INTERACTIONS AND INHIBITION OF BLOOD-COAGULATION FACTOR-VA INVOLVING RESIDUES 311-325 OF ACTIVATED PROTEIN-C

Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VI IIa. The synthetic peptide-(311-325) (KRNRTFVLNFIKIPV), derived from t he heavy chain sequence of APC, potently inhibited APC anticoagulant a ctivity in activated partial thromboplastin time (APTT) and Xa-1-stage coagulation assays in normal and in protein S-depleted plasma with 50 % inhibition at 13 muM peptide. In a system using purified clotting fa ctors, peptide-(311-325) inhibited APC-catalyzed inactivation of facto r Va in the presence or absence of phospholipids with 50% inhibition a t 6 muM peptide. However, peptide-(311-325) had no effect on APC amido lytic activity or on the reaction of APC with the serpin, recombinant [Arg358]alpha1-antitrypsin. Peptide-(311-325) surprisingly inhibited f actor Xa clotting activity in normal plasma, and in a purified system it inhibited prothrombinase activity in the presence but not in the ab sence of factor Va with 50% inhibition at 8 muM peptide. The peptide h ad no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, sugg esting it does not directly inhibit these enzymes. Factor Va bound in a dose-dependent manner to immobilized peptide-(311-325). Peptide-(311 -315) inhibited the binding of factor Va to immobilized APC or factor Xa. These data are consistent with the hypothesis that residues 311-32 5 in APC bind to factor Va at a site that can bind either APC or facto r Xa, and that peptide-(311-325) interferes with both APC inactivation of factor Va and expression of factor Xa activity in the prothrombina se complex by binding to this site.