A PLCγ2-independent pathway of GPVI-down-regulation. Wild-type (•) or PLCγ2^−/− (○) mice received 20 μg biotinylated JAQ1 intravenously and (A) platelet counts were determined. (B) Surface-bound JAQ1^biotin was detected by flow cytometric measurement of streptavidin^FITC binding and is given as percentage of maximal binding determined in vitro. (C) Detection of sGPVI/JAQ1^biotin complex in plasma at the indicated time points after antibody injection. (A-C) Results are expressed as the mean platelet count ± SD for groups of 6 mice each. (D) Western blot analysis of platelets at the indicated time points. The results are representative of 3 individual experiments. (E) JAQ1 does not alter thrombin responses in PLCγ2^−/− mice. Washed platelets were prepared at the indicated time points after JAQ1 treatment and stimulated with 0.1 (▾), 0.01 (○), or 0.001 (•) U/mL thrombin. Activation of integrin αIIbβ3 and surface expression of P-selectin were determined by flow cytometry and are given as percentage ± SD of the values obtained with untreated controls (n = 6 per group). Platelets were gated by FSC/SSC characteristics and FL-4 positivity (anti–GPIbα-Cy5).