Thethromboxane receptor (TP) also known as theprostanoid TP receptor is aprotein that in humans is encoded by theTBXA2Rgene, The thromboxane receptor is one among the five classes ofprostanoid receptors[5] and was the firsteicosanoid receptor cloned.[6] The TP receptor derives its name from its preferred endogenous ligandthromboxane A2.[5]
Thegene responsible for directing the synthesis of the thromboxane receptor,TBXA2R, is located on humanchromosome 19 at position p13.3, spans 15kilobases, and contains 5exons.[7]TBXA2R codes for a member of theG protein-coupled super family of seven-transmembrane receptors.[8][9]
Molecular biology findings have provided definitive evidence for two human TP receptor subtypes.[5] The originallycloned TP subtype from humanplacenta (343 amino acids in length) is known as the αisoform and the splice variant cloned fromendothelium (with 407 amino acids) is termed the β isoform.[9] The first 328 amino acids are the same for both isoforms, but the β isoform exhibits an extended C-terminal cytoplasmic domain.[10] Both isoforms stimulate cells in part by activating theGq family of G proteins.[6] In at least certain cell types, however, TPα also stimulates cells by activating the Gs family of G proteins while TPβ also stimulates cells by activating the Gi class of G proteins. This leads to the stimulation or inhibition, respectively, ofadenylate cyclase activity and thereby very different cellular responses.[6] Differences in their C-terminal tail sequence also allow for significant differences in the two receptors internalization and thereby desensitization (i.e. loss of G protein- and therefore cell-stimulating ability) after activation by an agonist; TPβ but not TPα undergoes agonist-induced internalization.[11]
The expression of α and β isoforms is not equal within or across different cell types.[9] For example,platelets express high concentrations of the α isoform (and possess residual RNA for the β isoform), while expression of the β isoform has not been documented in these cells.[9] The β isoform is expressed in humanendothelium.[11] Furthermore, each TP isoform can physically combine with:a) another of its isoforms to makeTPα-TPα or TPβ-TPβ homodimers that promote stronger cell signaling than achieved by their monomer counterparts;b) their opposite isoform to makeTPα-TPβ heterodimers that activate more cell signaling pathways than either isoform or homodimer; and c) with theprostacyclin receptor (i.e. IP receptor) to form TP-IP heterodimers that, with respect to TPα-IP heterodimers, trigger particularly intense activation ofadenyl cyclase. The latter effect on adenyl cyclase may serve to suppress TPα's cell stimulating actions and thereby some of its potentially deleterious actions.[12]
Mice and rats express only the TPα isoform. Since these rodents are used as animal models to define the functions of genes and their products, their failure to have two TP isoforms has limited understanding of the individual and different functions of each TP receptor isoform.[13]
Historically, TP receptor involvement in blood platelet function has received the greatest attention. However, it is now clear that TP receptors exhibit a wide distribution in different cell types and among different organ systems.[9] For example, TP receptors have been localized in cardiovascular, reproductive, immune, pulmonary and neurological tissues, among others.[9][14]
| Organ/Tissue | Cells/Cell lines | |
|---|---|---|
| TP Receptor Distribution[9] | Lung, Spleen, Uterus, Placenta, Aorta, Heart, Intestine, Liver, Eye, Thymus, Kidney, Spinal Cord, Brain | Platelets, Blood Monocytes, Glomerular mesangial cells, Oligodendrocytes, Cardiac myocytes, Afferent Sympathetic Nerve Endings in the Heart, Epithelial cells, Hela cells, Smooth muscle cells, Endothelial cells, Trophoblasts, Schwann cells, Astrocytes, Megakaryocytes, Kupffer cells, Human erythroleukemic megakaryocyte (HEL), K562 (Human chronic myelogenous leukemia) cells, Hepatoblastoma HepG2 cells, Immature thymocytes, EL-4 (mouse T cell line), astrocytoma cells |
