Effect of blood flow on the in vitro properties of rt-PA

We have previously shown that molecular mechanisms for platelet adhesion/aggregation are different under flow vs. static conditions. Currently used in vitro methods for the assessment of the properties/potencies of thrombolytic agents do not take into consideration the effect of blood flow on both the structural properties and the mechanical dislodging of the thrombi. In the present study, we examined the capacity of recombinant tissue-type plasminogen activator (rt-PA) to lyse mural thrombi formed from flowing blood onto fibrin-coated surfaces. Citrated human blood with fluorescentlylabeled platelets was recirculated through a parallel-plate perfusion chamber, one side of which was coated with fibrin, at wall shear rates of 1,000 s^-1 or 500 s^-1. Platelet deposition onto the fibrin was measured in real-time with a photomultipiier tube and video images were taken using a SIT camera. After 15 minutes of perfusion, a bolus of rt-PA was injected into the system and its effect on platelet deposition was observed throughout the 50 minutes of perfusion. At the high wall shear rate, rt-PA reduced the amount of platelets deposited in a dose-dependent manner by inducing fibrinolysis and dislodging the thrombi. The end values of platelet deposition normalized with their respective deposition at the point of injection were: control, 105±10; 0.1 μg rt-PA/ml, 88±6; and 1 μg rt-PA/ml, 48±13 (mean±SD; n=3). At the low wall shear rate, even 1 ug rt-PA/ml showed no significant difference from control values at all time points. The marked differences in the behavior of rt-PA depending on the fluid dynamics suggest that blood flow regulates the balance between thrombotic and thrombolytic events by affecting both the transport of platelets and t-PA to fibrin and the mechanical disruption of the platelet/fibrin thrombi.