Real-time measurement of lysis of mural platelet deposits by fibrinolytic agents under arterial flow

An in vitro whole blood reperfusion model was employed to quantify: (a) initial rates of lysis of mural platelet deposits from flowing blood onto fibrin-coated surfaces and (b) plasmin-mediated consumption of plasma plasminogen and fibrinogen, by recombinant tissue-type plasminogen activator (rt-PA) and two t-PA variants. KHRR 296-299 AAAA (K-tPA) and T103N, N117Q, KHRR 296-299 AAAA (TNK-tPA), at wall shear rates of either 500 or 1000 s^-1. K- and TNK-tPA are more fibrin-specific than rt-PA, and are also resistant to inactivation by plasminogen activator inhibitor-1 (PAI-1). At 500s^-1, no agent showed significant lysis of mural platelet deposits on fibrin, even at concentrations as high as 10 μg/ml of blood. At 1000 s^-1, each agent demonstrated a dose-dependent lysis of mural platelet deposits, due to plasmin-mediated lysis of the fibrin substrate (fibrinolysis). The local concentration of thrombolytic agents close to the fibrin-coated surface is probably higher than the concentration of released PAI-1 from the adherent and activated platelets. Hence, the initial rates of lysis achieved by K- and TNK-tPA were not significantly different from that by rt-PA, when each agent was tested at either 1 or 10 μg/ml of blood. However, TNK-tPA, at 1 μg/ml. caused the most extensive lysis at the end of the 50 min reperfusion period (50% vs 29% and 17% by rt-PA and K-tPA. respectively). K- and TNK-tPA, at concentrations as high as 10 μg/ml of blood, caused plasminogen activation that was controlled by the natural plasmin inhibitors, and, thus, no proteolytic degradation of plasma fibrinogen (fibrinogenolysis). On the contrary, rt-PA at 1 μg/ml revealed slight fibrinogenolysis that became extensive at 10 μg/ml. This study demonstrates the potential use of an in vitro model, that mimics the in vivo hemodynamic environment, in evaluating the performance of thrombolytic agents. The data suggest that: (a) adequate flow must accompany fibrinolysis for successful embolization, and (b) the TNK variant may lyse annular thrombi after recanalization, at least as efficiently as rt-PA does, while causing lesser defect of systemic hemostasis.