Standardprostanoids have the following relative efficacies asreceptor ligands in binding to and activating TP: TXA2=PGH2>>PGD2=PGE2=PGF2alpha=PGI2. Since TXA2 is highly unstable, receptor binding and biological studies on TP are conducted with stable TXA2 analogs such as I-BOP andU46619. These two analogs have one-half of their maximal binding capacity and cell-stimulating potency at ~1 and 10-20nanomolar, respectively; it is assumed that TXA2 and PGH2 (which also is unstable) have binding and cell-stimulating potencies within this range. PGD2, PGE2, PGF2alpha, and PGI2 have binding and stimulating potencies that are >1,000-fold weaker than I-BOP and therefore are assumed not to have appreciable ability to stimulate TP in vivo.20-Hydroxyeicosatetraenoic acid (20-HETE) is a full agonist and certainisoprostanes, e.g. 8-iso-PGF2 alpha and 8-iso-PGE2, arepartial agonists of the TP receptor. In animal models and human tissues, they act through TP to promote platelet responses and stimulate blood vessel contraction.[15] Synthetic analogs of TXA2 that activate TP but are relatively resistant to spontaneous and metabolic degradation include SQ 26655, AGN192093, and EP 171, all of which have binding and activating potencies for TP similar to I-BOP.[13][16][17]
Several synthetic compounds bind to, but do not activate, TP and thereby inhibit its activation by activating ligands. Thesereceptor antagonists include I-SAP, SQ-29548, S-145, domitroban, and vapiprost, all of which have affinities for binding TP similar to that of I-BOP. Other notable TP receptor antagonists areSeratrodast (AA-2414),Terutroban (S18886), PTA2, 13-APA, GR-32191, Sulotroban (BM-13177), SQ-29,548, SQ-28,668, ONO-3708, Bay U3405, EP-045, BMS-180,291, and S-145.[5][18] Many of these TP receptor antagonists have been evaluated as potential therapeutic agents forasthma,thrombosis andhypertension.[18] These evaluations indicate that TP receptor antagonists can be more effective than drugs which selectively block the production of TXA2thromboxane synthase inhibitors.[18] This seemingly paradoxical result may reflect the ability of PGH2, whose production is not blocked by the inhibitors, to substitute for TXA2 in activating TP.[13] Novel TP receptor antagonists that also have activity in reducing TXA2 production by inhibitingcyclooxygenases have been discovered and are in development for testing in animal models.[19]
TP is classified as a contractile type of prostenoid receptor based on its ability to contract diverse types of smooth muscle-containing tissues such as those of the lung, intestines, and uterus.[20] TP contracts smooth muscle and stimulates various response in a wide range of other cell types by coupling with and mobilizing one or more families of theG protein class of receptor-regulatedcell signaling molecules. When bound to TXA2, PGH2, or other of its agonists, TP mobilizes members of the:[14][21][22]
Following its activation of these pathways, the TP receptors's cell-stimulating ability rapidly reverses by a process termedhomologous desensitization, i.e. TP is no longer able to mobilize its G protein targets or further stimulate cell function. Subsequently, the β but not α isoform of TP undergoesreceptor internalization. Thesereceptor down regulating events are triggered by theG protein-coupled receptor kinases mobilized during TP receptor activation. TP receptor-independent agents that stimulate cells to activateprotein kinases C orprotein kinases A can also down-regulate TP in a process termedheterologous desensitization. For example,prostacyclin I2 (PGI2)-induced activation of itsprostacyclin receptor (IP) andprostaglandin D2-induced activation of itsprostaglandin DP1 receptor cause TP receptor desensitization by activating protein kinases A whileprostaglandin F2alpha-induced activation of itsprostaglandin F receptor andprostaglandin E2-induced activation of itsprostaglandin EP1 receptor receptor desensitizes TP by activating protein kinases C. These desensitization responses serve to limit the action of receptor agonists as well as the overall extent of cell excitation.[12]
In addition to its ability to down-regulate TPα, the IP receptor activates cell signaling pathways that counteract those activated by TP. Furthermore, the IP receptor can physically unite with the TPα receptor to form an IP-TPα heterodimer complex which, when bound by TXA2, activates predominantly IP-coupled cell signal pathways. The nature and extent of many cellular responses to TP receptor activation are thereby modulated by the IP receptor and this modulation may serve to limit the potentially deleterious effects of TP receptor activation (see following section on Functions).[12][13]
Studies using animals genetically engineered to lack the TP receptor and examining the actions of this receptor's agonists and antagonists in animals and on animal and human tissues indicate that TP has various functions in animals and that these functions also occur, or serve as a paradigm for further study, in humans.
