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 1

Characterization of YA-Abs over 15 years.

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Characterization of YA-Abs over 15 years. (A) YA’s serially diluted plas...
(A) YA’s serially diluted plasma stored over 15 years or control solutions containing known amounts of a human/mouse chimera anti-GPVI mAb were incubated with biotin-labeled hGPVI-Fc on streptavidin-coated plates. After washing, the bound Abs were detected using peroxidase-labeled anti-kappa and -lambda chain Abs and subsequent colorimetric reaction. The content of anti-GPVI auto-Abs in the plasma was expressed as an equivalent content of a human/mouse chimera anti-GPVI mAb bound to hGPVI-Fc. Each bar represents the mean of duplicate determinations. (B) Both YA-Abs-88 and -03 were purified from the patient’s plasma stored in 1988 and 2003, respectively, and quantified as described in Methods. Serially diluted YA-Abs-88 (triangles), YA-Abs-03 (circles), mF1201 (diamonds), mF1232 (squares), or mouse IgG (asterisks) as the competing Ab was applied to hGPVI-Fc–immobilized plates followed by the incubation with the peroxidase-labeled mF1201 (left panel) or mF1232 (right panel). After washing, the bound labeled Abs were detected by colorimetric reaction. Data are expressed as the percent of total binding of each peroxidase-labeled mAb without the competitor and are the mean of triplicate determinations.