Thromboxane Synthase Inhibitor Research Articles

Thrombin-induced platelet inhibition by 2′,4′-dihydroxychalcone and 2′,4′-dihydroxydihydrochalcone is mediated by inhibition of ROS production and Thromboxane synthase a activity: structure-activity relationship of polyphenol secondary metabolites from Empetrum nigrum

In this study, we expanded our previous work by testing compounds 1–12 for their ability to inhibit platelet activation at low (30 µM) concentration by inhibition of ROS production, thromboxane synthase (TxS) activity, and activation of cyclic nucleotide pathways. We also investigated whether some of these compounds could potentiate the effects of P2Y12 ADP receptor inhibitor action and discussed possible structure-activity relationships of the tested compounds. We showed that at this concentration only compounds 7 and 12 significantly inhibited thrombin-induced platelet activation which was accompanied by inhibition of ROS production and thromboxane synthase activity. Correspondingly, these compounds significantly potentiated the inhibitory effect of cangrelor on thrombin-induced platelet activation. In some other cases, inhibition of ROS production and thromboxane synthase activity did not correlate with platelet inhibition, indicating that these compounds could affect some, still unidentified, activatory pathways in platelets that counteract their inhibitory effects.

Li, P HY-021068 alleviates cerebral ischemia-reperfusion injury by inhibiting NLRP1 inflammasome and restoring autophagy function in mice

Cerebral ischemia-reperfusion injury (CIRI) is a severe pathological condition that involves oxidative stress, inflammatory response, and neuronal damage. HY-021068 belongs to a new drug of chemical class 1, which is a potential thromboxane synthase inhibitor. Our preliminary experiment found that HY-021068 has significant anti-neuroinflammatory and neuroprotective effects. However, the protective effect and mechanism of HY-021068 in CIRI remain unclear. To investigate the protective effect and mechanism of HY-021068 in CIRI mice. In mice, CIRI was induced by bilateral common carotid artery occlusion and reperfusion. Mice were treated with HY-021068 or LV-NLRP1-shRNA (lentivirus-mediated shRNA transfection to knock down NLRP1 expression). The locomotor activity, neuronal damage, pathological changes, postsynaptic density protein-95 (PSD-95) expression, NLRP1 inflammasome activation, autophagy markers, and apoptotic proteins were assessed in CIRI mice. In this study, treatment with HY-021065 and LV-NLRP1-shRNA significantly improved motor dysfunction and neuronal damage after CIRI in mice. HY-021065 and NLRP1 knockdown significantly ameliorated the pathological damage and increased PSD-95 expression in the cortex and hippocampus CA1 and CA3 regions. The further studies showed that compared with the CIRI model group, HY-021065 and NLRP1 knockdown treatment inhibited the expressions of NLRP1, ASC, caspase-1, and IL-1β, restored the expressions of p-AMPK/AMPK, Beclin1, LC3II/LC3I, p-mTOR/m-TOR and P62, and regulated the expressions of BCL-2, Caspase3, and BAX in brain tissues of CIRI mice in CIRI mice. These results suggest that HY-021068 exerts a protective role in CIRI mice by inhibiting NLRP1 inflammasome activation and regulating autophagy function and neuronal apoptosis. HY-021068 is expected to become a new therapeutic drug for CIRI.

Inhibition of TP signaling promotes endometriosis growth and neovascularization.

Endometriosis is highly dependent on angiogenesis and lymphangiogenesis. Prostaglandin E2, an arachidonic acid metabolite, has been shown to promote the formation of new blood and lymphatic vessels. However, the role of another arachidonic acid metabolite, thromboxane A2 (TXA2) in angiogenesis and lymphangiogenesis during endometriosis remains largely unexplored. Using a murine model of ectopic endometrial transplantation, fragments from the endometrium of WT donor mice were transplanted into the peritoneal walls of recipient WT mice (WT→WT), resulting in an increase in both the area and density of blood and lymphatic vessels. Upon transplantation of endometrial tissue from thromboxane prostanoid (TP) receptor (TXA2 receptor)‑deficient (TP‑/‑) mice into TP‑/‑ mice (TP‑/‑→TP‑/‑), an increase in implant growth, angiogenesis, and lymphangiogenesis were observed along with upregulation of pro‑angiogenic and lymphangiogenic factors, including vascular endothelial growth factors (VEGFs). Similar results were obtained using a thromboxane synthase (TXS) inhibitor in WT→WT mice. Furthermore, TP‑/‑→TP‑/‑ mice had a higher number of F4/80+ cells than that of WT→WT mice, with increased expression of genes related to the anti‑inflammatory macrophage phenotype in endometrial lesions. In cultured bone marrow (BM)‑derived macrophages, the levels of VEGF‑A, VEGF‑C, and VEGF‑D decreased in a TP‑dependent manner. Furthermore, TP signaling affected the polarization of cultured BM‑derived macrophages to the anti‑inflammatory phenotype. These findings imply that inhibition of TP signaling promotes endometrial implant growth and neovascularization.

