Thromboxane A2 Production Research Articles

Effects of antagonism of thromboxane-prostanoid receptor on alcohol-associated liver disease

Backgrounds: Alcohol-associated liver disease (ALD) is one of the leading causes of liver diseases. Thromboxane-prostanoid receptor (TP-R) is widely expressed in the liver and is activated by thromboxane A2 (TXA2). A previous study reported an increase in TP-R mRNA level in liver after ethanol feeding. Preclinical studies have documented that TP-R antagonists exert a protective effect against cardiovascular and liver diseases. In particular, TP-R antagonist is reported to attenuate ALD in rodents. However, the mechanism(s) by which antagonizing TP-R attenuates ALD remains unclear. Methods: Herein, we used the chronic plus binge ethanol feeding model. Briefly, male, C57BL/6 wild-type mice (8-week) were fed a Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by a single binge administration of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8) by oral administration. Results: Western blot analysis showed that the protein level of thromboxane A2 synthase 1 ( P<0.05), responsible for TXA2 production, was upregulated in the mouse liver upon ethanol exposure. Of note, ethanol feeding significantly increased plasma aspartate transferase (AST) level ( P<0.05), which was abolished by SQ administration ( P<0.05). Moreover, compared with CON mice, ET mice showed an increase in liver protein levels of pro-inflammatory markers, including TNFa ( P<0.05), cluster of differentiation 68 (CD68, P<0.001), and vascular cell adhesion protein 1 ( P<0.05). In addition, the pro-fibrogenic markers including collagen type 1 alpha 1 chain (COL1A1, P<0.05) and matrix metalloproteinase 9 (MMP9, P<0.01) were also increased in ET mice. However, these alterations were not found in ET+SQ mice compared to their controls. In particular, ET+SQ mice exhibited a marked reduction in hepatic protein levels of CD68 ( P<0.001), COL1A1 ( P<0.001), and MMP9 ( P<0.01) versus ET mice. Interestingly, we noted that ethanol consumption decreases the protein level of NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), a component of mitochondrial complex I, in mouse liver ( P<0.05), which was reversed by SQ administration( P<0.05). Conclusion: These findings suggest that blockade of TP-R activity attenuates ethanol-induced liver inflammation and fibrosis possibly through improving the function of mitochondrial complex I. This study was supported by the NIH-NIAAA P50 award-Alcohol Center of Research-Nebraska (P50AA030407-5130). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2097: Jak2 V617f Clonal Hematopoiesis Promotes Arterial Thrombosis Via Platelet Activation And Cross-talk

JAK2 V617F ( JAK2 VF ) clonal hematopoiesis (CH) is associated with a marked increase in atherothrombotic cardiovascular disease (CVD). The role of JAK2 VF CH in arterial thrombosis has not been assessed. Using mice simulating JAK2 VF CH, we showed accelerated carotid artery thrombosis, with increased platelet counts and activation and increased leukocytes and red blood cells. There was increased platelet activation in both Jak2 VF and wild type (WT) platelets in Jak2 VF CH suggesting cross-talk between mutant and WT platelets. To assess platelet-specific effects, we used Gp1ba-Cre (VFGp1ba) to selectively express Jak2 VF in platelets. VFGp1ba increased platelet counts to a similar level as in 20% Jak2 VF CH mice but had no effect on leukocyte counts. Like Jak2 VF CH, VFGp1ba increased platelet activation and accelerated arterial thrombosis. Mechanistically, Jak2 VF platelets showed 2.8-fold up-regulation of COX-1, increased activation of cytosolic phospholipase A2, an enzyme important for arachidonic acid generation, and increased thromboxane A2 production. Conditioned media from activated Jak2 VF platelets induced WT platelet activation that was reversed by a thromboxane receptor antagonist. Low dose aspirin ameliorated carotid thrombosis in VFGp1ba and Jak2 VF CH but not in WT control mice. This study shows increased arterial thrombosis in Jak2 VF CH related to Jak2 VF platelet activation and cross-talk with WT platelets via thromboxane, suggesting a potential beneficial role of aspirin treatment in JAK2 VF CH.

Jak2V617F clonal hematopoiesis promotes arterial thrombosis via platelet activation and cross talk

AbstractJAK2V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre–mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH.

