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

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

Platelets (2017-03-10)
Taryn N Green, Justin R Hamilton, Marie-Christine Morel-Kopp, Zhaohua Zheng, Ting-Yu T Chen, James I Hearn, Peng P Sun, Jack U Flanagan, Deborah Young, P Alan Barber, Matthew J During, Christopher M Ward, Maggie L Kalev-Zylinska
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ser-Phe-Leu-Leu-Arg-Asn-amide trifluoroacetate salt, ≥98% (HPLC), lyophilized powder
Sigma-Aldrich
Anti-NMDAR1 Antibody, clone 54.1, clone 54.1, Chemicon®, from mouse
Sigma-Aldrich
Anti-NMDAR1 Antibody, (all splice variants), clone R1JHL, clone R1JHL, Chemicon®, from mouse