Aspirin Inhibits the Inflammatory Response of Protease-Activated Receptor 1 in Pregnancy Neutrophils: Implications for Treating Women with Preeclampsia.

Neutrophils expressing cyclooxygenase-2 (COX-2) extensively infiltrate maternal blood vessels in preeclampsia, associated with vascular inflammation. Because pregnancy neutrophils also express protease-activated receptor 1 (PAR-1, F2R thrombin receptor), which they do not in non-pregnant subjects, they can be activated by proteases. We tested the hypothesis that aspirin at a dose sufficient to inhibit COX-2 would reduce inflammatory responses in preeclampsia neutrophils. Neutrophils were isolated from normal pregnant and preeclamptic women at approximately 30 weeks' gestation. Normal pregnancy neutrophils were treated with elastase, a protease elevated in preeclampsia, or elastase plus aspirin to inhibit COX-2, or elastase plus pinane thromboxane, a biologically active structural analog of thromboxane and a thromboxane synthase inhibitor. Preeclamptic pregnancy neutrophils were treated with the same doses of aspirin or pinane thromboxane. Confocal microscopy with immunofluorescence staining was used to determine the cellular localization of the p65 subunit of nuclear factor-kappa B (NF-κB) and media concentrations of thromboxane were measured to evaluate the inflammatory response. In untreated neutrophils of normal pregnant women, p65 was localized to the cytosol. Upon stimulation with elastase, p65 translocated from the cytosol to the nucleus coincident with increased thromboxane production. When neutrophils were co-treated with aspirin or pinane thromboxane, elastase was not able to cause nuclear translocation of p65 or increase thromboxane. In untreated neutrophils of preeclamptic women, the p65 subunit was present in the nucleus and thromboxane production was elevated, but when preeclamptic neutrophils were treated with aspirin or pinane thromboxane, p65 was cleared from the nucleus and returned to the cytosol along with decreased thromboxane production. These findings suggest that COX-2 is a downstream mediator of PAR-1 and demonstrate that PAR-1- mediated inflammation can be inhibited by aspirin. Given the extensive and ubiquitous expression of PAR-1 and COX-2 in preeclamptic women, consideration should be given to treating women with preeclampsia using a dose of aspirin sufficient to inhibit COX-2.

Abstract P1004: Macrophage Apoptotic Cell Receptor Mertk Triggers The Synthesis Of Arachidonic Acid Derivatives To Promote Cardiac Regeneration

