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An elusive receptor is finally caught: P2Y12, an important drug target in platelets
388
Research Update
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
Research News
An elusive receptor is finally caught: P2Y12, an important drug target in platelets Eric A. Barnard and Joseph Simon Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y12, that acts with the P2Y1 receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X1 receptor ion channel, contributes to platelet activation.
Activation and aggregation of platelets occur through a complex series of reactions initiated by ADP. An ADPselective receptor of the platelet was postulated and termed the ‘P2T receptor’ (‘T’ for thrombocyte), as one of the P2 receptors at which extracellular nucleotides act (reviewed in Refs 1,2). This P2T receptor has been widely reported1 to signal through several platelet messenger systems: (1) inhibition of stimulated adenylyl cyclase; (2) mobilization of intracellular Ca2+; (3) initial rapid influx of Ca2+; and (4) increase of inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] production. Thus, it was unclear1 whether the P2T receptor represented a single entity or a combination of receptors. Clarifications from recombinant receptors
A new perspective in this field came from the cloning of a series of G-protein-coupled nucleotide receptors3–5, designated P2Y1, P2Y2, etc. The human P2Y1 receptor6 protein was detected in platelets2,7. DNA cloning also identified another series of nucleotide receptors: seven P2X ion channels that are directly gated by ATP (Ref. 8). The P2X1 receptor was also detected in platelets2. Despite the fact that ADP is an extremely weak P2X1 receptor agonist, ADP-evoked rapid Ca2+ entry in platelets was attributed to this receptor. However, Mahaut-Smith et al.9 have now clarified this discrepancy: ADP, as used in http://tips.trends.com
earlier platelet studies, contains enough ATP to activate P2X1 receptors. This modifies the accepted model of ADP alone initiating physiological platelet activation because ATP might also be involved. Molecular characterization of the P2X1 receptor8 shows that this receptor cannot account for the inhibition of adenylyl cyclase, a nucleotide response that is always found in platelets. The P2Y1 receptor present in platelets generally couples, elsewhere, to Ins(1,4,5)P3, but its agonist specificity is also surprisingly similar to that of the adenylyl cyclase response10,11. However, receptor promiscuity was finally excluded by employing potent specific antagonists [i.e. ARC66096, its derivatives or (in vivo) clopidogrel for a separate cyclase-inhibitory P2Y receptor and adenosine bis-monophosphates for the P2Y1 receptor2,10–12]. It was concluded that the ‘P2T receptor’ is actually a composite, and the simultaneous activation of the P2Y1 receptor and the cyclase inhibitory receptor (and perhaps P2X1) is required for platelet aggregation2,7,12,13. With respect to the P2X1 receptor, it was reported that activation by its specific agonist α,β-methyleneATP did not elicit human platelet aggregation or the associated shape change (the manifestation of a fast reorganization of actin filaments), or affect the activation of P2X1 receptors by ADP (Ref. 2). However, during platelet isolation some ATP release occurs; it is known that the P2X1 receptor can be very rapidly desensitized8, and there is now evidence that, when this is avoided, α,β-methyleneATP evokes a P2X1-receptor-like Ca2+ influx and shape change in platelets14. Recently, a patient with a dominant-negative mutation in the platelet P2X1 receptor was reported15, with a bleeding disorder and impairment (although noted without detail) of ADP-induced platelet aggregation. Hence, a synergistic role for this receptor in platelet aggregation merits investigation.
Clarifications from the results of gene knockouts
Targeted gene disruption is currently widely used to test conclusions drawn on the in vivo roles of receptors. In such mice that lack the P2Y1 receptor, platelets do not aggregate in response to physiological concentrations of ADP, and ADP does not produce the usual shape change16,17. These mice also exhibit a prolonged bleeding time. It is of potential pharmaceutical interest that mice that lack the P2Y1 receptor acquired resistance to experimental thromboembolism. Furthermore, and crucially, ADP no longer induces intracellular Ca2+ mobilization in platelets, although its cyclase inhibition response is unimpaired. These results demonstrate a role in vivo of two platelet receptors, the P2Y1 receptor and a separate, but collaborating, cyclaseinhibitory ADP receptor16,17. Mutant mice that lack the α-subunit of the Gq protein have also been studied18. (Platelets do not contain the related G11, which can usually substitute for Gq.) Changes (similar to those noted above in mice that lack P2Y1 receptors) from the normal platelet responses to ADP are produced in the absence of Gq, and a total loss of the normal increase in Ins(1,4,5)P3 is also observed. Together, these results demonstrate that the P2Y1 receptor acts through Gαq and phospholipase C β to initiate aggregation. The missing receptor for ADP
What is the receptor that responds to ADP with inhibition of stimulated adenylyl cyclase and is regarded as one component of the previous P2T receptor? This receptor corresponds to none of the known P2Y receptors and, as a G-protein-coupled receptor for ADP known only through its function, a baffling series of names for the receptor – P2TAC,P2TAC, P2T , P2YT , P2YADP, P2Ycyc and P2YAC – have been coined by various authors. This receptor has frequently been described in the recent literature as an elusive protein. A recent meeting report in TiPS19 on P2Y receptors
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Research Update
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
389
Table 1. Agonist or antagonist potencies of adenosine diphosphates and triphosphates acting on native or recombinant P2Y12 receptorsa Agentb
2-MeSADP ADP 2-MeSATP ATP 2-ClATP ATPγS
Agonist (EC50, nM)
Antagonist (Ki, nM)
Rat B10 cellse Agonist (EC50, nM)
Rat C6-2B cellsf Agonist (EC50, nM)
hP2Y12 Agonist (EC50, nM)
100c 2000c − − − −
− − 100c 12 600c 31 000c −
2 3100 3.5 26 000 13 500 −
2 4500 4 30 000 8300 −
1g 300g − − − −
Human platelets
32d 470d − − − −
− − 63d 6200d 29 000d −
14h 60h 3.4h − 636h 110h
aNote
that the values from platelet aggregation will be determined by the human (h) P2Y12 plus the hP2Y1 receptor and hence are not comparable with any values from the cyclase inhibition.The potency values cannot be compared between the rat and human cases even for the same assay (cyclase inhibition), as a result of species and cellular environment differences, but they show that in both cases the triphosphates, outside the platelet, are agonists. bAbbreviations: 2-ClATP, 2-chloroATP; 2-MeSATP, 2-methylthioATP. cNucleotide-induced platelet aggregation. dAdenylyl cyclase inhibition or antagonism of the ADP-induced inhibition13,34. eAdenylyl cyclase inhibition in B10 cells11. fAdenylyl cyclase inhibition in C6-2B cells35. gIn Xenopus oocytes that are equipped with Kir3.1/3.4 channels to enable measurement of G -linked receptor expression and that contain heterologously expressed hP2Y i 12 receptors20. hAdenylyl cyclase inhibition in Chinese hamster ovary (CHO) cells that heterologously express the hP2Y receptor21. 12
