Mechanisms of Ca2+ regulation in platelets

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Changes in the cytosolic Ca2+ concentration regulate fundamental processes in virtually all cells. In platelets, the mechanisms underlying agonist-induced Ca2+ influx are only partially understood. We have shown that store operated calcium entry (SOCE) is the principal route of Ca2+ influx in platelets that is of major importance for ITAM/PLCgamma2-induced cellular activation and thus occlusive arterial thrombus formation and thrombo-inflammatory processes whereas it is not essential for normal hemostasis. We have identified the Ca2+ sensor STIM1 and the SOC channel protein Orai1 as key mediators of platelet SOCE and characterized the molecular interaction between SOCE and ROCE through the Ca2+-channel proteins Orai1 and TRPC6. Newly generated mice with a gain-of-function mutation in the STIM2 isoform (Stim2deltaEF) will be available soon. In a biochemical approach we have identified the adapter protein Bridging Integrator 2 (BIN2) as an interaction partner of STIM1 in platelets. Initial experiments with newly generated BIN2-deficient mice revealed a pronounced defect of agonist-induced Ca2+ store depletion, but also of SOCE in the platelets of the animals. Interestingly, BIN2 is efficiently cleaved in a calpain-dependent manner in the course of SOCE, but the functional relevance of this process is unknown. In this project we aim to study the function of BIN2 and BIN1, which is also expressed in platelets, in vitro and in vivo. We will utilize conditional BIN2 and BIN1 single and double KO mice for studies on megakaryopoiesis/platelet biogenesis and platelet function to identify the cellular functions of the BIN proteins and their (patho)physiological relevance. In further experiments, these mice will be crossed with mice carrying defined defects in the SOCE machinery to further analyze the functional role of the BIN isoforms in platelet Ca2+ signaling. Using biochemical and cell biological approaches, we want to identify and functionally characterize BIN2 (and BIN1) interaction partners and analyze the underlying mechanisms and functional significance of calpain-dependent BIN2 cleavage. We expect that these studies will provide fundamental new insights into the molecular mechanisms underlying Ca2+ signaling in platelets that may also serve as a basis for the development of novel antithrombotic therapeutics.