title: Inhibition of NMDA receptor function with an anti-GluN1-S2 antibody impairs human platelet function and thrombosis.
abstract: GluN1 is a mandatory component of N-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.
authors.affiliation: a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.
authors.forename: Taryn N
authors.initials: TN
authors.lastname: Green
authors.affiliation: b Australian Centre for Blood Diseases, Monash University , Melbourne , Australia.
authors.forename: Justin R
authors.identifier: http://orcid.org/0000-0001-7554-6727
authors.initials: JR
authors.lastname: Hamilton
authors.affiliation: c Department of Haematology and Transfusion Medicine , Royal North Shore Hospital , Sydney , Australia.|d Northern Blood Research Centre, Kolling Institute, University of Sydney , Sydney , Australia.
authors.forename: Marie-Christine
authors.identifier: http://orcid.org/0000-0003-4795-1746
authors.initials: MC
authors.lastname: Morel-Kopp
authors.affiliation: b Australian Centre for Blood Diseases, Monash University , Melbourne , Australia.
authors.forename: Zhaohua
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.|e Department of Pharmacology and Clinical Pharmacology , University of Auckland , Auckland , New Zealand.
authors.forename: Ting-Yu T
authors.initials: TT
authors.lastname: Chen
authors.affiliation: a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.
authors.forename: James I
authors.identifier: http://orcid.org/0000-0002-3988-2659
authors.initials: JI
authors.lastname: Hearn
authors.affiliation: a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.
authors.forename: Peng P
authors.initials: PP
authors.lastname: Sun
authors.affiliation: f Auckland Cancer Society Research Centre, University of Auckland , Auckland , New Zealand.|g Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland , Auckland , New Zealand.
authors.forename: Jack U
authors.initials: JU
authors.lastname: Flanagan
authors.affiliation: e Department of Pharmacology and Clinical Pharmacology , University of Auckland , Auckland , New Zealand.
authors.forename: Deborah
authors.identifier: http://orcid.org/0000-0003-1085-2824
authors.initials: D
authors.lastname: Young
authors.affiliation: h Department of Neurology , Auckland City Hospital , Auckland , New Zealand.|i Centre for Brain Research , University of Auckland , Auckland , New Zealand.
authors.forename: P Alan
authors.identifier: http://orcid.org/0000-0003-2469-9023
authors.initials: PA
authors.lastname: Barber
authors.affiliation: a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.|j Departments of Molecular Virology, Immunology and Medical Genetics , Neuroscience and Neurological Surgery, Ohio State University , Columbus , OH , USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: During
authors.affiliation: c Department of Haematology and Transfusion Medicine , Royal North Shore Hospital , Sydney , Australia.|d Northern Blood Research Centre, Kolling Institute, University of Sydney , Sydney , Australia.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Ward
authors.affiliation: a Department of Molecular Medicine and Pathology , University of Auckland , Auckland , New Zealand.|k LabPlus Haematology , Auckland City Hospital , Auckland , New Zealand.
authors.forename: Maggie L
authors.identifier: http://orcid.org/0000-0001-8378-8048
authors.initials: ML
authors.lastname: Kalev-Zylinska
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D006801:Humans; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors*; D013927:Thrombosis / Q000235:genetics*
chemical_list: D001323:Autoantibodies; D016194:Receptors, N-Methyl-D-Aspartate
keywords: Calcium; glutamate; ion channel; platelet inhibitor; stroke