Persistent Protease-activated Receptor 4 Signaling Mediates Thrombin-induced Microglial Activation

Abstract

We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-α (TNF-α) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-α. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca2+]i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFκB activation may be responsible for PAR4AP-induced TNF-α production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations. We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-α (TNF-α) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-α. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca2+]i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFκB activation may be responsible for PAR4AP-induced TNF-α production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations. The inflammatory response is part of the normal defensive processes that may produce both beneficial and detrimental effects in tissues. In the central nervous system (CNS) 1The abbreviations used are: CNS, central nervous system; GPCR, G-protein-coupled receptor; MAPK, mitogen-activated protein kinase; PAR, protease-activated receptor; PARAP, PAR agonist peptide; TNF-α, tumor necrosis factor-α; WB, Western blot; ELISA, enzyme-linked immunosorbent assay; HE, hematoxylin and eosin; mAb, monoclonal antibody; EMSA, electrophoretic mobility shift assay. microglia are the major immune effector cells and, reactive microgliosis has been implicated in the pathogenesis of a broad range of CNS disorders. These include not only infectious CNS diseases but also acute CNS injuries such as traumatic brain injury (TBI) (1Csuka E. Hans V.H. Ammann E. Trentz O. Kossmann T. Morganti-Kossmann M.C. Neuroreport. 2000; 11: 2587-2590Crossref PubMed Scopus (93) Google Scholar), spinal cord injury (2Popovich P.G. Prog. Brain Res. 2000; 128: 43-58Crossref PubMed Scopus (96) Google Scholar), stroke, and brain ischemia (3Stoll G. Jander S. Schroeter M. Prog. Neurobiol. 1998; 56: 149-171Crossref PubMed Scopus (709) Google Scholar). In addition, microgliosis is also intimately involved in several chronic neurodegenerative disorders such as Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) (4McGeer E.G. McGeer P.L. Exp. Gerontol. 1998; 33: 371-378Crossref PubMed Scopus (303) Google Scholar, 5Benveniste E.N. J. Mol. Med. 1997; 75: 165-173Crossref PubMed Scopus (475) Google Scholar). Accompanying the focal accumulation of activated microglia are elevated inflammatory mediators in the brain, such as tumor necrosis factor-α (TNF-α) and several classes of proteases, which are believed to contribute to the delayed or extended neuronal degeneration (1Csuka E. Hans V.H. Ammann E. Trentz O. Kossmann T. Morganti-Kossmann M.C. Neuroreport. 2000; 11: 2587-2590Crossref PubMed Scopus (93) Google Scholar, 2Popovich P.G. Prog. Brain Res. 2000; 128: 43-58Crossref PubMed Scopus (96) Google Scholar, 3Stoll G. Jander S. Schroeter M. Prog. Neurobiol. 1998; 56: 149-171Crossref PubMed Scopus (709) Google Scholar, 4McGeer E.G. McGeer P.L. Exp. Gerontol. 1998; 33: 371-378Crossref PubMed Scopus (303) Google Scholar, 5Benveniste E.N. J. Mol. Med. 1997; 75: 165-173Crossref PubMed Scopus (475) Google Scholar). Since neurons are largely irreplaceable, microglial-based inflammatory damage in the CNS has attracted considerable attention recently. Therefore, efforts to understand the fundamental mechanisms of microglial activation, and unique proinflammatory mediators, may identify novel therapeutic strategies to eliminate microglial deleterious effects. Among a myriad of microglial regulatory factors, the multi-functional serine protease, thrombin, is a recently discovered microglial activator. Prothrombin is highly concentrated in blood, circulating at micromolar levels (6Fenton J.W.d. Ann. N. Y. Acad. Sci. 1986; 485: 5-15Crossref PubMed Scopus (193) Google Scholar). Prothrombin activation and extravasation of active thrombin into CNS parenchyma have been implicated in a number of CNS disorders such as TBI, stroke, ischemia, AD, and ALS (7Akiyama H. Ikeda K. Kondo H. McGeer P.L. Neurosci. Lett. 1992; 146: 152-154Crossref PubMed Scopus (166) Google Scholar, 8Festoff B.W. Smith R.A. Handbook of Amyotrophic Lateral Sclerosis. Vol. 12. Marcel Dekker, New York1992: 661-685Google Scholar, 9Cunningham D.D. Pulliam L. Vaughan P.J. Thromb. Haemost. 