Platelet Kainate Receptor Signaling Promotes Thrombosis by Stimulating Cyclooxygenase Activation

Platelet Kainate Receptor Signaling Promotes Thrombosis byStimulating Cyclooxygenase Activation

Henry Sun1, AnneMarie Swaim2, Jesus Enrique Herrera1, Diane Becker1, Lewis Becker1,Kalyan Srivastava2,*, Laura E. Thompson2, Michelle R. Shero2, Alita Perez-Tamayo2, BhoomSuktitpat1, Rasika Mathias1, Anis Contractor3, Nauder Faraday4, and Craig N. Morrell2,*1 Department of Medicine, Johns Hopkins University School of Medicine, 733 North Broadway,Baltimore, MD 21205 USA.2 Department of Molecular and Comparative Pathobiology, Johns Hopkins University School ofMedicine, 733 North Broadway, Baltimore, MD 21205 USA.3 Department of Physiology, Northwestern University School of Medicine, Ward Building, Suite5-223, 303 E Chicago Ave. Chicago, IL 60611-30084 Department of Anesthesiology and Critical Care, Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, MD 21205 USA.

AbstractRationale—Glutamate is a major signaling molecule that binds to glutamate receptors includingthe ionotropic glutamate receptors; kainate (KA) receptor (KAR), the N-methyl-D-aspartate(NMDA) receptor (NMDAR), and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA) receptor (AMPAR). Each is well characterized in the central nervous system (CNS), butglutamate has important signaling roles in peripheral tissues as well, including a role in regulatingplatelet function.

Objective—Our previous work has demonstrated that glutamate is released by platelets in highconcentrations within a developing thrombus and increases platelet activation and thrombosis. Wenow show that platelets express a functional KAR that drives increased agonist induced plateletactivation.

Methods and Results—KAR induced increase in platelet activation is in part the result ofactivation of platelet cyclooxygenase (COX) in a Mitogen Activated Protein Kinase (MAPK)dependent manner. Platelets derived from KA receptor subunit knockout mice (GluR6−/−) areresistant to KA effects and have a prolonged time to thrombosis in vivo. Importantly, we have alsoidentified polymorphisms in KA receptor subunits that are associated with phenotypic changes inplatelet function in a large group of Caucasians and African Americans.

Conclusion—Our data demonstrate that glutamate regulation of platelet activation is in part COXdependent, and suggest that the KA receptor is a novel anti-thrombotic target.

Corresponding Author: Craig Morrell, Aab Cardiovascular Research Institute, University of Rochester School of Medicine & Dentistry,601 Elmwood Avenue, Box CVRI, Rochester, New York 14642 Phone: (585) 276-9921.*Current Address: Aab Cardiovascular Research Institute, University of Rochester School of Medicine & Dentistry, 601 ElmwoodAvenue, Box CVRI, Rochester, New York 14642.DisclosuresC.N.M has received funding from TorreyPines Therapeutics, but had no direct impact on this study.

NIH Public AccessAuthor ManuscriptCirc Res. Author manuscript; available in PMC 2010 September 11.

Published in final edited form as:Circ Res. 2009 September 11; 105(6): 595–603. doi:10.1161/CIRCRESAHA.109.198861.

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Keywordsplatelet; kainate; glutamate; thrombosis; cyclooxygenase; thromboxane; mitogen activated kinase;p38

IntroductionThrombosis is driven by complex interactions between platelets, endothelial cells, andcoagulation factors. Initial platelet activating events trigger intracellular signaling pathwaysnecessary for rapid and efficient thrombus formation. These include changes in intracellularion concentrations and pH, conformational changes in receptors such as Glycoprotein IIb/IIIa(GPIIb/IIIa), granule exocytosis, and secretion of vasoactive mediators. Many granuleconstituents are shared between platelets and neurons including Substance P, ADP, ATP,serotonin, and glutamate 1-3. Glutamate signaling is well explored in the brain, but glutamateeffects on platelet function are less well studied and only beginning to be understood.

