Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that impact multiple diverse processes required for megakaryopoiesis and the activation of platelets. We have recently published in Developmental Cell that phosphoinositides in neuronal cells initiate intracellular trafficking by recruiting effector proteins such as GOLPH3 that are involved in vesicular fusion and budding of plasma membranes during Golgi biogenesis. Since megakaryocyte α-granules are derived from the trans-Golgi network and Multi-Vesicular Bodies, I hypothesize that phosphoinositide signaling is necessary for the intracellular trafficking required for the biogenesis of α-granules. PhosphatidylInositol Transfer Proteins (PITPs) are members of a small protein family that bind and transfer phosphoinositide monomers from one cellular compartment to another and thereby enable phosphoinositide synthesis. We have made the unexpected observation that the two predominant PITP isoforms found within megakaryocytes, PITPα and PITPβ play previously unrecognized but essential roles in the trafficking of cargo from the Multi-Vesicular Body to α-granules. Loss of PITP-mediated phosphoinositide synthesis produces morphologic defects similar to what is seen in humans with Gray Platelet Syndrome. The overall hypothesis of this Proposal is that phosphoinositide signaling mediated by PITPs is necessary for the membrane dynamics and protein trafficking required for the biogenesis and maintenance of megakaryocyte α-granules. In Aim 1 of the Project, we will rigorously analyze the discrete biochemical properties of individual PITP isoforms in megakaryocytes. Our preliminary data shows that the two PITP isoforms control phosphoinositide signaling through biochemically distinct mechanisms. In Aim 2, we will determine how phosphoinositide signaling contributes to alpha granule biogenesis and function. In conjunction with Project 2, we will test the hypothesis that phosphoinositide synthesis within discrete microdomains of megakaryocytes and platelets regulates effector proteins such as NBEAL2 (the mutated protein responsible for the Gray Platelet Syndrome). This signaling cascade modulates NBEAL2’s ability to mediate membrane dynamics and protein trafficking. We will also analyze in detail the functional roles of α- granules with ex vivo rheologic and ultramicroscopy studies that will be performed with Project 2, in vivo thrombosis studies with Project 3, and in vivo inflammation studies with Project 4.