Promoting integrin engagement and Sema4D-mediated interactions increased Syk activation platelets. Washed platelets from Sema4D^(+/+) and Sema4D^(−/−) mice were supplemented with fibrinogen (150 μg/mL) and CaCl₂ (1mM). Where indicated, MnCl₂ (1mM) was added and the platelets were stirred for 7 minutes followed by a 2-minute incubation with or without collagen (2.5 μg/mL). (A) A representative immunoblot with anti-pSyk Y519/520 is shown, and the results of 4 experiments are summarized (mean ± SEM). (B) Representative aggregation traces for Mn²⁺-treated samples are shown. N.S. = not significant. (C) Washed platelets from Sema4D^(+/+) and Sema4D^(−/−) mice were incubated with 50 μg/mL rSema4D (recombinant Sema4D exodomain) for 10 minutes followed by collagen (2.5 μg/mL) for 2 minutes. A representative immunoblot with anti-pSyk Y519/520 is shown and results of 3 experiments are summarized (mean ± SEM). N.S. = not significant.