Chymase-independent vascular Ang-(1–12)/Ang II pathway and TXA2 generation are involved in endothelial dysfunction in the murine model of heart failure

The angiotensin (Ang)-(1–12)/Ang II pathway contributes to cardiac pathology. However, its involvement in the development of peripheral endothelial dysfunction associated with heart failure (HF) remains unknown. Therefore, this study aimed to characterise the effect of exogenous Ang-(1–12) and its conversion to Ang II on endothelial function using the murine model of HF (Tgαq*44 mice), focusing on the role of chymase and vascular-derived thromboxane A2 (TXA2).Ex vivo myographic assessments of isolated aorta showed impaired endothelium-dependent vasodilation in late-stage HF in 12-month-old Tgαq*44 mice. However, endothelium-dependent vasodilation was fully preserved in the early stage of HF in 4-month-old Tgαq*44 mice and 4- and 12-month-old FVB control mice. Ang-(1–12) impaired endothelium-dependent vasodilation in 4- and 12-month-old Tgαq*44 mice, that was associated with increased Ang II production. The chymase inhibitor chymostatin did not inhibit this response. Interestingly, TXA2 production reflected by TXB2 measurement was upregulated in response to Ang-(1–12) and Ang II in aortic rings isolated from 12-month-old Tgαq*44 mice but not from 4-month-old Tgαq*44 mice or age-matched FVB mice. Furthermore, in vivo magnetic resonance imaging showed that Ang-(1–12) impaired endothelium-dependent vasodilation in the aorta of Tgαq*44 mice and FVB mice. However, this response was inhibited by angiotensin I converting enzyme (ACE) inhibitor; perindopril, angiotensin II receptor type 1 (AT1) antagonist; losartan and TXA2 receptor (TP) antagonist-picotamide in 12-month-old-Tgαq*44 mice only.In conclusion, the chymase-independent vascular Ang-(1–12)/Ang II pathway and subsequent TXA2 overactivity contribute to systemic endothelial dysfunction in the late stage of HF in Tgαq*44 mice. Therefore, the vascular TXA2 receptor represents a pharmacotherapeutic target to improve peripheral endothelial dysfunction in chronic HF.

Thromboxane-induced cerebral microvascular rarefaction predicts depressive symptom emergence in metabolic disease.

Previous studies have suggested that the loss of microvessel density in the peripheral circulation with evolving metabolic disease severity represents a significant contributor to impaired skeletal muscle oxygenation and fatigue-resistance. Based on this and our recent work, we hypothesized that cerebral microvascular rarefaction was initiated from the increased prooxidant and proinflammatory environment with metabolic disease and is predictive of the severity of the emergence of depressive symptoms in obese Zucker rats (OZRs). In male OZR, cerebrovascular rarefaction followed the emergence of elevated oxidant and inflammatory environments characterized by increased vascular production of thromboxane A2 (TxA2). The subsequent emergence of depressive symptoms in OZR was associated with the timing and severity of the rarefaction. Chronic intervention with antioxidant (TEMPOL) or anti-inflammation (pentoxifylline) therapy blunted the severity of rarefaction and depressive symptoms, although the effectiveness was limited. Blockade of TxA2 production (dazmegrel) or action (SQ-29548) resulted in a stronger therapeutic effect, suggesting that vascular production and action represent a significant contributor to rarefaction and the emergence of depressive symptoms with chronic metabolic disease (although other pathways clearly contribute as well). A de novo biosimulation of cerebrovascular oxygenation in the face of progressive rarefaction demonstrates the increased probability of generating hypoxic regions within the microvascular networks, which could contribute to impaired neuronal metabolism and the emergence of depressive symptoms. The results of the present study also implicate the potential importance of aggressive prodromic intervention in reducing the severity of chronic complications arising from metabolic disease.NEW & NOTEWORTHY With clinical studies linking vascular disease risk to depressive symptom emergence, we used obese Zucker rats, a model of chronic metabolic disease, to identify potential mechanistic links between these two negative outcomes. Depressive symptom severity correlated with the extent of cerebrovascular rarefaction, after increased vascular oxidant stress/inflammation and TxA2 production. Anti-TxA2 interventions prevasculopathy blunted rarefaction and depressive symptoms, while biosimulation indicated that cerebrovascular rarefaction increased hypoxia within capillary networks as a potential contributing mechanism.

Daidzein Inhibits Human Platelet Activation by Downregulating Thromboxane A2 Production and Granule Release, Regardless of COX-1 Activity.

