Abstract 130: Platelet Rap1 Signaling, Mediated by CalDAG-GEFI and P2Y12, Contributes to Atherosclerotic Lesion Development in Mice

Background and Objective The development and progression of atherosclerotic lesions is mediated by various blood cell types, including platelets and neutrophils. We recently identified CalDAG-GEFI (CDGI) as a critical component of integrin-mediated adhesion of platelets and neutrophils at sites of vascular injury. In platelets, signaling by CDGI mediates the early activation of Rap1, while receptor for ADP, P2Y12, the target of the clinically used drug Plavix, is required for sustained Rap1 activation and thrombus stability. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr-/-) mice lacking CDGI and/or P2Y12 in hematopoietic cells. Methods and Results Ldlr-/- mice were irradiated and reconstituted with bone-marrow from wild-type (WT), CalDAG-GEFI-/- (CdgI), P2y12-/-, or CdgI-/- P2y12-/- (DKO) mice. Reconstituted mice were fed a fat and cholesterol enriched diet (21% fat, 0.2% cholesterol) for 12 weeks, ad libitum. Atherosclerotic lesions in the aortic sinus of Ldlr-/-;CdgI-/- chimeras were 42% smaller than those in Ldlr-/-;WT controls (0.18 ± 0.02 mm2 vs 0.31 ± 0.05 mm2, respectively; n = 13 each group, p < 0.001). Lesions in Ldlr-/-;P2y12-/- chimeras were also significantly smaller compared to controls (0.22 ± 0.10 mm2; n = 13, p < 0.05). Lesions in Ldlr-/-;DKO were reduced by 48% compared to WT controls (0.16 ± 0.02 mm2; n = 13, p < 0.001 ), but they were not statistically different from Ldlr-/-;CdgI-/- chimeras. Platelet adhesion and activation on collagen under flow was markedly impaired in Ldlr-/-;CdgI-/- and Ldlr-/-;DKO blood, but only partially impaired in Ldlr-/-;P2y12-/- blood. Importantly, firm neutrophil adhesion to collagen-bound platelets was significantly reduced in Ldlr-/-;CdgI-/- and Ldlr-/-;DKO blood, but not in Ldlr-/-;P2y12-/- blood. Total cholesterol and triglyceride levels were similar among groups. Conclusion Our findings reveal a critical role for CDGI and P2Y12, and Rap1-dependent platelet activation, in promoting atherosclerotic lesion development in hypercholesterolemic mice. Further studies are required to determine if the observed protection in Ldlr-/-;CdgI-/- chimeras is due to impaired neutrophil function.