Platelet RAP1 signaling minimally contributes to the maintenance of vascular integrity during development and inflammation. (A) Contribution to vascular integrity during development was determined by assessing blood lymphatic mixing (BLM) in embryos at ∼E16.5 by macroscopic observation, using a Leica MZ16FA dissecting stereoscope, and immunofluorescence staining of embryo sections. Embryos were sectioned at the jugular lymph sac (JLS), and lymphatic endothelial cells (green) were stained with primary antibody overnight (polyclonal rabbit anti-mouse LYVE-1), followed by donkey anti-rabbit Alexa Fluor 488 and 4′,6-diamidino-2-phenylindole. Red blood cells were visualized by autofluorescence (red). Images were acquired on a Nikon E800 microscope with a Hamamatsu camera with MetaMorph software (Molecular Devices). (B) Contribution to vascular integrity at sites of inflammation was determined by rpA reaction in the skin of control (Pf4-Cre-) mice depleted of all circulating platelets (by intravenous injection of antibodies against GPIbα) compared with nondepleted Rap1a/b-mKO or nondepleted Talin1-mKO mice. Representative images of rpA sites are shown (dashed circles) (left panels). Hemorrhage at sites of inflammation was quantified by measuring hemoglobin (Hb) levels in skin lesions 4 hours after rpA challenge (n = 5-7) (right panel). *P < .05, **P < .01. ns, not significant.