Pharmacodynamic Comparison of Ticagrelor Monotherapy Versus Ticagrelor and Aspirin in Patients After Percutaneous Coronary Intervention: The TEMPLATE (Ticagrelor Monotherapy and Platelet Reactivity) Randomized Controlled Trial.

Background To assess differences in platelet inhibition during ticagrelor monotherapy (TIC) or dual therapy with ticagrelor and aspirin (TIC+ASP) in patients after percutaneous coronary intervention using a comprehensive panel of functional tests. Methods and Results In a single-center parallel group, open label, randomized controlled trial, 110 participants were randomized to receive either TIC (n=55) or TIC+ASP (n=55) for 4 weeks. The primary outcome was the platelet aggregation response with 10 µmol/L thrombin receptor activation peptide-6 (TRAP-6). The secondary outcomes were platelet aggregation responses and binding of surface activation markers with a panel of other activators. The mean percentage aggregation for 10 µmol/L TRAP-6 was similar for the TIC and TIC+ASP groups (mean difference+4.29; 95% CI, -0.87 to +9.46). Aggregation was higher in the TIC group compared with the TIC+ASP group with 1 µg/mL (+6.47; +2.04 to +10.90) and 0.5 µg/mL (+14.00; +7.63 to +20.39) collagen related peptide. Aggregation responses with 5 µmol/L TRAP-6, 5 µmol/L or 2.5 µmol/L thromboxane A2 receptor agonist and surface activation marker binding with 5 µmol/L TRAP-6 or 0.5 µg/mL collagen related peptide were the same between the treatment groups. Conclusions Patients with PCI show similar levels of inhibition of most platelet activation pathways with TIC compared with dual therapy with TIC + ASP. However, the greater aggregation response with collagen related peptide during TIC indicates incomplete inhibition of glycoprotein VI (collagen) receptor-mediated platelet activation. This difference in pharmacodynamic response to anti-platelet medication may contribute to the lower bleeding rates observed with TIC compared with dual antiplatelet therapy in recent clinical trials. Registration Information URL: https://www.isrctn.com; Unique Identifier ISRCTN84335288.

TRPV4 (Transient Receptor Potential Vanilloid 4) Channel-Dependent Negative Feedback Mechanism Regulates Gq Protein-Coupled Receptor-Induced Vasoconstriction.

OBJECTIVE: Several physiological stimuli activate smooth muscle cell (SMC) GqPCRs (Gq protein-coupled receptors) to cause vasoconstriction. As a protective mechanism against excessive vasoconstriction, SMC GqPCR stimulation invokes endothelial cell vasodilatory signaling. Whether Ca2+ influx in endothelial cells contributes to the regulation of GqPCR-induced vasoconstriction remains unknown. Ca2+ influx through TRPV4 (transient receptor potential vanilloid 4) channels is a key regulator of endothelium-dependent vasodilation. We hypothesized that SMC GqPCR stimulation engages endothelial TRPV4 channels to limit vasoconstriction. APPROACH AND RESULTS: Using high-speed confocal microscopy to record unitary Ca2+ influx events through TRPV4 channels (TRPV4 sparklets), we report that activation of SMC α1ARs (alpha1-adrenergic receptors) with phenylephrine or thromboxane A2 receptors with U46619 stimulated TRPV4 sparklets in the native endothelium from mesenteric arteries. Activation of endothelial TRPV4 channels did not require an increase in Ca2+ as indicated by the lack of effect of L-type Ca2+ channel activator or chelator of intracellular Ca2+ EGTA-AM. However, gap junction communication between SMCs and endothelial cells was required for phenylephrine activation or U46619 activation of endothelial TRPV4 channels. Lowering inositol 1,4,5-trisphosphate levels with phospholipase C inhibitor or lithium chloride suppressed phenylephrine activation of endothelial TRPV4 sparklets. Moreover, uncaging inositol 1,4,5-trisphosphate profoundly increased TRPV4 sparklet activity. In pressurized arteries, phenylephrine-induced vasoconstriction was followed by a slow, TRPV4-dependent vasodilation, reflecting activation of negative regulatory mechanism. Consistent with these data, phenylephrine induced a significantly higher increase in blood pressure in TRPV4-/- mice. CONCLUSIONS: These results demonstrate that SMC GqPCR stimulation triggers inositol 1,4,5-trisphosphate-dependent activation of endothelial TRPV4 channels to limit vasoconstriction.

Sex differences in the role of phospholipase A2 -dependent arachidonic acid pathway in the perivascular adipose tissue function in pigs.

