JAQ1, JAQ2, and JAQ3 bind to different epitopes on mouse GPVI.

(A) Whole platelet proteins were separated by SDS-PAGE under nonreducing conditions and immunoblotted with the indicated antibodies. Bound mAb was detected by HRP-labeled rabbit antirat Ig and ECL (upper, WB). Surface biotinylated platelets were lysed and immunoprecipitation was performed with the indicated antibodies. Precipitated proteins were detected with streptavidin-HRP and ECL (lower, IP). (B) Heparinized prp was incubated with JAQ1, JAQ2, JAQ3, or irrelevant IgG2a (all 20 μg/mL) followed by addition of polyclonal rabbit antirat immunoglobulin antibody (10 μg/mL) and light transmission was recorded on a Fibrintimer 4 channel aggregometer. (C) Platelets were preincubated with the indicated antibodies (20 μg/mL, 30 minutes) and washed; binding of FITC-labeled JAQ1, JAQ2, or JAQ3 was detected by flow cytometry.