One of the earliest platelet activation events is reorganization of the actin cytoskeleton by the simultaneous disassembly and assembly of filamentous actin. The overall hypothesis of this proposal is that platelet actin reorganization plays a critical role in arterial hemostasis and thrombosis. The available data show that actin signaling pathways in platelets are not identical to those found in frequently studied fibroblast or yeast cell lines. Yet the literature suggests that the generation of phosphatidylinositol 4,5-bisphosphate (PIP2) by the lipid kinase, PIP5K, may be a critical event in platelet actin assembly initiated by either integrins or agonist receptors. PIP2 controls actin assembly at two steps: First, it is the substrate for polyphosphoinositide second messenger formation, and second, it directly interacts with actin-binding proteins and regulates their function. We have found that platelets have both PIP5K-beta and PIPSK-gamma. Furthermore, we have further found that Rho GTPase controls the intracellular localization of both PIP5K-beta and PIP5K-gamma in platelets, and thereby regulates the ability of these enzymes to synthesize PIP2 in platelets. The proposed experiments are designed to take a comprehensive and systematic study of the link between PIP2 production, actin reorganization, and stable platelet adhesion. I hypothesize that PIP5K-beta generates the pool of PIP2 required for polyphosphoinositide second messenger formation, and PIP5K-gamma generates the pool of PIP2 required for regulation of actin-binding proteins. In the first Aim, we will examine the regulation of PIP2 production by integrins and Rho family GTPases, and study the differences between the distinct platelet PIP5K isoforms. In the second Aim, we will formally address how PIP2 drives reorganization of platelet actin using a combination of biochemistry, murine genetics, and ex vivo platelet adhesion models.
| Lian, Lurong; Wang, Yanfeng; Flick, Matthew et al. (2009) Loss of pleckstrin defines a novel pathway for PKC-mediated exocytosis. Blood 113:3577-84 |
| Wang, Yanfeng; Chen, Xinsheng; Lian, Lurong et al. (2008) Loss of PIP5KIbeta demonstrates that PIP5KI isoform-specific PIP2 synthesis is required for IP3 formation. Proc Natl Acad Sci U S A 105:14064-9 |
