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Human and bovine factor Va (FVa) function similarly in the activation of prothrombin but differently in the activation of prethrombin-1 (Pre-1). Pre-1 activation with human FVa proceeds at about 22 percent of the rate with bovine FVa. The dependencies of initial rates on the FVa and Pre-1 concentrations indicate that the differential activity is ex...
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Context 1
... shown in Figure 7, the heavy chains of bovine and human FVa consist of two homologous repeated domains, designated A1 and A2, that comprise the bulk of the primary structure, plus two other regions of sequence that complete the primary structure. The A1 and A2 domains are highly homologous, both to one another within and across species, and to corresponding domains in the heavy chain of coagulation factor VIII, the A3 domains in the light chains of FVa and factor VIII(a), and the A1, A2, and A3 domains of ceruloplasmin (8–10,44). The A1 domain spans amino acid residues 1–303 in the heavy chains of both species and share 84.5% and 94.7% sequence homology and similarity, respectively. This is followed by thirteen amino acids in both species (residues 304–316) that differ only at positions 308, 311, and 314 (L->P, I->L, and E->D, human -> bovine). This is followed by the A2 domain. The A2 domain of the bovine heavy chain (residues 317–660) is four residues longer than the A2 domain of the human heavy chain by virtue of the lack of an arginine residue corresponding to position 439 in the human sequence, and the replacement of threonine residue corresponding to position 560 in the human sequence with the six residues NFTLPA at positions 559–564 in the bovine sequence. This latter insert has no counterpart in the human sequence. The A2 domains of the two species share 88.7% and 97.1% sequence homology and similarity, respectively. The A2 domain of the heavy chains of both species is followed by 53 amino acids, spanning residues 661–713 in the bovine heavy chain and residues 657–709 in the human heavy chain. In contrast to the high degree of similarity exhibited by the other regions of sequence of the two species, these latter 53 residues display species differences at 21 of the 53 positions when the sequences are aligned by discounting the glycine at position 667 of the bovine sequence and discounting the glutamate at position 691 of the human sequence. The functional differences observed in the current work can be attributed to one or more of the differences in primary structure between the two species of FVa. If one assumes that regions of substantial differences in sequence account for these functional differences, likely candidates comprise the inserts and deletions in the A2 domains and the regions spanning the 53 residues at the COOH-termini of the heavy chains. Recent studies have suggested that the pentapeptide sequence DYDYQ in the heavy chain of human FVa at positions 695–699 may be involved in the interaction of prothrombin with FVa in prothrombinase assembled on PCPS vesicles (27). A similar pentapeptide sequence DSDYQ is present in the heavy chain of bovine FVa at positions 699–703. These sequences of residues start 34 residues from the COOH-terminal end of the A2 domains in both species. These sequences are followed at their COOH-termini by ten amino acids, the last of which is the COOH-terminal residue of the heavy chain. In the human heavy chain, the sequence is NRLAAALGIR, whereas in the bovine heavy chain it is DELALILGLR. These are similar or identical except for the first two amino acids, which are NR and DE in human and bovine heavy chains, respectively. These two residues profoundly affect the calculated pI values of the two sequences. In human FVa, the pI value of the ten amino acids is 12.5, whereas in bovine FVa, it is 4.26. With bovine FVa, the net negative charge of these ten residues would augment the negative charge of the DSDYQ pentapeptide and perhaps specifically facilitate the interaction with Pre-1 required for cleavage at Arg320. Conversely, with human FVa, the net positive charge of these ten residues would partially negate the negative charge of the DYDYQ pentapeptide, and thereby would not facilitate the interaction with Pre-1. This is supported by the observations that the initial rates of Pre-2 accumulation, which reflects cleavage at Arg271, were nearly identical between the two species, whereas cleavage at Arg320 was 8.5-fold faster with bovine FVa. Toso and Camire recently published studies of prothrombin and Pre-1 activation by prothrombinase containing recombinant human FVa variants truncated from the COOH- terminus of the heavy chain (32). They found that the truncation of as many as 51 residues had no effect on prothrombin activation. In contrast, truncation of the ten COOH-terminal residues increased the K m for Pre-1 activation by 7-fold or more, with little effect on kcat. Their results imply that the ten amino acids of the COOH-terminus of the human FVa heavy chain influence the K m of Pre-1 activation, but not the kcat. Our results suggest that replacement of the ten amino acids of human FVa heavy chain with those of the bovine heavy chain do not affect K m , but increase kcat 6.4-fold. Both studies suggest that the identity of the last 51 residues of the heavy chain may influence the kinetics of Pre-1 ...
