Figure 6. In vitro internalization and endocytosis of GPVI-specific Abs with or without GPVI immunodepletion under various experimental conditions.

Cynomolgus PRP was incubated with 10 μg/ml CypHer5E-labeled (Cy5E-labeled) Ab in vitro under various experimental conditions as follows: 60 minutes incubation with cF1232 (black bars) or human IgG (white bars) at 37°C or 4°C (A), incubation with cF1232 (filled circles) or human IgG (open circles) for up to 120 minutes at 37°C in the presence of 3 μM PGE₁ (B), 60 minutes incubation with mF1232, mF1201, or mouse IgG (mIgG) at 37°C in the presence of 3 μM PGE₁ (C). Washed cynomolgus platelets were treated with the indicated Abs in the absence or presence of 3 μM PGE₁ for 60 minutes at 37°C (D). (A–D) The fluorescence in platelets (left panels) and surface GPVI expression (right panels) were analyzed by flow cytometry. Surface GPVI expression before incubation was normalized to 100. (E) Washed cynomolgus platelets were treated with 0.15 μg/ml convulxin, 10 μg/ml mF1232, 10 μg/ml mF1232, or 10 μg/ml mouse IgG for 60 minutes at 37°C in the absence or presence of PGE₁ and sedimented to separate supernatants. The supernatants were analyzed by western blotting with rabbit anti-human GPVI polyclonal Ab. The band density corresponding to soluble GPVI was quantified by a Typhoon Scanner 9410, and the value for convulxin treatment in the absence of PGE₁ was normalized to 100. All results represent mean ± SEM of 3 (A, D, and E) or 4 (B and C) separate experiments. *P < 0.05, **P < 0.01 versus control groups.