(749f) The Role of the Flux of Platelet Agonists on Clot Formation and Stability

Authors: 
Neeves, K. B., University of Pennsylvania
Diamond, S. L., University of Pennsylvania

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.