notes that ‘to the frustration of many researchers the P2TAC receptor has so far resisted all cloning efforts’. In part, this was due to the poverty, as a source, of the platelet, which has no mRNA synthesis. However, now two teams have independently reported the sequence and expression of a new P2Y receptor with the expected properties and presence in platelets20,21. This receptor is clearly the missing receptor of the P2T receptor system. When this receptor was expressed in Chinese hamster ovary (CHO) cells, with forskolin stimulation of cAMP accumulation, both studies showed a clear inhibition thereof by ADP. The response to ADP was abolished, in those cells and in an oocyte expression system (Table 1), by known antagonists of the cyclaseinhibitory ADP receptor of platelets, but not by an adenosine bis-monophosphate P2Y1-receptor-specific antagonist20,21. G-protein specificity was probed by Zhang et al.21: following coexpression of the receptor with chimeric Gα-subunits, coupling occurred strongly through Gαi, less through Gαo and not through the s, q, 12, 16 or z isoforms of Gα. This receptor has now been designated the P2Y12 receptor20 as the latest in the P2Y receptor numbering system, a system that is, in part, arbitrary because not all of the previous 11 are known to be functional nucleotide receptors; however, at present the system is used as a generally recognized reference. Hollopeter et al.20 started with rat platelet mRNA and used an expression cloning strategy in oocytes (Table 1) to obtain the rat P2Y12 receptor and hence http://tips.trends.com
the human P2Y12 receptor. Zhang et al.21 screened expressed orphan G-proteincoupled human receptors using fractionated tissue extracts to search for endogeneous matching ligands; the screen employed Gαq chimeras constructed with a range of Gα subtypes to detect by Ca2+ mobilization any type of coupling. An orphan receptor responded to a small molecule, eventually identified as ADP, and gave ‘P2TAC receptor’ behaviour (Table 1). The sequence is identical to that of the human P2Y12 receptor of Hollopeter et al.20 Ironically, the P2Y12 receptor sequence has in fact been available without recognition for several years, being patented by Human Genome Sciences Inc. in 1998 (patent WO 98/50549). Thus, three company laboratories (and academic collaborators) have arrived at this clone in different ways, which will doubtless make for an interesting outcome. P2Y receptors – a wider family than was previously thought
The P2Y12 receptor protein sequence is exceptionally divergent from the rest of the P2Y receptors (Fig. 1). It has only 17% identity with the P2Y1 receptor and, surprisingly, is much more closely related (45%) to a receptor22 for UDP-glucose, which had not previously been regarded as a member of the P2Y receptor family. This explains why P2Y12 receptor cloning using P2Y receptor family homology, although much attempted, had failed. Some non-nucleotide receptors [e.g. platelet-activating factor (PAF) and mu opioid peptide] are distinctly closer to the P2Y12 receptor than all except one (P2Y5)
of the other P2Y receptors (Fig. 1). We must now recognize that the P2Y receptor family is structurally very wide (but not without precedent, as in the equally great difference of the histamine H3 receptor23 from H1 and H2 receptors). Other nucleotide-sugar receptors and analogues thereof should now be considered for P2Y receptor properties. Two orphan receptors are also in the branch that contains the P2Y12 receptor (Fig. 1) and merit investigation for activity on any nucleotide-containing molecule. The nucleotide-binding P2Y5 receptor (of activated T cells24) had been questioned as being too dissimilar in sequence to be a P2Y receptor, but its sequence (human or chicken) is now also shown to lie in this new branch of the family (Fig. 1). In transfected cell lines, the P2Y5 receptor does not show a nucleotide response through Ins(1,4,5)P3 or cAMP but this does not exclude other types of transduction, and the human P2Y5 receptor has recently been shown25 to give a functional response to ATP, of atypical character, in oocyte expression. Furthermore, Hollopeter et al.20 mapped the gene encoding the P2Y12 receptor to the locus q24–25 on chromosome 3, which also contains the gene encoding the UDP-glucose receptor (its closest known relative)22 and the gene encoding the P2Y1 receptor6. This clustering suggests that a relatively recent gene duplication led to the evolution of the different activities of those receptors20. A family with a bleeding disorder and impaired platelet aggregation was shown20 to harbour a
Research Update
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TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
P2Y12 UDP-glucose Orphan 1 Orphan 2 PAF MOP receptor P2Y5 P2Y11 P2Y1 P2Y4 tp2y cP2Y3 P2Y6 P2Y2 A1 350
300
250
200
150
100
50
0
TRENDS in Pharmacological Sciences
Fig. 1. A dendrogram showing the relationships among cloned P2Y receptor subtypes (only human receptors are shown, except the avian P2Y3 and tp2y receptors). Comparison is also made with the functionally unrelated human G-protein-coupled receptors (GPCRs) closest in sequence to the P2Y12 receptor. The length of each pair of branches represents the amino acid sequence distance between those receptors. The horizontal axis plots the number of substitution events. The homology between P2Y12 and the other P2Y receptor subtypes is as low as 14% (with the P2Y11 receptor). Orphan 1 and orphan 2 represent the H963 (accession number: AF002986) and GPR34 (accession number: AF118670) GPCRs, respectively, which are not yet functionally identified. The human adenosine A1 receptor sequence is used as an outgroup comparator. The nomenclature of the receptors is as given by the original authors in each case. The chicken (c) P2Y3 and the human P2Y5 receptors are retained in upper-case letters because recently King and Townsend-Nicholson25 have found functional responses to ATP at the recombinant P2Y5 receptor expressed in Xenopus oocytes and the recombinant cP2Y3 receptor has been characterized functionally36, although its mammalian orthologue has not yet been studied. Note that the only P2Y receptor subtype other than P2Y12 that is known to couple through Gi negatively to adenylyl cyclase, tp2y from the turkey37, is nevertheless structurally very distant from the P2Y12 receptor. Abbreviations: MOP, mu opioid peptide; PAF, platelet-activating factor.
