C3G promotes angiogenesis through the regulation of the differential release of pro-/anti-angiogenic factors from thrombin-activated platelets

Abstract

C3G is a guanine nucleotide exchange factor (GEF) of several proteins within the Ras superfamily, including Rap1a/b and R-Ras. Rap1 is known to be involved in critical aspects of platelet function, including aggregation, adhesion and spreading, through the activation of the platelet integrin αIIbβ3. Using transgenic mouse models expressing C3G or C3GΔCat (a mutant lacking the catalytic domain) in megakaryocytes and platelets, our group has identified C3G as a mediator of Rap1 actions leading to platelet activation and aggregation. Furthermore, C3G promotes α-granule release, evidenced by the increase in P-selectin exposure on the platelet surface, following its activation. In addition, preliminary immunofluorescence analysis suggests that C3G modulates the release of VEGF and endostatin from thrombin-activated platelets. The goal of the present study was to further characterize the underlying mechanisms that regulate the function of C3G in the differential release of pro- and anti-angiogenic factors upon platelet activation, and to evaluate the angiogenic potential of the platelet releasate. We have found that VEGF release was up-regulated in thrombin-activated mouse platelets from both, C3G and C3GΔCat, transgenic mice. Additionally, thrombospondin release was also up-regulated in the latter. The releasate of TgC3G platelets had an overall pro-angiogenic effect as evidenced in a capillary-tube formation assay with HUVECs and in heterotopic tumor mouse models using B16 and 3LL cells. On the other hand, platelet spreading analysis revealed an up-regulation of this function in both transgenic mouse platelets upon thrombin activation, with TgC3G platelets showing the highest extension area. A preliminary co-IP analysis revealed an interaction between C3G and VAMP-7 upon activation, which could explain this result. Taken together, our data indicate the co-existence of RapGEF-dependent and independent mechanism through which C3G is regulating platelet secretion, and suggest an overall pro-angiogenic effect of platelet C3G.