Platelets play crucial roles in cardiovascular diseases (CVDs) by regulating hemostasis and blood coagulation at sites of blood vessel damage. Accumulating evidence indicates daidzein inhibits platelet activation, but the mechanism involved has not been elucidated. Thus, in this study, we investigated the mechanism responsible for the inhibition of collagen-induced platelet aggregation by daidzein. We found that in collagen-induced platelets, daidzein suppressed the production of thromboxane A2 (TXA2), a molecule involved in platelet activation and aggregation, by inhibiting the cytosolic phospholipase A2 (cPLA2) signaling pathway. However, daidzein did not affect cyclooxygenase-1 (COX-1). Furthermore, daidzein attenuated the PI3K/PDK1/Akt/GSK3αβ and MAPK (p38, ERK) signaling pathways, increased the phosphorylation of inositol trisphosphate receptor1 (IP3R1) and vasodilator-stimulated phosphoprotein (VASP), and increased the level of cyclic adenosine monophosphate (cAMP). These results suggest that daidzein inhibits granule release (ATP, serotonin, P-selectin), integrin αIIbβ3 activation, and clot retraction. Taken together, our study demonstrates that daidzein inhibits collagen-induced platelet aggregation and suggests that daidzein has therapeutic potential for the treatment of platelet aggregation-related diseases such as atherosclerosis and thrombosis.

Myeloid Bmal1 deletion suppresses the house dust mite-induced chronic lung allergy.

Asthma is the chronic pulmonary inflammatory response that could lead to respiratory failure when allergic reactions exacerbate. It is featured by type 2 immunity with eosinophilic inflammation, mucus, and IgE production, and Th2 cytokine secretion upon repeated challenge of allergens. The symptom severity of asthma displays an apparent circadian rhythm with aggravated airway resistance in the early morning in patients. Bmal1 is the core regulator of the circadian clock, while the regulatory role of Bmal1 in asthma remains unclear. Here, we investigate whether the myeloid Bmal1 is involved in the pathogenesis of house dust mite (HDM)-induced lung allergy. We found that knockdown of Bmal1 in macrophages suppressed the time-of-day variance of the eosinophil infiltration in the alveolar spaces in chronic asthmatic mice. This was accompanied by decreased bronchial mucus production, collagen deposition, and HDM-specific IgE production. However, the suppression effects of myeloid Bmal1 deletion did not alter the allergic responses in short-term exposure to HDM. The transcriptome profile of alveolar macrophages (AMs) showed that Bmal1-deficient AMs have enhanced phagocytosis and reduced production of allergy-mediating prostanoids thromboxane A2 and prostaglandin F2α synthesis. The attenuated thromboxane A2 and prostaglandin F2α may lead to less induction of the eosinophil chemokine Ccl11 expression in bronchial epithelial cells. In summary, our study demonstrates that Bmal1 ablation in macrophages attenuates eosinophilic inflammation in HDM-induced chronic lung allergy, which involves enhanced phagocytosis and reduced prostanoid secretion.

Pharmacological Efficacy and Gastrointestinal Safety of Different Aspirin Formulations for Cardiovascular Prevention: A Narrative Review

Aspirin inhibits platelet function by irreversibly inhibiting the synthesis of thromboxane A2 (TxA2). Aspirin, at low doses, is widely used for cardiovascular prevention. Gastrointestinal discomfort, mucosal erosions/ulcerations and bleeding are frequent complications of chronic treatment. To reduce these adverse effects, different formulations of aspirin have been developed, including enteric-coated (EC) aspirin, the most widely used aspirin formulation. However, EC aspirin is less effective than plain aspirin in inhibiting TxA2 production, especially in subjects with high body weight. The inadequate pharmacological efficacy of EC aspirin is mirrored by lower protection from cardiovascular events in subjects weighing >70 kg. Endoscopic studies showed that EC aspirin causes fewer erosions of the gastric mucosa compared to plain aspirin (which is absorbed in the stomach) but causes mucosal erosions in the small intestine, where it is absorbed. Several studies demonstrated that EC aspirin does not reduce the incidence of clinically relevant gastrointestinal ulceration and bleeding. Similar results were found for buffered aspirin. Although interesting, the results of experiments on the phospholipid-aspirin complex PL2200 are still preliminary. Considering its favorable pharmacological profile, plain aspirin should be the preferred formulation to be used for cardiovascular prevention.

Preeclampsia and aspirin.

