Thrombin induces GPIb‐IX‐mediated fibrin binding to αIIbβ3in a reconstituted Chinese hamster ovary cell model

Background:The interaction of thrombin with platelet glycoprotein (GP) Ib‐IX‐V has been recently suggested to induce fibrin‐dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease‐activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. Objectives:In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb‐IX. Methods:We used Chinese hamster ovary (CHO) cells stably expressing human αIIbβ3and/or GPIb‐IX complexes (CHO‐αIIbβ3‐IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate‐fibrinogen as precursor. Results:Thrombin induces, in a dose‐dependent manner, the binding of polymerizing fibrin to CHO‐αIIbβ3‐IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against αIIbβ3and GPIbα. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14‐3‐3ζadaptor and calcium–calmodulin‐dependent events are involved. Conclusions:The present data support a significant role of GPIb‐IX and αIIbβ3receptors in an alternative fibrin‐mediated pathway of platelet activation induced by thrombin.