Human and animalplatelets stimulated by various agents such as thrombin produce TXA2. Inhibition of this production greatly reduces the platelets finaladhesionaggregation anddegranulation (i.e. secretion of its granule contents) responses to the original stimulus. In addition, the platelets of mice lacking TP receptors have similarly defective adhesion, aggregation, and degranulation responses and these TP deficient mice cannot form stable blood clots and in consequence exhibit bleeding tendencies. TP, as studies show, is part of apositive feedback loop that functions to promote platelet adhesion, aggregation, degranulation, and platelet-induced blood clotting-responses in vitro and in vivo. The platelet-directed functions of TP are in many respects opposite to those of theIP receptor. This further indicates (see previous section) that the balance between the TXA2-TP and PGI2-IP axes contribute to regulating platelet function, blood clotting, and bleeding.[14][13]
Animal model studies indicate that TP receptor activation contracts vascular smooth muscle cells and acts on cardiac tissues to increase heart rate, triggerCardiac arrhythmias, and produce myocardialischemia. These effects may underlie, at least in part, the protective effects of TPgene knockout in mice. TP(-/-) mice are:a) resistant to thecardiogenic shock caused by infusion of the TP agonist, U46619, or the prostaglandin and thromboxane A2 precursor,arachidonic acid;b) partially protected from the cardiac damage caused by hypertension inIP-receptor deficient mice feed a high salt diet;c) prevented from developingangiotensin II-induced and N-Nitroarginine methyl ester-induced hypertension along with associated cardiac hypertrophy;d) resistant to the vascular damage caused by balloon catheter-induced injury of the external carotid artery;e) less likely to develop severe hepatic microcirculation dysfunction caused byTNFα as well as kidney damage caused by TNFα or bacteria-derivedendotoxin; andf) slow in developing vascularatherosclerosis inApoE gene knockout mice.[12][13][14][23] In addition, TP receptor antagonists lessen myocardial infarct size in various animal models of this disease and block the cardiac dysfunction caused by extensive tissue ischemia in animal models ofremote ischemic preconditioning.[24] TP thereby has wide-ranging functions that tend to be detrimental to the cardiovascular network in animals and, most likely, humans. However, TP functions are not uniformly injurious to the cardiovascular system: TP receptor-depleted mice show an increase in cardiac damage as well as mortality due totrypanosoma cruzi infection. The mechanisms behind this putative protective effect and its applicability to humans is not yet known.[14]
20-Hydroxyeicosatetraenoic acid (20-HETE), a product ofarachidonic acid formed byCytochrome P450 omega hydroxylases,[25] and certain isoprostanes, which form by non-enzymaticfree radical attack on arachidonic acid,[17] constrict rodent and human artery preparations by directly activating TP. While significantly less potent than thromboxane A2 in activating this receptor, studies on rat and human cerebral artery preparations indicate that increased blood flow through these arteries triggers production of 20-HETE which in turn binds TP receptors to constrict these vessels and thereby reduce their blood blow. Acting in the latter capacity, 20-HETE, it is proposed, functions as a TXA2 analog to regulate blood flow to the brain and possibly other organs.[15][26] Isoprostanes form in tissues undergoing acute or chronicoxidative stress such as occurs at sites of inflammation and the arteries of diabetic patients.[17] High levels of isoprostanes form in ischemic or otherwise injured blood vessels and acting through TP, can stimulate arterial inflammation and smooth muscle proliferation; this isoprostane-TP axis is proposed to contribute to the development of atherosclerosis and thereby heart attacks and strokes in humans.[17][19]
TP receptor activation contracts bronchial smooth muscle preparations obtained from animal models as well as humans and contracts airways in animal models.[14] In a mouse model of asthma (i.e. hypersensitivity to ovalabumin), a TP receptor antagonist decreased the number of eosinophils infiltrating lung as judged by their content inBronchoalveolar lavage fluid and in a mouse model of dust mite-induced astha, deletion ofTBXA2R prevented the development of airways contraction and pulmonary eosinophilia responses to allergen. Another TP receptor agonists likewise reduced airway bronchial reactivity to allergen as well as symptoms in volunteers with asthma.[27] The TP receptor appears to play and essential role in the pro-asthmatic actions ofleukotriene C4 (LTC4): in ovalbumin-sensitized mice,leukotriene C4 increased the number of eosinophils in bronchoalveolar lavage fluid and simultaneously decreased the percentages of eosinophils in blood but these responses did not occur inTBXA2R-deficient mice. LTC4 also stimulated lung expression of the pro-inflammatory intracellular adhesion molecules,ICAM-1 andVCAM-1 by a TP receptor-dependent mechanism.[28] These findings suggest that TP contributes to asthma in animal models at least in part by mediating the actions of LTC4. Further studies are required to determine if TP receptor antagonists might be useful for treating asthma and other airway constriction syndromes such aschronic obstructive lung diseases in humans.