Ionotropic glutamate receptors are classified as kainate (KA) receptors (KAR), N-methyl-D-aspartate (NMDA) receptors (NMDAR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) based on their sensitivity to the ligandthey are named for. NMDA receptors mediate the influx of calcium while AMPAR and KARprimarily mediate sodium influx upon ligand binding. AMPAR and KAR are closely relatedin their tetrameric structure (4 subunits), but are comprised of distinct receptor subunits.Kainate receptor subunits are commonly designated as GluR5, GluR6, GluR7, KA1, and KA2(genes are also designated GRIK1 through 5, respectively) 4, and each subunit type is theproduct of a distinct gene. The KA receptor was identified by its activation in response to thenaturally occurring marine toxin kainic acid and has similar activity to AMPAR, but is lesswell studied. GluR5-7 can form homomers (example, all GluR5) and heteromers (example, areceptor composed of both GluR5 and GluR6). KA1 and KA2 can only be part of functionalreceptors by combining with one of the GluR5-7 subunits 5, 6. Like AMPAR, when activatedthe KAR allows sodium influx at a rate similar to that of the AMPAR, but KARs desensitizemore rapidly following ligand binding.

Glutamate receptors are best described in the central nervous system (CNS), but are alsoexpressed and functional in many tissues outside of the CNS 7. For example, AMPA canstimulate insulin release from β-cells in the pancreas, and the AMPAR subunit GluR3 has beendescribed on circulating T-lymphocytes where it has a potential role in T-cell chemotaxis andadhesion8. Other glutamate receptors, such as the NMDA receptor, have been described inosteoblasts, osteoclasts, and megakaryocytes 7, 9. Most peripheral glutamate receptors whencloned and sequenced are identical to those of the CNS 7. We have previously demonstratedusing a real time glutamate sensitive enzymatic probe that glutamate reaches very highconcentrations (greater than 400 μM) within a developing thrombus1. We have alsodemonstrated that glutamate signaling has an important role in efficient thrombus formationby amplifying platelet activation, in part through platelet expressed AMPA receptors 1.

Glutamate signaling induces Mitogen Activated Protein Kinase (MAPK) signaling pathwaysin neurons 10. MAPK messengers, including p38, are well described platelet signalingmolecules 11-13. MAPK p38 is phosphorylated in agonist-stimulated platelets and dominantnegative MAPK signaling inhibition reduces functional outcomes of agonist stimulation, suchas GPIIb/IIIa activation 13. Platelet cyclooxygenase (COX) activation has been demonstratedto be downstream of MAPK in response to receptor stimulation 13, 14. COX activation and theproduction of prostaglandins (such as thromboxane), is an important step in efficient thrombusgeneration 15-17. Kainate induced seizures drives neuronal COX stimulation 18-20 and KAR

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signaling can induce activation of MAPK pathways in neurons, including p38 pathways 21,22. This provides a background for a possible role of KAR signaling through MAPK pathwaysin platelet COX activation.

We now demonstrate that glutamate signaling through the Kainate type of glutamate receptoris also an important regulator of platelet function, in part by stimulating cyclooxygenase, andthat this effect is dependent on MAPK signaling pathways. We also demonstrate that plateletsfrom mice lacking KAR subunits do not respond to KA and that these mice have prolongedtime to thrombosis in vivo. Furthermore, we have identified polymorphisms in the KA receptorthat results in platelet functional changes in human populations.

Materials and MethodsGluR6−/− mice are on a mixed 129 and C57Bl6/J background. Littermate wild-type mice wereused as controls. For thromboxane production studies platelet rich plasma (PRP) was pre-treated with inhibitors for at least 20 mins prior to stimulation. Platelets were pelleted aftertreating with indomethacin (20 μM, Sigma). Plasma was isolated for the TxB2 ELISA and theplatelet pellet lysed in p38 ELISA lysis buffer for measurement of P-p38. Plasma was furtherpurified using a C-18 SPE cartridge (Amersham). All other materials and studies are completelydescribed in the online supplement.

ResultsKAR signaling increases platelet activation

Our previous studies demonstrated that glutamate pre-treatment increased agonist inducedplatelet activation 1. Glutamate by itself does not directly alter the expression of surface markersof platelet activation, such as GPIIb/IIIa activation (PAC-1 antibody binding) and P-selectinexpression1. However, as shown in Figure 1A, pre-treatment of platelets with glutamate priorto agonist stimulation, such as with the thromboxane receptor agonist U46619 (1 μM), greatlyincreased platelet activation as determined by increase in PAC-1 antibody binding (Figure 1A,MFI=mean fluorescent intensity). Our previous work has also shown that glutamate effects arein part mediated by AMPAR 1. The NMDA receptor has also been described on platelets, butpre-treatment of platelets with NMDA does not increase agonist induced platelet activation(Supplementary Figure 1).

