- Platelets undergo morphological changes essential to hemostasis upon activation at sites of vascular injury. The Rho GTPases Rac1, Cdc42, and RhoA drive these changes. Rho guanine nucleotide dissociation inhibitors (RhoGDIs) bind to the Rho GTPases to block the GDP from dissociating from the Rho GTPases, as well as anchoring the Rho GTPase to a specific intracellular location, which allows the RhoGDIs to regulate and coordinate the activity of Rho GTPases within the cell. In this thesis, we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI–specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.
- Key Words: thrombosis, hemostasis, platelet, RhoGDI, Ly-GDI, Rho GTPases