Submitted: 17 July 2007; Accepted: 24 January 2008

Glutamate mediates platelet activation through the AMPA receptor

Craig N. Morrell1, Henry Sun2, Masahiro Ikeda8, Jean-Claude Beique3,4, Anne Marie Swaim1, Emily Mason1, Tanika V. Martin1, Laura E. Thompson1, Oguz Gozen5,9, David Ampagoomian1, Rolf Sprengel10, Jeffrey Rothstein3,5, Nauder Faraday6, Richard Huganir3,4, and Charles J. Lowenstein2,7

1--7Johns Hopkins University School of Medicine, Baltimore, MD 21205

1 Department of Molecular and Comparative Pathobiology

2 Department of Medicine

3 Department of Neuroscience

4 Howard Hughes Medical Institute

5 Department of Neurology

6 Department of Anesthesia and Critical Care

7 Department of Pathology

8 Department of Veterinary Pharmacology, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan

9 Department of Physiology, Ege University School of Medicine, 35100 Izmir, Turkey

10 Max Planck Institute for Medical Research, Department of Molecular Neurobiology, D-69120, Heidelberg, Germany

Materials And Methods

Reagents

L-glutamate, AMPA, CPP, ADP, CNQX:HBC, mouse thrombin, choline chloride, amiloride, U46619, and JST were purchased from Sigma-Aldrich. Antibody to GluR1 was purchased from Calbiochem; antibodies to GluR2, GluR3, and GluR4 were purchased from Santa Cruz Biotechnology. PAC-1 and P-Selectin antibodies were purchased from BD Biosciences. FITC-IgG secondary antibody for FACS, SBFI, and BCEF were purchased from Invitrogen. RT-PCR reagents were purchased from Invitrogen. TRAP-6 was purchased from Bachem.

Platelet isolation and ex vivo experiments

Human platelets were isolated from healthy volunteers who had not taken aspirin or nonsteroidal antiinflammatory drugs for 10 d, under a protocol approved by the Johns Hopkins University School of Medicine Institutional Review Board. Blood was collected into citrate anticoagulant, and platelets were isolated as PRPs by centrifugation at 180 g for 15 min and diluted in Tyrode's buffer at a dilution of 1:20 for activation flow cytometry studies. To determine surface GluR1, a Fc blocking antibody was added to reduce nonspecific background antibody binding. Mouse platelets were isolated by collection into heparinized Tyrode's buffer and isolated by centrifugation. Washed platelets were resuspended in Tyrode's.

Intracellular ion concentrations were performed after incubating platelets diluted 1:20 in Tyrode's buffer with SBFI for 45 min before experiments.

Glutamate concentration

Whole blood was isolated as described in the previous section and diluted 1:1 in Tyrode's buffer. A 1-mM glutamate-sensitive probe (Pinnacle Technology, Inc.) was used to determine real-time glutamate concentrations. The glutamate biosensor relies on the glutamate oxidase每catalyzed conversion of glutamate to 汐-ketoglutaric acid and hydrogen peroxide. The enzymatically produced hydrogen peroxide is then ampomerically detected by its oxidation at the probe's platinum-iridium electrode with an applied potential of 600 mV, with high sensitivity to glutamate at low (micromolar) concentrations, producing a linear response over a wide concentration range. The rapid response (1每4 s) is recorded using probe-specific software to monitor the time course of a physiological glutamate release. Probe calibration was performed by incubating the probe in a gradient of glutamate concentrations. Voltage output and glutamate concentration were then correlated. While stirring, the biosensor probe was placed in stirring blood and voltage recordings were initiated. Thrombin was added to induce platelet activation and aggregation.

Immunoblotting

Platelets purchased from HemaCare and rat brain cerebellar lysate were lysed with NP-40 lysis buffer, and supernatants were fractionated on a 4每15% gel. Membrane-transferred proteins were immunoblotted with antibodies against GluR1-4.

In vivo studies

Mouse experiments were performed as approved by the Johns Hopkins University School of Medicine Animal Care and Use Committee.

Tail bleeding

6-wk-old male mice were anesthetized with ketamine and xylazine (80/13 mg/kg) and injected i.v. with either PBS or CNQX:HBC, with 0.1 mg/kg of active CNQX. 20 min later, the distal 3 mm of the tail was amputated and immersed in 37~C saline, and the time to visual cessation of bleeding was recorded.

Intravital microscopy

Platelets were isolated from mice as above and resuspended in Tyrode's buffer at a concentration of 108/100 米l, fluorescently labeled with 10 米M calcein-AM, and 100 米l was injected intravenously into a mouse anesthetized with ketamine and xylazine. The mesentery was externalized, thrombosis was initiated by the addition of a 5-mm2 piece of Whatmann's paper soaked in 10% FeCl3, and thrombosis was recorded using a digital imaging camera and software (QCapture Pro; Retiga). GluR1?/? mice, on a C57Bl6/J background, were a gift from R. Sprengel (University of Heidelberg, Germany).

Electrophysiological experiments

Cell preparation and solutions.

