Patient plasma contains an antiplatelet antibody that binds to normal platelets and lacks soluble GPVI. (A) Flow cytometric analysis of patient antiplatelet IgG. Washed normal platelets were incubated with a 1:1 dilution of plasma derived from the patient (P) or a healthy human volunteer (Co) and probed with PE-conjugated goat anti–human IgG Fcγ fragment-specific secondary antibody. Note that patient plasma reactivity is approximately 3 times that of normal plasma. (B) Normal human plasma and patient plasma were incubated overnight with convulxin (CVX)-coated beads. Convulxin-bound proteins were separated on 8% SDS-PAGE, transferred to a PVDF membrane, and probed with cell culture supernatant containing the GPVI-specific mAb 6B12 (anti-GPVI) or media control (Control). Whole platelet lysate (107 human platelets/lane) was added to the first lane of each panel as a control for antibody reactivity and to mark the size of full-length 62-kDa GPVI. Used PVDF membranes were stained with 0.1% Coomassie blue to examine sample loading for normalization. Note that normal human plasma contains a prominent 52-kDa band corresponding to soluble GPVI that is absent from patient plasma.
