RAP1A and RAP1B are critical for platelet adhesion and thrombus formation under shear conditions ex vivo and in vivo. (A-C) Platelet adhesion to fibrillar collagen ex vivo. Anticoagulated whole blood from control (Rap1a^fl/flRap1b^fl/flPf4-Cre-; black), Rap1a^fl/flRap1b^+/flPf4-Cre⁺ (green), Rap1a^+/flRap1b^fl/flPf4-Cre⁺ (orange), or Rap1a^fl/flRap1b^fl/flPf4-Cre⁺ (red) mice was perfused over fibrillar collagen type I (200 μg/mL) at venous shear rates (400^−s). Adhesion of platelets was monitored continuously. Shown are representative images after 5 minutes of perfusion of Alexa Fluor 488–α-GPIX–labeled whole blood obtained using a Nikon TE300 (Nikon, Tokyo, Japan) microscope equipped with a QImaging Retiga EXi camera (Qimaging, Surrey, BC, Canada) (A), platelet adhesion determined as the area of adhesion coverage ± SEM (B), and thrombus buildup quantified as sum fluorescence intensity ± SEM (C). Analyses were performed using SlideBook 5.0 software (Intelligent Imaging Innovations, Denver, CO). Scale bars in A represent 10 μm. (D) FeCl₃-induced thrombosis in the carotid artery. Data shown are the scatter dot plot (line at median) of the time of occlusion (minutes). Blood flow velocity was monitored for 30 minutes with a 0.5-mm Doppler flow probe connected to a Transonic TS420 Flow Module (Transonic, Ithaca, NY); time to occlusion was recorded when blood velocity reached 25% of baseline velocity. All Rap1a/b-mKO vessels never dropped below 25% of baseline flow; thus, they were assigned a time of 30 minutes. *P < .05, **P < .01, ***P < .001. ns, not significant.