KEY POINTS: The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT-induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT-induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. ABSTRACT: Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase-derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re-added to the tissue bath and changes in tone measured over 1 h. Levels of PGF2α and thromboxane B2 (TXB2 ) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re-addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT-induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT-induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT-induced vasoconstriction in male and female PCAs, respectively.

Activation of thromboxane A2 receptors mediates endothelial dysfunction in diabetic mice.

BACKGROUND: Diabetes is one of high-risk factors for cardiovascular disease. Improvement of endothelial dysfunction in diabetes reduces vascular complications. However, the underlying mechanism needs to be uncovered. This study was conducted to elucidate whether and how thromboxane A2 receptor (TPr) activation contributes to endothelial dysfunction in diabetes. METHODS AND RESULTS: Exposure of human umbilical vein endothelial cells (HUVECs) to either TPr agonists, two structurally related thromboxane A2 (TxA2) mimetics, significantly reduced phosphorylations of endothelial nitric oxide synthase (eNOS) at Ser1177 and Akt at Ser473. These effects were abolished by pharmacological or genetic inhibitors of TPr. TPr-induced suppression of eNOS and Akt phosphorylation was accompanied by upregulation of PTEN (phosphatase and tension homolog deleted on chromosome 10) and Ser380/Thr382/383 PTEN phosphorylation. PTEN-specific siRNA restored Akt-eNOS signaling in the face of TPr activation. The small GTPase, Rho, was also activated by TPr stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase (ROCK) inhibitor, rescued TPr-impaired Akt-eNOS signaling. In mice, streptozotocin-induced diabetes was associated with aortic PTEN upregulation, PTEN-Ser380/Thr382/383 phosphorylation, and dephosphorylation of Akt (at Ser473) and eNOS (at Ser1177). Importantly, administration of TPr antagonist blocked these changes. CONCLUSION: We conclude that TPr activation impairs endothelial function by selectively inactivating the ROCK-PTEN-Akt-eNOS pathway in diabetic mice.

Thromboxane A2 Activates YAP/TAZ Protein to Induce Vascular Smooth Muscle Cell Proliferation and Migration.

The thromboxane A2 receptor (TP) has been implicated in restenosis after vascular injury, which induces vascular smooth muscle cell (VSMC) migration and proliferation. However, the mechanism for this process is largely unknown. In this study, we report that TP signaling induces VSMC migration and proliferation through activating YAP/TAZ, two major downstream effectors of the Hippo signaling pathway. The TP-specific agonists [1S-[1α,2α(Z),3ß(1E,3S*),4 α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) and 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619) induce YAP/TAZ activation in multiple cell lines, including VSMCs. YAP/TAZ activation induced by I-BOP is blocked by knockout of the receptor TP or knockdown of the downstream G proteins Gα12/13 Moreover, Rho inhibition or actin cytoskeleton disruption prevents I-BOP-induced YAP/TAZ activation. Importantly, TP activation promotes DNA synthesis and cell migration in VSMCs in a manner dependent on YAP/TAZ. Taken together, thromboxane A2 signaling activates YAP/TAZ to promote VSMC migration and proliferation, indicating YAP/TAZ as potential therapeutic targets for cardiovascular diseases.

Thromboxane A2 Regulates CXCL1 and CXCL8 Chemokine Expression in the Nasal Mucosa-Derived Fibroblasts of Chronic Rhinosinusitis Patients.

BACKGROUND: Chronic rhinosinusitis without nasal polyps (CRSsNP) is a common chronic disease and the etiology remains unclear. Thromboxane A2 (TXA2) participates in platelet aggregation and tissue inflammation. In this study, the CXCL1/8 chemokine and TXA2-TP receptor expression in the CRSsNP mucosa was investigated. EXPERIMENTAL APPROACH: Immunohistochemistry, chemokine release assay by ELISA, RT-PCR, Real-time PCR, Western blotting, pharmacological and siRNA knockdown analysis were applied in the CRSsNP tissue specimen and cultured nasal mucosa-derived fibroblasts. RESULTS: The immunohistochemistry results indicated that CXCL1 and CXCL8 were highly expressed in the CRSsNP mucosa compared with the controls; however, the TP receptors were expressed in both mucosa. Therefore, U46619 and IBOP, a TXA2 analog and TP agonist, were used to explore the role of TP activation in CXCL1/8 expression; both of these induced CXCL1/8 mRNA and protein expression in CRSsNP mucosa-derived fibroblasts. U46619 phosphorylated PI-3K, cyclic AMP (cAMP)/PKA, PKC, and cAMP response element (CREB). Activation of cAMP/PKA, PKC, and CREB was the major pathway for cxcl1/8 gene transcription. Pharmacological and siRNA knockdown analyses revealed that activation of cAMP/PKA and PKCµ/PKD pathways were required for CREB phosphorylation and PKA/C crosstalked with the PI-3K pathway. CONCLUSION AND IMPLICATIONS: Our study provides the first evidence for abundant TP receptor and CXCL1/8 expression in human CRSsNP mucosa and for TXA2 stimulation inducing CXCL1/8 expression in nasal fibroblasts primarily through TP receptor, cAMP/PKA, PKCµ/PKD, and CREB-related pathways.