Introduction: Macrophages (MΦs) are indispensable for regeneration of the mammalian neonatal heart. However, the underlying mechanisms by which neonatal MΦs sense the injured cardiac milieu and promote regeneration remain obscure. Utilizing single-cell transcriptomics, we previously observed a regenerative neonatal cardiac MΦ population that distinctly upregulates MerTK, an apoptotic cell receptor, compared to adult cardiac MΦs. Objective: We hypothesized that MerTK is required to program neonatal cardiac MΦs to secrete specialized pro-“regenerative” lipids to induce cardiomyocyte proliferation leading to heart regeneration. Methods: Postnatal day 1 and adult mice were subjected to cardiac injury via ligation of the left anterior descending artery. Single-cell transcriptomics, lipid-omics, and molecular approaches were utilized to determine the mechanisms by which neonatal MΦs regulate cardiac regeneration. Results: To determine the necessity of MΦ MerTK for cardiac regeneration, we employed a myeloid-specific knockout of MerTK ( mMertk -/- ) which failed to regain homeostatic cardiac function. We next performed single-cell transcriptomics on Mertk +/+ and Mertk -/- neonates to determine how MerTK activation reprograms neonatal MΦs for regeneration. Mertk +/+ MΦs significantly upregulated genes associated with arachidonic acid metabolism. Hypothesizing that arachidonic acid derivatives may be key signaling molecules for regeneration, we performed a targeted lipid-omics screen which revealed an increase in Thromboxane B 2 (TXB 2 ) in regenerating hearts but not within non-regenerative Mertk -/- hearts. Furthermore, addition of a TXA 2 agonist to neonatal cardiomyocytes in vitro significantly increases their proliferation by regulating the YAP/TAZ signaling. Alternatively, inhibition of thromboxane synthase in neonatal MΦs or thromboxane receptor on cardiomyocytes by small molecule inhibitors significantly abrogated the increase in cardiomyocyte proliferation by efferocytic Mertk +/+ neonatal MΦs. Conclusions: Altogether, our data suggest that MerTK programs neonatal cardiac MΦs to secrete TXA 2 to induce cardiomyocyte proliferation via the HIPPO-YAP/TAZ pathway prompting neonatal cardiac regeneration.

Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-β (transforming growth factor-β) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

THROMBOXANE-DEPENDENT CORONARY VASOCONSTRICTION IN OBESE MICE: ROLE OF PEROXYNITRITE

Objective: Obesity leads to chronic oxidative stress promoting the development of cardiovascular diseases including endothelial dysfunction, which might occur through the cyclooxygenase (COX) pathway. This study aimed to investigate the role of peroxynitrite in the generation of COX-derived products and its role in coronary vascular responses. Design and method: We evaluated arachidonic acid (AA)-dependent coronary vascular responses in isolated hearts from control and obese C57BL/6 mice and explored COX metabolism using the COX-2 inhibitor indomethacin and 1-benzylimidazole, inhibitor of thromboxane synthase (TXS). COX, TXS, and TXA2 receptor (TP) protein expression was assessed in heart homogenates. The release of TXA2 in the coronary circulation was also evaluated. In addition, the possible involvement of peroxynitrite on increased coronary response was explored by detecting peroxynitrite by coumarin boronic acid (CBA) and 3-nitrotyrosine protein expression. Alterations in TXS, and TP receptor protein expression were also evaluated in heart homogenates from obese mice treated with the peroxynitrite scavenger FeTMPyP. Results: Arachidonic acid (AA) dependent coronary vasoconstriction in isolated hearts, increased from 31.06 ± 2.67 mmHg in control mice to 88.99 ± 6.09 mmHg in obese mice. Both, indomethacin and 1-benzylimidazole reduced the vasoconstriction response in control and obese mice. TXA2 released during AA-induced vasoconstriction phase was increased in heart perfusates from 76.1 ± 18.42 pg/mL in control mice to 203.6 ± 29.8 pg/mL in obese mice. COX- 2, TXS and TP receptor protein expression was increased in hearts from obese mice (1.96 ± 0.25, 0.70 ± 0.07, 1.22 ± 0.19 arbitrary units, respectively) compared with control mice (0.17 ± 0.02; 0.06 ± 0.02; and 0.29 ± 0.06 arbitrary units, respectively). Moreover, nitrotyrosine and peroxynitrite levels were higher in obese mice, compared with control mice. Increase in coronary vasoconstriction, TXA2 release as well as protein expression was prevented with FeTMPyP treatment in obese mice. Conclusions: Peroxynitrite-dependent induction of COX-2 and TXS protein expression in heart tissue from obese mice may lead to increased TXA2 coronary circulation, thereby increasing coronary vasoconstriction.

Screening of Synthetic Heterocyclic Compounds as Antiplatelet Drugs.