two-base deletion in the coding region of the gene encoding the P2Y12 receptor, again confirming its function in vivo. Occurrence of the P2Y12 receptor outside the platelet
It has frequently been stated in the literature that the ‘P2T receptor’ is found only in the platelet lineage. However, the orphan clone that became the P2Y12 receptor21 was derived from human hypothalamus mRNA, and the P2Y12 receptor mRNA has now been shown to be highly expressed in both the spinal cord and the brain20,21. In situ hybridization suggested a glial location20, but its presence in neurones in some regions was not excluded. P2Y12 receptor mRNA occurs in most brain regions and is particularly strong in substantia nigra, caudate-putamen, thalamus and temporal cortex20,21. Interestingly, in these regions, the P2Y1 receptor transcript and protein are also strongly expressed in both neurones and glia5,26,27. The same association of these two receptors as occurs in the platelet might represent a common functional system. http://tips.trends.com
P2Y12 receptor mRNA was not detected in northern blots in other organs20,21, except at very low levels, which might be due to the blood content of these organs. However, a receptor with similar properties occurs in the endothelial cells of some blood vessels. Thus, a cyclaseinhibitory ADP response has been studied in capillary endothelial cells of the blood–brain barrier28. A clonal cell line (B10) formed spontaneously from those cells in culture is a good source of this activity and also expresses P2Y1 receptor29 mRNA and protein. The nucleotide specificity of the ADP-induced inhibition of stimulated adenylyl cyclase in B10 endothelial cells11 is similar to that now reported for the P2Y12 receptor (Table 1). In a rat glial cell line, C6-2B, known to contain a similar activity30, the nucleotide specificity is identical to that in B10 cells (Table 1). The potencies are distinctly lower in B10 and C6-2B cells than with heterologously expressed P2Y12 receptors for the weaker agonists, but this might reflect a difference in density of the same receptor11. In both cell lines, cloning has confirmed (J. Simon and E.A. Barnard,
unpublished) the expression of the rat P2Y12 receptor. The agony of ATP agonism
One of the defining characteristics of the ‘P2T receptor’ of platelets has historically been that the native agonist is ADP, whereas ATP and its triphosphate derivatives are antagonists. This has been reported consistently for the aggregation response and equally for the inhibition of adenylyl cyclase. Typical values for that agonist–antagonist division of adenine nucleotides are shown in Table 1. These values are not compromised by nucleotide metabolism or impurity because the behaviour on platelets is reproduced in recent studies where this is eliminated10,13. However, all of the triphosphates tested are agonists at the expressed P2Y12 receptor20,21 (Table 1). A striking case is that of 2-methylthioATP (2-MeSATP), which is a very potent antagonist of ADP-dependent cyclase inhibition in the platelet but is the strongest agonist at recombinant P2Y12 receptors. Such a difference is interesting because the same reversal from the behaviour observed in the platelet was recently reported for this receptor in its native expression in B10 endothelial cells11, and likewise occurs with this receptor in C6-2B glioma cells (Table 1). Hence, it is not an artefact of ectopic expression of the recombinant P2Y12 receptor. An explanation was proposed for the B10 cells11 in terms of a high sensitivity of this receptor activity to its density on the cell and a receptor reserve, as deduced in certain other cases31, including the P2Y1 receptor32,33, a phenomenon that is supported by similar findings with the cloned P2Y12 receptor. The density of the P2Y12 receptor in the platelet must be presumed to be lower than in its other locations considered here. However, alternatively a regulator of G-proteinlinked signalling might introduce the antagonism in the platelet. In summary, the question of the different responses evoked by ADP in the platelet is finally resolved by the identification of the missing player, the P2Y12 receptor. This resolution also embraces the problem of antagonism by ATP and the triphosphates in platelet responses to ADP: this phenomenon is not to be regarded now as an intrinsic property of the P2Y12 receptor.