Along withPGF2α acting through itsFP receptor, TXA2 acting through TP contracts uterine smooth muscle preparations from rodents and humans. Since the human uterus loses its sensitivity to PGP2α but not to TXA2 during theearly stages of labor in vaginal childbirth, TP agonists, it is suggested, might be useful for treating preterm labor failures.[14]
Activation of TP receptors stimulates vascular endothelial cell pro-inflammatory responses such as increased expression of cell surface adhesion proteins (i.e.ICAM-1,VCAM-1, andE-selectin); stimulatesapoptosis (i.e. cell death) ofCD4+ andCD8+ lymphocytes; causes thechemokinesis (i.e. cell movement) of nativeT cells; and impairs the adhesion ofdendritic cells toT cells thereby inhibiting dendritic cell-dependent proliferation of T cells. TP deficient mice exhibit an enhanced contact hypersensitivity response toDNFBthymocytes in thethymus of these deficient mice are resistant tolipopolysaccharide-induced apoptosis. TP receptor-depleted mice also gradually develop with age extensivelymphadenopathy and, associated with this, increased immune responses to foreign antigens. These studies indicate that TXA2-TP signaling functions as a negative regulator of DC-T cell interactions and possibly thereby the acquisition ofacquired immunity in mice. Further studies are needed to translate these mouse studies to humans.[14][29][30]
Increased expression ofcyclooxygenases and their potential involvement in the progression of various human cancers have been described. Some studies suggest that the TXA2 downstream metabolite of these cyclooxygenases along with its TP receptor contribute to mediating this progression. TP activation stimulates tumor cell proliferation, migration,neovascularization, invasiveness, and metastasis in animal models, animal and human cell models, and/or human tissue samples in cancers of the prostate, breast, lung, colon, brain, and bladder.[14][31] These findings, while suggestive, need translational studies to determine their relevancy to the cited human cancers.
Isolated cases of humans with mild to moderate bleeding tendencies have been found to have mutations in TP that are associated with defects in this receptors binding of TXA2 analogs, activating cell signal pathways, and/or platelet functional responses not only to TP agonists but also to agents that stimulate platelets by TP-independent mechanisms (see Genomics section below).[15]
TP receptor antagonistseratrodast is marketed in Japan and China for the treatment of asthma.Picotamide, a dual inhibitor of TP and TXA2 synthesis, is licensed in Italy for the treatment of clinical arterial thrombosis and peripheral artery disease.[15] These drugs are not yet licensed for use in other countries.
While functional roles for TP receptor signaling in diverse homeostatic and pathological processes have been demonstrated in animal models, in humans these roles have been demonstrated mainly with respect to platelet function, blood clotting, andhemostasis. TP has also been proposed to be involved in human: blood pressure and organ blood flow regulation;essential andpregnancy-induced hypertension; vascular complications due to sickle cell anemia; other cardiovascular diseases includingheart attack,stroke, andperipheral artery diseases; uterine contraction in childbirth; and modulation of innate and adaptiveimmune responses including those contributing to various allergic and inflammatory diseases of the intestine, lung, and kidney.[9] However, many of the animal model and tissue studies supporting these suggested functions have yet to be proven directly applicable to human diseases. Studies to supply these proofs rest primarily on determining if TP receptor antagonists are clinically useful. However, these studies face issues that drugs which indirectly target TP (e.g.Nonsteroidal anti-inflammatory drugs that block TXA2 production) or which circumvent TP (e.g.P2Y12 antagonists that inhibit platelet activation andcorticosteroids andcysteinyl leukotriene receptor 1 antagonists that suppress allergic and/or inflammatory reactions) are effective treatments for many putatively TP-dependent diseases. These drugs are likely to be cheaper and may prove to have more severe side effects that TP-targeting drugs.[14] These considerations may help to explain why relatively few studies have examined the clinical usefulness of TP-targeting drugs. The followingtranslation studies on TP antagonists have been conducted or are underway:[27][19]
In addition to the above TP antagonists, drugs that have dual inhibitory actions in that they block not only TP but also block the enzyme responsible for making TXA22,Thromboxane-A synthase, are in clinical development. These dual inhibitor studies include:[15]
Several isolated and/or inherited cases of patients suffering a mild to moderately severe bleeding diathesis have been found to be associated with mutations in theTBXA2R gene that lead to abnormalities in the expression, subcellular location, or function of its TP product. These cases include:[15][32]
Single nucleotide polymorphism (SNP) variations in theTBXA2R gene have been associated with allergic and cardiovascular diseases; these include:[33][34]