Antiplatelet drugs represent the keystone in the treatment and prevention of diseases of ischemic origin, including coronary artery disease. The current palette of drugs represents efficient modalities in most cases, but their effect can be limited in certain situations or associated with specific side effects. In this study, representatives of compounds selected from series having scaffolds with known or potential antiplatelet activity were tested. These compounds were previously synthetized by us, but their biological effects have not yet been reported. The aim of this study was to examine the antiplatelet and anticoagulation properties of selected compounds and determine their mechanism of action. Antiplatelet activity of compounds and their mechanisms of action were evaluated using human blood by impedance aggregometry and various aggregation inducers and inhibitors and compared to appropriate standards. Cytotoxicity was tested using breast adenocarcinoma cell cultures and potential anticoagulation activity was also determined. In total, four of 34 compounds tested were equally or more active than the standard antiplatelet drug Acetylsalicylic Acid (ASA). In contrast to ASA, all 4 active compounds decreased platelet aggregation triggered not only by collagen, but also partly by ADP. The major mechanism of action is based on antagonism at thromboxane receptors. In higher concentrations, inhibition of thromboxane synthase was also noted. In contrast to ASA, the tested compounds did not block cyclooxygenase- 1. The most active compound, 2-amino-4-(1H-indol-3-yl)-6-nitro-4H-chromene-3- carbonitrile (2-N), which is 4-5x times more potent than ASA, is a promising compound for the development of novel antiplatelet drugs.

Effects of human TFPI and CD47 expression and selectin and integrin inhibition during GalTKO.hCD46 pig lung perfusion with human blood.

Loss of barrier function when GalTKO.hCD46 porcine lungs are perfused with human blood is associated with coagulation pathway dysregulation, innate immune system activation, and rapid sequestration of human formed blood elements. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (hTFPI) and human CD47 (hCD47), alone or with combined selectin and integrin adhesion pathway inhibitors, delays GalTKO.hCD46 porcine lung injury or modulates neutrophil and platelet sequestration. In a well-established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic porcine lungs (hTFPI.hCD47, n=7) were compared to GalTKO.hCD46 lungs (reference, n=5). All lung donor pigs were treated with a thromboxane synthase inhibitor, anti-histamine, and anti-GPIb integrin-blocking Fab, and were pre-treated with Desmopressin. In both genotypes, one lung of each pair was additionally treated with PSGL-1 and GMI-1271 (P- and E-selectin) and IB4 (CD11b/18 integrin) adhesion inhibitors (n=6 hTFPI.hCD47, n=3 reference). All except for two reference lungs did not fail within 480 min when experiments were electively terminated. Selectin and integrin adhesion inhibitors moderately attenuated initial pulmonary vascular resistance (PVR) elevation in hTFPI.hCD47 lungs. Neutrophil sequestration was significantly delayed during the early time points following reperfusion and terminal platelet activation was attenuated in association with lungs expressing hTFPI.hCD47, but additional adhesion pathway inhibitors did not show further effects with either lung genotype. Expression of hTFPI.hCD47 on porcine lung may be useful as part of an integrated strategy to prevent neutrophil adhesion and platelet activation that are associated with xenograft injury. Additionally, targeting canonical selectin and integrin adhesion pathways reduced PVR elevation associated with hTFPI.hCD47 expression, but did not significantly attenuate neutrophil or platelet sequestration. We conclude that other adhesive mechanisms mediate the residual sequestration of human formed blood elements to pig endothelium that occurs even in the context of the multiple genetic modifications and drug treatments tested here.

Comparative study on the effect of aspirin, TP receptor antagonist and TxA2 synthase inhibitor on the vascular tone of human saphenous vein and internal mammary artery

AimsThromboxane (TxA2) is synthesized from arachidonic acid (AA) via thromboxane synthase (TxS) enzyme and induces vasoconstriction via TP receptor. Our aim is to compare the effects of aspirin, TxS inhibitor and TP receptor antagonist on vascular reactivity of bypass grafts (saphenous vein and internal mammary artery). Main methodsUsing isolated organ bath, saphenous vein and internal mammary artery preparations were incubated with TP receptor antagonist, TxS inhibitor, aspirin, IP or EP4 receptor antagonist. Then prostaglandin (PG)E2, PGF2α, phenylephrine and AA were administered in concentration-dependent manner. The expression of prostanoid receptor and PGI2 synthase (PGIS) enzyme was determined by Western Blot. Key findingsTP receptor antagonist inhibited the contraction induced by PGE2, PGF2α, and AA but not that induced by phenylephrine in both types of vessels. Aspirin increased phenylephrine-induced contraction only in internal mammary artery and decreased AA-induced contraction in saphenous vein. TxS inhibitor decreased both PGE2 and AA-induced contraction in both types of vessels. This decrease was reversed by co-incubation of TxS inhibitor and IP/EP4 receptor antagonists. The expressions of EP3 receptor and PGIS enzyme were greater in internal mammary artery compared to saphenous vein while IP and TP receptors expressed at similar levels. SignificanceTP receptor antagonist and TxS inhibitor are more effective to reduce contraction induced by different spasmogens in comparison to aspirin. Our results suggest that TP receptor antagonist and TxS inhibitor might have an advantage over aspirin due to their preventive effect on increased vascular reactivity observed in post-operative period of coronary artery bypass grafting.