Research Update
References 1 Boarder, M.R. and Hourani, S.M.O. (1998) The regulation of vascular function by P2 receptors: multiple sites and multiple receptors. Trends Pharmacol. Sci. 19, 99–107 2 Kunapuli, S.P. (1998) Multiple P2 receptor subtypes on platelets: a new interpretation of their function. Trends Pharmacol. Sci. 19, 391–394 3 Webb, T.E. et al. (1993) Cloning and functional expression of a brain G protein-coupled ATP receptor. FEBS Lett. 324, 219–225 4 Lustig, K.D. et al. (1993) Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc. Natl. Acad. Sci. U. S. A. 90, 5113–5117 5 Barnard, E.A. et al. (1997) Nucleotide receptors in the nervous system: an abundant component using diverse transduction mechanisms. Mol. Neurobiol. 15, 103–129 6 Ayyanathan, K. et al. (1996) Cloning and chromosomal localization of the human P2Y1 purinoceptor. Biochem. Biophys. Res. Commun. 218, 783–788 7 Jantzen, H-M. et al. (1999) Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb. Haemost. 81, 111–117 8 North, R.A. and Surprenant, A. (2000) Pharmacology of cloned P2X receptors. Annu. Rev. Pharmacol. 40, 563–580 9 Mahaut-Smith, M.P. et al. (2000) ADP is not an agonist at P2X1 receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets. Br. J. Pharmacol. 131, 108–114 10 Hechler, B. et al. (1998) ATP derivatives are antagonists of the P2Y1 receptor: similarities to the platelet ADP receptor. Mol. Pharmacol. 53, 727–733 11 Simon, J. et al. (2001) Activity of adenosine diphosphates and triphosphates on a P2YT receptor in brain capillary endothelial cells. Br. J. Pharmacol. 132, 173–182 12 Jarvis, G.E. et al. (2000) ADP can induce aggregation of human platelets via both P2Y1 and P2T receptors. Br. J. Pharmacol. 129, 275–282 13 Park, H.S. and Hourani, S.M.O. (1999) Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adenosine 5′-diphosphate (ADP) in human platelets. Br. J. Pharmacol. 127, 1359–1366 14 Rolf, M.G. et al. (2001) Platelet shape change evoked by selective activation of P2X1
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purinoceptors with α,β-methylene ATP. Thromb. Haemost. 85, 303–308 Oury, C. et al. (2000) A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue. J. Biol. Chem. 275, 22611–22614 Fabre, J.E. et al. (1999) Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat. Med. 5, 1199–1202 Leon, C. et al. (1999) Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y1 receptor-null mice. J. Clin. Invest. 104, 1731–1737 Offermanns, S. et al. (1997) Defective platelet activation in Gαq-deficient mice. Nature 389, 183–186 Boeynams, J.M. et al. (2000) P2Y receptors: in the middle of the road. Trends Pharmacol. Sci. 21, 1–3 Hollopeter, G. et al. (2001) Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409, 202–207 Zhang, F.L. et al. (2001) ADP is the cognate ligand for the orphan G-protein coupled receptor SP1999. J. Biol. Chem. 276, 8608–8615 Chambers, J.K. et al. (2000) A G protein-coupled receptor for UDP-glucose. J. Biol. Chem. 275, 10767–10771 Lovenberg, T.W. et al. (1999) Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 55, 1101–1107 Webb, T.E. et al. (1996) Identification of 6H1 as a P2Y purinoceptor: P2Y5. Biochem. Biophys. Res. Commun. 219, 105–110 King, B.F. and Townsend-Nicholson, A. (2000) Recombinant P2Y receptors: the UCL experience. J. Auton. Nerv. Syst. 81, 164–170 Moore, D. et al. (2000) Regional and cellular distribution of the P2Y1 purinergic receptor in the human brain: striking neuronal localization. J. Comp. Neurol. 421, 374–384 Morán-Jimenez, M-J. and Matute, C. (2000) Immunohistochemical localization of the P2Y1 purinergic receptor in neurons and glial cells of the central nervous system. Mol. Brain Res. 78, 50–58 Feolde, E. et al. (1995) ATP, a partial agonist of atypical P2Y purinoceptors in rat brain microvascular endothelial cells. Br. J. Pharmacol. 115, 1199–1203 Webb, T.E. et al. (1996) The P2Y purinoceptors in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase. Br. J. Pharmacol. 119, 1385–1392
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30 Boyer, J.L. et al. (1993) Identification of a P2Y-purinergic receptor that inhibits adenylyl cyclase. J. Pharmacol. Exp. Ther. 267, 1140–1146 31 Kenakin, T.P. (1997) Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stochiometry. Trends Pharmacol. Sci. 18, 456–464 32 Palmer, R.K. et al. (1998) Agonist action of adenosine triphosphates at the human P2Y1 receptor. Mol. Pharmacol. 54, 1118–1123 33 Filippov, A.K. et al. (2000) The P2Y1 receptor closes the N-type Ca2+ channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists. Br. J. Pharmacol. 129, 1063–1066 34 Cusack, N.J. and Hourani, S.M.O. (1982) Adenosine 5-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors. Br. J. Pharmacol. 76, 221–227 35 Simon, J. et al. (1999) Characterization of putative P2T receptor endogenously expressed in rat glioma and brain microvascular endothelial cells. Fundam. Clin. Pharmacol. 13, 264s 36 Webb, T.E. et al. (1996) A novel G protein-coupled P2 purinoceptor (P2Y3) activated preferentially by nucleoside diphosphatases. Mol. Pharmacol. 50, 258–265 37 Boyer, J.L. et al. (2000) A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleotide triphosphates. Mol. Pharmacol. 57, 805–810
Eric A. Barnard* Joseph Simon Dept of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QJ. *e-mail: [email protected]
Chemical name ARC66096: 2-propylthioadenosine-5′(β,γ-difluoromethylene)triphosphonate
Lipoxins: revelations on resolution Blaithin McMahon, Siobhan Mitchell, Hugh R. Brady and Catherine Godson Lipoxins (LXs) are endogenously produced eicosanoids typically generated during cell–cell interactions. In this article, the compelling evidence from in vitro and in vivo model systems that LXs actively promote the resolution of inflammation is reviewed. Of particular interest are indications that stable http://tips.trends.com
synthetic analogues of LXs and aspirin-triggered 15-epi-LXs can mimic many of the desirable antiinflammatory, ‘pro-resolution’ actions of native LXs. Given the enhanced stability and efficacy of these compounds a role as novel anti-inflammatory therapeutics is proposed.
The dynamic regulation of leukocyte trafficking is an essential component of an effective host defence that can be subverted in inflammatory diseases and ischaemic injury. The resolution of inflammation depends on multiple processes that include the generation of endogenous ‘braking signals’ that
0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01771-5
Research Update
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
Research News
An elusive receptor is finally caught: P2Y12, an important drug target in platelets Eric A. Barnard and Joseph Simon Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y12, that acts with the P2Y1 receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X1 receptor ion channel, contributes to platelet activation.
