Binding site for blood coagulation factor Xa involving residues 311-325 in factor Va

Factor Va inactivation by activated protein C is associated with cleavages at Arg306, Arg506, and Arg679 with Arg306 cleavage causing the major activity loss. To study functional roles of the Arg306 region, overlapping 15-mer peptides representing the sequence of factor Va residues 271-345 were synthesized and screened for anticoagulant activities. The peptide containing residues 311-325 (VP311) noncompetitively inhibited prothrombin activation by factor Xa, but only in the presence of factor Va. Fluorescence studies showed that VP311 bound to fluorescence-labeled 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg factor Xa in solution with a Kd of 70 microM. Diisopropylphosphoryl factor Xa and factor Xa but not factor VII/VIIa or prothrombin bound to immobilized VP311 with relatively high affinity. These results support the hypothesis that residues 311-325, which are positioned between the A1 and A2 domains of factor Va and likely exposed to solvent, contribute to the binding of factor Xa by factor Va. Based on this hypothesis, it is suggested that cleavage by activated protein C at Arg306 in factor Va not only severs the covalent connection between the A1 and A2 domains but also disrupts the environment and structure of residues 311-325, thereby down-regulating the binding of factor Xa to factor Va.