Shear-induced shedding of GPVI occurs in the absence of VWF. Citrated PRP or washed platelets were prepared from blood isolated from a patient with VWD type 3 or a healthy donor. (A) To assess levels of VWF and GPIbα in each sample, equivalent concentrations of washed platelets were lysed in nonreducing sample loading buffer and subjected to SDS-PAGE and Western blot using 1 μg/mL anti-VWF mAb, 6G1 (top panel) or 1 μg/mL anti-glycocalicin antibody (bottom panel). (B) To assess VWF function, citrated PRP from the same patient or a healthy donor was subjected to ristocetin-induced platelet aggregation using light transmission aggregometry. Arrow indicates the time at which 1.5 mg/mL ristocetin was added. Absent ristocetin-induced aggregation in the patient sample was rescued by inclusion of 10 μg/mL purified VWF before addition of ristocetin. (C-D) Citrated PRP from the patient or a healthy donor was subjected to 10 000-second⁻¹ shear for 5 minutes, and samples were then analyzed for (C) platelet aggregation by particle counting or (D) sGPVI levels by ELISA. (E) VWD type 3 washed platelets were either left untreated (NT) or treated for 15 minutes with 5mM NEM, or exposed to 10 000 seconds⁻¹ shear for 5 minutes in the presence of TS buffer alone, or containing 10 μg/mL AK2, 10 μg/mL purified VWF, or 100μM GM6001. Samples were then lysed in nonreducing sample loading buffer and subjected to SDS-PAGE and Western blot using 1 μg/mL anti-GPVI cytoplasmic tail antibody. A vertical line indicates a repositioned lane.