The other class of glutamate receptor that is similar in structure and function to AMPAR isKAR. To begin to explore the role of KAR in platelet activation, platelets were incubated withKA (250 μM) prior to platelet activation with U46619 (1 μM). Like glutamate, KA alone hasno effect on PAC-1 antibody binding (Figure 1B, white bars). However, KA pre-treatmentincreased platelet activation approximately 3 times that of control treated platelets (Figure 1B,black bars). A KA dose response curve was performed and as little and as 100 μM of KAsignificantly increased platelet activation (Figure 1C). These are physiologically relevantglutamate and KA concentrations, as we have shown in the past that the concentration ofglutamate in a developing thrombus is greater than 400 μM 1.

KA receptors are made of subunits that assemble to form a tetrameric receptor complex. Todemonstrate that platelets express KAR subunits, human platelets and mouse brain lysates wereimmunoblotted. Platelets express both GluR5 and GluR6 protein (Figure 2A, top). In addition,mRNA was isolated from rat primary neurons and the megakaryocyte cell line MEG-01, andRT-PCR performed with primers specific for GluR5 and GluR6. MEG-01 cells also expressGluR5 and GluR6 mRNA (Figure 2A, bottom).

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Kainate, particularly at higher concentrations, can have some AMPAR affinity, although it ismuch less than its KAR affinity. To demonstrate that the KA induced increase in plateletactivation was specific to KAR, we employed both pharmacologic and genetic methods. ATPAis an agonist that only acts on GluR5-containing receptors. At 100 μM ATPA nearly doubledTRAP induced platelet activation, and as little as 10 μM also significantly increased PAC-1binding (Figure 2B, lower concentrations had no effect, not shown). To confirm KA inducedeffects are mediated by KAR signaling, platelets were isolated from GluR6−/− mice andlittermate control wild-type (WT) mice, incubated with control buffer or KA (250 μM), andthen activated with thrombin (0.1 U/mL). WT and GluR6−/− platelets have similar thrombinresponses (Supplemental Figure 2). KA pre-treatment nearly doubled WT platelet activation,but had no effect on GluR6−/− platelets as measured by GPIIb/IIIa activation using JON/Aantibody binding (Figure 2C).

To further demonstrate that KAR has an important role in platelet activation we performedplatelet aggregation studies in the presence of control buffer (PBS) or the KAR antagonistUBP302. Platelets rich plasma (PRP) was incubated with UBP302 (50 μM) for 20 mins, andthen activated by addition of TRAP (2 μM). At the same concentration of UBP302, KARantagonism either reduced by about 50% (green line), or completely blocked (blue line),platelet aggregation as compared to control PBS treated platelets (Figure 2D, quantification at5 min post TRAP).

KAR signaling increases platelet COX activationKainate induced seizures and neuronal loss are in part due to induction and expression ofcyclooxygenases COX-1 and COX-2 20. Platelets primarily express COX-1 under normalphysiologic conditions, but COX-2 expression can be induced in pathophysiologic processes23. We determined whether or not KAR signaling in platelets stimulated COX. We firstincubated platelets with the endogenous ligand for KAR, glutamate, to see if this led to theproduction and release of thromboxane from platelets. Human PRP (approximately 1×108 totalplatelets per reaction) were treated with control buffer, glutamate alone, or glutamate in thepresence of the COX inhibitor indomethacin (20 μM) as a negative control. Platelets were alsostimulated with low dose TRAP (0.5 μM) as a positive control. After 10 mins platelets werepelleted in the presence of 20 μM of indomethacin to block any further COX activation.Thromboxane (Tbx) release into the supernatant was determined using an EIA that measuresthe stable TxA2 breakdown product, TxB2 (plasma was first purified by column purificationprior to concentration determination to increase EIA TxB2 specificity). Glutamate inducedplatelet thromboxane production, and this was completely blocked by indomethacin pre-treatment (Figure 3A). Similar results were also found when washed platelets were used insteadof PRP (Supplemental Figure 3). To determine whether glutamate induced thromboxaneproduction may in part be due to signaling through the KA receptor, platelets were incubatedwith control buffer (PBS), KA (250 μM) or KA after treatment with the KAR antagonistUBP301 (200 μM). Similar to glutamate, KA increased TxB2 production (Figure 3B, white vsblack bars) and receptor antagonism inhibited KA induced increase in TxB2 (Figure 3B, blackvs grey bars). COX activation was confirmed by measuring PGE2 production (SupplementalFigure 4) and AMPAR signaling also has a similar effect on thromboxane production(Supplemental Figure 5). Glutamate stimulation of thromboxane production may in partaccount for the ‘priming’ effect of glutamate receptor signaling that drives an increase inplatelet activation in response to agonist stimulation.

