Prothrombin residues 473-487 contribute to factor Va binding in the prothrombinase complex

To identify sequences in prothrombin (fII) involved in prothrombinase complex (fXa . fVa . fII . phospholipids) assembly, synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit factor Xa (fXa)-induced clotting of normal plasma. The fII peptide (PT473 - 487, homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity, with 50% inhibition of 12 and 10 muM peptide, respectively. Prothrombinase inhibition by PT473 - 487 was factor Va (fVa)-dependent and sequence-specific, because the peptide did not inhibit fII activation in the absence of fVa, and a scrambled sequence peptide, PT473 - 487SCR, was not inhibitory. Peptide PT473 - 487 did not inhibit the amidolytic activities of fXa and thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine whether PT473 - 487 interacted directly with fVa, fluorescein-labeled fVa (Fl-fVa) was prepared. When PT473 - 487 was titrated into samples containing phospholipid-bound Fl-fVa, the peptide increased fluorescein anisotropy (EC50 at 3 muM peptide), whereas the control peptide PT473 - 487SCR did not alter the anisotropy, suggesting a direct binding interaction between PT473 487 and Fl-fVa. These functional and spectroscopic data suggest that fII residues 473 - 487 provide fVa-binding sites and mediate interactions between fVa and fII in the prothrombinase complex.