We have recently shown that amino acid region 307-348 of factor Va heavy chain (42 amino acids, N42R) is critical for cofactor activity and may contain a binding site for factor Xa and/or prothrombin [(2001) J. Biol. Chem. 276, 18614-18623]. To ascertain the importance of this region for factor Va cofactor activity, we have synthesized eight overlapping peptides (10 amino acid each) spanning amino acid region 307-351 of the heavy chain of factor Va and tested them for inhibition of prothrombinase activity. The peptides were also tested for the inhibition of the binding of factor Va to membrane-bound active site fluorescent labeled Glu-Gly-Arg human factor Xa ([OG488]-EGR-hXa). Factor Va binds specifically to membrane-bound [OG488]-EGR-hXa (10nM) with half-maximum saturation reached at approximately 6 nM. N42R was also found to interact with [OG488]-EGR-hXa with half-maximal saturation observed at approximately 230 nM peptide. N42R was found to inhibit prothrombinase activity with an IC50 of approximately 250 nM. A nonapeptide containing amino acid region 323-331 of factor Va (AP4') was found to be a potent inhibitor of prothrombinase. Kinetic analyses revealed that AP4' is a noncompetitive inhibitor of prothrombinase with respect to prothrombin, with a K(i) of 5.7 microM. Thus, the peptide interferes with the factor Va-factor Xa interaction. Displacement experiments revealed that the nonapeptide inhibits the direct interaction of factor Va with [OG488]-EGR-hXa (IC50 approximately 7.5 microM). The nonapeptide was also found to bind directly to [OG488]-EGR-hXa and to increase the catalytic efficiency of factor Xa toward prothrombin in the absence of factor Va. In contrast, a peptadecapeptide from N42R encompassing amino acid region 337-351 of factor Va (P15H) had no effect on either prothrombinase activity or the ability of the cofactor to interact with [OG488]-EGR-hXa. Our data demonstrate that amino acid sequence 323-331 of factor Va heavy chain contains a binding site for factor Xa.

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690 -699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC 50 values of ϳ12 and ϳ10 M, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K i values of ϳ6.3 and ϳ5.3 M, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase.

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....

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4) contains a binding site for factor Xa The maintenance of hemostasis is a complex event requiring the controlled interaction of proteases, zymogens, cofactors, and inhibitors on surfaces provided by platelets and endothelial cells, culminating in timely ␣-thrombin formation at the site of a vascular injury. The enzymatic complex prothrombinase, which activates prothrombin, is required for normal blood clotting and is composed of the enzyme, factor Xa, and the protein cofactor, factor Va, associated on a cell surface in the presence of divalent metal ions (1, 2). Whereas factor Xa possesses the enzymatic capability to activate prothrombin, the incorporation of factor Va into prothrombinase and its interaction with factor Xa increase the catalytic efficiency of the enzyme by 5 orders of magnitude as compared with factor Xa alone (3). The increase in the catalytic efficiency of prothrombinase as compared with factor Xa alone is believed to arise from a 100-fold decrease in the K m and a 3000-fold increase in the k cat of the enzyme (4 -7). The decrease in the K m of the reaction appears to be the result of the interaction of the complex with the cell surface, resulting in higher local concentration of substrate. The increase in the k cat of prothrombinase is attributed to the binding of factor Va to factor Xa. The bulk of the data generated suggest that incorporation of factor Va into prothrombinase and its interaction with factor Xa and prothrombin do not have any significant effect on the catalytic site of the enzyme (8 -10). The accumulated data suggest that binding of factor Va to factor Xa induces a conformational transition in the structure of the enzyme exposing a prothrombin-binding exosite on the enzyme, resulting in more efficient catalysis. However, the cofactor itself may also represent a portion of the exosite for prothrombin. Evidence for a direct interaction of factor Va with prothrombin has been repetitively provided (11)(12)(13)(14). The amino acids responsible for this dual effect of factor Va on factor Xa remain to be identified.Human factor V circulates in plasma as a single chain glycoprotein of M r 330,000 consisting of multiple domains A1-A2-B-A3-C1-C2, at a concentration of ϳ20 nM (15-18). The factor V gene is 80 kb in length and contains 24 introns, whereas the mRNA is 6.9 kb long (19). Single chain factor V does not appear to interact with factor Xa and to have cofactor activity. Factor V is cleaved sequentially by ␣-thrombin at Arg 709 , Arg 1018 , and Arg 1545 to produce the active cofactor, factor Va, that consists of a heavy chain (M r 105,000) and a light chain (M r 74,000). The heavy chain derives from the NH 2 -terminal part of factor V (A1-A2 domains, residues 1-709), whereas the light chain represents the COOH-terminal end of the procofactor (A3-C1-C2 domains, residues 1546 -2196) (17). The heavy and light chains are noncovalently associated via divalent metal ions (20). Act...