Figure 8. Role of Rap1b in C3aR mediated thrombus formation.

(A) Generation of Spa.1^−/− x C3aR^−/− double knockout (DKO) mice. Rap1 is activated (Rap1*GTP) by different ligands binding to G-Protein coupled receptors such as C3aR. Rap1 is inhibited by specific GTPase activating proteins (Rap1GAPs) such as Spa.1. In Spa.1^−/− mice, lack of this Rap1GAP results in constantly active Rap1. In C3aR^−/− mice, we observed a phenotype of reduced thrombosis and increased bleeding. To address any intersection point between these observations, we crossed Spa.1 knockout mice with C3aR^−/− mice to generate double knockout (DKO) mice (C3aR^−/− x Spa.1^−/− mice). (B) Rap1 pull down assay for detection of activated Rap1 in murine platelet lysates of WT, C3aR^−/− x Spa.1^+/+, C3aR^−/− x Spa.1^−/− and Spa.1^−/− mice stimulated with C3a. Activated Rap1 in murine platelet lysates was isolated with GST-tagged sepharose, subjected to SDS-PAGE, blotted and detected with an activation specific Rap1 antibody. Total Rap1 in platelet lysates was analyzed by western blot. One representative blot of three experiments is depicted. (C) Tail-bleeding time was measured in C3aR^−/− x Spa.1^−/− DKO mice, WT mice and C3aR^−/− Spa.1^+/+ mice. In C3aR^−/− x Spa.1^−/− DKO mice, bleeding time was significantly reduced compared to C3aR^−/− x Spa.1^+/+ mice. Data represent mean ± SD, n=15 (WT), n=9 (C3aR^−/− x Spa.1^+/+) and n=8 (C3aR^−/− x Spa.1^−/−). *p<0.05 (D) Similarly, FeCl₃-induced thrombus formation was analyzed in these mice. After injury of mesenteric arterioles, time to vessel occlusion was significantly shorter in C3aR^−/− x Spa.1^−/− DKO mice compared to C3aR^−/− x Spa.1^+/+ littermate controls. Data represent mean ± SD, n=5 (WT) and n=7 (C3aR^−/− x Spa.1^+/+ and C3aR^−/− x Spa.1^-/-),*p<0.05.