Prothrombin Residues 473 to 487 Contribute To Factor Va Binding in the Prothrombinase Complex.

To identify sequences in prothrombin (fII) involved in assembly of the prothrombinase complex (fXa:factor Va:fII:phospholipids), synthetic peptides based on fII sequences were prepared and screened for their ability to inhibit fXa-induced clotting of normal plasma. The fII peptide comprising residues 473–487 (designated PT473–487 that are homologous to chymotrypsin residues 149D-163) potently inhibited plasma clotting assays and prothrombinase activity with 50% inhibition observed at 12 microM and 10 microM peptide, respectively. Prothrombinase inhibition by PT473–487 was fVa-dependent and sequence-specific since 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 also inhibited cleavage at Arg271 in meizo-thrombin, showing that it similarly inhibited fXa cleavage at both Arg320 and Arg271. Peptide PT473–487 did not inhibit the amidolytic activities of fXa or thrombin, suggesting that the peptide did not alter the integrity of their active sites. To determine if 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 microM peptide) whereas the control peptide PT473–487SCR peptide 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 that contribute to cleavages at both Arg271 and Arg320 by fXa.