We also determined the kinetics of KA induced TbxB2 production by incubating platelets withPBS or KA and measuring TbxB2 after 5, 10, 20 and 30 mins. At 5 mins post KA there wasno change in TbxB2, but after 10 mins a significant increase in TbxB2 was found (Figure 3

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C). There was an additional increase noted after 20 and 30 mins, but the greatest increase wasseen between 5 and 10 mins post KA (Figure 3 C).

To confirm these pharmacologic data we used platelets isolated from WT and GluR6−/− mice.Employing methods similar to the human platelet studies, washed WT and GluR6−/− plateletswere incubated with PBS or KA (250 μM) and TxB2 measured by EIA. KA increased WTmouse thromboxane production (Figure 3D, left side) but had no effect on thromboxaneproduction from GluR6−/− platelets (Figure 3D, right side). This provides complementarygenetic evidence that KA induced COX activation is through the KAR.

KAR signaling activates COX in a MAPK dependent mannerMAPK signaling has been implicated as a pathway leading to COX activation in many celltypes, including renal glomeruli, neurons, and platelets 13, 14, 22, 24, 25. KAR signaling is alsoknown to activate MAPK pathways in neurons 22. We therefore explored whether KARsignaling drives thromboxane production in a MAPK dependent manner.

The MAPK p38 pathway is induced in response to numerous platelet agonists including vWF,collagen, and thrombin 14, 24, and mice lacking p38 have prolonged time to thrombus formation26. We determined whether KAR signaling activates platelet p38 MAPK pathway by incubatingplatelets with PBS, KA (250 μM), or KA after the KAR specific antagonist UBP302. TRAPstimulation (0.5 μM) was used as a positive control. p38 phosphorylation (P-p38) wasquantified by ELISA. KA treatment increased P-p38 approximately twice that of restingplatelet levels and this was blocked by KAR antagonism (Figure 4A). We confirmed these datausing platelets from WT and GluR6−/− mice. WT and KO platelets express equal total p38 byWestern blot (Figure 4B), and platelets were incubated with buffer or KA. KA induced anincrease in WT platelet P-p38, but KA had no effect on GluR6−/− platelet P-p38 (Figure 4C).

Although the downstream signaling effects of MAPK pathways are sustained, the actualphosphorylation of p38 is often transient. To determine the time course of KAR signalinginduced platelet P-p38, we incubated platelets with KA as above, and P-p38 was determinedat multiple time points. Similar to TbxB2 production, we found that P-p38 peaked at 10 minspost KA addition (Figure 4 D). This is a transient event, as P-p38 declined to baseline levelsby 20 mins post KA addition (Figure 4 D), confirmed by Western blot (Supplemental Figure6).

To place p38 in the KAR induced COX activation pathway we incubated platelets with the p38inhibitor SB203580 prior to the addition of KA. The KAR antagonist UBP302 was used topre-treat samples prior to the addition of KA as a negative control. As expected KA increasedplatelet thromboxane production (Figure 4E, black vs white bars). This was blocked by pre-incubation of platelets with the p38 inhibitor (Figure 4E, black vs light grey bars), implicatingKAR induced P-p38 as a mechanism to increase platelet COX activation. KAR signaling alsoinduced phosphorylation of ERK1/2 (Supplemental Figure 7), but ERK inhibition did not blockKA induced thromboxane production (Supplemental Figure 8).

Taken together, these data demonstrate that KAR signaling drives low level thromboxaneproduction in a manner dependent on p38 MAPK pathway activation. The induction of MAPKactivation may in part account for glutamate mediated increase in agonist induced plateletactivation by ‘priming’ signaling pathways, thereby amplifying agonist induced activation.This is analogous to what has been noted in other important platelet signaling pathways; forexample native Low Density Lipoprotein (LDL) is known to sensitize platelets to other agonistsin a manner that also involves MAPK 27, 28.

