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Ischaemic brain damage induces autoimmune responses, including the production of autoantibodies with potential neuroprotective effects. Platelets share unexplained similarities with neurons, and the formation of anti-platelet antibodies has been documented in neurological disorders. The aim of this study was to investigate the presence of anti-platelet antibodies in the peripheral blood of patients after ischaemic stroke and determine any clinical correlations. Using a flow cytometry-based platelet immunofluorescence method, we detected platelet-reactive antibodies in 15 of 48 (31%) stroke patients and two of 50 (4%) controls ( p < 0.001). Western blotting revealed heterogeneous reactivities with platelet proteins, some of which overlapped with brain proteins. Stroke patients who carried anti-platelet antibodies presented with larger infarcts and more severe neurological dysfunction, which manifested as higher scores on the National Institutes of Health Stroke Scale (NIHSS; p = 0.009), but they had a greater recovery in the NIHSS by the time of hospital discharge (day 7 ± 2) compared with antibody-negative patients ( p = 0.043). Antibodies from stroke sera reacted more strongly with activated platelets ( p = 0.031) and inhibited platelet aggregation by up to 30.1 ± 2.8% ( p < 0.001), suggesting the potential to interfere with thrombus formation. In conclusion, platelet-reactive antibodies can be found in patients soon after ischaemic stroke and correlate with better short-term outcomes, suggesting a potential novel mechanism limiting thrombosis.

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.

We aimed to assess the utility of HitAlert flow cytometry as a diagnostic functional heparin-induced thrombocytopenia (HIT) assay in a tertiary care hospital. The 4Ts score was used to assess pretest probability of HIT in 37 patients. Serum was analysed for HIT antibodies by the flow cytometry HitAlert assay. Results were compared with an antigenic assay, the particle gel immunoassay, PaGIA ID PF4/Hep Ab assay; and two functional assays, the Multiplate whole blood impedance aggregometry assay (WBIA), and the serotonin release assay (SRA). Flow cytometry was positive in 14 out of 37 patients, including zero out of eight, five out of 19 and nine out of 10 in the low, intermediate and high-risk groups by 4Ts score, respectively. Using the SRA as a 'gold standard', flow cytometry has a sensitivity of 81% and a specificity of 100% for the diagnosis of HIT. The other functional assay (WBIA) had similar sensitivity (81%) and specificity (90%) to flow cytometry. In contrast, the PaGIA maintained a high sensitivity of 100% but a specificity of only 20%. The improved specificity of flow cytometry over the antigenic assay was most marked in the 4T intermediate-risk group in which similar results were obtained between all three functional assays. We demonstrate that compared with an immunological assay (PaGIA), flow cytometry can improve the specificity of laboratory diagnosis of HIT without loss of sensitivity using SRA as a standard. Flow cytometry may have a role in the first-line laboratory diagnosis of HIT, especially when combined with an immunological assay such as PaGIA.

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin use. IgG antibodies to complexes of platelet factor 4 (PF4) and heparin trigger the clinical manifestations of HIT. Only a subset of these antibodies will activate platelets and these can only be identified with platelet aggregation (functional) assays. Heparin-induced platelet aggregation (HIPA) and (14)C-serotonin release (SRA) assays for HIT are time-consuming and complex to perform. We have developed a whole blood impedance (WBI) test using the new Multiplate analyser. All samples referred to our laboratory over a 10 month period were screened for heparin-PF4 antibodies by an ELISA method (Zymutest HIA IgG). The 4T's score was used to assess HIT pretest probability. Twenty antibody positive samples were further tested by all three functional assays: light transmission aggregometry (LTA), SRA and WBI. Thirteen out of twenty samples were positive by LTA (10 patients) and 15 by WBI (11 patients). SRA, considered to be the gold standard, was used as a confirmatory test and 11 were found to be positive (10 patients); four discrepant samples were weakly positive by WBI. The prevalence of a positive functional test was strongly correlated with the 4T's clinical risk score, but a small number of low-risk patients had positive functional assays. In this study, the WBI assay detected all SRA positive patients and was positive for two others suggesting greater sensitivity. The rapid and easy to perform assay may be a useful tool for haematology laboratories to detect platelet-activating HIT antibodies., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)