Clinical studies have investigated the combination of glycoprotein (GP) IIb/IIIa inhibitors and thrombolytic agents for acute myocardial infarction. However, thrombolytic agents alone may possess direct antiplatelet properties that could affect reperfusion. Blood from 11 patients with coronary disease and five healthy subjects was incubated for 30 min with tenecteplase (4, 12, and 24 μg/ml), alteplase (1, 4, and 10 μg/ml), reteplase (1, 5, and 10 μg/ml) or control buffer. Platelet aggregation induced by 1, 20 and 50 μmol/l adenosine diphosphate (ADP), the stimulated expression of GP IIb/IIIa and P-selectin, and plasma fibrinogen levels were determined. Platelet aggregation in patients was inhibited by medium and high concentrations of alteplase when induced by 1 μmol/l ADP [1.6 ± 0.5%, P = 0.001 and 0.9 ± 0.2%, P = 0.002 versus 8.3 ± 1.6% (control)] and 20 μmol/l ADP [46.9 ± 3.9%, P = 0.001 and 46.2 ± 4.8%, P = 0.001 versus 65.7 ± 2.7% (control)]. High concentration tenecteplase was associated with lower aggregation by 20 μmol/l ADP (58 ± 2.1% versus control, P = 0.033). There were no changes in GP IIb/IIIa activation or P-selectin expression in patients or healthy subjects. Platelet aggregation (1 μmol/l ADP) in healthy subjects was inhibited only by high doses of alteplase (P = 0.001). Plasma fibrinogen levels were significantly decreased after treatment with reteplase at 1 μg/ml (1.53 ± 0.21 versus 2.65 ± 0.31, P = .009) and 5 μg/ml (1.55 ± 0.16 versus 2.65 ± 0.31, P = .005). Alteplase inhibits platelet aggregation more than tenecteplase and reteplase. The attenuation of platelet aggregation by alteplase is dissociated from the expression of activated GP IIb/IIIa and P-selectin, and by fibrinogen degradation. These results suggest that alteplase exerts its antiplatelet effect independent of GP IIb/IIIa and P-selectin expressions and fibrinogen degradation. These findings may be directly relevant to the effect of alteplase on reperfusion and to future studies using combined platelet inhibitors and thrombolytic therapy.