Shear-induced GPVI shedding does not require platelet aggregation, intracellular signaling, or platelet activation. Citrated PRP was either untreated or preincubated with TS buffer alone or containing 1mM RGD peptide, 1mM control peptide, 10 μg/mL AK2, 10 μg/mL SZ2, 10μM PP1, 10μM PP2, 100 nM wortmannin, or 10μM LY294002 as indicated and then subjected to a shear rate of 10 000 seconds⁻¹ for 5 minutes. Aggregation was measured in samples by obtaining platelet particle count (A-C), and then plasma was generated and assayed for sGPVI by ELISA in triplicate (D-F). Panels A and D, B and E, and C and F contain data from 3 different donors that are representative of 3 independent experiments.