In vivo depletion of GPVI by monovalent F(ab) fragments of JAQ1, JAQ2, and JAQ3.

(A) Platelet counts were monitored for 10 days after injection of the indicated F(ab) fragments or control IgG (all 100 μg). Results are expressed as mean ± SD of 6 mice/group. (B) Flow cytometric analysis of GPVI expression on platelets from mice treated with JAQ1, JAQ2, JAQ3 F(ab) fragments or control IgG. Diluted blood collected at the indicated time points was incubated with FITC-labeled JAQ1 (5 μg/mL) for 15 minutes at RT and analyzed directly. Results are presented as mean ± SD of 6 mice/group. (C) Western blot analysis of GPVI expression on day 3 after injection of anti-GPVI F(ab) fragments. Whole platelet proteins were separated by SDS-PAGE under nonreducing conditions and immunoblotted with HRP-labeled JAQ1 (anti-GPVI) or EDL1 (anti-GPIIIa) followed by ECL. (D) Platelets from F(ab) fragment-treated or control mice were stimulated with CRP (5 μg/mL) and analyzed for P-selectin expression and GPIIb/IIIa activation by flow cytometry. The results shown are representative of 6 mice/group.