Thromboxane Release Research Articles

Carbamylation Is Instrumental in End-Stage Kidney Disease Coagulopathies: The Impact on von Willebrand Factor and Platelet Functionality.

Chronic kidney disease (CKD) is a progressive, irreversible, and incurable condition characterized by high morbidity and mortality, affecting approximately one-tenth of the global population. Rise of urea-derived cyanate levels in CKD patients, severalfold higher in comparison to those found in healthy individuals, leads to an increased rate of carbamylation of lysine residues of proteins and peptides. This posttranslational modification plays an important role in the progression of kidney failure but also in the onset of CKD-related complications, including previously reported coagulopathies. In this study, we have explored the impact of carbamylation on the functionality of von Willebrand factor (vWF), a pivotal player in hemostasis, and its implications for platelet adhesion. We have explored carbamylated vWF's interactions with its partner proteins via ELISA. Mass spectrometry was employed to identify modified lysine residues. Blood platelets isolated from healthy donors were carbamylated, and their activation, binding to endothelium and thromboxane release were evaluated using flow cytometry, adhesion assays and ELISA, respectively. Using mass spectrometry we detected the vWF's lysine residue smost susceptible to carbamylation. This modification has in turn affected vWF's interactions with its key binding partners: decreased binding to collagen types I/III but increased the affinity to factor FVIII, while its binding to fibrinogen remained unchanged. Carbamylation of vWF impeded vWF-blood platelet binding, but carbamylation of platelets led to their increased thrombin-dependent activation as observed by enhanced phosphatidylserine exposure, improved their binding to vascular endothelium, at the same time decreasing the production of the prothrombotic mediator, thromboxane A2. Our findings highlight the multifaceted impact of carbamylation on vWF and platelets, disturbing the delicate balance of coagulation cascade. These alterations could contribute to the complex hemostatic imbalance in ESKD, underscoring the need for further research to fully understand these mechanisms and their clinical implications.

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Testosterone-to-estradiol ratio and platelet thromboxane release in ischemic heart disease: the EVA project

BackgroundData on the interplay between sexual hormones balance, platelet function and clinical outcomes of adults with ischemic heart disease (IHD) are still lacking.ObjectiveTo assess the association between the Testosterone (T)-to-Estradiol (E2) Ratio (T/E2) and platelet activation biomarkers in IHD and its predictive value on adverse outcomes.MethodsThe EVA study is a prospective observational study of consecutive hospitalized adults with IHD undergoing coronary angiography and/or percutaneous coronary interventions. Serum T/E2 ratios E2, levels of thromboxane B2 (TxB2) and nitrates (NO), were measured at admission and major adverse events, including all-cause mortality, were collected during a long-term follow-up.ResultsAmong 509 adults with IHD (mean age 67 ± 11 years, 30% females), males were older with a more adverse cluster of cardiovascular risk factors than females. Acute coronary syndrome and non-obstructive coronary artery disease were more prevalent in females versus males. The lower sex-specific T/E2 ratios identified adults with the highest level of serum TxB2 and the lowest NO levels. During a median follow-up of 23.7 months, the lower sex-specific T/E2 was associated with higher all-cause mortality (HR 3.49; 95% CI 1.24–9.80; p = 0.018). In in vitro, platelets incubated with T/E2 ratios comparable to those measured in vivo in the lowest quartile showed increased platelet activation as indicated by higher levels of aggregation and TxB2 production.ConclusionAmong adults with IHD, higher T/E2 ratio was associated with a lower long-term risk of fatal events. The effect of sex hormones on the platelet thromboxane release may partially explain such finding.

Prostaglandin D2 inhibits mediator release and antigen induced bronchoconstriction in the Guinea pig trachea by activation of DP1 receptors

The mechanism by which cyclooxygenase (COX) inhibition increases antigen-induced responses in airways remains unknown.Male albino guinea pigs were sensitized to ovalbumin (OVA). Intact rings of the trachea were isolated and mounted in organ baths for either force measurements or lipid mediator release analysis by UPLC-MS/MS or EIA following relevant pharmacological interventions.First, challenge with OVA increased the release of all primary prostanoids (prostaglandin (PG) D2/E2/F2α/I2 and thromboxanes). This release was eliminated by unselective COX inhibition (indomethacin) whereas selective inhibition of COX-2 (lumiracoxib) did not inhibit release of PGD2 or thromboxanes. Additionally, the increased levels of leukotriene B4 and E4 after OVA were further amplified by unselective COX inhibition. Second, unselective inhibition of COX and selective inhibition of the prostaglandin D synthase (2-Phenyl-Pyrimidine-5-Carboxylic Acid (2,3-dihydro-indol-1-yl)-amide) amplified the antigen-induced bronchoconstriction which was reversed by exogenous PGD2. Third, a DP1 receptor agonist (BW 245c) concentration-dependently reduced the antigen-induced constriction as well as reducing released histamine and cysteinyl-leukotrienes, a response inhibited by the DP1 receptor antagonist (MK-524). In contrast, a DP2 receptor agonist (15(R)-15-methyl PGD2) failed to modulate the OVA-induced constriction.In the guinea pig trachea, endogenous PGD2 is generated via COX-1 and mediates an inhibitory effect of the antigen-induced bronchoconstriction via DP1 receptors inhibiting mast cell release of bronchoconstrictive mediators. Removal of this protective function by COX-inhibition results in increased release of mast cell mediators and enhanced bronchoconstriction.