1993; 70: 168-171Crossref PubMed Scopus (61) Google Scholar, 10Nishino A. Suzuki M. Ohtani H. Motohashi O. Umezawa K. Nagura H. Yoshimoto T. J. Neurotrauma. 1993; 10: 167-179Crossref PubMed Scopus (212) Google Scholar, 11Ho G.J. Smirnova I.V. Akaaboune M. Hantai D. Festoff B.W. Biomed. Pharmacother. 1994; 48: 296-304Crossref PubMed Scopus (31) Google Scholar, 12Lee K.R. Kawai N. Kim S. Sagher O. Hoff J.T. J. Neurosurg. 1997; 86: 272-278Crossref PubMed Scopus (346) Google Scholar). It has long been known that thrombin is a proinflammatory agent in other tissues (13Strukova S.M. Biochemistry (Mosc). 2001; 66: 8-18Crossref PubMed Scopus (79) Google Scholar) but it also can induce infiltration of inflammatory cells, edema, and reactive gliosis in the CNS in vivo (10Nishino A. Suzuki M. Ohtani H. Motohashi O. Umezawa K. Nagura H. Yoshimoto T. J. Neurotrauma. 1993; 10: 167-179Crossref PubMed Scopus (212) Google Scholar). However, specific demonstration of direct activation of microglial cells by thrombin was only reported recently (14Moller T. Hanisch U.K. Ransom B.R. J. Neurochem. 2000; 75: 1539-1547Crossref PubMed Scopus (160) Google Scholar, 15Ryu J. Pyo H. Jou I. Joe E. J. Biol. Chem. 2000; 275: 29955-29959Abstract Full Text Full Text PDF PubMed Google Scholar, 16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). From the therapeutic point of view, systemic inhibition of the thrombin proteolytic activity is certain to induce severe anti-coagulation side effects, possibly even fatal bleeding in brain and elsewhere (17Festoff B.W. Mattson M.P. Neuroprotective Signal Transduction. Humana Press, Totowa, NJ1997: 221-241Google Scholar). Therefore, identification of specific cellular/molecular target(s) mediating thrombin effect holds the key for developing safe and effective alternative therapeutic strategies. Thrombin-induced cellular effects are primarily mediated by means of G-protein-coupled receptors (GPCRs) known as protease-activated receptors (PARs) (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar). Among four PARs (PAR1–4) identified so far, thrombin activates PAR1, PAR3, and PAR4, but not PAR2 (19Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2680) Google Scholar, 20Ishihara H. Connolly A.J. Zeng D. Kahn M.L. Zheng Y.W. Timmons C. Tram T. Coughlin S.R. Nature. 1997; 386: 502-506Crossref PubMed Scopus (804) Google Scholar, 21Xu W.F. Andersen H. Whitmore T.E. Presnell S.R. Yee D.P. Ching A. Gilbert T. Davie E.W. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6642-6646Crossref PubMed Scopus (759) Google Scholar, 22Nystedt S. Emilsson K. Wahlestedt C. Sundelin J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9208-9212Crossref PubMed Scopus (833) Google Scholar). Regarding molecular mechanisms underlying thrombin-induced microglial activation, others have suggested that PAR1 is not involved in activation of microglia, using parameters such as inducible nitric-acid synthase (15Ryu J. Pyo H. Jou I. Joe E. J. Biol. Chem. 2000; 275: 29955-29959Abstract Full Text Full Text PDF PubMed Google Scholar). Our recent study demonstrated that although the prototypic PAR1 participates in thrombin-induced microglial proliferation, it does not mediate release of thrombin-induced potentially detrimental cytokines, such as TNF-α. This has cast in doubt the perspectives of PAR1 as the therapeutic target to exploit and suggested that more specific therapeutic target(s) remained to be identified. Since PAR3 has been suggested to function primarily as a chaperone for PAR4 rather than transducing signals on its own (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar, 23Sambrano G.R. Weiss E.J. Zheng Y.W. Huang W. Coughlin S.R. Nature. 2001; 413: 74-78Crossref PubMed Scopus (457) Google Scholar), we have concentrated on determining just how PAR4 might participate in thrombin-induced microglial activation in this study. The evidence provided here indicates that persistent PAR4 intracellular signaling is largely responsible for thrombin-induced TNF-α release in microglia. The mechanisms involved include prolonged phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs) and subsequent NFκB activation. Materials—Recombinant human α-thrombin was a gift from John Fenton, II, Ph.D. (Wadsworth Public Health Laboratories, Albany, NY). Synthetic PAR1AP (SFFLRN) and from PAR4 and by to PAR4 was from and p44/42 from and from TNF-α and a TNF-α monoclonal from to was a