A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor γ-chain complex
Hiroshi Takayama, … , Michiru Kurihara, Shoji Furusako
Hiroshi Takayama, … , Michiru Kurihara, Shoji Furusako
Published April 1, 2008
Citation Information: J Clin Invest. 2008;118(5):1785-1795. https://doi.org/10.1172/JCI32513.
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Research Article Hematology

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor γ-chain complex

Abstract

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor γ-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI–specific mAbs with characteristics similar to YA-Abs, we generated human GPVI–specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI–specific mAb as what we believe to be a novel antiplatelet therapy.

Authors

Hiroshi Takayama, Yoshitaka Hosaka, Kazuyuki Nakayama, Kamon Shirakawa, Katsuki Naitoh, Tomokazu Matsusue, Mikihiko Shinozaki, Motoyasu Honda, Yukiko Yatagai, Tetsushi Kawahara, Jiro Hirose, Tooru Yokoyama, Michiru Kurihara, Shoji Furusako

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Figure 6

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

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In vitro internalization and endocytosis of GPVI-specific Abs with or wi...
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.