Platelet activation and aggregation, and thrombus formation on collagen under blood flow. (A) Washed platelets from WT (black line) or PN-1–deficient mice (gray line) were activated by increasing doses of thrombin, and P-selectin was measured by flow cytometry. (B-C) Washed platelets from WT mice (black line) or PN-1–deficient mice (gray line) were activated by 0.1nM thrombin. Time courses of (B) P-selectin and (C) phosphatidylserine exposure on platelets were measured by flow cytometry using FITC-labeled rat anti–mouse P-selectin IgG and FITC–annexin V. (D) Washed platelets from WT mice (black line) or PN-1–deficient mice (gray line) were activated by different doses of thrombin, and platelet aggregation was monitored. Data are from 1 experiment representative of at least 3 separate experiments. (E) Rhodamine 6G–labeled mouse platelets in whole blood were perfused in procoagulant conditions (2pM TF and 5mM Ca²⁺) or in anticoagulant conditions (80μM PPACK), at 1000/s over a collagen surface. After 2 minutes of perfusion, the formation of thrombi was observed at original magnification ×20 under an epifluorescent microscope (Nikon Eclipse TE2000-U; Champigny sur Marne, France), coupled to Metamorph 7.0r1 software (Universal Imaging Corporation). The mean percentage of the area covered by platelets ± SD of 3 independent experiments was calculated and expressed as the mean percentage of the total area covered by thrombi. Data are presented as mean ± SD. *Significant difference (P < .05) versus WT. Bars represent 100 μm.