Thrombin mutant W215A/E217A acts as a platelet GPIb antagonist.

OBJECTIVE Thrombin containing the mutations Trp215Ala and Glu217Ala (WE) selectively activates protein C and has potent antithrombotic effects in primates. The aim of this study was to delineate the molecular mechanism of direct WE-platelet interactions under static and shear conditions. METHODS AND RESULTS Purified platelets under static conditions bound and spread on immobilized wild-type but not WE thrombin. In PPACK-anticoagulated blood under shear flow conditions, platelets tethered and rolled on both wild-type and WE thrombin, and these interactions were abrogated by the presence of a glycoprotein Ib (GPIb)-blocking antibody. Platelet deposition on collagen was blocked in the presence of WE, but not wild-type thrombin or prothrombin. WE also abrogated platelet tethering and rolling on immobilized von Willebrand factor in whole blood under shear flow. CONCLUSIONS These observations demonstrate that the thrombin mutant WE, while not activating platelets, retains the ability to interact with platelets through GPIb, and inhibits GPIb-dependent binding to von Willebrand factor-collagen under shear.