The mechanisms that underlie reocclusion during thrombolytic therapy have not yet been clarified. The purpose of this study was to investigate the activating effects of tissue-type plasminogen activator and urokinase and the inhibitory effects of acetylsalicylic acid by measuring platelet surface P-selectin as a marker of platelet activation. After addition of urokinase (final concentration 192 U/ml, 1920 U/ml, or 19,200 U/ml) or tissue-type plasminogen activator (final concentration 120 U/ml, 1200 U/ml, or 12,000 U/ml) to platelet-rich plasma from 12 healthy persons, platelet surface P-selectin expression was measured by means of flow cytometry with an anti-CD62 monoclonal antibody. The presence of urokinase and tissue-type plasminogen activator increased platelet surface P-selectin expression in a concentration-dependent manner. In the next step, either 160 mg/day (n = 6) or 660 mg/day (n = 6) acetylsalicylic acid was administered to the 12 healthy persons, and venous blood samples were collected after 7 days of treatment. Platelet surface P-selectin expression was measured with the method used earlier and after addition of tissue-type plasminogen activator or urokinase. Although the effect of acetylsalicylic acid at 160 mg/day on P-selectin expression was minimal, a dose of 660 mg/day suppressed platelet P-selectin expression and inhibited the platelet activating effects of tissue-type plasminogen activator and urokinase in a statistically significant way. Platelets were activated by tissue-type plasminogen activator or urokinase, and this platelet activation was suppressed with administration of acetylsalicylic acid at 660 mg/day.