Figure 2.

Analysis of platelet spreading and F-actin organization. Washed platelets (2 × 10⁷ platelets/ml) from WT and HS1^-/- mice were added to cover slips coated with collagen (100 μg/ml), CRP (10 μg/ml) or fibrinogen (100 μg/ml) ± thrombin (1 U/ml) and allowed to settle for 45 or 90 min at 37°C. Spread platelets were fixed in formalin and imaged using DIC. Representative images of platelets at 45 min are shown in (A). Mean platelet area was measured. No significant difference was observed between WT and HS1^-/- platelets on any surface at 45 min (B). Platelets spread on fibrinogen ± thrombin for 45 min (C) were labeled with α-p34 for Arp2/3 complex (top panel), and rhodamine phalloidin for F-actin (middle panel). The merged images are shown in the bottom panel. (D) Spread platelets were also labeled with α-cortactin (top panel) and rhodamine phalloidin (middle panel). Merged images are shown in the bottom panel. Protein extracts from WT and HS1^-/- mice were blotted (E) for cortactin, WASp, Scar/WAVE1, Scar/WAVE 2 and N-WASp. Blots were also probed for tubulin to check loading. Scale bars = 5 μM.