Platelet adhesion to fibrin(ogen) in an in vitro (re)perfusion model: Effects of rt-PA, c7E3 Fab, and the GP IIb/IIIa receptor Pl(A) polymorphism.

摘要

The use of recombinant tissue-type plasminogen activator (rt-PA) is well established in treating acute myocardial infarction to dissolve blood clots occluding coronary arteries and restore blood flow to the heart. The problems associated with this therapy include resistance to lysis in spite of accelerated doses and early treatment, reocclusion of the vessel after successful reperfusion, and cerebral hemorrhage.; The presence of platelets in a thrombus increases its lytic resistance. Activated platelets bind fibrin in the thrombus and secrete plasminogen activator inhibitor-1 (PAI-1) and α₂-antiplasmin that inactivate rt-PA and plasmin, respectively. Platelets bind fibrin via their α_IIb β₃ (GP IIb/IIIa) receptors and cause clot retraction, which extrudes loosely bound plasminogen and t-PA from the thrombus and attenuates the diffusion of fibrinolytic proteins through the thrombus.; Flow regulates the delivery of cells/species in the thrombolytic process. It determines how rt-PA penetrates the thrombus. We developed an in vitro recirculation model that mimicked blood flow in an injured vessel to quantify platelet adhesion and fibrinolysis by rt-PA in “real-time”. Using this model we characterized the effects of rt-PA on mural thrombi formed onto fibrin from whole blood flowing at different wall shear rates. rt-PA caused a dose-dependent lysis of the fibrin substrates at 1,000 s−1 but not at 500 s−1.; We used the model to characterize the effect of c7E3 Fab (abciximab, ReoPro™) on platelet adhesion to fibrin and showed that c7E3 Fab inhibited this process dose-dependently at 100 s−1 and 500 s −1 with inhibition being higher at 500 s−1. Further, we tested the hypothesis that c7E3 Fab enhances the fibrinolytic efficiency of rt-PA and attempted to delineate the mode of action. Our results confirmed that c7E3 Fab does enhance the efficacy of rt-PA by limiting platelet participation in forming platelet-rich highly resistant residual thrombi.; Finally, we used a version of the model to study the effect of the Pl A2 polymorphism of GP IIb/IIIa on the adhesion to immobilized fibrinogen of CHO cells homozygous for PlA1 or PlA2. We tested the hypothesis that PlA2 represents an increased risk for vascular thrombosis. Results showed that PlA2 cells adhered more to fibrinogen than did PlA1 cells at 25, 50 and 100 s −1 implying that PlA2 platelets are hyperactive and maybe a risk factor for myocardial infarction.; Having measured the efficacy of rt-PA to lyse mural thrombi under flow conditions, either by itself, or in combination with c7E3 Fab, we can use our data to provide guidelines for a better treatment of thromboembolic diseases. Having compared the adhesiveness of CHO cells expressing the platelet GP IIb/IIIa receptor, with or without the PlA2 polymorphism, we can propose how to adjust the fibrinolytic therapy according to each patient's genotype.