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KAR signaling is a mediator of platelet function in vivoIt is often difficult to extrapolate in vitro platelet function tests to the in vivo relevance of aparticular platelet signaling pathway. To address this we used an intravital microscopy modelof arterial thrombosis with WT and GluR6−/− mice in which mesenteric arterioles weredamaged with ferric chloride and the time to vessel occlusion determined by visualizingfluorescently labeled platelets (Figure 5A). WT mice on average form occlusive thrombi 500sec after vessel damage (Figure 5B). The time to vessel occlusion in GluR6−/− mice isapproximately twice as long as WT mice (Figure 5B). To investigate if KAR antagonists mayhave anti-thrombotic therapeutic potential, mice were injected intravenous with control bufferor the KAR antagonist UBP302 and bleeding time determined after amputating the distal 3mm of the tail. We also determined the time to vessel occlusion using the intravital microscopymesenteric thrombosis model. Mice treated with KAR antagonists have prolonged bleedingtime (Figure 5C) and prolonged time to mesenteric arteriole occlusion following ferric chloridedamage (Figure 5D). These data show that the KAR modulates thrombosis in vivo.

Gene variants in KAR subunits modify platelet response to aspirin treatmentLaboratory experiments evaluating GluR5 (GRIK1) and GluR6 (GRIK2) expression andfunction were followed by gene association studies in humans. Using an established cohort ofCaucasians and African Americans, we examined the association of single nucleotidepolymorphisms (SNPs) in the GluR5 (located on chromosome 21q22.11) and GluR6 (locatedon chromosome 6q16.3-q21) genes, with platelet function after aspirin treatment. A block ofhighly correlated SNPs in intron 1 of GluR5 was associated with urinary excretion of 11-dehydro thromboxane B2 (Tx-M) in whites. Several of these SNPs were associated with plateletaggregation in African Americans (Table 1A, top). Significant associations were also observedafter adjustments were made for baseline platelet function (for RS465566, P = 3.5 × 10−5 forTx-M in Caucasians, and P = 0.004 for collagen aggregation in African Americans). Similartrends were observed for platelet aggregation in whites and Tx-M in African Americans. Theclinical relevance of these phenotypes is underscored by the fact that these measures areassociated with greater risk of myocardial infarction (MI), stroke, and cardiovascular death inaspirin-treated patients with coronary artery disease (CAD) 29-31. The adenine allele of arepresentative SNP in GluR5, rs465566, was associated with less residual platelet functionafter exposure to aspirin (i.e., greater aspirin inhibition) for aggregation and Tx-M excretion(Table 1B). A SNP in intron 13 of GluR6 was also associated with Tx-M in whites, but wewere unable to reinforce this by finding an association in African Americans (Table 1A,bottom). Taken together, these data identify polymorphisms in KAR that are associated withimportant changes in platelet function.

DiscussionOur work demonstrates that platelets express functional KA receptors that have an importantrole in platelet activation and thrombosis. We have now defined a pathway in which glutamatereleased from activated platelets binds to KAR, driving MAPK pathway activation andthromboxane production. Mice lacking KAR subunits no longer respond to KA and exhibitdelayed thrombus formation after vascular injury. Furthermore, we have identifiedpolymorphisms in KAR that are associated with changes in platelet function, validating thepotential physiologic relevance of the KAR and platelet glutamate signaling pathways inhuman health and disease.

Glutamate has an important role in helping drive thrombus formation. While glutamate doesnot directly increase the surface expression of markers of platelet activation such as P-selectinand active GPIIb/IIIa, it has a key function in amplifying the effects of agonist stimulation onplatelet activation and aggregation. This and our previous studies demonstrate that glutamate

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receptor signaling makes thrombus formation more efficient in vivo. Mice lacking AMPARor KAR receptor subunits still form occlusive thrombi; however, it is significantly delayed.Glutamate's function in platelet activation can be seen as analogous to a rheostat in regulatingthe outcome of platelet agonist stimulation. Glutamate receptor signaling may in part serve toalter the ‘set point’ for stimulation by other platelet agonists and thus augment and amplifyplatelet responses during thrombus generation. This is an important concept when consideringthat many of the important steps in platelet aggregation take place in close contact betweenplatelets as a thrombus develops. The local increase in glutamate concentration as plateletactivation proceeds serves to make other agonists such as thromboxane, thrombin, and ADPmore effective. This may also help to focus thrombus generation locally to the site of vascularinjury and lesion development.