Preeclampsia (PE) is a multisystem disorder that is an important cause of maternal and perinatal deaths. Currently, delivery is the only final treatment for PE. This practice is usually accompanied by premature birth, which inevitably increases neonatal morbidities. Aspirin is a non-selective NSAID that irreversibly inhibits cyclooxygenase enzymes involved in converting arachidonic acid to prostaglandins and thromboxane. Aspirin inhibits thromboxane A2 production via platelet aggregation, thereby increasing the prostacyclin/thromboxane A2 ratio and reducing platelet aggregation. Since the first case report of aspirin's potential use during pregnancy was reported in 1978, many studies have attempted to confirm the effect of aspirin on preeclampsia, and the results have been controversial. However, this preventive strategy is generally accepted in clinical practice. As evidence for aspirin's prevention of pre-eclampsia has been accumulating, a recent study investigated the effectiveness of aspirin at high doses of 150 mg, which is higher than before. However, there is an ongoing debate about how much aspirin should be used during pregnancy and when to start aspirin therapy. Guidelines for the use of prophylactic aspirin during pregnancy vary slightly among countries and groups. In this article, we review and summarize the evidence regarding the use of aspirin for PE prevention.

Effect of low-dose aspirin on urinary 11-dehydro-thromboxane B2 in the ASCEND (A Study of Cardiovascular Events iN Diabetes) randomized controlled trial

BackgroundAspirin is widely used for cardioprotection with its antiplatelet effects due to the blocking of thromboxane A2 production. However, it has been suggested that platelet abnormalities in those with diabetes prevent adequate suppression with once daily aspirin.MethodsIn the ASCEND randomized double-blind trial of aspirin 100 mg once daily versus placebo in participants with diabetes but no history of cardiovascular disease, suppression was assessed by measuring 11-dehydro-thromboxane B2 excretion in urine (U-TXM) in a randomly selected sample of 152 participants (76 aspirin arm, 74 placebo arm), plus 198 (93 aspirin arm, 105 placebo arm) adherent to study drugs and selected to maximize the numbers ingesting their last tablet 12–24 h before urine sampling. U-TXM was assayed using a competitive ELISA assay in samples mailed a mean of 2 years after randomization, with time since taking last aspirin/placebo tablet recorded at the time of sample provision. Effective suppression (U-TXM < 1500 pg/mg creatinine) and percentage reductions in U-TXM by aspirin allocation were compared.ResultsIn the random sample, U-TXM was 71% (95% CI 64–76%) lower among aspirin vs placebo-allocated participants. Among adherent participants in the aspirin arm, U-TXM was 72% (95% CI 69–75%) lower than in the placebo arm and 77% achieved effective suppression overall. Suppression was similar among those who ingested their last tablet more than 12 h before urine sampling with levels in the aspirin arm 72% (95% CI 67–77%) lower than in the placebo arm and 70% achieving effective suppression.ConclusionsDaily aspirin significantly reduces U-TXM in participants with diabetes, including at 12–24 h after ingestion.Trial registrationISRCTN ISRCTN60635500. Registered on 1 Sept 2005; ClinicalTrials.gov NCT00135226. Registered on 24 Aug 2005.

Increased Thromboxane A2 Levels in Pulmonary Artery Smooth Muscle Cells Isolated from Patients with Chronic Obstructive Pulmonary Disease.

Introduction: Pulmonary hypertension due to chronic obstructive pulmonary disease (COPD) is classified as Group 3 pulmonary hypertension, with no current proven targeted therapies. It has been shown that cigarette smoke, the main risk factor for COPD, can increase thromboxane A2 production in healthy human pulmonary artery smooth muscle cells and pulmonary artery endothelial cells, and that blocking the effect of increased thromboxane A2 using daltroban, a thromboxane A2 receptor antagonist, can inhibit cigarette smoke-induced pulmonary artery cell proliferation. However, it is largely unknown whether thromboxane A2 is increased in smokers with COPD. Therefore, the aim of this study was to assess the level of thromboxane A2 production in patients with COPD who smoke. Methods: Pulmonary artery smooth muscle cells from three smokers with COPD and three healthy donors were cultured in cell culture medium. The culture medium was collected and the thromboxane B2 (a stable metabolite of thromboxane A2) released in the culture medium was quantified using an ELISA kit. The data were normalised with the total protein concentration and then expressed in pg/mg protein. Demographic data were collected and baseline pulmonary function tests of patients with COPD were conducted. Results: The mean age of patients with COPD was 69 ± 7 years. All patients were smokers and had a mean smoking history of 39.66 ± 9.50 packs per year. The mean forced expiratory volume in one second, that is, FEV1%, and the ratio of forced vital capacity (FVC) to FEV1% of COPD patients were 63.33 ± 19.60% and 52.66 ± 14.64%, respectively. The results revealed that thromboxane A2 production was significantly increased in pulmonary artery smooth muscle cells from smokers with COPD (434.56 ± 82.88 pg/mg protein) compared with the thromboxane A2 levels in pulmonary artery smooth muscle cells from healthy donors (160 ± 59.3 pg/mg protein). Conclusions: This is the first report of increased thromboxane A2 production in pulmonary artery smooth muscle cells from smokers with COPD. This observation strongly suggests that thromboxane A2 can be used as a novel therapeutic target for the treatment of patients with COPD-associated pulmonary hypertension.

