Abstract
Prothrombin activation is a pivotal event in thrombosis and hemostasis because thrombin can mediate fibrin formation and can activate and aggregate platelets. Platelet aggregation depends upon the binding of adhesive proteins to integrin alphaIIbbeta3 on the platelet surface. In the present study, a novel interface between the blood coagulation system and platelets is demonstrated by showing that 1) prothrombin binds to alphaIIbbeta3 and 2) this interaction accelerates prothrombin activation. Prothrombin bound to purified alphaIIbbeta3 in a specific, saturable, and divalent cation-dependent manner. This interaction was inhibited by certain monoclonal antibodies to alphaIIbbeta3, by the alphaIIbbeta3 ligands fibrinogen and RGD peptides, but not by thrombin or unrelated proteins. Prothrombin also interacted with alphaIIbbeta3 on resting and stimulated platelets as demonstrated by soluble ligand binding and platelet adhesion assays. Activation of prothrombin by Factor Xa alone or Factor Xa-Va was accelerated by alphaIIbbeta3, and this enhancement was blocked by a monoclonal antibody that inhibited prothrombin binding to the receptor. Taken together, these data identify a previously unrecognized linkage between platelets and the blood coagulation system that may have a significant regulatory consequence.
Highlights
Activation of prothrombin by Factor Xa alone or Factor Xa-Va was accelerated by ␣IIb3, and this enhancement was blocked by a monoclonal antibody that inhibited prothrombin binding to the receptor
Lets by showing that certain antiplatelet agents could inhibit thrombin generation. These agents included a chimeric monoclonal antibody1 7E3, which binds to ␣IIb3, blocks ligand binding to this receptor and is currently being used as an antithrombotic drug in humans [2, 3], and peptides containing an Arg-Gly-Asp (RGD) sequence that interact with ␣IIb3 [4] and inhibit the binding of adhesive proteins to the receptor [5,6,7]
After post-coating with Bovine serum albumin (BSA), prothrombin was added at concentrations of 20 –50 mg/ml. monoclonal antibody (mAb) 7E3 (20 g/ml) was used to inhibit prothrombin binding to the receptor, and nonspecific mouse IgG at the same concentration served as control
Summary
A novel interface between the blood coagulation system and platelets is demonstrated by showing that 1) prothrombin binds to ␣IIb3 and 2) this interaction accelerates prothrombin activation. MAb 7E3 (20 g/ml) was used to inhibit prothrombin binding to the receptor, and nonspecific mouse IgG at the same concentration served as control. Prothrombin was incubated in wells coated with ␣IIb3 in the presence of mAb 7E3 (20 g/ml) or the same concentration of mouse IgG.
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