Vasodilators for primary Raynaud's phenomenon.

Vasodilators for primary Raynaud's phenomenon.

Ex Vivo Perfusion and In Vivo Xenotransplantation of Pig Lungs with Humanized Von Willebrand Factor Demonstrate Reduced Platelet Sequestration

<h3>Purpose</h3> In normal hemostasis, activated von Willebrand factor (vWF) stimulates platelet adhesion and aggregation, but porcine vWF (pvWF) is "always active" when exposed to human blood. We hypothesized that humanized vWF (h*pvWF) would reduce this nonphysiologic platelet adhesion in lung xenotransplantation models. <h3>Methods</h3> GTKO.hCD46.h*pvWF pig lungs were perfused with human blood <i>ex vivo</i> and compared to reference GTKO.hCD46 lungs. Anti-GPIb Fab, 1-BIA (thromboxane synthase inhibitor), and antihistamines were used in all perfusions. One lung from each pair (n=5 h*pvWF, n=5 ref) also received integrin (H52/IB4) and selectin (GMI-1271, PSGL-1) blockade. Lungs surviving 8 hrs of perfusion were terminated electively. Platelets were measured by flow cytometry. h*pvWF lungs were also transplanted into baboon recipients (n=3 h*pvWF, n=10 ref), with platelets measured by CBC. <h3>Results</h3> All <i>ex vivo</i> groups had similar survival, with most electively terminated. Pulmonary vascular resistance was very low and constant in all groups. Platelet sequestration was significantly reduced and delayed in h*pvWF lungs compared to pvWF (p<0.01), not further modulated by selectin and integrin blockade (Fig.1). No significant differences were seen in BTG (platelet activation) levels, F1+2 (thrombosis) levels, or neutrophil counts. h*pvWF lung baboon recipients trended toward longer survival (p=0.07) with attenuated thrombocytopenia compared to reference lungs (p<0.01)(Fig.2). <h3>Conclusion</h3> When perfused with human blood, h*pvWF lungs had reduced sequestration of platelets compared to pvWF lungs, with similar reductions in lung xenotransplant recipients. "Humanization" of porcine vWF attenuates the nonphysiologic platelet adhesion and aggregation associated with xenograft rejection. Further study will focus on Fc-receptors, adenosine and coagulation pathway dysregulation, and activated neutrophils through additional drugs and genetic modifications.

XENOGENEIC LUNG PERFUSION USING GALTKO.HCD46 LUNGS EXPRESSING HTFPI AND HCD47

Purpose: To address known xenogeneic rejection pathways, various genetic modifications have been introduced into donor pig lines. Here we evaluated whether tissue factor pathway inhibitor (TFPI), a protease inhibitor which blocks initial steps of the extrinsic coagulation cascade, and human CD47, a “don’t eat me” immunoregulatory protein, lessen inflammation and promote GalTKO.hCD46 lung xenograft survival. Methods: In a well-established ex vivo lung perfusion model, 8 GalTKO.hCD46 and 9 GalTKO.hCD46.hTFPI.hCD47 lungs were perfused with freshly collected heparinized human blood. All lungs were treated with an anti-GPIb Fab, 1-BIA, a thromboxane synthase inhibitor and Famotidine; and donor pigs were pre-treated with Desmopressin to reduce vWF expression. In both groups, 3 or 4 lungs in addition received selectin and integrin blockers. Organs were perfused for up to 8 hours or until the lung failed. Results: All except two GalTKO.hCD46 organs (183, 440 min) ‘survived’ until elective termination after 8 hours of perfusion. While the pulmonary vascular resistance (PVR) remained low with both genotypes, additional selectin and integrin blockade lowered PVRs in both groups. Neutrophil but not platelet sequestration and activation were reduced during the first 2 hours in association with hTFPI.hCD47 expression; this trend was more pronounced in the absence of selectin and integrin blockade, reaching significance at the 1 and 2 hour time point. Further biochemical analyses are underway. Conclusion: While the expression of hTFPI and hCD47 showed a trend toward protective effects (improved survival, delayed neutrophil sequestration), overall lung physiology and hematological analyses did not demonstrate significant improvements. Lack of improvement of parameters other than PVR with addition of selectin and integrin blockade suggests that other injury pathways (e.g. non-gal antibody; cytokines; coagulation pathway dysregulation; GPIIb/IIIa-driven platelet activation) contribute to xenolung injury. Identifying and addressing these factors may be necessary to reveal effects attributable to hTFPI and hCD47.NIH U19 AI090959. Unrestricted educational gifts from United Therapeutics/ Lung Biotechnology LLC.

