A Thromboxane A2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis.

BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.

Roles of Thromboxane Receptor Signaling in Enhancement of Lipopolysaccharide-Induced Lymphangiogenesis and Lymphatic Drainage Function in Diaphragm.

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Prenatal endothelin or thromboxane receptor antagonism surpasses sympathoinhibition in improving cardiorenal malfunctions in preeclamptic rats.

Current therapies for preeclampsia (PE) and its complications are limited and defective. Considering the importance of endothelin (ET) and thromboxane A2 (TXA2) signaling in PE pathophysiology, we tested the hypothesis that prenatal blockade of endothelin ETA or thromboxane TXA2 receptors favorably reprograms preeclamptic cardiovascular and renal insults. PE was induced by daily oral administration of L-NAME (50 mg/kg) to pregnant rats for 7 consecutive days starting from gestational day 14. The effects of co-exposure to atrasentan (ETA receptor blocker, 10 mg/kg/day) or terutroban (TXA2 receptor blocker, 10 mg/kg/day) on cardiovascular and renal anomalies induced by PE were assessed on gestational day 20 (GD20) and at weaning time and compared with those evoked by the sympatholytic drug α-methyldopa (α-MD, 100 mg/kg/day), a prototypic therapy for PE management. Among all drugs, terutroban was basically the most potent in ameliorating PE-evoked increments in blood pressure and decrements in creatinine clearance. Cardiorenal tissues of PE rats exhibited significant increases in ETA and TXA2 receptor expressions and these effects disappeared after treatment with atrasentan and to a lesser extent by terutroban or α-MD. Atrasentan was also the most effective in reversing the reduced ETB receptor expression in renal tissues of PE rats. Signs of histopathological damage in cardiac and renal tissues of PE rats were mostly improved by all therapies. Together, pharmacologic elimination of ETA or TXA2 receptors offers a relatively better prospect than α-MD in controlling perinatal cardiorenal irregularities sparked by PE.

Antiplatelet properties of Pim kinase inhibition are mediated through disruption of thromboxane A2 receptor signaling.

Pim kinases are upregulated in several forms of cancer, contributing to cell survival and tumour development, but their role in platelet function and thrombotic disease has not been explored. We report for the first time that Pim-1 is expressed in human and mouse platelets. Genetic deletion or pharmacological inhibition of Pim kinase results in reduced thrombus formation but is not associated with impaired haemostasis. Attenuation of thrombus formation was found to be due to inhibition of the thromboxane A2 receptor as effects on platelet function was non-additive to inhibition caused by the cyclooxygenase inhibitor indomethacin or thromboxane A2 receptor antagonist GR32191. Treatment with Pim kinase inhibitors caused reduced surface expression of the thromboxane A2 receptor and resulted in reduced responses to thromboxane A2 receptor agonists, indicating a role for Pim kinase in the regulation of thromboxane A2 receptor function. Our research identifies a novel, Pim kinase dependent regulatory mechanism for the thromboxane A2 receptor and represents a new targeting strategy that is independent of COX-1 inhibition or direct antagonism of the thromboxane A2 receptor that whilst attenuating thrombosis does not increase bleeding.

Platelet dysfunction caused by a novel thromboxane A2 receptor mutation and congenital thrombocytopenia in a case of mild bleeding.

Chronic hemorrhagic diathesis in patients showing normal levels of plasmatic clotting factors strongly suggests for congenital platelet disorders. We report on a pediatric patient (male, 3 years, D1) with mild bleeding. A sibling (D2), his mother (D3) and father (D4) were included for laboratory investigation. Platelet counts in D1, D2 and D4 indicated mild thrombocytopenia (100 Gpt/L). D1 and D3 platelets showed significantly diminished aggregation response on arachidonic acid and U46619 stimulation. Immunostaining for platelet proteins on blood smears of D1 and D2 indicated defects in ß1-tubulin. Exon sequencing of TBXA2R and TUBB1 revealed heterozygosity for the novel TBXA2R*c.908T>C (p.L303P) mutation in D1 and D3. TUBB1 was either wild type (D2, D3) or heterozygous (D1, D4) for the common polymorphism TUBB1*c.920G>A (rs6070697; p.R307H). In conclusion, the bleeding phenotype of the index patient can be explained by a diminished platelet function caused by the TBXA2R*c.908T>C mutation inherited from the mother and a mild thrombocytopenia with unknown molecular basis that is inherited from the father.