Activation and aggregation of platelets occur through a complex series of reactions initiated by ADP. An ADPselective receptor of the platelet was postulated and termed the ‘P2T receptor’ (‘T’ for thrombocyte), as one of the P2 receptors at which extracellular nucleotides act (reviewed in Refs 1,2). This P2T receptor has been widely reported1 to signal through several platelet messenger systems: (1) inhibition of stimulated adenylyl cyclase; (2) mobilization of intracellular Ca2+; (3) initial rapid influx of Ca2+; and (4) increase of inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] production. Thus, it was unclear1 whether the P2T receptor represented a single entity or a combination of receptors. Clarifications from recombinant receptors
A new perspective in this field came from the cloning of a series of G-protein-coupled nucleotide receptors3–5, designated P2Y1, P2Y2, etc. The human P2Y1 receptor6 protein was detected in platelets2,7. DNA cloning also identified another series of nucleotide receptors: seven P2X ion channels that are directly gated by ATP (Ref. 8). The P2X1 receptor was also detected in platelets2. Despite the fact that ADP is an extremely weak P2X1 receptor agonist, ADP-evoked rapid Ca2+ entry in platelets was attributed to this receptor. However, Mahaut-Smith et al.9 have now clarified this discrepancy: ADP, as used in http://tips.trends.com
earlier platelet studies, contains enough ATP to activate P2X1 receptors. This modifies the accepted model of ADP alone initiating physiological platelet activation because ATP might also be involved. Molecular characterization of the P2X1 receptor8 shows that this receptor cannot account for the inhibition of adenylyl cyclase, a nucleotide response that is always found in platelets. The P2Y1 receptor present in platelets generally couples, elsewhere, to Ins(1,4,5)P3, but its agonist specificity is also surprisingly similar to that of the adenylyl cyclase response10,11. However, receptor promiscuity was finally excluded by employing potent specific antagonists [i.e. ARC66096, its derivatives or (in vivo) clopidogrel for a separate cyclase-inhibitory P2Y receptor and adenosine bis-monophosphates for the P2Y1 receptor2,10–12]. It was concluded that the ‘P2T receptor’ is actually a composite, and the simultaneous activation of the P2Y1 receptor and the cyclase inhibitory receptor (and perhaps P2X1) is required for platelet aggregation2,7,12,13. With respect to the P2X1 receptor, it was reported that activation by its specific agonist α,β-methyleneATP did not elicit human platelet aggregation or the associated shape change (the manifestation of a fast reorganization of actin filaments), or affect the activation of P2X1 receptors by ADP (Ref. 2). However, during platelet isolation some ATP release occurs; it is known that the P2X1 receptor can be very rapidly desensitized8, and there is now evidence that, when this is avoided, α,β-methyleneATP evokes a P2X1-receptor-like Ca2+ influx and shape change in platelets14. Recently, a patient with a dominant-negative mutation in the platelet P2X1 receptor was reported15, with a bleeding disorder and impairment (although noted without detail) of ADP-induced platelet aggregation. Hence, a synergistic role for this receptor in platelet aggregation merits investigation.
Clarifications from the results of gene knockouts
Targeted gene disruption is currently widely used to test conclusions drawn on the in vivo roles of receptors. In such mice that lack the P2Y1 receptor, platelets do not aggregate in response to physiological concentrations of ADP, and ADP does not produce the usual shape change16,17. These mice also exhibit a prolonged bleeding time. It is of potential pharmaceutical interest that mice that lack the P2Y1 receptor acquired resistance to experimental thromboembolism. Furthermore, and crucially, ADP no longer induces intracellular Ca2+ mobilization in platelets, although its cyclase inhibition response is unimpaired. These results demonstrate a role in vivo of two platelet receptors, the P2Y1 receptor and a separate, but collaborating, cyclaseinhibitory ADP receptor16,17. Mutant mice that lack the α-subunit of the Gq protein have also been studied18. (Platelets do not contain the related G11, which can usually substitute for Gq.) Changes (similar to those noted above in mice that lack P2Y1 receptors) from the normal platelet responses to ADP are produced in the absence of Gq, and a total loss of the normal increase in Ins(1,4,5)P3 is also observed. Together, these results demonstrate that the P2Y1 receptor acts through Gαq and phospholipase C β to initiate aggregation. The missing receptor for ADP
What is the receptor that responds to ADP with inhibition of stimulated adenylyl cyclase and is regarded as one component of the previous P2T receptor? This receptor corresponds to none of the known P2Y receptors and, as a G-protein-coupled receptor for ADP known only through its function, a baffling series of names for the receptor – P2TAC,P2TAC, P2T , P2YT , P2YADP, P2Ycyc and P2YAC – have been coined by various authors. This receptor has frequently been described in the recent literature as an elusive protein. A recent meeting report in TiPS19 on P2Y receptors
0165-6147/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(00)01759-4
Research Update
TRENDS in Pharmacological Sciences Vol.22 No.8 August 2001
389
Table 1. Agonist or antagonist potencies of adenosine diphosphates and triphosphates acting on native or recombinant P2Y12 receptorsa Agentb
2-MeSADP ADP 2-MeSATP ATP 2-ClATP ATPγS
Agonist (EC50, nM)
Antagonist (Ki, nM)
Rat B10 cellse Agonist (EC50, nM)
Rat C6-2B cellsf Agonist (EC50, nM)
hP2Y12 Agonist (EC50, nM)
100c 2000c − − − −
− − 100c 12 600c 31 000c −
2 3100 3.5 26 000 13 500 −
2 4500 4 30 000 8300 −
1g 300g − − − −
Human platelets
32d 470d − − − −
− − 63d 6200d 29 000d −
14h 60h 3.4h − 636h 110h
aNote
that the values from platelet aggregation will be determined by the human (h) P2Y12 plus the hP2Y1 receptor and hence are not comparable with any values from the cyclase inhibition.The potency values cannot be compared between the rat and human cases even for the same assay (cyclase inhibition), as a result of species and cellular environment differences, but they show that in both cases the triphosphates, outside the platelet, are agonists. bAbbreviations: 2-ClATP, 2-chloroATP; 2-MeSATP, 2-methylthioATP. cNucleotide-induced platelet aggregation. dAdenylyl cyclase inhibition or antagonism of the ADP-induced inhibition13,34. eAdenylyl cyclase inhibition in B10 cells11. fAdenylyl cyclase inhibition in C6-2B cells35. gIn Xenopus oocytes that are equipped with Kir3.1/3.4 channels to enable measurement of G -linked receptor expression and that contain heterologously expressed hP2Y i 12 receptors20. hAdenylyl cyclase inhibition in Chinese hamster ovary (CHO) cells that heterologously express the hP2Y receptor21. 12