Enhanced Myogenic Constriction in the SHR Preglomerular Vessels Is Mediated by Thromboxane A2 Synthesis.

BackgroundSpontaneously Hypertensive Rats (SHR) have chronically elevated blood pressures at 30 weeks of age (systolic: 191.0 ± 1.0, diastolic: 128.8 ± 0.9). However, despite this chronic malignant hypertension, SHR kidneys remain relatively free of pathology due to having an augmented myogenic constriction (MC). We hypothesized that the enhanced MC in the SHR preglomerular vessels was due to increased prostaglandin and decreased nitric oxide (NO) synthesis, providing renal protection.MethodsSHR and Wistar Kyoto (WKY) arcuate and mesenteric arteries were treated with indomethacin (prostaglandin synthesis inhibitor), N omega-nitro-L-arginine (L-NNA, NO synthase inhibitor), and nifedipine (L-type calcium channel blocker); and MC was measured in these vessels. The role of endothelium in MC was examined by removing endothelium from WKY and SHR preglomerular and mesenteric arteries using human hair, and measuring MC. We also studied the source of prostaglandin in the SHR by treating endothelium-removed arcuate arteries with indomethacin and furegrelate (thromboxane synthase inhibitor).ResultsMC was enhanced in the SHR preglomerular vessels but not the mesenteric arteries. Indomethacin and LNNA removed the enhanced MC in the SHR. Nifedipine also inhibited MC in both WKY and SHR arcuate and mesenteric arteries. Removing endothelium did not change MC in either arcuate or mesenteric arteries of WKY and SHR rats; and did not remove the augmented MC in the SHR arcuate arteries. Indomethacin and furegrelate decreased MC in endothelium-removed SHR arcuate arteries and obliterated the enhanced MC in the SHR.ConclusionThe enhanced MC in the SHR arcuate arteries was due to thromboxane A2 synthesis from the tunica media and/or adventitia layers. MC was not dependent on endothelium, but was dependent on L-type calcium channels. Nevertheless, SHR arcuate arteries displayed differential intracellular calcium signaling compared to the WKYs.

Thromboxane A2 in the paraventricular hypothalamic nucleus mediates glucoprivation-induced adrenomedullary outflow

Glucoprivation stimulates a rapid sympathetic response to release and/or secrete catecholamines into the bloodstream. However, the central regulatory mechanisms involving adrenoceptors and prostanoids production in the paraventricular hypothalamic nucleus (PVN) that are responsible for the glucoprivation-induced elevation of plasma catecholamines are still unresolved. In this study, we aimed to clarify whether glucoprivation-induced activation of noradrenergic neurons projecting to the PVN can induce α- and/or β-adrenergic receptor activation and prostanoids production in the PVN to elevate plasma catecholamine levels. We examined the effects of α- and β-adrenergic receptor antagonists, a cyclooxygenase inhibitor, a thromboxane A synthase inhibitor, and a PGE2 subtype EP3 receptor antagonist on intravenously administered 2-deoxy-D-glucose (2-DG)-induced elevation of noradrenaline in the PVN and plasma levels of catecholamine in freely moving rats. In addition, we examined whether intravenously administered 2-DG can increase prostanoids levels in the PVN microdialysates. Intracerebroventricular (i.c.v.) pretreatment with phentolamine (a non-selective α-adrenergic receptor antagonist) suppressed the 2-DG-induced increase in the plasma level of adrenaline, whereas i.c.v. pretreatment with propranolol (a non-selective β-adrenergic receptor antagonist) suppressed the 2-DG-induced elevation of the plasma level of noradrenaline. I.c.v. pretreatment with indomethacin (a cyclooxygenase inhibitor) and furegrelate (a thromboxane synthase inhibitor) attenuated the 2-DG-induced elevations of both noradrenaline and adrenaline levels. Furthermore, 2-DG administration elevated the thromboxane B2 level, a metabolite of thromboxane A2 in PVN microdialysates. Our results suggest that glucoprivation-induced activation of α- and β-adrenergic receptor in the brain including the PVN and then thromboxane A2 production in the PVN, which are essential for the 2-DG-induced elevations of both plasma adrenaline and noradrenaline levels.

