Pairwise platelet activation in flow cytometry

Platelet activation during thrombosis is vital to minimizing blood loss through formation of the platelet aggregate and enhancement of the coagulation cascade. This process is controlled by many signals that vary both spatially and temporally within the growing thrombus and surrounding lumen. While the platelet response to each signal is usually studied individually, we have utilized high throughput flow cytometry to analyze the effects of pairs of signals on integrin activation, degranulation, and phosphatidylserine exposure. In examining all pairwise combinations of 6 major platelet agonists (convulxin, ADP, U466219, SFLLRN, AYPGKF, and PGE2), we developed a method for measuring three donor-specific markers of platelet activation. Additionally, a similar method testing the effects of anti-platelet agents was developed that produces donor-specific barcodes of data that could be integrated into models of coagulation. This also led to the discovery of a novel V241G mutation in the thromboxane receptor. In addition to the effect of a second agonist, we found that whether the agonists are added simultaneously or sequentially, as well as the order of addition and delay between additions, can have dramatic effects on platelet activation. In general, platelet response is lower for sequential addition than simultaneous, and addition of high concentrations of thrombin prevents subsequent calcium signaling, but has no effect on extracellular measures of activation. Finally, we established that GPVI signaling is important for production of procoagulant platelets, but its expression does not determine which platelets expose phosphatidylserine. Also, we found that reticulated platelets are less likely to become these procoagulant coated platelets, contrary to previous reports, while aged platelets are more procoagulant