Gp96-FITC binding to human platelets can be specifically competed by unlabeled Gp96 and by antibodies against CD36 and CD91.

Platelets were fixed and incubated with 50 μg/mL Gp96-FITC after preincubation with competitor for 30 minutes on ice. Fluorescence of stained cells was analyzed by flow cytometry. (A) Different amounts of unlabeled Gp96 or ovalbumin were used for competition on thrombin-activated cells. Data shown are representative for 3 independent experiments. (B) Nonstimulated and thrombin-activated platelets were stained using a 10-fold excess of the indicated competitors. Fluorescence intensity without competitor was set as 100%. Means of triplicates are shown, and error bars represent SEM. (C) Unlabeled Gp96 (500 μg/mL), isotypic controls, and monoclonal antibodies against CD36 (CB38, SM0; both IgM isotype) or against the 85-kd subunit of CD91 (IgG1 isotype) were used for competition before staining of thrombin-activated platelets with 50 μg/mL Gp96-FITC (concentration of competing antibodies: 50 μg/mL). Autofluorescence is indicated by the dotted line, and uncompeted and competed staining are indicated by the solid line and the filled histogram. Experiments were performed in triplicate.