Glutamate is released in thrombus formation. (A) Whole blood was diluted in Tyrode's buffer (1:1), and glutamate concentration was measured with a voltage-sensitive enzymatic probe at rest after addition of PBS (no change) or 0.5 U/ml thrombin (representative tracing). (B) Quantification. Baseline and peak glutamate concentrations during thrombus formation. n = 3–4. *, P < 0.05.

AMPAR signaling mediates platelet aggregation. (A) AMPAR inhibition decreases platelet aggregation. Platelets were incubated with control or CNQX and activated with 10 μM TRAP. Platelet aggregation was measured by an aggregometer. (B) Quantification. AMPAR inhibition decreases platelet aggregation. n = 5–12 ± the SD. *, P < 0.01. (C) Exogenous AMPA increases early platelet aggregation. n = 5 ± the SD. *, P = 0.03; NS, P = 0.09.

Glutamate mediates an increase in GPCR signaling. (A) Glutamate increases epinephrine-mediated platelet activation. Epinephrine-induced (10 μM) platelet activation was measured by PAC-1 antibody binding. n = 5 ± the SD. *, P < 0.01 versus 0 μM. (B) Glutamate does not effect collagen-mediated platelet activation. Platelets were incubated with glutamate and activated with collagen. Activation was measured by FACS analysis for surface PAC-1 antibody binding. n = 5 ± the SD. (C) Glutamate does not increase ATP-induced platelet activation. ATP induced platelet activation was measured by PAC-1 antibody binding. n = 5 ± the SD. (D) Glutamate increases P2Y-mediated platelet activation. The P2Y agonist 2-Me-ADP-induced platelet activation was measured by PAC-1 antibody binding. n = 5 ± the SD. *, P < 0.01 versus 0 μM.

AMPAR signaling mediates platelet activation. (A) AMPA dose dependently increases agonist-induced platelet activation. Platelets were incubated with AMPA and activated with TRAP. Platelet activation was measured by FACS analysis for surface PAC-1 antibody binding. n = 5 ± the SD. *, P < 0.05 versus 0 mM. (B) AMPAR antagonist inhibits AMPA-mediated increase in platelet activation. Platelets were incubated with control, CNQX, AMPA, or AMPA after CNQX, and then activated with TRAP. n = 3 ± the SD. *, P < 0.05. (C) AMPAR antagonist decreases platelet activation. Platelets were incubated with control or CNQX, and then activated or not with TRAP. n = 5 ± the SD. *, P < 0.05. (D) GluR1^−/− platelets do not respond to AMPA. Platelets from WT and GluR1^−/− mice were incubated with control or AMPA, and activated with 0.25 U/ml thrombin (Thr). n = 3 ± SD. *, P < 0.05.

Platelets express the AMPAR. (A) Glutamate does not act through the NMDA receptor. Platelets were incubated with control, glutamate, or glutamate after the NMDA receptor inhibitor CPP, and then activated or not with TRAP. n = 3 ± the SD. *, P < 0.05 versus 0 mM. (B) Platelets express AMPAR subunits. Human platelet and mouse brain lysates were immunoblotted for AMPAR subunit proteins GluR1–4. (C) GluR1 is localized to the surface of platelets. Platelets were incubated with control IgG or GluR1 antibody and surface expression assayed by FACS (representative of three experiments with similar results).

AMPAR mediates platelet function in vivo. (A) AMPAR inhibition with 0.1 mg/kg CNQX prolongs bleeding time in vivo. *, P < 0.01 versus control. (B) FeCl₃ was placed on externalized mesenteric arterioles, and platelets were imaged using intravital microscopy (representative images 400 s after FeCl₃). Bars, 25 μm. (C) AMPAR inhibition with 0.1 mg/kg CNQX prolongs platelet adherence, thrombus formation, and vessel occlusion in vivo. *, P < 0.05; **, P < 0.01 versus control. (D) GluR1^−/− mice have prolonged time to vessel occlusion in vivo. *, P < 0.02 versus WT. Error bars represent the mean ± the SD.

Glutamate mediates platelet activation. (A) Glutamate increases platelet activation. Platelets were incubated with glutamate and activated with a moderate concentration of TRAP (5 μM) or high concentration of TRAP (20 μM). Activation was measured by FACS analysis for surface PAC-1 antibody binding. n = 4 ± the SD. *, P < 0.02 versus 0.5 μM TRAP. (B) Dose response. Platelets were incubated with glutamate and activated with a moderate concentration of TRAP (5 μM). Activation was measured by FACS analysis for surface PAC-1 antibody binding and expressed as the percentage of increase in fluorescence versus resting platelets. n = 5–7 ± the SD. *, P < 0.01 versus 0. (C) AMPAR regulates platelet activation. Glutamate increases thromboxane mimetic-induced (U46619; 10 μM) platelet activation measured by PAC-1 antibody binding. n = 3 ± the SD. *, P < 0.01 versus 0 μM. (D) Glutamate is highly effective at low-dose agonist stimulation. Platelets were incubated with 150 μM glutamate and activated with 1 μM TRAP or U46619. n = 5 ± the SD. *, P < 0.05; **, P < 0.01 versus control.

Platelet AMPAR mediates sodium transport. (A) AMPA increases intracellular sodium. Platelets were loaded with SBFI; treated with control, AMPA, CNQX, or AMPA after CNQX; and activated with TRAP. Change in intracellular sodium was measured by FACS. n = 3 ± the SD. *, P < 0.05 versus control. (B) AMPAR increases Na⁺ influx independent of NHE. Platelets were treated with control, amiloride, AMPA, or amiloride before AMPA. Intracellular Na⁺ was determined by FACS. n = 3 ± the SD. *, P < 0.05. (C) Fast application of AMPA to megakaryocytes induces a rapid inward current that reverses close to 0 mV. (top) A current trace showing an inward current induced by fast application of AMPA (time of application shown by the solid line). Rapid current steps for determining I–V relationships were applied before (not depicted) and during application of AMPA (rapid current deflection, black dots). (bottom) The I–V relationship obtained for the AMPA-induced current. The plot represents the averaged I–V curves obtained from 5 different cells, normalized to the current obtained at −60 mV. (D) Fast application of kainate to megakaryocytes induced a rapid inward current that reversed close to 0 mV. (top) A current trace and the averaged I–V curves for kainate application are shown on the bottom. (E) The inward current induced by 100 μM AMPA was blocked by administration of the AMPAR antagonist CNQX (30 μM; ± the SD) *, P < 0.05. (F) JST had no effect on the AMPA- triggered increase in platelet activation. Platelets were incubated with 1 μM JST and activated or not with TRAP. n = 3 ± the SD.