Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
Lawrence F. Brass
G protein-coupled receptors (GPCRs) are critical mediators of platelet activation whose signaling is limited in part by members of the regulator of G protein signaling (RGS) family. To better understand how individual RGS proteins impact the optimal balance between activation and inhibition, the most abundant RGS in platelets, RGS10 and RGS18, were deleted both individually and simultaneously in mice. Loss of RGS10 causes increased platelet activation and accumulation following hemostatic injury, due to an expansion of the P-selectin(─) shell, driven by thromboxane A2 and ADP, rather than the P-selectin(+) core, driven by thrombin. Loss of RGS18 results in milder increases in GPCR signaling, primarily thrombin-dependent, and causes moderate thrombocytopenia, due to decreased platelet production. Loss of both RGS10 and RGS18 results in dramatically increased platelet activation and accumulation in vivo, with an expansion of both the P-selectin(+) core and the P-selectin(─) shell, and uninhibited growth that increases the occurrence of vascular occlusion. Furthermore, dual deletion of RGS10 and RGS18 results in reduced platelet survival due to premature activation in circulation and subsequent clearance. Additionally, in efforts to explore RGS-mediated regulation of Gq signaling in platelets, we induced a homozygous RGS-insensitive G188S mutation in mouse Gq(alpha) (Gq[alpha] G188S). Unexpectedly, Gq(alpha) G188S mice had dramatically reduced platelet accumulation in vivo, which was due to decreased Gq signaling via disrupted PLC(beta) interactions. Structural and computational analyses revealed substantial overlap between RGS and effector binding interfaces, but provided candidate mutations predicted to specifically disrupt RGS interactions. Finally, to corroborate our results with mouse models, we sought to identify predicted loss-of-function RGS10 and RGS18 variants in human patients and analyze their platelet function. We identified 16 variants in 101 patients and have plans to recall them to analyze platelet reactivity and RGS expression levels. Viable candidates will also be selected for in vivo hemostatic analysis using iPSC-derived megakaryocytes and a humanized mouse model. Overall, these studies demonstrate how RGS-mediated regulation of platelet GPCRs is important for platelet production, survival and hemostatic reactivity. Furthermore, it suggests that a delicate equilibrium between negative and positive platelet activation regulators is necessary to promote rapid responsivity while restraining unwarranted signaling.
Dehelian, Daniel John, "Regulators Of G Protein Signaling Modulate Platelet Function To Impact Normal Physiology And The Hemostatic Response" (2020). Publicly Accessible Penn Dissertations. 3802.