CD148-deficient platelets exhibit impaired GPVI-mediated platelet aggregation, secretion, and integrin activation. (A) Washed platelets (2 × 10⁸/mL) prepared from wild-type (WT), CD148 transmembrane-knockout (CD148 TM-KO), and FcR γ-chain heterozygous-deficient (γ-chain^+/−) mice were stimulated with low, intermediate, and high doses of: (Ai) collagen-related peptide (CRP; 1, 3, and 10 μg/mL); (Aii) collagen (1, 3, and 10 μg/mL); and (Aiii) thrombin (0.03, 0.09, and 0.3 U/mL). Platelet-rich plasma prepared from WT and CD148 TM-KO mice was stimulated with low, intermediate, and high doses of (Aiv) thromboxane A2 analog U46619 (1, 3, and 10 μM) and (Av) ADP (1, 3, and 10 μM). Platelet aggregation was measured as a change in light transmission, and ATP secretion was measured as luciferin/luciferase-mediated luminescence, using a lumi-aggregometer. Representative images are shown (n = 3-6 mice per condition). (B) Surface expression of P-selectin and (C) “active” integrin αIIbβ3 was quantified on resting and activated (10 μg/mL CRP or 0.06 U/mL thrombin) platelets from litter-matched WT and CD148 TM-KO (KO) mice by flow cytometry. Resting (black lines) and activated (gray lines) platelets were stained with either (Bi) FITC-conjugated rat anti–mouse P-selectin antibody or (Ci) PE-conjugated JON/A antibody to “active” integrin αIIbβ3. Data presented as (Bii) P-selectin geometric mean fluorescence intensity (MFI) and (Cii) fold increase in JON/A binding relative to total αIIbβ3 staining. Representative histograms are shown (n = 3 mice per condition). Bar height and error bars represent mean ± SEM (*P < .05, **P < .01).