Are Aspirin and Apixaban Sufficient to Prevent Immunothrombosis in COVID-19?

Severe COVID-19 disease is associated with respiratory and vascular injury and microvascular immunothrombosis mediated by complement activation, inflammatory lipid storm, platelet activation, platelet-leukocyte adhesion and activation of coagulation cascade. Amongst the inflammatory lipids, thromboxane A2 (TxA2) is a known key mediator of microvascular thrombosis. The levels of TxB2, a stable metabolite of TxA2 are markedly increased in the bronchoalveolar lavage fluid and plasma in severe COVID-19 patients. Low-dose aspirin mitigates the generation of prostanoids including TxA2 by irreversible inactivation of the constitutive enzyme cyclooxygenase (COX)-1. The anti-thrombotic action of aspirin is currently being investigated in outpatient and hospitalized COVID-19 patients in the global RECOVERY and ACTIV-4 clinical trials. Several lines of investigations suggest that COX-2 plays an important and critical role in the immunothrombotic effects mediated by COVID-19. Pharmacologic inhibition of either COX-1 or COX-2 can prevent a plethora of lipid mediators of inflammation that are both pro- and anti-inflammatory in function. Thus, a more definitive approach to prevent immunothrombotic events in COVID-19 will be to directly block the prothrombotic effects of TxA2. Although thromboxane synthase (TS) inhibitors suppress TxA2 formation, accumulation of the substrate PGH2 is known to interact with the platelet and vessel wall TxA2 prostanoid receptor (TPR), thus reducing the antiplatelet effects of TS inhibitors. TPR antagonists block the activity of both TxA2 and PGH2 on platelets and vessels but do not block TxA2 production, which leads to increased generation of 11-dehydro-thromboxane B2, a stable metabolite of TxA2, and a potent agonist of the DP2 (CRTH2) receptor for prostaglandin D2 (PGD2). PGD2/DP2 receptor signaling has been implicated in immune dysregulation in viral infections including COVID-19. Ramatroban, an orally bioavailable, potent, dual TxA2/TP and PGD2/DP2 receptor antagonist, has demonstrated efficacy in a variety of animal models of atherosclerosis, thrombosis and sepsis. Ramatroban has a proven safety profile, having been used in Japan over the past 20 years as a treatment of allergic rhinitis and therefore, merits investigation as a promising antithrombotic and immunomodulator agent for chemoprophylaxis and treatment in COVID-19 patients.

Anti‐platelet agents: past, present and future

The discovery of the role of platelets in cardiovascular disease led to the introduction of aspirin as a therapeutic agent for heart disease. Aspirin has proven to be effective in preventing cardiovascular events but the fact that not all patients benefit from aspirin has led to the search for more effective agents. Numerous agents have been discovered including thromboxane synthase inhibitors, phosphodiesterase inhibitors, GPIIb/IIIa antagonists, P2Y12 antagonists and PAR‐1 antagonists. However, all faced two challenges – their cost‐benefit relationship in comparison with aspirin and the risk of bleeding that is often associated with greater efficacy. In this paper, I review the performances of these different classes of anti‐platelet agent and discuss the potential future for novel anti‐thrombotics. I also highlight the role of platelets in immunology and discuss the potential for targeting the immune properties of platelets.

