Figure 5.
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.

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.

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