A Preliminary Study On Platelet Hyperreactivity Induced By High Plasma Glutamate Via Excitatory Amino Acid Transporters In Acute Ischemic Stroke

Background and ObjectivePlatelet hyperreactivity leads to pathological thrombosis and is strongly associated with the occurrence and progress of acute ischemic stroke(AIS).Yet the mechanisms responsible for platelet hyperreactivity have not been fully clarified.Plasma glutamate levels increased after AIS and high plasma glutamate was an independent risk factor for poor prognosis,which may be related to platelet hyperreactivity.Because basic studies have shown that incubation of exogenous high glutamate enhanced platelets stimulation by various agonists,and other studies have found that extracellular high glutamate upregulated platelet membrane protein excitatory amino acid transporters(EAATs)which mediated glutamate uptake by platelets.Therefore,we speculate that high plasma glutamate via the EAATs pathway may lead to platelet hyperreactivity in the acute phase after AIS.Therefore,this study preliminarily explored the role of EAATs-mediated glutamate uptake in platelet activation and its association with platelet hyperreactivity in AIS,aiming to discover the potential causes and mechanisms for platelet hyperreactivity in AIS.MethodsPlatelet-rich plasma(PRP)and platelet-poor plasma(PPP)were collected from AIS patients admitted to the hospital within 24 hours after onset and healthy adult volunteers.Plasma glutamate and glutamine levels were measured by liquid chromatography-tandem mass spectrometry(LC-MS);platelet activation markers including CD62P and PAC-1 expression were analyzed by flow cytometry;and the maximum platelet aggregation rate was recorded in 5 min by Light Transmission Aggregation(LTA).Results1.EAATs inhibitors(DL-TBOA)significantly inhibited thrombin-induced platelet aggregation function and CD62P and PAC-1 expression in healthy volunteers(P<0.05);2.Compared to healthy volunteers,the AIS patients had significantly higher plasma glutamate and glutamine levels in the acute phase(on admission and the second day after admission)(P<0.05);3.Compared to healthy volunteers,the AIS patients had platelet hyperreactivity with platelet count(PLT)decreased,mean platelet volume(MPV)and platelet distribution width(PDW)increased as well as expression of platelet activation markers(CD62P and PAC-1)increased(P<0.05);4.Preincubation with 250 μM glutamate increased platelet activation in healthy volunteers(P<0.05),and the inhibitory effect of DL-TBOA on platelet activation under glutamate incubation conditions decreased;5.The inhibitory effect of DL-TBOA on platelet activation decreased in AIS patients,manifested that 4 mM DL-TBOA didn’t inhibit thrombin-induced platelet aggregation and the expression of CD62P and PAC-1 in AIS(P>0.05),while 8 mM DL-TBOA did inhibit platelet aggregation in AIS(P<0.05).Conclusion1.Glutamate uptake via EAATs is involved in thrombin-induced platelet activation.2.High plasma glutamate via EAATs pathway may promote platelet hyperreactivity in the acute phase of AIS patients.