L-arginine supplementation and thromboxane synthase inhibition increases cerebral blood flow in experimental cerebral malaria

Cerebral malaria pathogenesis involves vascular dysfunction with low nitric oxide (NO) bioavailability, vasoconstriction and impaired vasodilation, leading to ischemia, tissue hypoxia and ultimately death. Cerebral blood flow (CBF) involves NO and other pathways, including arachidonic acid (AA)-derived metabolites. Here we show that mice with experimental cerebral malaria (ECM) by P. berghei ANKA showed marked decreases in CBF (as assessed by laser speckle contrast imaging - LSCI) and that administration of L-arginine supplementation (50 mg/kg) and/or of the thromboxane synthase inhibitor Ozagrel (100 mg/kg) induced immediate increases in CBF. L-arginine in combination with artesunate (32 mg/kg) induced immediate reversal of brain ischemia in the short-term (1 hour), but the effect subsided after 3 and 6 hours. Neither L-arginine nor Ozagrel reversed blood brain barrier breakdown. Mice with ECM showed brain levels of selected AA-derived metabolites with a vasoconstrictor profile, with increased levels of 8-isoprostanes, 20-HETE and 14,15-DHET, whereas mice infected with a non-ECM-inducing strain of P. berghei (NK65) showed a vasodilator profile, with normal levels of 20-HETE and 14,15-DHET and increased levels of PGE2. L-arginine is capable of partially reversing cerebral ischemia and AA metabolites may play a role in the cerebrovascular dysfunction in ECM.

31 Day Xeno Lung Recipient Survival - Progress towards the Clinic

Purpose Improved gene editing techniques have accelerated generation of genetically engineered (GE) pigs with multiple knock-outs (KO) and expression of human “transgenes” that address xenograft rejection pathways. The association between various transgene combinations and prevention of lung xenograft injury was assessed in a rigorous life-supporting pig-to-baboon lung xenograft model. Methods GalTKO.hCD46 pigs with up to 5 additional modifications, including hCD55, hTBM, hEPCR, hTFPI, hCD47, hCD39, HO-1, HLA-E, A20, β4GalKO, Neu5GcKO, and humanized vWF (hvWF) were used in our last 48 left single lung transplants into baboons. A rationally designed “platform drug regimen” consisted of steroids, sC1Inh, thromboxane synthase inhibitor, histamine receptor blockers, and anti-GPIb Fab, and DDAVP donor pretreatment. Immunosuppression consisted of αCD20, ATG, MMF, αCD40, αIL6R, αIL8, and/or αTNF. Results While 3- or 4-GE lungs generally were not life-supporting and recipients survived for Conclusion The combination of multi-GE lungs with mechanism-directed drugs significantly prolongs life-supporting lung xenograft recipient survival. In addition to anti-non-Gal antibody, recipient NK cells (HLA-E), pvWF-GPIb platelet binding, and donor macrophages (CD47, thromboxane) each drive residual inflammation. Controlling these injury pathways with further mechanism-based GE and/or drugs coupled with improved IS appear likely to advance lung xenotransplantation towards clinical application.

Diverse associations between oxidative stress and thromboxane A2 in hypertensive glomerular injury

We examined the potential contributions of oxidative stress and thromboxane A2 (TXA2) to the development of regional heterogeneity in hypertensive glomerular injury using stroke-prone spontaneously hypertensive rats (SHRSP), an animal model of human essential hypertension. We also examined the effect of antioxidant treatment on the regional expression of thromboxane synthase (TXAS) mRNA using a microdissection method. Increases in the glomerular expression of TXAS mRNA were observed in the SHRSP at 15 weeks of age compared with those in the age-matched normotensive control Wistar–Kyoto (WKY) rats: 2.4-fold and 3.1-fold in the superficial and juxtamedullary glomeruli, respectively (P < 0.05). The heme oxygenase-1 mRNA expression was markedly increased (greater than eightfold, P < 0.05) in both the superficial and juxtamedullary glomeruli in the SHRSP compared with the expression in the WKY rats. In contrast to our expectations, the treatment of SHRSP with tempol (a superoxide dismutase mimetic) significantly (P < 0.05) increased the TXAS mRNA expression in the superficial glomeruli and did not improve the histological injury or albuminuria, which were both aggravated. Moreover, ozagrel (a TXAS inhibitor) had a suppressive effect on the TXAS mRNA expression and significantly (P < 0.05) improved the histological injury. These results indicated that although TXA2 and oxidative stress are linked to each other, TXA2 rather than oxidative stress may be a better therapeutic target to improve hypertensive glomerular injury.