Structural Requirements for Expression of Factor Va Activity

Abstract

Thrombin activated factor Va (factor V IIa , residues 1-709 and 1546 -2196) has an apparent dissociation constant (K d,app ) for factor Xa within prothrombinase of ϳ0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp 697 , Asp 1509 , and Asp 1514 to produce a molecule (factor V NN ) that is composed of a M r 100,000 heavy chain (amino acid residues 1-696) and a M r 80,000 light chain (amino acid residues 1509/1514 -2196). Factor V NN , has a K d,app for factor Xa of 4 nM and reduced clotting activity. Cleavage of factor V IIa by NN at Asp 697 results in a cofactor that loses ϳ60 -80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg 1018 and Arg 1545 to produce a M r 150,000 heavy chain and M r 74,000 light chain (factor V RVV , residues 1-1018 and 1546 -2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor V NN at Arg 1545 by ␣-thrombin (factor V NN/IIa ) or RVV (factor V NN/RVV ) leads to enhanced affinity of the cofactor for factor Xa (K d,app ϳ 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg 1545 and formation of the light chain of factor V IIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.The prothrombinase complex responsible for the generation of ␣-thrombin in the hemostatic process is composed of factor Va and factor Xa associated on a phospholipid membrane in the presence of Ca 2ϩ (1, 2). Although factor Xa alone can convert prothrombin to ␣-thrombin, the prothrombinase complex has a catalytic efficiency five orders of magnitude greater than factor Xa acting alone (3). Plasma factor V circulates as a large single chain protein of M r 330,000 (4 -6). The cDNA sequences for human, murine, porcine, and bovine factor V have been reported previously (7-11). The factor V molecule is composed of triplicated "A" domains, duplicated "C" domains, and a "B" region. Human factor V is cleaved by ␣-thrombin at Arg 709 , Arg 1018 , and Arg 1545 and generates the active cofactor factor Va, which is composed of a heavy chain (A1-A2 domains, M r 105,000, amino acid residues 1-709) non-covalently associated with the light chain (A3-C1-C2 domains, M r 74,000, amino acid residues 1546 -2196). The interaction between the two chains is promoted by divalent cations (12, 13).Activation of factor V by ␣-thrombin is required for the interaction of the cofactor with factor Xa and prothrombin. Factor Va and factor Xa interact stoichiometrically in the absence of phospholipids with a K d of 0....

The Contribution of Amino Acid Region Asp695-Tyr698 of Factor V to Procofactor Activation and Factor Va Function

“…Molecules-Cofactor activity of wild type and mutant molecules was measured in a clotting assay using factor V-deficient plasma prior and after activation by thrombin (15 min, 37°C) and RVV-V activator (2 h, 37°C) as described (43,46). The values were standardized to the percentage of control (55).…”

“…This amino acid region, which is highly acidic in nature and contains the tyrosine residues that were previously shown to have the potential to be involved in both procofactor activation by thrombin as well as cofactor function (47,48), may possess residues that are directly involved in the interaction of the cofactor with positively charged amino acids provided by one of the protein components of prothrombinase. Further, we have recently demonstrated that a binding site for prothrombin is located on the last 13 amino acids of the factor Va heavy chain (46). The present study was undertaken to identify the specific amino acid residues from the acidic COOH-terminal region of factor Va heavy chain that are important for cofactor function and the molecular mechanisms underlying their contribution to prothrombinase function.…”

A Bipartite Autoinhibitory Region within the B-domain Suppresses Function in Factor V

“…Broadly, these studies found that variable amounts of cofactor activity are observed depending on which region of the B-domain is cleaved and which assay is used to evaluate activity. A clear consensus is that maximal activity correlates with cleavage at Arg 1545 (7,13,(15)(16)(17)(18)(19)(20)(21)(22). Although these studies have advanced the field, such correlative studies have not provided mechanistic insight into how FV activation unfolds.…”

Further Evidence for Two Functional Forms of Prothrombinase Each Specific for Either of the Two Prothrombin Activation Cleavages

“…It also enhances the turnover number for prothrombin activation by a factor of about 3000 (7,8). Several groups have shown that factor Va interacts with factor Xa (13,(15)(16)(17). Interactions between factor Va and prothrombin have also been characterized (17)(18)(19)(20).…”