Brief exposure to high shear triggers significant GPVI shedding over time. Washed platelets (5 × 10⁸/mL) from a healthy donor were resuspended in (A) Tyrode buffer alone or (B) containing 50μM DM-BAPTA or 1 U/mL apyrase and then subjected to a shear rate of 10 000 seconds⁻¹ for 1 minute. Samples were then left at room temperature for the indicated time before addition of EDTA followed by centrifugation and lysis of platelet pellets. Platelet lysates were analyzed by SDS-PAGE and Western blot using 1 μg/mL anti-GPVI cytoplasmic tail antibody. Data are representative of 3 independent experiments with different donors. (C) PRP from healthy donors was subjected to the indicated level of shear for 1 minute before addition of EDTA (dark bars) or incubated at room temperature for 30 minutes followed by addition of EDTA (light bars) as shown in the pictogram. All samples were then processed to isolate plasma and analyzed for sGPVI levels by ELISA. Data are representative of 4 independent experiments with different donors. (D) In the presence of an inhibitor of αIIbβ3, washed platelets in Tyrode buffer were exposed to 10 000-second⁻¹ shear for 2 minutes and then mixed with equal amounts of buffer or unsheared platelets for up to 30 minutes (left panel), or PRP was subjected to 7500 seconds⁻¹ for 1 minute and then mixed with equal amounts of buffer or unsheared PRP for up to 30 minutes (right panel). Aliquots of supernatant and PRP were isolated for measurement of sGPVI by ELISA (right panel) and platelet surface levels of GPVI by flow cytometry (left panel), respectively.