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 2

mF1201, but not mF1232, induces both platelet aggregation and shedding of surface GPVI, while mF1232 inhibits collagen-induced, but not CRP- or ADP-induced, platelet aggregation.

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mF1201, but not mF1232, induces both platelet aggregation and shedding o...
(A) Platelet aggregation was monitored after the addition of the indicated concentrations of mF1201 or mF1232 into human PRP. (B) After human PRP was incubated without (none) or with the indicated concentrations of CRP, mF1232, mF1201, or mIgG for 30 minutes at 37°C, the platelets were fixed and stained with PE-labeled anti-human CD62P Ab for flow cytometry analysis. Data represent mean ± SEM of triplicate determinations. MFI, mean fluorescence intensity. (C) Human PRP was preincubated with the indicated concentrations of mF1232 for 5 minutes at 37°C and stimulated with 1 μg/ml collagen, 4 μg/ml CRP, or 5 μM ADP as indicated by arrows. Traces are representatives of 2 or 3 separate experiments from different donors. (D) Washed human platelets were treated without (control) or with 0.15 μg/ml convulxin (Cvx), 10 μg/ml mF1232, or 10 μg/ml mF1201 for 60 minutes at 37°C, and centrifuged to isolate platelet pellets from supernatants. Platelet pellets (cell lysate) and supernatants were analyzed by western blotting using rabbit anti-GPVI polyclonal Abs. Data are representatives of 3 separate experiments from different donors.