notes that ‘to the frustration of many researchers the P2TAC receptor has so far resisted all cloning efforts’. In part, this was due to the poverty, as a source, of the platelet, which has no mRNA synthesis. However, now two teams have independently reported the sequence and expression of a new P2Y receptor with the expected properties and presence in platelets20,21. This receptor is clearly the missing receptor of the P2T receptor system. When this receptor was expressed in Chinese hamster ovary (CHO) cells, with forskolin stimulation of cAMP accumulation, both studies showed a clear inhibition thereof by ADP. The response to ADP was abolished, in those cells and in an oocyte expression system (Table 1), by known antagonists of the cyclaseinhibitory ADP receptor of platelets, but not by an adenosine bis-monophosphate P2Y1-receptor-specific antagonist20,21. G-protein specificity was probed by Zhang et al.21: following coexpression of the receptor with chimeric Gα-subunits, coupling occurred strongly through Gαi, less through Gαo and not through the s, q, 12, 16 or z isoforms of Gα. This receptor has now been designated the P2Y12 receptor20 as the latest in the P2Y receptor numbering system, a system that is, in part, arbitrary because not all of the previous 11 are known to be functional nucleotide receptors; however, at present the system is used as a generally recognized reference. Hollopeter et al.20 started with rat platelet mRNA and used an expression cloning strategy in oocytes (Table 1) to obtain the rat P2Y12 receptor and hence http://tips.trends.com
the human P2Y12 receptor. Zhang et al.21 screened expressed orphan G-proteincoupled human receptors using fractionated tissue extracts to search for endogeneous matching ligands; the screen employed Gαq chimeras constructed with a range of Gα subtypes to detect by Ca2+ mobilization any type of coupling. An orphan receptor responded to a small molecule, eventually identified as ADP, and gave ‘P2TAC receptor’ behaviour (Table 1). The sequence is identical to that of the human P2Y12 receptor of Hollopeter et al.20 Ironically, the P2Y12 receptor sequence has in fact been available without recognition for several years, being patented by Human Genome Sciences Inc. in 1998 (patent WO 98/50549). Thus, three company laboratories (and academic collaborators) have arrived at this clone in different ways, which will doubtless make for an interesting outcome. P2Y receptors – a wider family than was previously thought
The P2Y12 receptor protein sequence is exceptionally divergent from the rest of the P2Y receptors (Fig. 1). It has only 17% identity with the P2Y1 receptor and, surprisingly, is much more closely related (45%) to a receptor22 for UDP-glucose, which had not previously been regarded as a member of the P2Y receptor family. This explains why P2Y12 receptor cloning using P2Y receptor family homology, although much attempted, had failed. Some non-nucleotide receptors [e.g. platelet-activating factor (PAF) and mu opioid peptide] are distinctly closer to the P2Y12 receptor than all except one (P2Y5)
of the other P2Y receptors (Fig. 1). We must now recognize that the P2Y receptor family is structurally very wide (but not without precedent, as in the equally great difference of the histamine H3 receptor23 from H1 and H2 receptors). Other nucleotide-sugar receptors and analogues thereof should now be considered for P2Y receptor properties. Two orphan receptors are also in the branch that contains the P2Y12 receptor (Fig. 1) and merit investigation for activity on any nucleotide-containing molecule. The nucleotide-binding P2Y5 receptor (of activated T cells24) had been questioned as being too dissimilar in sequence to be a P2Y receptor, but its sequence (human or chicken) is now also shown to lie in this new branch of the family (Fig. 1). In transfected cell lines, the P2Y5 receptor does not show a nucleotide response through Ins(1,4,5)P3 or cAMP but this does not exclude other types of transduction, and the human P2Y5 receptor has recently been shown25 to give a functional response to ATP, of atypical character, in oocyte expression. Furthermore, Hollopeter et al.20 mapped the gene encoding the P2Y12 receptor to the locus q24–25 on chromosome 3, which also contains the gene encoding the UDP-glucose receptor (its closest known relative)22 and the gene encoding the P2Y1 receptor6. This clustering suggests that a relatively recent gene duplication led to the evolution of the different activities of those receptors20. A family with a bleeding disorder and impaired platelet aggregation was shown20 to harbour a
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P2Y12 UDP-glucose Orphan 1 Orphan 2 PAF MOP receptor P2Y5 P2Y11 P2Y1 P2Y4 tp2y cP2Y3 P2Y6 P2Y2 A1 350
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Fig. 1. A dendrogram showing the relationships among cloned P2Y receptor subtypes (only human receptors are shown, except the avian P2Y3 and tp2y receptors). Comparison is also made with the functionally unrelated human G-protein-coupled receptors (GPCRs) closest in sequence to the P2Y12 receptor. The length of each pair of branches represents the amino acid sequence distance between those receptors. The horizontal axis plots the number of substitution events. The homology between P2Y12 and the other P2Y receptor subtypes is as low as 14% (with the P2Y11 receptor). Orphan 1 and orphan 2 represent the H963 (accession number: AF002986) and GPR34 (accession number: AF118670) GPCRs, respectively, which are not yet functionally identified. The human adenosine A1 receptor sequence is used as an outgroup comparator. The nomenclature of the receptors is as given by the original authors in each case. The chicken (c) P2Y3 and the human P2Y5 receptors are retained in upper-case letters because recently King and Townsend-Nicholson25 have found functional responses to ATP at the recombinant P2Y5 receptor expressed in Xenopus oocytes and the recombinant cP2Y3 receptor has been characterized functionally36, although its mammalian orthologue has not yet been studied. Note that the only P2Y receptor subtype other than P2Y12 that is known to couple through Gi negatively to adenylyl cyclase, tp2y from the turkey37, is nevertheless structurally very distant from the P2Y12 receptor. Abbreviations: MOP, mu opioid peptide; PAF, platelet-activating factor.