Differential expression of the TPα and TPß isoforms of the human T Prostanoid receptor during chronic inflammation of the prostate: Role for FOXP1 in the transcriptional regulation of TPß during monocyte-macrophage differentiation.

Inflammation is linked to prostate cancer (PCa) and to other diseases of the prostate. The prostanoid thromboxane (TX)A2 is a pro-inflammatory mediator implicated in several prostatic diseases, including PCa. TXA2 signals through the TPα and TPß isoforms of the T Prostanoid receptor (TP) which exhibit several functional differences and transcriptionally regulated by distinct promoters Prm1 and Prm3, respectively, within the TBXA2R gene. This study examined the expression of TPα and TPß in inflammatory infiltrates within human prostate tissue. Strikingly, TPß expression was detected in 94% of infiltrates, including in B- and T-lymphocytes and macrophages. In contrast, TPα was more variably expressed and, where present, expression was mainly confined to macrophages. To gain molecular insight into these findings, expression of TPα and TPß was evaluated as a function of monocyte-to-macrophage differentiation in THP-1 cells. Expression of both TPα and TPß was upregulated following phorbol-12-myristate-13-acetate (PMA)-induced differentiation of monocytic THP-1 to their macrophage lineage. Furthermore, FOXP1, an essential transcriptional regulator down-regulated during monocyte-to-macrophage differentiation, was identified as a key trans-acting factor regulating TPß expression through Prm3 in THP-1 cells. Knockdown of FOXP1 increased TPß, but not TPα, expression in THP-1 cells, while genetic reporter and chromatin immunoprecipitation (ChIP) analyses established that FOXP1 exerts its repressive effect on TPß through binding to four cis-elements within Prm3. Collectively, FOXP1 functions as a transcriptional repressor of TPß in monocytes. This repression is lifted in differentiated macrophages, allowing for upregulation of TPß expression and possibly accounting for the prominent expression of TPß in prostate tissue-resident macrophages.

The TXA2R rs1131882, P2Y1 rs1371097 and GPIIIa rs2317676 three-loci interactions may increase the risk of carotid stenosis in patients with ischemic stroke.

BACKGROUND: The genetic risk factors for carotid stenosis are not fully understood. The aim of this study is to investigate the relationship between variants in platelet activation-relevant genes and carotid stenosis in patients with ischemic stroke (IS). METHODS: Eleven variants of platelet activation-relevant genes, aggregates of platelet-leukocyte, and platelet aggregation were examined in 236 IS patients with carotid stenosis and 378 patients without carotid stenosis. High-resolution B-mode ultrasound was used to assess carotid stenosis. Generalized multifactor dimensionality reduction (GMDR) methods were applied in analyzing gene-gene interactions to determine whether there was any interactive role of assessed variants in affecting risk of carotid stenosis. RESULTS: Platelet aggregation and aggregates of platelet-leukocyte showed higher value in patients with carotid stenosis, compared with patients without carotid stenosis. Excluding potential disturbance variables, these 11 variants were not associated with carotid stenosis. However, according to the GMDR analysis, gene-gene interactions among TXA2R rs1131882, P2Y1 rs1371097 and GPIIIa rs2317676 had a synergistic influence on carotid stenosis. The high-risk interactions between the three variants showed a relationship with higher platelet activation, and have independent associations with risk of carotid stenosis (OR = 2.72, 95% CI: 1.28-7.82, P = 0.001). CONCLUSION: The interactions among rs1131882, rs1371097 and rs2317676 perhaps increase the risk of symptomatic carotid stenosis, and maybe a potential marker for carotid stenosis. In this study, the combinatorial analysis made good use in elucidating complex risk factors in the heredity of carotid stenosis.

