Influence of Vortex Speed on Fresh Versus Stored Platelet Aggregation in the Absence and Presence of Extracellular ATP

Platelets are subjected to vastly differing shear forces under laminar and nonlaminar flow patterns throughout the tortuous cardiovascular system. Different activation pathways appear to be associated with platelet adhesion and aggregation under high shear rates vs. low shear rates. We found that platelets continue to aggregate at very low stirring rates (100 RPM) and low shear forces although significantly less than at high stirring rates (1000 RPM). These conditions may model vortices encountered in vivo, such as downstream of partially occluded blood vessels. The extent of agonist-induced platelet aggregation, at varying stir rates, remained essentially unchanged between 1200 and 600 RPM. This was true for both freshly prepared and stored platelets even though the extent of aggregation was significantly reduced with stored platelets. Agonists used were thrombin, thrombin receptor activating peptide (TRAP), SFLLRNP, the thromboxane A2 mimetic, U46619, plus epinephrine and ADP+epinephrine. At lower stir rates (100–400 RPM), little or no difference in aggregation levels was observed between fresh and stored platelets, depending upon agonist used. This may indicate that old and young platelets, in vivo, would be equally active at vessel walls exposed to blood flowing through a slow vortex at low shear rates. ATP, released from activated platelets, may act as a potent regulator of platelet aggregation within a vortex where the resident time of platelets and bioactive molecules is greater than in laminar flow regions. High levels of extracellular ATP (100 μM) inhibited agonist-induced aggregation of fresh platelets to a greater extent than stored platelets, except with ADP+epinephrine where the converse was observed. Inhibition, in general, appeared to be inversely related to stir rates. Low levels of extracellular ATP (10 nM, 1 μM) generally stimulated agonist-induced aggregations independent of stir rates and to a greater extent with stored platelets than fresh platelets. Unraveling how hemostasis functions within microenvironments may facilitate ways to further regulate this process.