Year of Publication

2015

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Molecular and Cellular Biochemistry

First Advisor

Dr. Sidney W. Whiteheart

Abstract

Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets.

Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets.

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