TBXA2R rs4523 G allele is associated with decreased susceptibility to Kawasaki disease.

Kawasaki disease is a multi-system vasculitis and a primary cause of acquired heart disease among children. Genetic factors may increase susceptibility to Kawasaki disease. TBXA2R is a G-protein-coupled receptor that participates in tissue inflammation and is associated with susceptibility to several diseases, but its relevance in Kawasaki disease is unclear. We genotyped TBXA2R (rs1131882 and rs4523) in 694 Kawasaki disease cases and 657 healthy controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the intensity of the associations. We found a significantly decreased risk of Kawasaki disease associated with TBXA2R rs4523 G variant genotypes (AG vs AA: adjusted OR = 0.788, 95%CI = 0.626-0.993; GG vs AA: adjusted OR = 0.459, 95%CI = 0.258-0.815; AG/GG vs AA: adjusted OR = 0.744, 95%CI = 0.595-0.929; GG vs AG/AA: adjusted OR = 0.497, 95% CI = 0.281-0.879). In the combined analysis of the two single-nucleotide polymorphisms (SNPs), we found that individuals with two unfavorable genotypes exhibited decreased risk for Kawasaki disease (adjusted OR = 0.754, 95%CI = 0.577-0.985) compared with those who did not have or one unfavorable genotypes. This cumulative effect on protection is effect-genotype dose-dependent (ptrend = 0.022). Moreover, the combined analysis indicated that the two unfavorable genotypes were associated with a decreased risk of Kawasaki disease in children 12-60 months of age, females and the subgroup with non-coronary artery lesion (NCAL) formation compared with those who did not have or one unfavorable genotypes. In conclusion, the TBXA2R rs4523 G allele may contribute to protection against Kawasaki disease and decreased risk of coronary artery aneurysm complications in a southern Chinese population.

Differential TBXA2 receptor transcript stability is dependent on the C924T polymorphism.

BACKGROUND: In order to better characterize the molecular mechanisms involved in processing mutated transcripts, we investigated the post-transcriptional role of the C924T polymorphism (rs4523) located in the 3' region of the TBXA2R gene. METHODS AND RESULTS: Experiments of dose response with Actinomycin D on MEG-01 human cell line showed a significant decrease on cell viability that was more evident on cells treated for 24h. In addition, we showed that treatments with 5-10µM, 15µM and 20µM of actinomycin D reduced cell viability by 44%, 72% and 75%, respectively, compared to the control group. Conversely, the samples treated with 1µM of actinomycin D did not show significant difference on cell viability as compared to the control group. Analysis of the steady state mRNA level of TBXA2R by qRT-PCR evidenced an increase in mRNA stability for the wild type (C) compared to the mutant (T) allele. Furthermore, the expression levels of TBXA2R on wild type (CC) and mutant type (TT) patients, based on C924T polymorphism, were analyzed. The wild type showed a higher expression of TBXA2 receptor also with two different degrees of glycosylation (55 and 64kDa), when compared to the mutant. These observations correlated with platelet aggregation, which was reduced in TT, independently of the platelet aggregation stimuli. CONCLUSIONS: The instability of the TBXA2R transcript and the lack of effect on platelet aggregation might suggest a protective role for the TBXA2R TT genotype against atherothrombosis and its complications in high-risk aspirin-treated patients.

TBXA2R gene variants associated with bleeding.

Platelet activity is regulated by a number of surface expressed G protein-coupled receptors (GPCRs) including the α isoform of the thromboxane receptor (TPα receptor). With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare GPCR variants including in the TBXA2R gene, which encodes the TPα receptor. The study of patients with naturally occurring variants within TBXA2R associated with bleeding and abnormal TPα receptor function has provided a powerful insight in defining the critical role of TPα in thrombus formation. This review will highlight how the identification of these function-disrupting variants of the platelet TPα has contributed important structure-function information about these GPCRs. Further we discuss the potential implications these findings have for understanding the molecular basis of mild platelet based bleeding disorders.

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.

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 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.

Regulated expression of the TPß isoform of the human T prostanoid receptor by the tumour suppressors FOXP1 and NKX3.1: Implications for the role of thromboxane in prostate cancer.

