Cofactor Control of a Vital Enzymatic Reaction;the Effect of Factor Va on Thrombin Formation During Blood Coagulation

The LONG-TERM goal of our research is to study and analyze the structure and function of the factor V molecule in order to understand its regulatory effects on the natural process of hemostasis and its role in the life-threatening development of deep venous thrombosis. The SHORT-TERM goal of our research is to identify the amino acid residue(s) of factor V that interact with prothrombin during the assembly and function of the prothrombinase complex in order to fully understand its particular role in maintaining the integrity of the blood coagulation cascade. The final goal of the coagulation cascade is the formation of a fibrin clot that is catalyzed by the serine protease, thrombin. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa, its cofactor, factor Va, assembled on a membrane surface in the presence of divalent metal ions. Although factor Xa alone can activate prothrombin, it is at a rate that is not compatible for survival. Incorporation of factor Va into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of factor Xa for thrombin generation and plays an important role in regulating prothrombin activation. Thus, it is crucial to identify which specific amino acid residue(s) are responsible for the interaction of factor Va with prothombin and factor Xa in the formation of thrombin, which is absolutely vital for maintaining normal hemostasis in a healthy individual. The specific aims of the present study are To identify the specific amino acid region of the factor Va heavy chain that promotes optimal cofactor function during prothrombin activation To identify key acidic amino acid sequences in the factor Va molecule that control the rate of prothrombin cleavage To identify the specific amino acids of the factor V/Va heavy chain that regulate enzyme-substrate interaction during prothrombin activatio

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

Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg 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 Asn-Arg 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 NR→DE. rhfV together with the wild-type molecule (rhfV) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the of rhfVa for human fXa as well as the and of prothrombinase made with rhfVa for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVa. 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 rhfVa was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVa. 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 NR regulate at least in part the enzyme-substrate/product interaction during fibrin clot formation

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser478–Val479–Leu480–Gln481–Val482. Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu480 and Gln481 as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIaS478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIaSLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIaΔSLQ (where rIIaΔSLQ is recombinant human α-thrombin with amino acids Ser478/Leu480/Gln481 deleted). These data provide new evidence demonstrating that amino acid sequence Leu480–Gln481: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg320; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa

Role of the Acidic Hirudin-like COOH-Terminal Amino Acid Region of Factor Va Heavy Chain in the Enhanced Function of Prothrombinase†

Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680-709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Delta680-709)) and a mutant molecule with the (695)DYDY (698) --\u3e AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va (Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Delta680-709) displayed an approximately 39% increase in k cat, while prothrombinase assembled with factor Va(4A) exhibited an approximately 20% increase in k cat for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase

Contribution of Amino Acid Region 659−663 of Factor Va Heavy Chain to the Activity of Factor Xa within Prothrombinase†,‡

ABSTRACT: Factor Va, the cofactor of prothrombinase, is composed of heavy and light chains associated noncovalently in the presence of divalent metal ions. The COOH-terminal region of the heavy chain contains acidic amino acid clusters that are important for cofactor activity. In this work, we have investigated the role of amino acid region 659-663, which contains five consecutive acidic amino acid residues, by site-directed mutagenesis. We have generated factor V molecules in which all residues were mutated to either lysine (factor V 5K) or alanine (factor V 5A). We have also constructed a mutant molecule with this region deleted (factor V Δ659-663). The recombinant molecules along with wild-type factor V (factor V WT) were transiently expressed in mammalian cells, purified, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to that of factor Va WT. Kinetic analyses of prothrombinase assembled with the mutant molecules demonstrated diminished k cat values, while the affinity of all mutant molecules for factor Xa was similar to that for factor Va WT. Gel electrophoresis analyses of plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg 320 and Arg 271 by prothrombinase assembled with the mutant molecules, resulting i

Cofactor Control of a Vital Enzymatic Reaction;the Effect of Factor Va on Thrombin Formation During Blood Coagulation

The LONG-TERM goal of our research is to study and analyze the structure and function of the factor V molecule in order to understand its regulatory effects on the natural process of hemostasis and its role in the life-threatening development of deep venous thrombosis. The SHORT-TERM goal of our research is to identify the amino acid residue(s) of factor V that interact with prothrombin during the assembly and function of the prothrombinase complex in order to fully understand its particular role in maintaining the integrity of the blood coagulation cascade. The final goal of the coagulation cascade is the formation of a fibrin clot that is catalyzed by the serine protease, thrombin. The proteolytic conversion of prothrombin to thrombin is catalyzed by the prothrombinase complex composed of the enzyme, factor Xa, its cofactor, factor Va, assembled on a membrane surface in the presence of divalent metal ions. Although factor Xa alone can activate prothrombin, it is at a rate that is not compatible for survival. Incorporation of factor Va into the prothrombinase complex results in a 300,000-fold increase in the catalytic efficiency of factor Xa for thrombin generation and plays an important role in regulating prothrombin activation. Thus, it is crucial to identify which specific amino acid residue(s) are responsible for the interaction of factor Va with prothombin and factor Xa in the formation of thrombin, which is absolutely vital for maintaining normal hemostasis in a healthy individual. The specific aims of the present study are To identify the specific amino acid region of the factor Va heavy chain that promotes optimal cofactor function during prothrombin activation To identify key acidic amino acid sequences in the factor Va molecule that control the rate of prothrombin cleavage To identify the specific amino acids of the factor V/Va heavy chain that regulate enzyme-substrate interaction during prothrombin activatio

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

China - Linhsien, Red Flag Canal, aqueduct across valleyThe Red Flag Canal (Hongqi Canal) is a 'canal and irrigation system in northern Henan and in Shanxi provinces, eastern China, constructed in 1960-69 to irrigate the poor and infertile area of Linxian county (now Linzhou municipality) in the foothills of the Taihang Mountains west of Anyang.'Hongqi Canal. (2007). Encyclopædia Britannica, Inc.ColorVolume 56, Page