A binding site expressed on the surface of activated human platelets is shared by factor X and prothrombin

We have demonstrated the presence of a saturable, reversible, and Ca(2+)-dependent binding site for 125I-labeled factor X ([125I]factor X) on human platelets (16000 +/- 2000 sites per platelet, Kd = 320 +/- 40 nM, n = 12) activated with either thrombin or the thrombin receptor agonist peptide, SFLLRN-amide, but not with ADP. Bound [125I]factor X could be completely removed by the addition of a Ca2+ chelator or an excess of unlabeled factor X. Antibodies that inhibit binding of factor X to the MAC-1 integrin receptor of monocytes and those directed against human factor V, failed to disrupt [125I]factor X binding to platelets. Prothrombin, but neither factor VII, factor IX, protein C, nor protein S, was an effective competitor of [125I]factor X binding with a K1 approximately Kd. [125I]Prothrombin also binds to activated (but not unactivated) platelets in a saturable, reversible, and Ca(2+)-dependent manner (20500 +/- 1500 sites, Kd = 470 +/- 110 nM, n = 3). Annexin V potently inhibited the binding of both [125I]factor X and [125I]prothrombin (IC50 approximately 3 nM). Factor X, prothrombin, and prothrombin fragment 1 (residues 1-155) were equipotent inhibitors of [125I]prothrombin and [125I]factor X binding, whereas Gla-domain-less factor X was unable to compete with [125I]factor X for platelet binding sites. Thus, it is the Gla-domains of factor X and prothrombin that appear to contain the regions necessary for platelet binding. The results of studies utilizing artificial phospholipid surfaces have led to the hypothesis that the substrates (FX and prothrombin) for the intrinsic pathway FXase and prothrombinase complexes are bound to the phospholipid surface. The factor X/prothrombin binding site we have described on the surface of activated platelets permits the utilization of surface-bound substrates by these complexes when they are assembled on a physiologic surface.