Hemin activates human platelet through the SFK-SYK-PLCγ2 pathway. (A) Hemin-induced platelet aggregation in humans. Washed human platelets were stimulated with 6 concentrations of hemin (0, 1.0, 2.5, 5.0, 7.5, and 10 μg/mL). The representative curves (left) and quantifications of maximum light transmission (right) are shown. (B) Blocking effect of 0.2 mM RGDS, 50 μM PP2, 10 µM SU6656, and 1 µM R406 on hemin (7.5 μg/mL)-, CRP (0.25 µg/mL)-, or rhodocytin (Rhod) (10 nM)-induced platelet aggregation. The representative curves and quantifications of maximum light transmission are shown. (C) Western blot analysis of phospho-human SYK (Y525/526), phospho-human PLCγ2 (Y759), human SYK, and human PLCγ2. Representative images of hemin-induced (7.5 μg/mL) phosphorylation of SYK and PLCγ2 and its inhibition by PP2 in human platelets are shown. (D) Quantitative analysis of SYK (left) and PLCγ2 (right) phosphorylation normalized by total SYK and PLCγ2, respectively. Data are the mean ± standard deviation (panels A, B, and D); n = 3 to 4. *P < .05, **P < .01, ***P < .001. Tukey’s multiple-comparison test for panels A and D, Student t test for panel B.