Afebrile and Leukopenic Sweet's Syndrome Following Partial Ileectomy in an Elderly Woman

Afebrile and Leukopenic Sweet's Syndrome Following Partial Ileectomy in an Elderly Woman

A new mechanism of the protamine-dependent hypotension after cardiopulmonary bypass and the role of calcium

Heparin and protamine are two indispensable agents of cardiopulmonary bypass surgery with effects on the cardiovascular and hematological system. Heparin is used as an anticoagulant in open heart surgery; whereas protamine is used to neutralize heparin effects when surgery is terminated. Protamine is given in order to neutralize heparin effects after cardiopulmonary bypass surgery and it causes hypotension in patients. However, the mechanism of this side effect is not clearly known. Current mechanism is that hypotension occurs after the administration of protamine due to the conformational change in the calcium channels or anaphylactoid thromboxane release or serum ionized calcium levels. The present study was to explain how protamine causes hypotension in evidence-based medicine indexed in PubMed and Web of Science. In addition to above mechanisms, possibly the infused protamine binds heparin and causes the coagulation cascade to activate heparin-AT complex on thrombin beside activating FXIa, FXa and FIXa and causing the re-use of Ca2+. The re-use of Ca2+ at the coagulation cascade initiates an anion gap and it is assumed that hypotension develops because of the Ca2+ deficiency. Ca2+ ions are trapped in the thrombus by the resumption of thrombus formation. Ca2+ ions trapped in the thrombus cannot be used, so that Ca2+ ion deficit will develop in circulation and hypotension occurs due to the insufficiency of Ca2+ ions. The administration of Ca2+ ions together with the protamine might help to eliminate the side effect of the protamine (hypotension) while neutralizing heparin after open heart surgery in light of the information provided in the literature.

Leukotriene E4 induces airflow obstruction and mast cell activation through the cysteinyl leukotriene type 1 receptor

Leukotriene (LT) E4 is the final active metabolite among the cysteinyl leukotrienes (CysLTs). Animal studies have identified a distinct LTE4 receptor, suggesting that current cysteinyl leukotriene type 1 (CysLT1) receptor antagonists can provide incomplete inhibition of CysLT responses. We tested this hypothesis by assessing the influence of the CysLT1 antagonist montelukast on responses induced by means of inhalation of LTE4 in asthmatic patients. Fourteen patients with mild intermittent asthma and 2 patients with aspirin-exacerbated respiratory disease received 20mg of montelukast twice daily and placebo for 5 to 7days in a randomized, double-blind, crossover study (NCT01841164). The PD20 value was determined at the end of each treatment period based on an increasing dose challenge. Measurements included lipid mediators in urine and sputum cells 4hours after LTE4 challenge. Montelukast completely blocked LTE4-induced bronchoconstriction. Despite tolerating an at least 10 times higher dose of LTE4 after montelukast, there was no difference in the percentage of eosinophils in sputum. Urinary excretion of all major lipid mediators increased after LTE4 inhalation. Montelukast blocked release of the mast cell product prostaglandin (PG) D2, as well as release of PGF2α and thromboxane (Tx) A2, but not increased excretion of PGE2 and its metabolites or isoprostanes. LTE4 induces airflow obstruction and mast cell activation through the CysLT1 receptor.

Evidence for autocrine and paracrine regulation of allergen-induced mast cell mediator release in the guinea pig airways

Mast cells play an essential role in immediate type hypersensitivity reactions and in chronic allergic diseases of the airways, including asthma. Mast cell mediator release can be modulated by locally released autacoids and circulating hormones, but surprisingly little is known about the autocrine effects of mediators released upon mast cell activation. We thus set out to characterize the autocrine and paracrine effects of mast cell mediators on mast cell activation in the guinea pig airways. By direct measures of histamine, cysteinyl-leukotriene and thromboxane release and with studies of allergen-evoked contractions of airway smooth muscle, we describe a complex interplay amongst these autacoids. Notably, we observed an autocrine effect of the cysteinyl-leukotrienes acting through cysLT1 receptors on mast cell leukotriene release. We confirmed the results of previous studies demonstrating a marked enhancement of mast cell mediator release following cyclooxygenase inhibition, but we have extended these results by showing that COX-2 derived eicosanoids inhibit cysteinyl-leukotriene release and yet are without effect on histamine release. Given the prominent role of COX-1 inhibition in aspirin-sensitive asthma, these data implicate preformed mediators stored in granules as the initial drivers of these adverse reactions. Finally, we describe the paracrine signaling cascade leading to thromboxane synthesis in the guinea pig airways following allergen challenge, which occurs indirectly, secondary to cysLT1 receptor activation on structural cells and/ or leukocytes within the airway wall, and a COX-2 dependent synthesis of the eicosanoid. The results highlight the importance of cell-cell and autocrine interactions in regulating allergic responses in the airways.