Aspirin in essential thrombocythemia. For whom? What formulation? What regimen?

Essential thrombocythemia (ET) is a BCR-ABL1-negative myeloproliferative neoplasm, the most common clinical manifestations of which include arterial and venous thrombosis, bleeding and vasomotor/microvascular disturbances. Low-dose (81-100 mg) aspirin once daily, which irreversibly inhibits platelet thromboxane A2 (TxA2) production by acetylating cyclo-oxygenase-1, is the recommended treatment for the control of vascular events in all ET risk categories, except patients at very low risk, who need aspirin for treatment of vasomotor/microvascular disturbances only. Simple observation should be preferred over aspirin prophylaxis in low-risk patients with platelet counts >1,000x109/L or harboring CALR mutations. Plain aspirin should be preferred over enteric coated aspirin because some ET patients display poor responsiveness ("resistance") to the latter. When treated with a once daily aspirin regimen, adequate inhibition of platelet TxA2 production (measured as serum thromboxane B2 level) does not persist for 24 h in most patients. This phenomenon is associated with the patients' platelet count and the number (but not the fraction) of circulating immature reticulated platelets with non-acetylated cyclo-oxygenase-1 and is therefore consequent to high platelet production (the hallmark of ET), rather than increased platelet turnover (which is normal in ET). Twice daily aspirin administration overcame this problem and proved safe in small studies. Although additional data on gastrointestinal tolerability will be useful, the twice daily regimen could already be implemented in clinical practice, considering its favorable risk/benefit profile. However, patients whose platelet count has been normalized could still be treated with the once daily regimen, because they would otherwise be unnecessarily exposed to a potential small risk of gastrointestinal discomfort.

Aspirin Suppresses Hepatic Glucagon Signaling Through Decreasing Production of Thromboxane A2.

Excessive hepatic glucose production (HGP) is a major cause of fasting hyperglycemia in diabetes, and antihyperglycemic therapy takes center stage. Nonsteroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), reduce hyperglycemia caused by unrestrained gluconeogenesis in diabetes, but its mechanism is incompletely understood. Here, we reported that aspirin lowers fasting blood glucose and hepatic gluconeogenesis, corresponds with lower thromboxane A2 (TXA2) levels, and the hypoglycemic effect of aspirin could be rescued by TP agonist treatment. On fasting and diabetes stress, the cyclooxygenase (COX)/TXA2/thromboxane A2 receptor (TP) axis was increased in the livers. TP deficiency suppressed starvation-induced hepatic glucose output, thus inhibiting the progression of diabetes, whereas TP activation promoted gluconeogenesis. Aspirin restrains glucagon signaling and gluconeogenic gene expression (phosphoenolpyruvate carboxykinase [PCK1] and glucose-6-phosphatase [G6Pase]) through the TXA2/TP axis. TP mediates hepatic gluconeogenesis by activating PLC/IP3/IP3R signaling, which subsequently enhances CREB phosphorylation via facilitating CRTC2 nuclear translocation. Thus, our findings demonstrate that TXA2/TP plays a crucial role in aspirin's inhibition of hepatic glucose metabolism, and TP may represent a therapeutic target for diabetes.

