- 12:35 PM

The Role of the Flux of Platelet Agonists on Clot Formation and Stability

Keith B. Neeves and Scott L. Diamond. Chemical and Biomolecular Engineering, University of Pennsylvania, 3340 Smith Walk, 1150 Vagelos Laboratories, Philadelphia, PA 19104

The flux of platelet agonists into flowing blood is a critical event in thrombosis and hemostasis. However, few in vitro method exist for examining and controlling the role of platelet agonists on clot formation and stability under hemodynamic conditions. In this work, 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). As a proof-of-concept, ADP was introduced into whole blood flowing at 250 sec-1 at three fluxes (1.5, 2.4, and 4.4 x 10-18 mol mm-2 sec-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 mm-2 sec-1), we observed little to no aggregation. At the higher fluxes, we observed monolayer (2.4 x 10-18 mol mm-2 sec-1) and multilayer (4.4 x 10-18 mol mm-2 sec-1) aggregates of platelets and found that the platelet density within an aggregate increased with increasing ADP flux.  This is the first demonstration of the threshold ADP flux required to trigger intense platelet deposition under flow.  Preliminary results on the deposition of fibrin under flow show a similar dependence on thrombin flux.  We expect this device to be a useful tool in unraveling the role of platelet agonists on clot formation and stability.