Adhesion and spreading of human and mouse platelets on laminin. (A) Washed murine platelets (3 × 10⁷/mL; left column) or washed human platelets (2 × 10⁷/mL; right column) were pretreated with 1 mM GRGDS peptide in the presence (bottom row) or absence (top row) of 20 μg/mL anti–integrin α₆β₁ antibody. Coverslips were coated with 50 μg/mL laminin from human placenta overnight at 4°C and then blocked with 1% fatty acid–free BSA for 2 hours at 4°C. After rinsing with Tyrode buffer, platelets were seeded on the coverslips and incubated at room temperature. After 30 minutes of incubation, unbound platelets were removed and adherent platelets were fixed by 3% paraformaldehyde, permeabilized with 0.3% Triton X-100, and F-actin stained using TRITC-labeled phalloidin. After F-actin staining, platelets were visualized using confocal fluorescent microscopy. (B) Murine platelets, washed as described in “Materials and methods” (3 × 10⁷/mL), were pretreated with or without 20 μg/mL anti–integrin α₂-blocking antibody (middle column) or anti–integrin α₆ antibody (right column) for 10 minutes and then seeded on the BSA-(top row), 50 μg/mL laminin-(middle row), or 50 μg/mL collagen-coated (bottom row) surfaces. Adherent platelets were fixed and visualized as described in “Materials and methods.” (C) Changes in morphology of adherent washed murine platelets (3 × 10⁷/mL; left column) or washed human platelets (2 × 10⁷/mL; right column) were visualized by time-lapse real-time imaging using videomicroscopy with DIC optics. The results are representative of 3 to 12 experiments. LM indicates laminin; col, collagen.