The prostanoid thromboxane (TX)A2 signals through the TPα and TPß isoforms of T Prostanoid receptor (TP) that are transcriptionally regulated by distinct promoters termed Prm1 and Prm3, respectively, within the TBXA2R gene. We recently demonstrated that expression of TPα and TPß is increased in PCa, differentially correlating with Gleason grade and with altered CpG methylation of the individual Prm1/Prm3 regions within the TBXA2R. The current study sought to localise the sites of CpG methylation within Prm1 and Prm3, and to identify the main transcription factors regulating TPß expression through Prm3 in the prostate adenocarcinoma PC-3 and LNCaP cell lines. Bisulfite sequencing revealed extensive differences in the pattern and status of CpG methylation of the individual Prm1 and Prm3 regions that regulate TPα and TPß expression, respectively, within the TBXA2R. More specifically, Prm1 is predominantly hypomethylated while Prm3 is hypermethylated across its entire sequence in PC-3 and LNCaP cells. Furthermore, the tumour suppressors FOXP1 and NKX3.1, strongly implicated in PCa development, were identified as key transcription factors regulating TPß expression through Prm3 in both PCa cell lines. Specific siRNA-disruption of FOXP1 and NKX3.1 each coincided with up-regulated TPß protein and mRNA expression, while genetic-reporter and chromatin immunoprecipitation (ChIP) analyses confirmed that both FOXP1 and NKX3.1 bind to cis­elements within Prm3 to transcriptionally repress TPß in the PCa lines. Collectively these data identify Prm3/TPß as a bona fide target of FOXP1 and NKX3.1 regulation, providing a mechanistic basis, at least in part, for the highly significant upregulation of TPß expression in PCa.

Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPß isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer.

The prostanoid thromboxane (TX) A2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPß form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA2/TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA2-TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA2/TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA2/TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPß mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPß can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA2/TP signalling axis in PCa, including potentially in CRPC.

Role of ADP ribosylation factor6- Cytohesin1-PhospholipaseD signaling axis in U46619 induced activation of NADPH oxidase in pulmonary artery smooth muscle cell membrane.

Treatment of human pulmonary artery smooth muscle cells (HPASMCs) with the thromboxane A2 receptor antagonist, SQ29548 inhibited U46619 stimulation of phospholipase D (PLD) and NADPH oxidase activities in the cell membrane. Pretreatment with apocynin inhibited U46619 induced increase in NADPH oxidase activity. The cell membrane contains predominantly PLD2 along with PLD1 isoforms of PLD. Pretreatment with pharmacological and genetic inhibitors of PLD2, but not PLD1, attenuated U46619 stimulation of NADPH oxidase activity. U46619 stimulation of PLD and NADPH oxidase activities were insensitive to BFA and Clostridium botulinum C3 toxin; however, pretreatment with secinH3 inhibited U46619 induced increase in PLD and NADPH oxidase activities suggesting a major role of cytohesin in U46619-induced increase in PLD and NADPH oxidase activities. Arf-1, Arf-6, cytohesin-1 and cytohesin-2 were observed in the cytosolic fraction, but only Arf-6 and cytohesin-1 were translocated to the cell membrane upon treatment with U46619. Coimmunoprecipitation study showed association of Arf-6 with cytohesin-1 in the cell membrane fraction. In vitro binding of GTPγS with Arf-6 required the presence of cytohesin-1 and that occurs in BFA insensitive manner. Overall, BFA insensitive Arf6-cytohesin1 signaling axis plays a pivotal role in U46619-mediated activation of PLD leading to stimulation of NADPH oxidase activity in HPASMCs.

Glycated albumin modifies platelet adhesion and aggregation responses.