Context 2
... shown in Figure 7, the heavy chains of bovine and human FVa consist of two homologous repeated domains, designated A1 and A2, that comprise the bulk of the primary structure, plus two other regions of sequence that complete the primary structure. The A1 and A2 domains are highly homologous, both to one another within and across species, and to corresponding domains in the heavy chain of coagulation factor VIII, the A3 domains in the light chains of FVa and factor VIII(a), and the A1, A2, and A3 domains of ceruloplasmin (8)(9)(10)44). ...
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Citations
... The main function of FVa in the prothrombinase complex is to promote the efficient conversion of prothrombin into thrombin 25 , in part by mediating the interaction of a mature exosite I in meizothrombin 43 with a negatively-charged C-terminal region of the FVa-HC [44][45][46] . Binding between prothrombin and FVa is also thought to involve the F2 domain 26,37,47 and the F1 domain 10,39 . ...
... was prepared from purified bovine FVa, following a procedure as described previously 45 . Briefly, FV was activated to FVa by the addition of thrombin (1.96 units/mL) at 37 °C for 25 min and stopped with Phe-Pro-Argchloromethylketone (FPRck, 1 μM). ...
The fragment 2 domain (F2) of prothrombin and its interaction with factor (F) Va is known to contribute significantly to prothrombinase-catalyzed activation of prothrombin. The extent to which the F2-FVa interaction affects the overall thrombin generation, however, is uncertain. To study this interaction, nuclear magnetic resonance spectroscopy of recombinant F2 was used to identify seven residues within F2 that are significantly responsive to FVa binding. The functional role of this region in interacting with FVa during prothrombin activation was verified by the FVa-dependent inhibition of thrombin generation using peptides that mimic the same region of F2. Because six of the seven residues were within a 9-residue span, these were mutated to generate a prothrombin derivative (PT6). These mutations led to a decreased affinity for FVa as determined by surface plasmon resonance. When thrombin generation by an array of FXa containing prothrombinase components was monitored, a 54% decrease in thrombin generation was observed with PT6 compared with the wild-type, only when FVa was present. The functional significance of the specific low-affinity binding between F2 and FVa is discussed within the context of a dynamic model of molecular interactions between prothrombin and FVa engaging multiple contact sites.
... A substantial difference was demonstrated when bovine factor Va (bfVa) or hfVa were used to activate prethrombin-1 (Pre1) by prothrombinase. 33 The reason for the different effect of the cofactor on Pre1 was shown to be confined within the very last portion of the COOH-terminus of the hfVa heavy chain. A prominent difference in amino acid between the two cofactor molecules in that region is restricted to positions 700−701 where an Asn−Arg dipeptide in hfVa is replaced by the Asp−Glu sequence in bfVa, 34−36 resulting in a total replacement of the net positive charge with two negative charges. ...
... 26 Adjacent to this region are also the amino acids Asn 700 and Arg 701 , which are important for cofactor activity. 33 The amino acid substitutions within the heavy chain are indicated together with the designation for the recombinant mutant hfV molecule created and used throughout the article. Right panel; electrophoretic analyses of rhfV NR→DE and rhfVa NR→DE molecules. ...
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.
Activated protein C (aPC) proteolytically inactivates factor Va (FVa) and thereby downregulates prothrombinase. Although FVa inactivation by aPC has been studied extensively, the inactivation of prothrombinase during prothrombin activation has not. Therefore, prothrombin activation initiated both without and with aPC (5.0, 7.5 or 10.0 nM) was monitored over time by fluorescence. The experiments were performed with 0.075 nM FVa and 1.0 nM FXa, and with these concentrations reversed. The time courses of the residual prothrombinase activity with aPC, determined from the slopes of fluorescence over time, were pseudo first order with both limiting and excess FVa. With FVa limiting or in excess, the second rate constants for inactivation of prothrombinase were 1.98 +/- 0.09 x 10(5) M(-1)s(-1) and 2.54 +/- 0.13 x 10(5) M(-1)s(-1), respectively. The former value is 101-fold smaller than that for FVa inactivation by aPC alone. Since with limiting FVa the second order rate constants for prothrombinase inactivation and FVa inactivation are equal, FVa is protected 101-fold, presumably by both FXa and prothrombin. In contrast, with excess FVa, the calculated rate constant for FVa inactivation exceeds that for prothrombinase inactivation 17.3-fold, which reflects a loss of protection by FXa. Since the protective effects of the two proteins are theoretically multiplicative, FXa protected 17.3-fold and prothrombin protected 5.8-fold. With 150 nM protein S and limiting FVa, prothrombinase inactivation was two-fold faster, yet it was still protected 91-fold. These studies show that FVa is down-regulated by aPC during prothrombin activation, but both FXa and prothrombin protect FVa in a multiplicative way, with or without protein S.