Augmentation by resveratrol of the inhibitory effect of ethanol on platelet aggregation

Ethanol and resveratrol have been shown to inhibit platelet aggregation. The aim of this study was to determine whether resveratrol has an additional effect on ethanol-induced inhibition of platelet aggregation. Ca2+ entry and subsequent aggregation of human platelets were measured by the fluorescence method and light transmittance method, respectively. Thromboxane B2 concentrations in media containing platelets were measured by using the enzyme-linked immunosorbent assay. Platelet aggregation induced by thrombin (0.025 U/ml) was significantly inhibited by preincubation of platelets with ethanol (0.5%). Preincubation with resveratrol (3.125 µM), which did not affect thrombin-induced platelet aggregation, significantly augmented the inhibitory effect of ethanol on platelet aggregation. Similar synergic effects of ethanol and resveratrol were found on aggregatory responses to collagen (2 µg/ml) and arachidonic acid (0.25 mM). On the other hand, the thrombin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was not affected by ethanol alone, resveratrol alone or both ethanol and resveratrol together. In nominally Ca2+-free medium, arachidonic acid (0.75 mM) caused a potent platelet aggregation, which was not affected by the presence of ethanol alone, resveratrol alone, or both of them together. Thromboxane B2 formation induced by thrombin was significantly inhibited by ethanol (0.5%) alone and resveratrol (3.125 µM) alone, and these inhibitory effects were significantly augmented in the presence of both ethanol and resveratrol together. Resveratrol shows an additive effect on ethanol-induced inhibition of platelet aggregation. This effect by resveratrol is partly explained by its inhibitory action on thromboxane A2 production in platelets. In addition, both ethanol and resveratrol attenuate platelet aggregation through acting on the Ca2+-dependent intra-platelet pathway after an increase in [Ca2+]i induced by thrombin.

Pharmacological Actions of 5-Hydroxyindolin-2 on Modulation of Platelet Functions and Thrombus Formation via Thromboxane A2 Inhibition and cAMP Production

Platelets play a very significant role in hemostasis while simultaneously posing a risk for the development of various cardiovascular diseases. Platelet-mediated issues can occur in blood vessels and trigger various medical problems. Therefore, controlling platelet function is important in the prevention of thrombosis. In this regard, we need to find compounds that provide potent antiplatelet activity with minimum side effects. Therefore, we examined the effect of 5-hydroxyindolin-2-one isolated from Protaetia brevitarsis larvae having antiplatelet properties and investigated different pathways that mediate the antiplatelet activity. We examined the effect of 5-hydroxyindolin-2-one (5-HI) on the regulation of phosphoproteins, thromboxane A2 generation, and integrin αIIbβ3 action. Our data showed that human platelet aggregation was inhibited by 5-HI (75, 100, 150, 200 μM) without cytotoxicity, and it suppressed intracellular Ca2+ concentration through the regulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756) and extracellular signal-regulated kinase (ERK). Moreover, collagen-elevated thromboxane A2 production and αIIbβ3 action were inhibited by 5-HI through the regulation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinase p38 (p38MAPK), vasodilator-stimulated phosphoprotein (VASP), phosphoinositide 3-kinase (PI3K), and Akt (protein kinase B). Therefore, we suggested that 5-HI could be a potential substance for the prevention of thrombosis-mediated thrombosis.

DPP4 Promotes Human Endothelial Cell Senescence and Dysfunction via the PAR2-COX-2-TP Axis and NLRP3 Inflammasome Activation.

Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. Human endothelial cell senescence was assessed by senescence-associated β-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA2 (thromboxane A2) acting over TP (thromboxane receptor) receptors (PAR2-COX-2-TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.

Aspirin resistance among patients with new and recurrent ischemic heart disease episodes in Qassim region, Saudi Arabia.

Aspirin resistance is described as the failure of aspirin to decrease the production of thromboxane A2 by platelets, which is the mechanism by which aspirin decreases platelet activation and aggregation. This study was performed to assess the prevalence of aspirin resistance among cardiovascular patients in al-Qassim, Saudi Arabia. The study used a survey of patients with first and recurrent attacks of ischemic heart disease (IHD) and available data from blood samples processed using a VerifyNow® kit, which measures aspirin reaction units (ARUs). A total of 119 patients were included: 45 with their first IHD episodes and 74 with recurrent episodes. Of the surveyed patients, 40% with a first episode were younger than 50 years old, and 75.6% of them have been diagnosed with IHD during the previous 5 years. Of the patients with recurrent attacks, 45.9% were older than 60 years, and 54.1% of them have been diagnosed more than 5 years before. The group with first episodes of IHD had 133.2 ARUs, whereas the group with recurrent episodes had 168.5 ARUs (p=0.105). In the recurrent-episode group, 77% had diabetes; in the first-episode group, only 37.8% had diabetes (p≤0.001). Overall, 46.2% were overweight, 54.6% were nonsmokers, and 82.4% underwent percutaneous coronary intervention. The study participants in both the new and recurrent IHD groups showed no sign of aspirin resistance. The presence of cardiovascular risk factors increased the likelihood of episode recurrence.