Sensitivity to the thromboxane A2 analog U46619 varies with inner diameter in human stem villous arteries.

INTRODUCTION: The vascular resistance of stem villous arteries is determined by the balance between different contractile and relaxant agents and in the utero-placental circulation. Thromboxane A2 (TxA2), prostaglandin F2α (PGF2α) and endothelin-1 (ET-1) are considered to be among the most important contractile factors. However, it is not known if their contractile effects are consistent along the villous tree. We hypothesized that the sensitivity to different agonists could be influenced by artery diameter and thus that their contribution to placental vascular resistance may differ. METHODS: Using an isometric wire myograph, the contractility and sensitivity (pD2) to the thromboxane A2 mimetic U46619, PGF2α and ET-1 were investigated in isolated human stem villous arteries and human uterine fundus and isthmus arteries obtained from healthy, pregnant women who had experienced uncomplicated pregnancy. RESULTS: In fetal arteries, the pD2 values for U46619 correlated positively with arterial diameter with no such dependence observed for ET-1 and PGF2α. In maternal arteries, pD2 remained constant for all the agonists tested despite highly variable vessel diameter. DISCUSSION: A selective decrease in sensitivity to TxA2 receptor stimulation was observed with decreasing vascular diameter in human stem villous arteries. The contractile factors PGF2α and ET-1 show no such relationship.

Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

Mechanisms Involved in Thromboxane A2 -induced Vasoconstriction of Rat Intracavernous Small Penile Arteries.

Diabetes is associated with erectile dysfunction and with hypercontractility in erectile tissue and this is in part ascribed to increased formation of thromboxane. Rho kinase (ROCK) is a key regulator of calcium sensitization and contraction in vascular smooth muscle. This study investigated the role of calcium and ROCK in contraction evoked by activation of the thromboxane receptors. Rat intracavernous penile arteries were mounted for isometric tension and intracellular calcium ([Ca2+ ]i ) recording and corpus cavernosum for measurements of MYPT1 phosphorylation. In penile arteries, U46619 by activation of thromboxane receptors concentration dependently increased calcium and contraction. U46619-induced calcium influx was blocked by nifedipine, a blocker of L-type calcium channels, and by 2-aminoethoxydiphenyl borate, a blocker of transient receptor potential (TRP) channels. Inhibitors of ROCK, Y27632 and glycyl-H1152P, concentration dependently reduced U46619-induced contraction, but only Y27632 reduced [Ca2+ ]i levels in the penile arteries activated with either high extracellular potassium or U46619. MYPT-Thr850 phosphorylation in corpus cavernous strips was increased in response to U46619 through activation of TP receptors and was found to be a direct result of phosphorylation by ROCK. Y27632 induced less relaxation in mesenteric arteries, H1152P induced equipotent relaxations, and a protein kinase C inhibitor, Ro-318220, failed to relax intracavernous penile arteries, but induced full relaxation in rat mesenteric arteries. Our findings suggest that U46619 contraction depends on Ca2+ influx through L-type and TRP channels, and ROCK-dependent mechanisms in penile arteries. Inhibition of the ROCK pathway is a potential approach for the treatment of erectile dysfunction associated with hypertension and diabetes.

Protein kinase C-related kinase 1 and 2 play an essential role in thromboxane-mediated neoplastic responses in prostate cancer.

The prostanoid thromboxane (TX) A2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TPα and TPß isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TPα/TPß-induced PCa cell migration but also enabled the TXA2-TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenetic marker both essential for and previously exclusively associated with androgen-induced chromatin remodelling and transcriptional activation. PRK1 is a member of a subfamily of three structurally related kinases comprising PRK1/PKNα, PRK2/PKNγ and PRK3/PKNß that are widely yet differentially implicated in various cancers. Hence, focusing on the setting of prostate cancer, this study investigated whether TPα and/or TPß might also complex with PRK2 and PRK3 to regulate their activity and neoplastic responses. While TPα and TPß were found in immune complexes with PRK1, PRK2 and PRK3 to regulate their activation and signalling, they do so differentially and in a TP agonist-regulated manner dependent on the T-loop activation status of the PRKs but independent of their kinase activity. Furthermore, TXA2-mediated neoplastic responses in prostate adenocarcinoma PC-3 cells, including histone H3Thr11 phosphorylation, was found to occur through a PRK1- and PRK2-, but not PRK3-, dependent mechanism. Collectively, these data suggest that TXA2 acts as both a neoplastic and epigenetic regulator and provides a mechanistic explanation, at least in part, for the prophylactic benefits of Aspirin in reducing the risk of certain cancers.

