg¹⁵⁴⁵ Optimizes Cofactor Function by Facilitating Factor Xa Binding

Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function

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Functional properties of human factor Va lacking the Asp683-Arg709 domain of the heavy chain.

Cited by 45 publications

References 32 publications

Characterization of the P2‘ and P3‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis,

Characterization of the P2‘ and P3‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis,

Proteolysis of Factor V by Cathepsin G and Elastase Indicates That Cleavage at Arg1545 Optimizes Cofactor Function by Facilitating Factor Xa Binding

Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function

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Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Characterization of the P₂‘ and P₃‘ Specificities of Thrombin Using Fluorescence-Quenched Substrates and Mapping of the Subsites by Mutagenesis^,

Proteolysis of Factor V by Cathepsin G and Elastase Indicates That Cleavage at Arg¹⁵⁴⁵ Optimizes Cofactor Function by Facilitating Factor Xa Binding