Fibrinogen binding, spreading on collagen and the formation of an initial platelet monolayer on a collagen-coated surface under flow proceeds normally in Sema4D^(−/−) platelets. (A) Fibrinogen binding. Platelets from Sema4D^(+/+) and Sema4D^(−/−) mice were incubated with Alexa Fluor 488–labeled fibrinogen (100 μg/mL) and convulxin (CVX). Fibrinogen binding was measured by flow cytometry (mean ± SEM, N = 4-8). (B) Spreading of individual platelets on collagen. Platelets from Sema4D^(+/+) and Sema4D^(−/−) mice were deposited on glass coverslips coated with acid soluble type I collagen (0.1 mg/mL). Platelet spreading was quantified by reflection interference contrast microscopy (RICM). Representative images are shown and the results of 3 experiments are summarized (mean ± SEM). Scale bar equals 3 μm. (C-E) Platelets in PPACK-treated whole blood obtained from Sema4D^(−/−) and matched Sema4D^(+/+) mice were labeled with Alexa Fluor 488–conjugated anti-CD41 (α_IIb) and perfused through a microfluidic flow chamber at 800 second⁻¹. Platelet accumulation was detected in real time. Where indicated, a second, anti-CD41 antibody (Leo.H4, unlabeled) was used to block α_IIbβ₃ and prevent platelet aggregates from forming. (C-D) Video captures after 4 minutes of platelet accumulation. Fluorescence intensities for Sema4D^(+/+) and Sema4D^(−/−) platelets have been adjusted equally for presentation. (E) Changes in fluorescence intensity over time as platelets accumulated on the collagen-coated surface (mean ± SEM, N = 4). See supplemental videos.