N-methyl-D-aspartate receptors amplify activation and aggregation of human platelets

Background: Glutamate is stored in platelet dense granules and large amounts (>400 mu M) are released during thrombus formation. N-methyl-D-aspartate glutamate receptors (NMDARs) have been shown in platelets but their roles are unclear. Materials and methods: Platelet activation indices (CD62P expression and PAC-1 binding) and platelet aggregation were tested in the presence of well-characterized agonists (glutamate, NMDA, glycine) and antagonists (MK-801, memantine, AP5) of neuronal NMDARs. Expression of NMDAR subunits in platelets was determined. Results: NMDAR agonists facilitated and NMDAR antagonists inhibited platelet activation and aggregation. Low concentrations (100 mu M) of MK-801 and memantine reduced adrenaline-induced CD62P expression by 47 +/- 5 and 42 +/- 3%, respectively, and inhibited adrenaline-induced platelet aggregation by 17 +/- 6 and 25 +/- 5%, respectively (P < 0.05). AP5 caused less inhibition of platelet function, requiring concentrations of at least 250 mu M to inhibit aggregation. NMDAR agonists did not aggregate platelets by themselves but enhanced aggregation initiated by low concentrations of ADP. Exogenous glutamate helped reverse inhibition of platelet aggregation by riluzole (inhibitor of glutamate release). Compared with seven possible NMDAR subunits in neurons, human platelets contained four: GluN1, GluN2A, GluN2D and GluN3A, a combination rarely seen in neurons. The presence of NMDAR transcripts in platelets implied platelet ability to regulate NMDAR expression presumably 'on demand'. Flow cytometry and electron microscopy demonstrated that in non-activated platelets, NMDAR subunits were contained inside platelets but relocated onto platelet blebs, filopodia and microparticles after platelet activation. Conclusions: Our results support an active role for NMDARs in platelets, in a process that involves activationdependent receptor relocation towards the platelet surface. (C) 2014 Elsevier Ltd. All rights reserved.