One important downstream signaling event of KAR in platelets that may be important inreducing the set point to subsequent agonist stimulation is the production of low, sub-activationthreshold levels of thromboxane that on its own is not sufficient to increase measureablephenotypic markers of platelet activation. Rather, this low level p38 dependent thromboxaneproduction makes platelets more sensitive to subsequent stimuli, thus amplifying agonistresponse and making thrombus formation more efficient. This is similar to previousdescriptions of other important platelet signaling pathways. For example, native Low DensityLipoprotein (LDL) is known to sensitize platelets to other agonists in a manner that alsoinvolves MAPK. Serotonin, a small neurotransmitter similar to glutamate, is best understoodin neurobiology. Serotonin also has little direct platelet stimulatory effects, but can increaseplatelet response to other agonists in ways that are only beginning to be appreciated 3, 11, 32,33.

Platelets have many activation pathways that become integrated during thrombus generation.Hemostasis is vital to survival, likely leading to evolutionary pressures resulting in redundantactivation signaling pathways. Ensuring that thrombosis is efficient and localized isadvantageous, and this is accomplished through the elaboration of activation molecules withinthe developing thrombus (such as glutamate) that help to sustain and amplify platelet activationand thrombus formation in a localized manner. These messenger molecules and signalingpathways provide attractive targets for platelet directed anti-thrombotic therapies. Acutevascular thrombosis is the major pathophysiologic process underlying acute myocardialinfarctions and ischemic strokes. Antiplatelet therapies have been cornerstones of the medicalarmamentarium in acute coronary syndromes, cerebrovascular accidents, and now coronarystent thrombosis and restenosis. The ideal antithrombotic and antiplatelet agent should inhibitpathologic platelet aggregation while minimizing the risk of bleeding. Our results indicate thatglutamate signaling pathways may provide such a therapeutic target. Because glutamate is nota ‘traditional’ platelet agonist and serves primarily to amplify agonist-induced plateletresponse, mice lacking glutamate receptor subunits do not exhibit spontaneous bleeding orhemorrhagic phenotypes. Rather, thrombus formation in mice treated with glutamate receptorantagonists and mice lacking glutamate receptor subunits is left intact, although it is delayedand less efficient. Our mouse bleeding time studies with a KA receptor inhibitor howeverindicate that any potential use of these compounds in humans would have to be approachedwith caution to prevent adverse bleeding events. We do not yet fully appreciate thepharmacokinetics of these compounds. Nonetheless, glutamate antagonists make an attractivepotential adjunctive therapy in the treatment of patients with acute coronary syndromes andstrokes that warrants further consideration and study.

Our results have consistently demonstrated that glutamate amplifies platelet activation via theAMPAR and KAR receptors, but acute NMDAR signaling has little effect on plateletactivation. A major difference in these types of ionotropic glutamate receptors is that the KARand AMPAR both mediate the influx of sodium whereas the NMDAR forms primarily a

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calcium permeable channel. Calcium signaling is obviously very important in plateletactivation; however, there are multiple other pathways that provide for calcium influx duringplatelet activation thus blocking or triggering the NMDAR may not exert a significant effecton platelet activation. There are limited cellular mechanisms to rapidly depolarize plateletmembranes, underscoring the notion that glutamate signaling through the KAR and AMPARmay have relatively greater effect on platelet activation in acute settings. This does not ruleout the possibility that the NMDAR is important in other aspects of platelet biology. NMDARhas been shown to have a role in regulating platelet production and megakaryopoesis 9 and inthe brain NMDAR signaling can also alter AMPAR and KAR receptor densities and function.It is possible that because the effects we are observing are very acute, NMDAR has otherimportant platelet functions that are unappreciated due to the constraints of our ex vivo and invivo systems.

