The actin cytoskeleton mediates spatial regulation of platelet spreading and α-granule secretion in a Rac1- and Rho-dependent manner. (A) Latrunculin A inhibits platelet (red, cell membrane stain) spreading and P-selectin expression (green) beyond the microdot patterns, whereas nocodazole has no effect. (B) Phalloidin binding (red) shows thick bundles of filamentous actin (f-actin) at the geometric boundaries of the micropatterns and at platelet-platelet interconnections. The thick f-actin “rings” are observed on smaller microdots (4 µm), but f-actin is dispersed over the entire microdot region as microdot diameter increases (5 µm). P-selectin binding is mutually excluded from these thick f-actin bundles but colocalize with thinner strands of filopodial f-actin (arrow), where platelets extend beyond the microdot boundaries. (C) Platelets (red) adhere and spread onto 5-µm fibrinogen microdot arrays (blue). Platelets treated with Y27632 exhibit more spreading beyond the microdot boundaries and formed more platelet connections, whereas platelets treated with ML-7 and NSC23766 exhibit less spreading beyond the microdot boundaries and decreased platelet connections. F-actin bundles (green) are observed at platelet-platelet connections in the control condition and Y27632-treated platelets, but not in ML-7– and NSC23766-treated platelets, although actin stress fiber expression within the microdot boundaries appears intact. (D) Platelet-platelet connections on collagen (left) and fibrinogen (right) microdot arrays (2700-dot arrays per condition) were quantified and normalized by vehicle control. All conditions are significantly different compared with the controls (P < .0005) using the Mann-Whitney U test. All samples are permeabilized upon staining. The scale bars = 5 µm; error bars indicate SE.