A vasoconstrictor response to COX-1-mediated prostacyclin synthesis in young rat renal arteries that increases in prehypertensive conditions.

This study aimed to determine whether prostacyclin (PGI2) functions as an endothelium-derived contracting factor (EDCF) in young rat renal arteries, and, if so, we wanted to examine the underlying mechanism(s) and how it changes in prehypertensive conditions. Vessels from Wistar-Kyoto (WKY) and prehypertensive spontaneously hypertensive rats (SHRs) of 25-28 days of age were isolated for functional and biochemical analyses. Result showed that following NO synthase (NOS) inhibition PGI2 and the thromboxane-prostanoid (TP) receptor agonist U-46619 evoked contractions in young WKY renal arteries that were similar to those in prehypertensive SHRs. Meanwhile, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent contraction under NOS-inhibited conditions and a production of the PGI2 metabolite 6-keto-PGF1α; both were sensitive to cyclooxygenase (COX) and/or COX-1 inhibition but higher in prehypertensive SHRs than in young WKYs. Interestingly, in WKY renal arteries PGI2 did not evoke relaxation even after TP receptor antagonism that diminished the contraction evoked by the agonist. Indeed, PGI2 (IP) receptors were not detected in the vessel with Western blot. Moreover, we noted that treatment with the nonselective COX inhibitor indomethacin, which was started at the prehypertensive stage, blunted the elevation of systolic blood pressure and reduced the heart-to-body ratio in SHR within 2 mo of treatment. These results demonstrate that due to scarcity of IP receptors, PGI2, which is derived mainly from COX-1-mediated metabolism, acts as an EDCF in young WKY renal arteries, and it increases in prehypertensive conditions. Also, our data revealed that COX inhibition starting from the prehypertensive stage has an antihypertensive effect in young SHRs.

PKCα Deficiency in Mice Is Associated with Pulmonary Vascular Hyperresponsiveness to Thromboxane A2 and Increased Thromboxane Receptor Expression.

Pulmonary vascular hyperresponsiveness is a main characteristic of pulmonary arterial hypertension (PAH). In PAH patients, elevated levels of the vasoconstrictors thromboxane A2 (TXA2), endothelin (ET)-1 and serotonin further contribute to pulmonary hypertension. Protein kinase C (PKC) isozyme alpha (PKCα) is a known modulator of smooth muscle cell contraction. However, the effects of PKCα deficiency on pulmonary vasoconstriction have not yet been investigated. Thus, the role of PKCα in pulmonary vascular responsiveness to the TXA2 analog U46619, ET-1, serotonin and acute hypoxia was investigated in isolated lungs of PKCα-/- mice and corresponding wild-type mice, with or without prior administration of the PKC inhibitor bisindolylmaleimide I or Gö6976. mRNA was quantified from microdissected intrapulmonary arteries. We found that broad-spectrum PKC inhibition reduced pulmonary vascular responsiveness to ET-1 and acute hypoxia and, by trend, to U46619. Analogously, selective inhibition of conventional PKC isozymes or PKCα deficiency reduced ET-1-evoked pulmonary vasoconstriction. The pulmonary vasopressor response to serotonin was unaffected by either broad PKC inhibition or PKCα deficiency. Surprisingly, PKCα-/- mice showed pulmonary vascular hyperresponsiveness to U46619 and increased TXA2 receptor (TP receptor) expression in the intrapulmonary arteries. To conclude, PKCα regulates ET-1-induced pulmonary vasoconstriction. However, PKCα deficiency leads to pulmonary vascular hyperresponsiveness to TXA2, possibly via increased pulmonary arterial TP receptor expression.

Thromboxane A(2) receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

Ductus arteriosus (DA) closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP) stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324.

BACKGROUND AND PURPOSE: Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. EXPERIMENTAL APPROACH: Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. KEY RESULTS: The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. CONCLUSIONS AND IMPLICATIONS: In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity.

Inverse agonism of SQ 29,548 and Ramatroban on Thromboxane A2 receptor.

