Flow cytometric analysis of the prevention of platelet activation by tissue type plasminogen activator and streptokinase

The therapeutic success of plasminogen activators in recanalizing occluded coronary arteries may be impaired by their action on blood platelets, and some conflicting reports claim both platelet activation and inhibition. To elucidate the interactions responsible for inhibition of platelets activation in whole EDTA-anticoagulated blood, preincubated with pharmacological and subpharmacological doses of recombinant tissue type plasminogen activator (rt-PA) or streptokinase (SK), 35 healthy donors were selected for the experiments in which we employed flow cytometry to monitor the exposure of the glycoprotein complex α_IIbβ₃ and PADGEM-140 antigen in surface membranes of platelets. The EDTA-induced increase in the expression of the latter antigen, which is a commonly known marker of the increased platelet activation and release reaction, became greatly depressed when blood cells were incubated with either rt-PA (by up to 60%, P<0.0001) or SK (by 58%, P<0.0001). The effects of the highest protection of platelet activation by rt-PA and SK occurred at their bolus injection doses (2 μg/ml and 600 U/ml blood, respectively). Likewise, both activators significantly reduced the expression of PADGEM-140 antigen (by 12%, P<0.0004 and 16%, P<0.003, respectively) and the platelet membrane integrin α_IIbβ₃ (by up to 34%, P<0.00002 and 9%, P<0.001, respectively). Also, the lowerings in the fractions of platelet aggregates were noted in the presence of the increasing concentrations of rt-PA and SK (P<0.04 and P<0.05). The spontaneous EDTA-induced platelet activation was even augmented in the presence of apyrase (up to 1.0 U/ml) and became significantly reduced by the addition of rt-PA. Furthermore, the addition of apyrase notwithstanding, the plasminogen activators added or not, significantly augmented the fraction of platelet microparticles (rt-PA P<0.04, SK P<0.05) and reduced platelet aggregates (rt-PA P<0.023, SK P<0.04). We conclude that both rt-PA and SK prevent platelet activation and release reaction in a whole blood system. These effects of plasminogen activators seem to occur via plasmin generation rather than the action of plasminogen activators themselves, although the detailed mechanism of plasmin interaction with platelets membrane receptors remains unclear. Our findings suggest that the pharmacological doses of either t-PA or SK are not directly responsible for platelet activation observed as the result of a thrombolytic treatment. Since both plasminogen activators inhibit the spontaneous platelet activation, it seems that the activation associated with the use of plasminogen activators might be rather attributed to thrombin released in the vicinity of superimposed thrombi. Hence, in the effective prevention of reocclusions the strategies directed towards thrombin inhibition, such as the use of hirudins, might provide better and more promising results.