We examined the relationship between SNPs in two gene components of the human KAR(subunits GluR5 and GluR6) and platelet functional responses to aspirin. The goal of thisanalysis was to determine whether gene variants in components of human KAR have clinicallyrelevant platelet functional phenotypes (such as in this case, aspirin response). These datademonstrated that there are important SNPs in KAR subunits that result in variability in aspirinresponse, a clinically defined and poorly understood phenomenon. Significant SNPs wereapproximately replicated in each ethnic group examined adding great significance of theresults. Mechanistic insight into how the block of SNPs in GluR5 in particular, results in thisinteresting phenotypic change in platelet function is not within the scope of this initialdescription of the role of KAR in platelet activation and thrombus formation, but the data doeslend itself to speculation for future study. The variant allele block (minor allele) is associatedwith less platelet aggregation and urinary thromboxane metabolite post aspirin in both whitesand African Americans. These data imply that the allelic variant confers greater aspirin inducedsuppression of platelet activation. Aspirin effectively blunts COX activity and our resultsdefine a KAR mediated signaling pathway that augments platelet activation in part throughCOX stimulation. Does this mean that a reduction in KAR signaling in these individuals resultsin the loss of the KAR signaling pathway that leads to COX activation, thereby conferringgreater platelet aspirin sensitivity? Does this imply an alternative pathway in COX stimulationthrough KAR in platelets that is yet to be understood? At this time any answer is purelyspeculative and will require further investigation. However, the importance of the genetic datais to demonstrate for the first time the functional and clinical relevance of glutamate receptorsignaling in a large well described cohort. It should also be emphasized that because regulationof thrombosis is so critical, platelets have many redundant signaling pathways. Phenotypes inhuman populations where genes and protein products are not totally disrupted are often onlynoted under physiologic stress or pharmacologic treatment response. These data mayemphasize this principal.

Platelet glutamate receptor signaling is an exciting and relatively unexplored area of plateletbiology. Our data indicate it may have a significant role in platelet function that impactsvascular disease pathogenesis. Although much work remains to be done to fully validate theimportance of the platelet glutamate receptor pathway, our study advances our currentunderstanding of the novel role of glutamate receptors in platelet biology and in potentialcontribution to the variabilities in response to antiplatelet therapy.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

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AcknowledgmentsThis work was supported by NIH grants K08HL74945 and R01HL093179 to C.N.M., U01HL72518 and HL65229 toL.C.B., by M01-RR000052 to The Johns Hopkins General Clinical Research Center, and by an Intramural ResearchProgram of the National Human Genome Research Institute.

Non-standard Abbreviations and AcronymsKA, kainate; KAR, kainate receptor; NMDA, N-methyl-D-aspartate; NMDAR, NMDAreceptor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAR, AMPAreceptor; COX, cyclooxygenase; MAPK, Mitogen Activated Protein Kinase; GPIIb/IIIa,Glycoprotein IIb/IIIa; CNS, Central Nervous System.

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20. Kim EJ, Lee JE, Kwon KJ, Lee SH, Moon CH, Baik EJ. Differential roles of cyclooxygenase isoformsafter kainic acid-induced prostaglandin E(2) production and neurodegeneration in cortical andhippocampal cell cultures. Brain Res 2001;908:1–9. [PubMed: 11457426]

21. Chen J, Li C, Pei DS, Han D, Liu XM, Jiang HX, Wang XT, Guan QH, Wen XR, Hou XY, ZhangGY. GluR6-containing KA receptor mediates the activation of p38 MAP kinase in rat hippocampalCA1 region during brain ischemia injury. Hippocampus 2009;19:79–89. [PubMed: 18680160]

22. Ferrer I, Blanco R, Carmona M, Puig B, Dominguez I, Vinals F. Active, phosphorylation-dependentMAP kinases, MAPK/ERK, SAPK/JNK and p38, and specific transcription factor substrates aredifferentially expressed following systemic administration of kainic acid to the adult rat. ActaNeuropathol 2002;103:391–407. [PubMed: 11904760]

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26. Dangelmaier C, Jin J, Daniel JL, Smith JB, Kunapuli SP. The P2Y1 receptor mediates ADP-inducedp38 kinase-activating factor generation in human platelets. Eur J Biochem 2000;267:2283–2289.[PubMed: 10759852]

27. Hackeng CM, Franke B, Relou IA, Gorter G, Bos JL, van Rijn HJ, Akkerman JW. Low-densitylipoprotein activates the small GTPases Rap1 and Ral in human platelets. Biochem J 2000;349:231–238. [PubMed: 10861233]

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Figure 1.Kainic Acid Increases Platelet Activation. (A) PRP was diluted in Tyrode's buffer, pre-treatedwith glutamate and stimulated with U46619 (1 μM). Platelet activation was determined byPAC-1 antibody binding (n=4± S.D., *P<0.01 vs 0). (B) Diluted PRP was pre-treated with 250μM KA and stimulated with U46619 (1 μM). Platelet activation was determined by FACS withPAC-1 antibody (n=5± S.D., *P<0.01). (C) KA Dose Response. Diluted PRP was treated withKA and stimulated with TRAP (5 μM). Platelet activation was determined by FACS withPAC-1 antibody (n=5± S.D., *P<0.01 vs 0).

