Inhibition of NMDA receptor function with an anti-GluN1-S2 antibody impairs human platelet function and thrombosis

GluN1 is a mandatory component of <i>N</i>-methyl-D-aspartate receptors (NMDARs) best known for their roles in the brain, but with increasing evidence for relevance in peripheral tissues, including platelets. Certain anti-GluN1 antibodies reduce brain infarcts in rodent models of ischaemic stroke. There is also evidence that human anti-GluN1 autoantibodies reduce neuronal damage in stroke patients, but the underlying mechanism is unclear. This study investigated whether anti-GluN1-mediated neuroprotection involves inhibition of platelet function. Four commercial anti-GluN1 antibodies were screened for their abilities to inhibit human platelet aggregation. Haematological parameters were examined in rats vaccinated with GluN1. Platelet effects of a mouse monoclonal antibody targeting the glycine-binding region of GluN1 (GluN1-S2) were tested in assays of platelet activation, aggregation and thrombus formation. The epitope of anti-GluN1-S2 was mapped and the mechanism of antibody action modelled using crystal structures of GluN1. Our work found that rats vaccinated with GluN1 had a mildly prolonged bleeding time and carried antibodies targeting mostly GluN1-S2. The monoclonal anti-GluN1-S2 antibody (from BD Biosciences) inhibited activation and aggregation of human platelets in the presence of adrenaline, adenosine diphosphate, collagen, thrombin and a protease-activated receptor 1-activating peptide. When human blood was flowed over collagen-coated surfaces, anti-GluN1-S2 impaired thrombus growth and stability. The epitope of anti-GluN1-S2 was mapped to α-helix H located within the glycine-binding clamshell of GluN1, where the antibody binding was computationally predicted to impair opening of the NMDAR channel. Our results indicate that anti-GluN1-S2 inhibits function of human platelets, including dense granule release and thrombus growth. Findings add to the evidence that platelet NMDARs regulate thrombus formation and suggest a novel mechanism by which anti-GluN1 autoantibodies limit stroke-induced neuronal damage.

GluN1是N-甲基-D-天冬氨酸受体（N-methyl-D-aspartate receptors, NMDARs）的必需亚基，该受体以在大脑中的功能最为人熟知，但越来越多证据表明其在包括血小板在内的外周组织中也具有生理相关性。某些抗GluN1抗体可在缺血性脑卒中的啮齿动物模型中缩小脑梗死体积。另有研究证据显示，人类抗GluN1自身抗体可减轻脑卒中患者的神经元损伤，但其潜在分子机制尚未阐明。本研究旨在探究抗GluN1介导的神经保护作用是否涉及血小板功能的抑制。研究团队筛选了4种商用抗GluN1抗体抑制人血小板聚集的能力，对经GluN1免疫接种的大鼠开展了血液学参数检测，并针对靶向GluN1甘氨酸结合区域的小鼠单克隆抗体（GluN1-S2），通过血小板活化、聚集及血栓形成实验体系评估了其对血小板的生物学效应。本研究对GluN1-S2的抗原表位进行了定位，并借助GluN1的晶体结构对该抗体的作用机制进行了计算机建模。实验结果显示，经GluN1免疫接种的大鼠出血时间轻度延长，且其体内诱导产生的抗体主要靶向GluN1-S2区域。来自BD Biosciences的单克隆抗GluN1-S2抗体，可在肾上腺素、二磷酸腺苷（adenosine diphosphate, ADP）、胶原蛋白、凝血酶及蛋白酶激活受体1激活肽存在的条件下，有效抑制人血小板的活化与聚集。当人体血液流经胶原蛋白包被的表面时，抗GluN1-S2可显著损害血栓的生长过程与结构稳定性。抗GluN1-S2的表位被定位至GluN1甘氨酸结合贝壳状结构域内的α螺旋H区域，经计算机模拟预测，该抗体的结合会阻碍NMDAR通道的开放。本研究结果表明，抗GluN1-S2可抑制人血小板的多项功能，包括致密颗粒释放与血栓生长。该研究进一步佐证了血小板NMDAR可调控血栓形成，并揭示了抗GluN1自身抗体减轻脑卒中诱导神经元损伤的全新潜在机制。