G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.

Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

The intrahelical salt bridge between E/D(3.49) and R(3.50) within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30) in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

Platelet dysfunction associated with the novel Trp29Cys thromboxane A2 receptor variant.

BACKGROUND: Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. OBJECTIVES: To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. PATIENTS/METHODS: We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. RESULTS: Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. CONCLUSION: These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding.

Effects of the experimental subarachnoid hemorrhage on the eicosanoid receptors in nicotine-induced contraction of the rat basilar artery.

BACKGROUND: Smoking is one of the most important risk factors for subarachnoid hemorrhage (SAH). The purpose of this study was to investigate the influence of experimental SAH and arachidonic acid metabolites on nicotine-induced contraction in the rat basilar artery. METHODS: Rats were killed at 1 hour and 1 week after SAH, and the basilar artery was isolated and cut into a spiral strip. The effects of various eicosanoid receptor antagonists on nicotine-induced contraction in the rat basilar artery were investigated. RESULTS: Antagonists of thromboxane A2 (TXA2) and cysteinyl leukotriene (CysLT) receptors did not affect nicotine-induced contraction. In contrast, the antagonists of leukotriene B4 (LTB4) receptors (BLT1 and BLT2) attenuated the nicotine-induced contraction in the rat basilar artery. We also observed that SAH did not influence the effect of TXA2, LTB4, and CysLTs receptor antagonists on the nicotine-induced contraction. These results suggest that TXA2 and CysLTs are not involved in nicotine-induced contraction, while LTB4 potentates this contraction in rat basilar artery. CONCLUSIONS: BLT2 receptor seemed to be more involved in the nicotine-induced contraction than the BLT1 receptor. SAH did not affect the involvement of eicosanoids in the nicotine-induced contraction of the rat basilar artery. The present study shows the involvement of some of the arachidonic acid metabolites into signaling pathways of nicotine-induced contraction. It will serve to improve therapeutic interventions of SAH and suggests a promising approach to protect the cerebral vasculature of cigarette smokers.

Low-dose thromboxane A2 receptor stimulation promotes closure of the rat ductus arteriosus with minimal adverse effects.

BACKGROUND: Patent ductus arteriosus (PDA) is a common life-threatening complication among premature infants. Although cyclooxygenase inhibitors are frequently used to treat PDA, as they inhibit the synthesis of prostaglandin E(2), the most potent vasodilator in the ductus arteriosus (DA), their efficacy is often limited. As thromboxane A(2) (TXA(2)) induces vascular contraction via the TXA(2) receptor (TP), we hypothesized that TP stimulation would promote DA closure. METHOD: To measure the inner diameter of the vessels, a rapid whole-body freezing method was used. RESULTS: Injection of the selective TP agonists U46619 and I-BOP constricted the fetal DA at embryonic day 19 (e19) and e21 in a dose-dependent manner. Of note, U46619 also exerted a vasoconstrictive effect on two different types of postnatal PDA models: premature PDA and hypoxia-induced PDA. We also found that U46619 constricted the ex vivo DA ring to a greater extent than it constricted the ex vivo aorta. Furthermore, we found that U46619 at lower concentrations (up to 0.05 mg/g of body weight) had a minimal vasoconstrictive effect on other vessels and did not induce microthrombosis in the pulmonary capillary arteries. CONCLUSION: Low-dose TP stimulation constricts the DA with minimal adverse effects at least in rat neonates and our results could point to an alternative potent vasoconstrictor for PDA.

Increased expression of matrix metalloproteinases mediates thromboxane A2-induced invasion in lung cancer cells.

Thromboxane A(2) receptor (TP) has been shown to play an important role in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Here we report that TP mediates cancer cell invasion by inducing expression of matrix metalloproteinases (MMPs). TP agonist, I-BOP, significantly elevated MMP-1, MMP-3, MMP-9 and MMP-10 mRNA levels in A549 human lung adenocarcinoma cells overexpressing TPα or TPß. The secretion of MMP-1 and MMP-9 in conditioned media was determined using Western blot analysis and zymographic assay. Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein ß (C/EBPß) pathways. I-BOP-induced cellular invasiveness of A549 cells expressing TPα or TPß was determined by invasion assay. GM6001, a general inhibitor of MMPs, decreased basal and I-BOP-induced cell invasion. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated cell invasion suggesting that other MMPs induced by I-BOP were also involved. Our studies establish the relationship between TP and MMPs in cancer cell invasion and suggest that the thromboxane A(2) (TXA(2))-TP signaling is a potential therapeutic target for cancer invasion and metastasis.