Abstract
The flux of platelet agonists into flowing blood is a critical event in thrombosis and hemostasis. However, few in vitro methods exist for examining and controlling the role of platelet agonists on clot formation and stability under hemodynamic conditions. In this paper, we describe a membrane-based method for introducing a solute into flowing blood at a defined flux. The device consisted of a track-etched polycarbonate membrane reversibly sealed between two microfluidic channels; one channel contained blood flowing at a physiologically relevant shear rate, and the other channel contained the agonist(s). An analytical model described the solute flux as a function of the membrane permeability and transmembrane pressure. The model was validated using luciferase as a model solute for transmembrane pressures of 50-400 Pa. As a proof-of-concept, the weak platelet agonist ADP was introduced into whole blood flowing at 250 s(-1) at three fluxes (1.5, 2.4, and 4.4 x 10(-18) mol microm(-2) s(-1)). Platelet aggregation was monitored by fluorescence microscopy during the experiment and the morphology of aggregates was determined by post hoc confocal and electron microscopy. At the lowest flux (1.5 x 10(-18) mol microm(-2) s(-1)), we observed little to no aggregation. At the higher fluxes, we observed monolayer (2.4 x 10(-18) mol microm(-2) s(-1)) and multilayer (4.4 x 10(-18) mol microm(-2) s(-1)) aggregates of platelets and found that the platelet density within an aggregate increased with increasing ADP flux. We expect this device to be a useful tool in unraveling the role of platelet agonists on clot formation and stability.