Pharmacokinetic and Pharmacodynamic Profile of a Novel Phospholipid Aspirin Formulation.

Aspirin is one of the most widely used medicines. Although aspirin is commonly utilized for the treatment of several medical conditions, its broadest uptake is for the prevention of recurrent ischemic events in patients with atherosclerotic disease. Its mechanism of action of inhibiting platelet activation via blockade of thromboxane A2 production is unique and is not covered by any other antiplatelet agents. While plain, uncoated, immediate-release aspirin is used in acute settings to help assure rapid absorption, enteric-coated aspirin formulations dominate current chronic use, particularly in North America, including for secondary prevention of cardiovascular events. The unmet needs with current aspirin formulations include a high risk of gastrointestinal (GI) adverse events with plain aspirin, which enteric-coated formulations are not able to overcome, and subject to erratic absorption leading to reduced drug bioavailability. These observations underscore the need for aspirin formulations with a more favorable safety and efficacy profile. Phospholipid-aspirin complex (PL-ASA) is a novel formulation designed to address these needs. It is associated with reduced local acute GI injury compared with plain aspirin, and predictable absorption resulting in more reliable platelet inhibition compared with enteric-coated tablets. This review explores the rationale and pharmacologic profile of PL-ASA intended to address the unmet needs for aspirin therapy.

Protein disulfide isomerase‐A1 regulates intraplatelet reactive oxygen species–thromboxane A2‐dependent pathway in human platelets

BackgroundPlatelet‐derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood. AimsThe aim of this study was to characterize the role of PDIA1 in human platelets. MethodsProteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP‐14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A2 (TxA2), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate‐based fluorescent assay, respectively. ResultsPDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP‐14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP‐14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA2 and ROS production in collagen‐ or thrombin‐stimulated platelets. Furthermore, bepristat reduced the activation of αIIbβ3 integrin and expression of P‐selectin. ConclusionsPDIA1 acts as an intraplatelet regulator of the ROS‐TxA2 pathway in collagen‐GP VI receptor‐mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbβ3 integrin by PDIA3.

Association of Platelet Thromboxane Inhibition by Low-Dose Aspirin With Platelet Count and Cytoreductive Therapy in Essential Thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by enhanced platelet production and thrombotic complications. The inhibition of platelet cyclooxygenase (COX) activity by the standard once‐daily aspirin is mostly incomplete due to accelerated thrombopoiesis. The phase II Aspirin Regimens in EsSential thrombocythemia (ARES) trial has recently compared the efficacy of once‐ vs. twice‐ or three‐times daily low‐dose aspirin in inhibiting platelet thromboxane (TX) A2 production, as reflected by serum (s) TXB2 measurements. The present substudy characterized the determinants of the highly variable response to the standard aspirin 100 mg once‐daily regimen in fully compliant patients with ET and the effects of the experimental dosing regimens on response variability. By multivariable analysis, the platelet count (directly) and cytoreductive treatment (inversely) were significantly associated with sTXB2 values in 218 patients with ET. However, the platelet count positively correlated with sTXB2 in patients not being treated with cytoreductive drugs (ρ = 0.51, P < 0.01, n = 84), but not in patients on cytoreduction. Patients in the lowest sTXB2 quartile were older, more often on cytoreductive drugs, had lower platelet count and Janus‐Associated Kinase2 (JAK2)‐V617F allele frequency as compared with patients in the upper sTXB2 quartiles. After 2 weeks of a twice‐ or 3‐times daily aspirin regimen, the association between the platelet count and sTXB2 became similar in cytoreduced and non‐cytoreduced patients. In conclusion, the platelet count appears the strongest determinant of TXA2 inhibition by once‐daily low‐dose aspirin in ET, with different patterns depending of cytoreductive treatment. More frequent aspirin dosing restores adequate platelet inhibition and reduces interindividual variability, independently of cytoreduction.