BACKGROUND: Secretory granules are key elements for platelet functions. Their biogenesis and integrity are regulated by fine-tuned mechanisms that need to be fully characterized. Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, PtdIns5P (phosphatidylinositol 5 monophosphate) and PtdIns(3,5)P 2 (phosphatidylinositol (3,5) bisphosphate) in granule homeostasis in megakaryocytes and platelets. METHODS: For that, we invalidated PIKfyve by pharmacological inhibition or gene silencing in megakaryocytic cell models (human MEG-01 cell line, human imMKCLs, mouse primary megakaryocytes) and in human platelets. RESULTS: We unveiled that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P 2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P 2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Permeant PtdIns5P and PtdIns(3,5)P 2 and the cation channel TRPML1 (transient receptor potential mucolipins) and TPC2 activation were able to accelerate alpha and dense granule integrity recovery following release of PIKfyve pharmacological inhibition. In platelets, PIKfyve inhibition specifically impaired the integrity of dense granules culminating in defects in their secretion, platelet aggregation, and thrombus formation. CONCLUSIONS: These data demonstrated that PIKfyve and its lipid products PtdIns5P and PtdIns(3,5)P 2 control granule integrity both in megakaryocytes and platelets.
Phosphoinositides are lipid second messengers regulating in time and place the formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking, and cytoskeleton/membrane dynamics. One of these lipids, phosphatidylinositol 3 monophosphate (PtdIns3P), is present in small amounts in mammalian cells and is involved in the control of endocytic/endosomal trafficking and in autophagy. Its metabolism is finely regulated by specific kinases and phosphatases including class II phosphoinositide 3‐kinases (PI3KC2s) and the class III PI3K, Vps34. Recently, PtdIns3P has emerged as an important regulator of megakaryocyte/platelet structure and functions. Here, we summarize the current knowledge in the role of different pools of PtdIns3P regulated by class II and III PI3Ks in platelet production and thrombosis. Potential new antithrombotic therapeutic perspectives based on the use of inhibitors targeting specifically PtdIns3P‐metabolizing enzymes will also be discussed. Finally, we provide report of new research in this area presented at the International Society of Thrombosis and Haemostasis 2019 Annual Congress.
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