Phosphoinositides, cytoskeleton and platelet activation – PHOSPHOINOPLAT

Platelets are critical for normal haemostasis but are also centrally involved in pathological processes like thrombosis and atherosclerosis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Recently, they have also been implicated in angiogenesis, dissemination of metastases and a variety of inflammatory and immune reactions within the vasculature. In view of the pivotal role played by platelet activation and platelet-vessel wall interactions in these different pathological situations, the inhibition of platelet functions is a relevant therapeutic strategy to prevent ischemic diseases such as stroke, myocardial infarction or peripheral arterial diseases. There is now accumulating evidence that phosphoinositide metabolism plays a major role in platelet activation. Our project focus on D3-phosphoinositides and their roles in signaling pathways implicated in the induction of adhesive / aggregatory platelet properties following integrin activation. Their role in platelet secretion and in the reorganization of the cytoskeleton required for platelet shape change, adhesion and spreading will be investigated as well. Our previous results suggest that the class IA PI3K p110 plays a major role in platelet activation. To evaluate the potential interest of this isoform of PI3K as a new target for antithrombotic therapy, we will generate a mouse model with a PI3K p110 inactive catalytic subunit specifically in megakaryocytes and platelets. The characterization of this mouse model is challenging and will allow a detailed investigation of the role of this isoform of PI3K in platelet activation and thrombus formation in vivo. Moreover, this model will permit to address topical question in the PI3K signaling field. Furthermore, platelets from phosphoinositide 5-phosphatase SHIP1-deficient mice display a dramatic accumulation of PtdIns(3,4,5)P3 and a defect in PtdIns(3,4)P2 production correlated to an abnormal cytoskeleton organization / contractile apparatus. To gain insights in the understanding of this platelet phenotype and to clarify the link between D3-phosphoinositides and cytoskeleton, we plan to investigate in detail the cytoskeleton organization in the absence of SHIP1. Because SHIP1 can act as a 5-phosphatase or as a docking protein, its role as an adaptator protein as well as the effectors of its product PtdIns(3,4)P2, will be investigated via proteomic approaches. This project combines in vivo and in vitro approaches to investigate in detail the role of two key enzymes of the phosphoinositide metabolism and their products in platelet functions. Besides a the better knowledge on D3-phosphoinositide role and signaling, this project will lead to the identification of new molecular mechanisms and new pharmacologic targets to improve the treatment of thrombosis and platelet-related pathologies in general.