The overall hypothesis of this proposal is that platelet actin reorganization plays a critical role in arterialhemostasis and thrombosis. The reorganization of filamentous actin is one of the earliest events of plateletactivation. Our recent data has focused on two critical aspects of platelet actin dynamics, phospholipidsignaling and post-translational actin modifications. Phosphatidylinositol bisphosphate (PIP2) is critical forthe regulation of the platelet cytoskeleton and is required for several steps of actin assembly. This includesserving as a substrate for phosphoinositide second messenger formation, as well as directly binding to andthereby regulating actin-binding proteins. Platelets have both the (3 and y isoforms of phosphatidylinositolphosphate 5-kinase I (PIP5KI) that are each capable of converting PI4P to PI4.5P2 (PIP2), so it is unclearwhy platelets require more than one isoform to perform this single biochemical reaction. To test thehypothesis that these isoforms have non-overlapping functions, we have generated mice containing nullmutations in either the PIP5KI3 or the PIPSKIy genes, which encode for the dominant PIP5KI isoforms inplatelets. Our preliminary studies of platelets from these mice suggest that production of PIP2 by PIP5KI3 isrequired to generate second messengers, while synthesis of PIP2 by PIP5Kly is required to maintain theintegrity of the membrane skeleton. We have also recently reported that at the leading edge of migratingfibroblasts, actin must have an arginine post-translationally added onto its N-terminus. Absence of thismodification impairs fibroblast adhesion and migration, demonstrating the essential role of this modificationin actin assembly. Our preliminary studies indicate that in response to agonist stimulation, platelet actin isalso arginylated. We hypothesize that this recently discovered post-translational modification is critical forplatelet actin dynamics. The goal of this application is to understand on a molecular basis the in vivo plateletactin changes, and to determine whether actin reorganization is vital for stable platelet adhesion.
In Aim 1, we will define the biologic roles of PIP5KI in platelets using our recently developed murine models.
In Aim 2, we will determine how integrins regulate PIP2 production by a combination of biochemistry, murine genetics,and platelet adhesion models.
In Aim 3, we will address the contribution of actin arginylation on plateletadhesion using biochemistry and mice genetically altered to lack the ability to arginylate platelet actin.
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