two-base deletion in the coding region of the gene encoding the P2Y12 receptor, again confirming its function in vivo. Occurrence of the P2Y12 receptor outside the platelet
It has frequently been stated in the literature that the ‘P2T receptor’ is found only in the platelet lineage. However, the orphan clone that became the P2Y12 receptor21 was derived from human hypothalamus mRNA, and the P2Y12 receptor mRNA has now been shown to be highly expressed in both the spinal cord and the brain20,21. In situ hybridization suggested a glial location20, but its presence in neurones in some regions was not excluded. P2Y12 receptor mRNA occurs in most brain regions and is particularly strong in substantia nigra, caudate-putamen, thalamus and temporal cortex20,21. Interestingly, in these regions, the P2Y1 receptor transcript and protein are also strongly expressed in both neurones and glia5,26,27. The same association of these two receptors as occurs in the platelet might represent a common functional system. http://tips.trends.com
P2Y12 receptor mRNA was not detected in northern blots in other organs20,21, except at very low levels, which might be due to the blood content of these organs. However, a receptor with similar properties occurs in the endothelial cells of some blood vessels. Thus, a cyclaseinhibitory ADP response has been studied in capillary endothelial cells of the blood–brain barrier28. A clonal cell line (B10) formed spontaneously from those cells in culture is a good source of this activity and also expresses P2Y1 receptor29 mRNA and protein. The nucleotide specificity of the ADP-induced inhibition of stimulated adenylyl cyclase in B10 endothelial cells11 is similar to that now reported for the P2Y12 receptor (Table 1). In a rat glial cell line, C6-2B, known to contain a similar activity30, the nucleotide specificity is identical to that in B10 cells (Table 1). The potencies are distinctly lower in B10 and C6-2B cells than with heterologously expressed P2Y12 receptors for the weaker agonists, but this might reflect a difference in density of the same receptor11. In both cell lines, cloning has confirmed (J. Simon and E.A. Barnard,
unpublished) the expression of the rat P2Y12 receptor. The agony of ATP agonism
One of the defining characteristics of the ‘P2T receptor’ of platelets has historically been that the native agonist is ADP, whereas ATP and its triphosphate derivatives are antagonists. This has been reported consistently for the aggregation response and equally for the inhibition of adenylyl cyclase. Typical values for that agonist–antagonist division of adenine nucleotides are shown in Table 1. These values are not compromised by nucleotide metabolism or impurity because the behaviour on platelets is reproduced in recent studies where this is eliminated10,13. However, all of the triphosphates tested are agonists at the expressed P2Y12 receptor20,21 (Table 1). A striking case is that of 2-methylthioATP (2-MeSATP), which is a very potent antagonist of ADP-dependent cyclase inhibition in the platelet but is the strongest agonist at recombinant P2Y12 receptors. Such a difference is interesting because the same reversal from the behaviour observed in the platelet was recently reported for this receptor in its native expression in B10 endothelial cells11, and likewise occurs with this receptor in C6-2B glioma cells (Table 1). Hence, it is not an artefact of ectopic expression of the recombinant P2Y12 receptor. An explanation was proposed for the B10 cells11 in terms of a high sensitivity of this receptor activity to its density on the cell and a receptor reserve, as deduced in certain other cases31, including the P2Y1 receptor32,33, a phenomenon that is supported by similar findings with the cloned P2Y12 receptor. The density of the P2Y12 receptor in the platelet must be presumed to be lower than in its other locations considered here. However, alternatively a regulator of G-proteinlinked signalling might introduce the antagonism in the platelet. In summary, the question of the different responses evoked by ADP in the platelet is finally resolved by the identification of the missing player, the P2Y12 receptor. This resolution also embraces the problem of antagonism by ATP and the triphosphates in platelet responses to ADP: this phenomenon is not to be regarded now as an intrinsic property of the P2Y12 receptor.