A diabetic vasculature is detrimental to cardiovascular health through the actions of advanced glycation end products (AGEs) on endothelial cells and platelets. Platelets activated by AGEs agonize endothelial responses promoting cardiovascular disease (CVD) development. While it has been established that AGEs can alter platelet functions, little is known about the specific platelet pathways that AGEs modify. Therefore, we evaluated the effects of AGEs on specific salient platelet pathways related to CVDs and whether the effects that AGEs elicit are dependent on glycation extent. To accomplish our objective, platelets were incubated with reversibly or irreversibly glycated albumin. A time course for adhesion and aggregation agonist receptor expression was assessed. Optical platelet aggregometry was used to confirm the functional activity of platelets after AGE exposure. In general, platelets subjected to glycated albumin had a significantly enhanced adhesion and aggregation potential. Furthermore, we observed an enhancement in dense body secretion and intracellular calcium concentration. This was especially prevalent for platelets exposed to irreversibly glycated albumin. Additionally, functional aggregation correlated well with receptor expression, suggesting that AGE-induced altered receptor sensitivity translated to altered platelet functions. Our findings indicate that under diabetic vascular conditions platelets become more susceptible to activation and aggregation due to an overall enhanced receptor expression, which may act to promote CVD development.

Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay.

Up to 1% of the population have mild bleeding disorders, but these remain poorly characterized, particularly with regard to the roles of platelets. We have compared the usefulness of Optimul, a 96-well plate-based assay of 7 distinct pathways of platelet activation to characterize inherited platelet defects in comparison with light transmission aggregometry (LTA). Using Optimul and LTA, concentration-response curves were generated for arachidonic acid, ADP, collagen, epinephrine, Thrombin receptor activating-peptide, U46619, and ristocetin in samples from (1) healthy volunteers (n = 50), (2) healthy volunteers treated with antiplatelet agents in vitro (n = 10), and (3) patients with bleeding of unknown origin (n = 65). The assays gave concordant results in 82% of cases (κ = 0.62, P < .0001). Normal platelet function results were particularly predictive (sensitivity, 94%; negative predictive value, 91%), whereas a positive result was not always substantiated by LTA (specificity, 67%; positive predictive value, 77%). The Optimul assay was significantly more sensitive at characterizing defects in the thromboxane pathway, which presented with normal responses with LTA. The Optimul assay is sensitive to mild platelet defects, could be used as a rapid screening assay in patients presenting with bleeding symptoms, and detects changes in platelet function more readily than LTA. This trial was registered at www.isrctn.org as #ISRCTN 77951167.

Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway.

Association between thromboxane A2 receptor polymorphisms and asthma risk: A meta-analysis.

OBJECTIVE: To determine whether there is an association between thromboxane A2 receptor (TBXA2R) gene polymorphisms (+924C/T and +795C/T) and asthma risk by conducting a meta-analysis. DATA SOURCES: Pubmed, Embase, Chinese National Knowledge Infrastructure (CNKI) and Wanfang database were searched (updated May 1, 2015). STUDY SELECTIONS: Articles evaluating the association between TBXA2R gene polymorphisms and asthma risk were selected. RESULTS: A total of 7 studies on +924C/T polymorphism and 6 studies on +795C/T polymorphism were included in this meta-analysis. There was a significant association between TBXA2R +924C/T polymorphism and asthma risk in the recessive model (OR = 1.33, 95% CI = 1.01-1.75, P = 0.045). No significant association between +795C/T polymorphism and asthma risk in the overall population was demonstrated. In subgroup analyzes, significant association was observed in atopic asthma risk in the recessive model (OR = 1.43, 95% CI = 1.01-2.01, P = 0.043), but no significant association was found between TBXA2R +924C/T polymorphism and asthma risk in Asians (OR = 1.14, 95% CI = 0.80-1.63, P = 0.457). TBXA2R +795C/T polymorphism was associated with aspirin-intolerant asthma (AIA) risk when stratified by asthma subphenotype in the allelic model (OR = 1.30, 95% CI = 1.05-1.60, P = 0.014) and dominant model (OR = 1.50, 95% CI = 1.11-2.03, P = 0.008). CONCLUSION: Our results suggested that TBXA2R +924C/T polymorphism is associated with asthma risk, and +795C/T polymorphism may be a risk factor for AIA. Larger-scale and well-designed studies are required to validate the association identified in the current meta-analysis.