Cleavage Requirements for Activation of Factor V by Factor Xa

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Abstract
Coagulation factor V circulates in plasma as a single chain protein which expresses little procoagulant activity. After its activation by limited proteolysis by thrombin or factor Xa, factor Va functions as cofactor to factor Xa in the activation of prothrombin. Thrombin cleaves human factor V at Arg709, Arg1018 and Arg1545 and factor Va is formed by the heavy and light chains, which correspond to the N‐terminal and C‐terminal fragments, respectively. Factor Xa has been shown to cleave factor V at Arg1018 and at a second undefined position close to Arg709. The factor‐Xa‐mediated cleavage at Arg1018 has been proposed to be sufficient for expression of full factor Va activity. To study the activation of factor V by factor Xa, site‐directed mutagenesis was used to convert Arg709 to Gln, Arg1018 to Ile, and Arg1545 to Gln. Constructs containing all possible combinations of native and mutated residues in these positions were expressed transiently in COS 1 cells. The various factor‐V mutants were incubated with factor Xa or thrombin. The proteolytic cleavage pattern was analyzed by Western blotting, and the specific factor‐Va activities determined in a prothrombinase assay. Control experiments using thrombin gave results which were in agreement with those on record, i.e. cleavages at both Arg709 and Arg1545 were required for expression of full factor‐Va activity, whereas the cleavage at Arg1018 enhanced the rate of cleavage at Arg1545. Factor Xa was found to cleave factor V at all three thrombin cleavage sites, i.e. at Arg709, Arg1018 and Arg1545. An additional factor‐Xa‐cleavage site was found in the light chain region at Arg1765. Cleavage at Arg1018 by factor Xa was not sufficient for expression of full factor‐Va activity. Full factor‐Va activity was only obtained after cleavage at both Arg709 and Arg1545. The factor‐Xa‐mediated cleavage at Arg709 was kinetically favourable over that at Arg1545. Factor V which was mutated at all three sites (at positions 709, 1018 and 1545) was resistant to activation by thrombin. However, treatment with factor Xa yielded an increased factor‐Va activity which was associated with the cleavage at Arg1765. Our study extends previously results on thrombin activation of factor V and elucidates the relative importance of the different cleavage sites for activation of factor V by factor Xa.