Research Update
References 1 Boarder, M.R. and Hourani, S.M.O. (1998) The regulation of vascular function by P2 receptors: multiple sites and multiple receptors. Trends Pharmacol. Sci. 19, 99–107 2 Kunapuli, S.P. (1998) Multiple P2 receptor subtypes on platelets: a new interpretation of their function. Trends Pharmacol. Sci. 19, 391–394 3 Webb, T.E. et al. (1993) Cloning and functional expression of a brain G protein-coupled ATP receptor. FEBS Lett. 324, 219–225 4 Lustig, K.D. et al. (1993) Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc. Natl. Acad. Sci. U. S. A. 90, 5113–5117 5 Barnard, E.A. et al. (1997) Nucleotide receptors in the nervous system: an abundant component using diverse transduction mechanisms. Mol. Neurobiol. 15, 103–129 6 Ayyanathan, K. et al. (1996) Cloning and chromosomal localization of the human P2Y1 purinoceptor. Biochem. Biophys. Res. Commun. 218, 783–788 7 Jantzen, H-M. et al. (1999) Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb. Haemost. 81, 111–117 8 North, R.A. and Surprenant, A. (2000) Pharmacology of cloned P2X receptors. Annu. Rev. Pharmacol. 40, 563–580 9 Mahaut-Smith, M.P. et al. (2000) ADP is not an agonist at P2X1 receptors: evidence for separate receptors stimulated by ATP and ADP on human platelets. Br. J. Pharmacol. 131, 108–114 10 Hechler, B. et al. (1998) ATP derivatives are antagonists of the P2Y1 receptor: similarities to the platelet ADP receptor. Mol. Pharmacol. 53, 727–733 11 Simon, J. et al. (2001) Activity of adenosine diphosphates and triphosphates on a P2YT receptor in brain capillary endothelial cells. Br. J. Pharmacol. 132, 173–182 12 Jarvis, G.E. et al. (2000) ADP can induce aggregation of human platelets via both P2Y1 and P2T receptors. Br. J. Pharmacol. 129, 275–282 13 Park, H.S. and Hourani, S.M.O. (1999) Differential effects of adenine nucleotide analogues on shape change and aggregation induced by adenosine 5′-diphosphate (ADP) in human platelets. Br. J. Pharmacol. 127, 1359–1366 14 Rolf, M.G. et al. (2001) Platelet shape change evoked by selective activation of P2X1
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purinoceptors with α,β-methylene ATP. Thromb. Haemost. 85, 303–308 Oury, C. et al. (2000) A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue. J. Biol. Chem. 275, 22611–22614 Fabre, J.E. et al. (1999) Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat. Med. 5, 1199–1202 Leon, C. et al. (1999) Defective platelet aggregation and increased resistance to thrombosis in purinergic P2Y1 receptor-null mice. J. Clin. Invest. 104, 1731–1737 Offermanns, S. et al. (1997) Defective platelet activation in Gαq-deficient mice. Nature 389, 183–186 Boeynams, J.M. et al. (2000) P2Y receptors: in the middle of the road. Trends Pharmacol. Sci. 21, 1–3 Hollopeter, G. et al. (2001) Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409, 202–207 Zhang, F.L. et al. (2001) ADP is the cognate ligand for the orphan G-protein coupled receptor SP1999. J. Biol. Chem. 276, 8608–8615 Chambers, J.K. et al. (2000) A G protein-coupled receptor for UDP-glucose. J. Biol. Chem. 275, 10767–10771 Lovenberg, T.W. et al. (1999) Cloning and functional expression of the human histamine H3 receptor. Mol. Pharmacol. 55, 1101–1107 Webb, T.E. et al. (1996) Identification of 6H1 as a P2Y purinoceptor: P2Y5. Biochem. Biophys. Res. Commun. 219, 105–110 King, B.F. and Townsend-Nicholson, A. (2000) Recombinant P2Y receptors: the UCL experience. J. Auton. Nerv. Syst. 81, 164–170 Moore, D. et al. (2000) Regional and cellular distribution of the P2Y1 purinergic receptor in the human brain: striking neuronal localization. J. Comp. Neurol. 421, 374–384 Morán-Jimenez, M-J. and Matute, C. (2000) Immunohistochemical localization of the P2Y1 purinergic receptor in neurons and glial cells of the central nervous system. Mol. Brain Res. 78, 50–58 Feolde, E. et al. (1995) ATP, a partial agonist of atypical P2Y purinoceptors in rat brain microvascular endothelial cells. Br. J. Pharmacol. 115, 1199–1203 Webb, T.E. et al. (1996) The P2Y purinoceptors in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase. Br. J. Pharmacol. 119, 1385–1392
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30 Boyer, J.L. et al. (1993) Identification of a P2Y-purinergic receptor that inhibits adenylyl cyclase. J. Pharmacol. Exp. Ther. 267, 1140–1146 31 Kenakin, T.P. (1997) Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stochiometry. Trends Pharmacol. Sci. 18, 456–464 32 Palmer, R.K. et al. (1998) Agonist action of adenosine triphosphates at the human P2Y1 receptor. Mol. Pharmacol. 54, 1118–1123 33 Filippov, A.K. et al. (2000) The P2Y1 receptor closes the N-type Ca2+ channel in neurones, with both adenosine triphosphates and diphosphates as potent agonists. Br. J. Pharmacol. 129, 1063–1066 34 Cusack, N.J. and Hourani, S.M.O. (1982) Adenosine 5-diphosphate antagonists and human platelets: no evidence that aggregation and inhibition of stimulated adenylate cyclase are mediated by different receptors. Br. J. Pharmacol. 76, 221–227 35 Simon, J. et al. (1999) Characterization of putative P2T receptor endogenously expressed in rat glioma and brain microvascular endothelial cells. Fundam. Clin. Pharmacol. 13, 264s 36 Webb, T.E. et al. (1996) A novel G protein-coupled P2 purinoceptor (P2Y3) activated preferentially by nucleoside diphosphatases. Mol. Pharmacol. 50, 258–265 37 Boyer, J.L. et al. (2000) A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleotide triphosphates. Mol. Pharmacol. 57, 805–810
Eric A. Barnard* Joseph Simon Dept of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QJ. *e-mail: [email protected]
Chemical name ARC66096: 2-propylthioadenosine-5′(β,γ-difluoromethylene)triphosphonate
Lipoxins: revelations on resolution Blaithin McMahon, Siobhan Mitchell, Hugh R. Brady and Catherine Godson Lipoxins (LXs) are endogenously produced eicosanoids typically generated during cell–cell interactions. In this article, the compelling evidence from in vitro and in vivo model systems that LXs actively promote the resolution of inflammation is reviewed. Of particular interest are indications that stable http://tips.trends.com
synthetic analogues of LXs and aspirin-triggered 15-epi-LXs can mimic many of the desirable antiinflammatory, ‘pro-resolution’ actions of native LXs. Given the enhanced stability and efficacy of these compounds a role as novel anti-inflammatory therapeutics is proposed.
The dynamic regulation of leukocyte trafficking is an essential component of an effective host defence that can be subverted in inflammatory diseases and ischaemic injury. The resolution of inflammation depends on multiple processes that include the generation of endogenous ‘braking signals’ that
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