CLP36 Is a Negative Regulator of Glycoprotein VI Signaling in Platelets

Rationale: At sites of vascular injury, exposed subendothelial collagens not only trigger sudden platelet adhesion and aggregation, thereby initiating normal hemostasis, but also can lead to acute ischemic diseases, such as myocardial infarction or stroke. The glycoprotein (GP) VI/Fc receptor gamma-chain complex is a central regulator of these processes because it mediates platelet activation on collagens through a series of tyrosine phosphorylation events downstream of the Fc receptor gamma-chain-associated immunoreceptor tyrosine-based activation motif. GPVI signaling has to be tightly regulated to prevent uncontrolled intravascular platelet activation, but the underlying mechanisms are not fully understood. Objective: We studied the role of PDZ and LIM domain family member CLP36 in platelet physiology in vitro and in vivo. Methods and Results: We report that CLP36 acts as a major inhibitor of GPVI immunoreceptor tyrosinebased activation motif signaling in platelets. Platelets from mice either expressing a low amount of a truncated form of CLP36 lacking the LIM domain (Clp36(Delta LIM)) or lacking the whole protein (Clp36(-/-)) displayed profound hyperactivation in response to GPVI agonists, whereas other signaling pathways were unaffected. This was associated with hyperphosphorylation of signaling proteins and enhanced Ca2+ mobilization, granule secretion, and integrin activation downstream of GPVI. The lack of functional CLP36 translated into accelerated thrombus formation and enhanced procoagulant activity, assembling a prothrombotic phenotype in vivo. Conclusions: These data reveal an inhibitory function of CLP36 in GPVI immunoreceptor tyrosine-based activation motif signaling and establish it as a key regulator of arterial thrombosis. (Circ Res. 2012;111:1410-1420.)