gift from E. of and from from from The was from from and and other used from and microglial was a gift from and microglial cultures from the of and as we previously Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). we in and microglial cells with and also conditions in the cells and in and to the on at and was with of and in a with of Foster at for for by of for 1 and for 1 and with and and with the of a and to the 1 in for at and with and The of in to the and the with human α-thrombin PAR1AP and thrombin, and with with and in a rat the and the a was by a into the using of and to Brain G. C. The Brain in Press, Scholar). The to the was the and with by the for and with a of and with to TNF-α by and The with and a and was as previously Z. C. M. Brain Res. 1997; PubMed Scopus Google Scholar). Western for PAR4 of the PAR4 and MAPK, and of protein as previously Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). [Ca2+]i in response to of thrombin, PAR1AP and in microglial cultures with a as previously Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). and from microglial cells as previously Mol. PubMed Scopus Google Scholar). cells in and by in with protease and and by with a at at for and in of of in was and by with at protein of by the assay. EMSA, for at with and by of of oligonucleotide K. M. D. G. U. A. J. 2000; PubMed Scopus Google Scholar) specific for and at for by 1 of and the on a and by a of TNF-α production into microglial was and as previously Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). was at from TNF-α levels in for and was are as and by using of means using or PAR4 or not PAR4 is involved in thrombin-induced microglial activation, we PAR4 was in microglia. this we used both microglial cultures from with a of as we previously reported Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar), as well as a microglial and microglia cultured in normal and the by with The results in 1 indicated that both and microglial cells levels of PAR4 In addition, we have also PAR4 expression in the microglial cells of other others have recently reported in that and other and microglial cells PAR4 T. S. Y. J. Ransom B.R. Hanisch U.K. for Neurosci. 2002; Scholar). Therefore, these studies show that murine microglial cells PAR4 at both and protein Thrombin-induced TNF-α in that murine microglia PAR4, we the effects of on microglial TNF-α this microglial cultures with of human or for as indicated TNF-α production in the by that both thrombin and but not induced TNF-α release from the microglial peptide the as but in a not induce increase of TNF-α even at in a not These results indicated that but not was to the thrombin-induced microglial TNF-α production in and in this in vitro and it in we α-thrombin PAR1AP or into rat with the of a to the the brain for with a microglial with a to TNF-α We that thrombin induced microgliosis with to the of the reactive microglial cells for TNF-α. In although PAR1AP induced a microglial proliferation, we to cells with this the other we to that induced a even than that with indicated that PAR1AP induced a increase of microglial both thrombin and induced increase of microglial cells with These results that both thrombin and but not to induce microgliosis in vivo. with in vitro Therefore, these novel that can thrombin-induced microglial TNF-α production both in vitro and in vivo. Down-regulation of PAR4 and PAR4AP-induced has been to PAR4 in other Weiss E.J. Huang W. Coughlin S.R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), to that PAR4AP-induced TNF-α production in microglial cells was mediated activation of PAR4, we to PAR4 and TNF-α this we oligonucleotide that the of the murine PAR4 as well as a oligonucleotide with a to of microglial cells, of PAR4 a of PAR4 expression induced by the In the at a to the used for to show effect we the of the to effect on PAR4 PAR4 remained 1 not was In addition, with a of expression The results that the of PAR4 expression is not to or response to the In with the PAR4 the PAR4 microglial cells with a of thrombin or for TNF-α production in the was by We that the PAR4 induced a of TNF-α induced by thrombin and In the PAR4 effects on TNF-α induced by thrombin or These results that activation of PAR4 mediated effects of thrombin and on microglial TNF-α PAR4 and p44/42 both PAR1 and PAR4 to be and in microglia, we to these thrombin receptors might mediate