Flow cytometric and Western blot analysis of the GPVI/FcRγ-chain complex. (A) Washed platelets from the patient or from a healthy human donor were incubated for 60 minutes with monoclonal Abs specific for GPIb, GPIIb-IIIa, PECAM-1, integrin α2β1, and GPVI. The median fluorescence intensity is shown near each peak for the patient (P) and a healthy control sample (Co). As shown, the patient expresses 10% to 15% of normal levels of GPVI, whereas other platelet membrane glycoproteins examined are expressed at normal levels. PE-conjugated goat anti–mouse IgG in the presence of an isotype-matched mouse mAb control IgG gave median fluorescence values of approximately 3 arbitrary units (not shown). (B-C) Whole-cell lysates from the patient and a healthy volunteer were subjected to Western blot analysis using the indicated antibodies. Lysates were prepared from either washed platelets (B) or mononuclear cells (C) isolated using Polymorphprep (Axis-Shield, Oslo, Norway). Note that only trace amounts of GPVI and the FcRγ chain are present in patient platelet detergent lysates but that the FcRγ chain is expressed at normal levels in patient mononuclear cells.
