PB0882 - Rab GTPase Protein localization, regulation and function in platelets

Background: Rab GTPases serve as master regulators of vesicle biogenesis, trafficking, and fusion in eukaryotic cells. Platelets express over 40 different Rab proteins with potential roles in granule secretion, receptor trafficking and other cell physiological process critical to platelet function.In previous work, we examined Rab localization and distribution in platelets using fluorescent microscopy. However, the specific localization and distribution of Rab and the mechanisms by which Rabs and their effectors regulate complex vesicular trafficking in platelets remain to be elucidated.

Aims: Study the regulation and function of Rab GTPases in platelets.

Methods: Through a combination of immunofluorescence microscopy, time-lapse microscopy, Western blot analysis, and pharmacological inhibition, we quantified the regulation of >10 different Rab GTPases in both resting and platelets stimulated with the collagen receptor GPVI/ITAM agonist crosslinked collagen-related peptide (CRP-XL).

Results: Through conventional fluorescent microscopy, we observed that Rab proteins dynamically localized to distinct vesicle populations upon activation of platelets. Phos-tag gel electrophoresis and Western blot analysis revealed that Rab7, Rab8, and other Rab associated proteins were phosphorylated following platelet activation by CRP-XL; phosphorylation of Rab proteins was reduced by pharmacological inhibitors of protein kinase C (PKC), as well as kinases known to target Rabs in other cell types including TAK1 and TBK1. Moreover, our preliminary experiments suggest that select platelet functions were inhibited by novel pharmacological inhibitors of Rab family members including the Rab7 GTPase inhibitor CID 1067700 and the Rab8-binding stapled peptide StRIP16.

Conclusion(s): Platelets express a variety of Rab GTPase family members that localize to distinct vesicular compartments in quiescent and activating platelets. Future work will determine how different Rabs orchestrate intracellular vesicular trafficking activities associated with platelet function.