All procedures using animals were approved by the University of Miyazaki Institutional Animal Care and Use Committee. Megakaryocyte preparation was performed as previously reported (43). In brief, mouse bone marrow was flushed with Na+-rich external solution (Na+-ES, 150 mM NaCl, 1 mM CaCl2, and 10 mM Hepes, pH adjusted to 7.2 using NMDG-OH). Cells were dispersed by repetitive pipetting, washed three times by gentle centrifugation, resuspended in Na+-ES, and used within 90 min of isolation.

Electrophysiological studies.

Whole-cell recordings were obtained from freshly isolated megakaryocytes using an intracellular solution of the following composition: 150 mM CsCl, 1 mM EGTA, and 10 mM Hepes. The pH was adjusted to 7.3 using NMDG-OH. The patch-clamp procedures were similar to those previously described (43). In brief, the experiments were performed in the tight-seal whole-cell configuration at room temperature, and electrical signals were acquired with an EPC-7 (HEKA). The pipette resistances ranged between 2 and 3 M次.

Current每voltage relationships (I每V curves) were obtained by clamping the cell to different potentials in 30-mV increments (?30, 0, and + 30 mV; each for 35 ms) from a holding potential of ?60 mV. I每V curves obtained before the application of the agonists were subtracted from those (typically two) obtained in the presence of the agonists. The current responses were monitored on a chart recorder (WR7700; Graphtec) through a low-pass filter (200 Hz). Chemicals and reagents used were purchased from either Sigma-Aldrich or Wako Pure Chemicals.

Rapid perfusion of AMPA and Kainate was achieved by puff application from a nearby pipette of 30每35 mm diam to an isolated single megakaryocyte. The perfusion pipette was located within 30 mm of the cell, and the puff pressure was adjusted to obtain rapid and effective agonist application (< 10 ms).

Fig. 5E was performed using two bathing chambers (test and control) placed on the stage of an inverted microscope. One chamber contained a megakaryocytes immersed in Na+-ES每containing CNQX (test chamber), and the other contained the same batch of megakaryocytes immersed in Na+-ES alone (control chamber). Traces used in the I每V curve were those which had good signal-to-noise ratio during the voltage steps.

Data analysis

Data are expressed as the mean ㊣ the SD, unless otherwise stated. Statistical comparisons between two groups were performed using the Student's t test.

Online supplemental material

Figure S1.?Additional data in support of Figs. 1每8.?(A) Calibration of glutamate probe. (B) Glutamate increases agonist-induced platelet activation. P-selectin. Platelets were incubated with control or glutamate and activated or not with TRAP. P-selectin expression was measured by FACS (n = 3 ㊣ the SD; *, P < 0.05 vs. 0 mM). (C) AMPA increases agonist-induced platelet activation at moderate agonist concentrations. Platelets were incubated with AMPA (250 uM) and then activated or not with thromboxane mimetic. Platelet activation was measured by FACS analysis for surface increase in PAC-1 antibody binding versus resting platelets (n = 5 ㊣ the SD; *, P < 0.01 vs. control). (D) AMPAR antagonist decreases platelet activation. P-selectin. Platelets were incubated with control or CNQX, and then activated or not with TRAP. Platelet activation was measured by FACS for P-selectin expression (n = 5 ㊣ the SD; *, P < 0.05 vs. 0 mM). (E) AMPAR antagonist decreases platelet activation. Platelets were incubated with control or NBQX and activated or not with TRAP. Platelet activation was measured by FACS for PAC-1 antibody binding (n = 3 ㊣ the SD; *, P < 0.05). (F) Amiloride inhibits NHE. Platelets were preincubated with the pH-sensitive dye BCEF, and the change in intracellular pH with TRAP activation was measured by FACS. (G) Platelet AMPA receptor mediates ion transport. AMPAR activity is independent of NHE. Platelets were incubated with control, AMPA, amiloride (Amil), or amiloride before AMPA and activated or not with TRAP (n = 3 ㊣ the SD; *, P < 0.05). (H) Representative current traces from multiple megakaryocyte isolations. (left) AMPA (100 uM) evoked currents. Top three traces are from a single isolation also demonstrated in J. (right) KA (100 uM) evoked currents. (I) Representative current traces showing pre- and post-agonist/antagonist application with and without cyclothiazide (CTZ). (J) CNQX (30 uM) blocks AMPA-induced current. Pair run, controlled AMPA evoked currents corresponding to the data in Fig. 5 E. All of the megakaryocytes used in this study were isolated at same time. Numbers to the right of each trace correspond to the peak amplitude.

Abbreviations used

AMPA, 汐-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid;

AMPAR, AMPA receptor;

CNS, central nervous system;

CPP, (㊣)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid;

GPCR, G protein每coupled receptor;

JST, Joro spider toxin;

NHE, Na+/H+ exchanger;

NMDA, N-methyl-D-aspartate;

NMDAR, NMDA receptor;

PRP, platelet-rich plasma;

SBFI, sodium-binding benzofuran isophthalate;

TRAP, thrombin receptor每activating peptide.

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