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Figure 2.Platelets Express a Functional KAR. (A) Expression. Platelet (100 μg) and brain (50 μg) lysatewere fractionated and immunoblotted for GluR5 and GluR6 (top). RT-PCR with MEG-01 celland rat primary neuron cDNA (bottom). (B) Diluted PRP was incubated with the KAR specificagonist ATPA and activated with TRAP (5 μM) (n=5±S.D., *P<0.05, **P<0.01 vs 0). (C)Platelets from WT and GluR6−/− mice was incubated with KA (250 μM) and activated with0.1 U/mL of thrombin (n=5±S.D. *P<0.02). (D) KAR inhibitor reduces platelet aggregation.PRP was incubated with UBP302 and aggregation initiated with TRAP (n=7±SEM *P<0.01vs Control).

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Figure 3.Glutamate Receptor Signaling Induces Platelet COX Activation. (A) Glutamate increasesTxB2. PRP was incubated with PBS, glutamate (250 μM) or glutamate after indomethacin (20μM). TRAP (0.5 μM) was used as a positive control. TxB2 in the supernatant was measuredby EIA (n=4 ±S.D. *P<0.01 vs Control). (B) KAR signaling increased TxB2. PRP was treatedwith control PBS, KA (250 μM) or pre-treated with KAR inhibitor (100 μM) prior to KA. (n=5± S.D. *P<0.01 vs Control). (C) Time Course. PRP was incubate with control PBS or KA(250μM) and TbxB2 production determined by EIA at 5, 10, 20, and 30 mins. KA increasedTbxB2 over time (n=4±S.D. *P<0.01 vs Control). (D) KA does not increase GluR6−/− platelet

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TxB2 production. Washed platelets from WT and GluR6−/− mice were incubated with controlbuffer or KA (250 μM). TxB2 determined by EIA (n=5±S.D. *P<0.01 vs Control).

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Figure 4.KAR Signaling Induces p38 MAPK Activation. (A) KA increases p38 phosphorylation. PRPwas incubated with control buffer, KA (250 μM), or KA after UBP301 and P-p38 determinedby ELISA. TRAP (0.5 μM) stimulation as positive control (n=4 ± S.D. *P<0.01 vs Control)(B) WT and GluR6−/− platelets express equal total p38. Platelet lysate (50 μg each) wasfractionated and immunoblotted for total p38. (C) KA does not increase GluR6−/− platelet P-p38. WT and GluR6−/− washed platelets were incubated with control buffer or KA (250 μM)and P-p38 measured by ELISA (n=4 ± S.D. *P<0.01 vs Control) (D) P-p38 time course. PRPwas incubated with KA (250 μM) and P-p38 measured by ELISA over time. P-p38 peaks at10 mins and then declines (n=4 ± S.D. *P<0.01 vs Control). (E) Inhibiting p38 blocks KA

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induced increase in TxB2. PRP was treated with p38 inhibitor or KAR inhibitor prior to KA(250 μM) (n=4 ± S.D. *P<0.02 vs Control).

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Figure 5.KAR is a Mediator of Platelet Function In Vivo. (A) Mesenteric ferric chloride injury model.(B) WT and GluR6−/− mesenteric thrombosis. Time to vessel occlusion (± S.D. *P<0.01 vsWT). (C) Bleeding Time. Mice were treated with KAR inhibitor and tail bleeding timedetermined (*P<0.01 vs Control). (D) Thrombosis. Mice were treated with UBP302,mesenteric vessel damaged and the time to vessel occlusion determined (± S.D. *P<0.05 vsControl).

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Platelet Kainate Receptor Signaling Promotes Thrombosis by Stimulating Cyclooxygenase Activation

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Transcript of Platelet Kainate Receptor Signaling Promotes Thrombosis by Stimulating Cyclooxygenase Activation