TNF-α from indicated that activation, PAR1 was PAR4 was much more than PAR1 Weiss E.J. Coughlin S.R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). We have recently that, microglial PAR1 is by G-protein-coupled receptor and a to agonist but PAR4 may in microglial cells. M. A. G. T. and W. in the of these receptors might be responsible for we PAR1 and PAR4 signaling in of [Ca2+]i is such PAR activation. microglial cells with a increase of [Ca2+]i was by and of [Ca2+]i to with induced a although increase of [Ca2+]i by a to It remained at the of the The induced by PAR1AP and indicated that the for the to receptors was not However, the that these receptors of [Ca2+]i induced by thrombin, which are to both PAR1 and PAR4, a that to a of effects of both PAR1 and PAR4 a and a of a by a these suggested that PAR4 activation induced a prolonged [Ca2+]i increase as to PAR1 activation, the that PAR4 is than In to [Ca2+]i we have previously that PAR1 activation in microglial cells induced a p44/42 activation with the at Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). PAR1 and PAR4 signaling, we levels of and p44/42 in microglial cells at We that thrombin and PAR1AP as well as induced of p44/42 at However, p44/42 phosphorylation to and PAR4AP-induced p44/42 phosphorylation remained elevated at These results suggested that as with PAR1 signaling, PAR4 activation not only induced a prolonged [Ca2+]i increase but also a of p44/42 activation, which the of PAR4 in to PAR1 in microglial cells. PAR4 p44/42 NFκB activation has previously been implicated in thrombin-induced rat microglial production (15Ryu J. Pyo H. Jou I. Joe E. J. Biol. Chem. 2000; 275: 29955-29959Abstract Full Text Full Text PDF PubMed Google Scholar). the potential of PAR1 and PAR4 signaling on NFκB activation, microglial cells with thrombin PAR1AP and for NFκB activation was by We that both thrombin and but not activated the of p44/42 in the NFκB activation induced by thrombin or microglial cells with thrombin or with the p44/42 from 1 to of for the We that inhibition of p44/42 by was to NFκB activation induced by thrombin or These results implicated that thrombin and activated NFκB at in part the prolonged p44/42 activation. of p44/42 or NFκB and PAR4AP-induced TNF-α in with the NFκB activation the of p44/42 and NFκB in thrombin-induced microglial activation at the with TNF-α as the this microglial cells with thrombin or with the NFκB or with from 1 to of TNF-α into the at was by We that inhibition of NFκB by TNF-α induced by thrombin or In addition, inhibition of p44/42 by at but not at and reduced TNF-α induced by thrombin or These results suggested that PAR4 signaling prolonged p44/42 activation and subsequent NFκB activation to the thrombin-induced microglial TNF-α In this study, we demonstrate that murine microglial cells PAR4 at both protein and a agonist peptide the thrombin-induced TNF-α production both in vitro and in vivo. The of the on TNF-α induction the of the The inhibition of the effects of both thrombin and on TNF-α induction of PAR4 with oligonucleotide demonstrated the of PAR4 in and PAR4AP-induced TNF-α Therefore, we that PAR4 is at of the receptors that mediates the thrombin-induced TNF-α production in both and rat microglia. of PAR4 with the PAR4 oligonucleotide at 1 for of the thrombin-induced TNF-α increase This that PAR4 is to be the not the receptor that mediates the thrombin-induced microglial TNF-α to for PAR4 in this such as microglial cells from M. Zheng Y.W. Weiss E.J. Coughlin S.R. Nature. 2000; PubMed Scopus Google Scholar). From report on PAR1 in microglia Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar), and the results with PAR4, we have now demonstrated that both PARs are and in murine microglial cells. Our indicates that PAR3 is also present in murine microglial cells as well not others have reported for four PARs in murine microglia T. S. Y. J. Ransom B.R. Hanisch U.K. for Neurosci. 2002; Scholar), and results this Since thrombin is known to of these PARs (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar), the of thrombin in microglial cells may on both thrombin that might from a or by brain M. M. E. D. D. 1991; Full Text PDF PubMed Scopus Google Scholar, M.C. Brain Res. Mol. Brain Res. 1991; 11: PubMed Scopus Google Scholar), and PAR1, PAR3 and PAR4. Since the thrombin and PARs are of thrombin may only PAR1 and PAR3 thrombin may PARs (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar, S.