New approaches for the assessment of platelet activation status in thrombus under flow condition using confocal microscopy.

The goal of the study was the assessment of heterogeneous platelet activation status in thrombus. In a ferric(III) chloride (FeCl3) thrombosis (intravital) model of C57BL/6 J mice, the area of irreversibly activated (phosphatidylserine (PS)-positive) platelets was assessed after 1-s exposure of a vessel to FeCl3. In a laser-induced thrombosis (intravital) model of GFP mice, the area of the thrombus composed of PS-negative platelets was evaluated. The ratio of the area of PECAM-1 to the area of the thrombus was used as a marker to assess the activity of PS-negative platelets. In the in vitro flow chamber model, the thrombus area (PS-negative and PS-positive platelets) and the platelet activation index (ratio of the area of PS-positive platelets to the area of thrombus) were determined. To assess platelet activation status with these models, acetylsalicylic acid (ASA) and iloprost (Ilo) were used. In the FeCl3 thrombosis, ASA (10 mg/kg, 100 mg/kg) decreased the area of PS-positive platelets. In the laser thrombosis, ASA (10 mg/kg) decreased the thrombus area, but the decrease in platelet activity was evident even at 3 mg/kg by an increased PECAM-1/thrombus ratio. In the flow chamber, ASA (0.02 mg/ml, 0.2 mg/ml) equally decreased the platelet activation index, whereas only at 0.2 mg/ml, it decreased the thrombus area. Ilo (3.6 ng/ml, 36 ng/ml) decreased the thrombus area but at 36 ng/ml increased the platelet activation index. We showed that intravital models and flow chamber provide a detailed assessment of platelet activation status and the mechanism of drug action.