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). activation of PAR3 does not to intracellular but rather (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar, 23Sambrano G.R. Weiss E.J. Zheng Y.W. Huang W. Coughlin S.R. Nature. 2001; 413: 74-78Crossref PubMed Scopus (457) Google Scholar, M. Zheng Y.W. Weiss E.J. Coughlin S.R. Nature. 2000; PubMed Scopus Google Scholar), the of thrombin in microglial cells may be primarily by PAR1 and PAR4 signaling we previously PAR1 appears to primarily participate in thrombin-induced microglial proliferation Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar). In this study, of PAR1AP into rat brain only induced a increase of microglial cells that for TNF-α Therefore, we that activation of microglial PAR1 may not produce inflammatory damage to the the the of induced a microgliosis and neuronal to that PAR4 is the principal for potentially detrimental effects of These results may have therapeutic that for cellular in systemic inhibition of the thrombin proteolytic activity even bleeding for potential therapeutic effect (17Festoff B.W. Mattson M.P. Neuroprotective Signal Transduction. Humana Press, Totowa, NJ1997: 221-241Google Scholar). PAR1 is the PAR to thrombin, point to PAR4 as the and the specific proinflammatory receptor to using PAR4 In addition, PAR3 may thrombin to PAR4 it may also have a potential therapeutic a although is that it not ultimate expression for PARs have been for human and (18Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2149) Google Scholar, S.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Therefore, potential for microglial of PARs from murine results to be to human microglia. be Our in vitro results show that thrombin induced more TNF-α production than and However, thrombin than that used in vitro induced a which was much than the effect by This that the effects of thrombin in vivo may be This inhibition of the thrombin effect may be to several the of protease such as protease and other in may the we used may for the in vivo mechanisms to or a of other potential thrombin or receptors that are in microglial cultures may with PARs for to thrombin in vivo. such receptor is the that thrombin with a that we active on brain A. Hantai D. M. Festoff B.W. 1997; PubMed Scopus Google Scholar) and which may be for neurons K. J. M. T. T. I. I. Thromb. Haemost. PubMed Scopus Google Scholar). may be much thrombin to and PAR4. of these not have on which may induced more microgliosis than thrombin in vivo in this study. a although induced prolonged [Ca2+]i increase than PAR1AP in microglial cells we also that previously reported in other such as to be more delayed and prolonged Weiss E.J. Coughlin S.R. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). might from PAR4 expression levels in microglial and cells might be the mechanisms of PAR4 in these cells might also the the study indicated that PAR4 activation in in of PAR4 phosphorylation to be by or other However, results show that PAR4 activation in microglial cells induced a and rather than increase of PAR4 to and This indicates that microglial PAR4, PAR4, may be by the involved cellular thrombin study also that, with PAR1, the PAR4 signaling in microglia is with prolonged [Ca2+]i increase and p44/42 activation as well as subsequent NFκB activation. This may PAR1 primarily to promoting microglial proliferation PAR4 is in microglia and These results are with previously reported thrombin signaling in microglia (14Moller T. Hanisch U.K. Ransom B.R. J. Neurochem. 2000; 75: 1539-1547Crossref PubMed Scopus (160) Google Scholar, 15Ryu J. Pyo H. Jou I. Joe E. J. Biol. Chem. 2000; 275: 29955-29959Abstract Full Text Full Text PDF PubMed Google Scholar, 16Suo Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar), more and identification of mediators. with study for PAR1 Z. Wu M. Ameenuddin S. Anderson H.E. Zoloty J.E. Citron B.A. Andrade-Gordon P. Festoff B.W. J. Neurochem. 2002; 80: 655-666Crossref PubMed Scopus (165) Google Scholar), we now a for how thrombin microglial proliferation and activation. Since thrombin is involved in the pathogenesis of a of CNS disorders and potentially involved in certain disorders such as Alzheimer's these not only the underlying mechanisms of thrombin-induced microglial activation, but also in therapeutic