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

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. 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 1h. 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.

Unlocking Aspirin's Chemopreventive Activity: Role of Irreversibly Inhibiting Platelet Cyclooxygenase-1.

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically induced mouse model of colon cancer) evidence that this profound antineoplastic action may be associated with aspirin's ability to irreversibly inhibit COX-1-mediated platelet activation, thereby blocking platelet-cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced epithelial-mesenchymal transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, β-catenin, and SNAIL. We also provide evidence that a novel, gastrointestinal-safe phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions versus unmodified aspirin with regard to antiplatelet effects (in vitro: reducing platelet activation as determined by measuring the release of thromboxane and VEGF in culture medium; in vivo: inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro: inhibiting colonic cell growth and invasive activity; in vivo: inhibiting colonic dysplasia, inflammation, and tumor mass). These results suggest that aspirin's chemopreventive effects may be due, in part, to the drug blocking the proneoplastic action of platelets, and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers. Cancer Prev Res; 10(2); 142-52. ©2016 AACR.

All too common: bleeding and genetic variation

Inherited bleeding disorders are both common and challenging to diagnose, particularly when the disorder is mild (reviewed in references [1, 2]). Most mild bleeding disorder patients are not routinely bleeding in their daily lives, and it can be difficult to differentiate bleeding events common in healthy populations from events which rise to the level of pathology. Fundamental to the diagnosis of bleeding disorders is a detailed assessment of the history of the patient and the family. This article is protected by copyright. All rights reserved.

Combined variants in factor VIII and prostaglandin synthase‐1 amplify hemorrhage severity across three generations of descendants

Background Inherited human variants that concurrently cause disorders of primary hemostasis and coagulation are uncommon. Nevertheless, rare cases of co-existent damaging variants are likely to cause more severe bleeding and may go undiagnosed. Objective We prospectively sought to determine pathogenic variants in a three-generational pedigree with excessive bleeding. Patients/methods Platelet number, size and light transmission aggregometry to multiple agonists were evaluated in pedigree members. Transmission electron microscopy determined platelet morphology and granule content. Thromboxane release studies and light transmission aggregometry in the presence or absence of prostaglandin G2 assessed specific functional defects in the arachidonic acid pathway. Whole exome sequencing (WES) and targeted nucleotide sequence analysis identified potentially deleterious variants. Results Pedigree members with excessive bleeding had impaired platelet aggregation with arachidonic acid, epinephrine and low-dose ADP, as well as reduced platelet thromboxane B2 release. Impaired platelet aggregation in response to 2MesADP was rescued with prostaglandin G2 , a prostaglandin intermediate downstream of prostaglandin synthase-1 (PTGS-1) that aids in the production of thromboxane. WES identified a non-synonymous variant in the signal peptide of PTGS-1 (rs3842787; c.50C>T; p.Pro17Leu) that completely co-segregated with disease phenotype. A variant in the F8 gene causing hemophilia A (rs28935203; c.5096A>T; p.Y1699F) was also identified. Individuals with both variants had more severe bleeding manifestations than characteristic of mild hemophilia A alone. Conclusion We provide the first report of co-existing variants in both F8 and PTGS-1 genes in a three-generation pedigree. The PTGS-1 variant was associated with specific functional defects in the arachidonic acid pathway and more severe hemorrhage.

The proteasome regulates collagen-induced platelet aggregation via nuclear-factor-kappa-B (NFĸB) activation

IntroductionPlatelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. Material and methodsUsing fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. ResultsWe show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. ConclusionsThis work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.

PP.29.02] METFORMIN REDUCES HIGH-FAT DIET EFFECTS ON BLOOD PRESSURE, METABOLIC PARAMETERS AND VASCULAR PROSTANOIDS IN RATS

Objective: Metformin (Mf) is an antidiabetic drug used in type 2 diabetes and metabolic syndrome treatment. A high-fat (HF) diet produces insulin resistance in the rat which is related to cardiovascular alterations such as hypertension. Prostanoids (PR), cyclooxygenase-derived arachidonic acid metabolites, have vasoactive properties and mediate inflammation. The aim of this study was to analyze the effect of Mf on blood pressure (BP), metabolic parameters, and mesenteric vascular bed (MVB) PR production in male Sprague-Dawley rats under a HF diet. Design and method: Four groups were studied during 12 weeks (n = 4 each): Control (C), standard diet (SD) and tap water to drink; HF diet (HF), 50% (w/w) bovine fat added to SD and tap water; C + metformin (CMf), SD + 500 mg/Kg/day Mf in the drinking water; and HF + Mf (HFMf) 50% (w/w) bovine fat added to SD + 500 mg/Kg/day Mf. PR were determined by HPLC. Results: HF diet increased body weight (BW: g, HF, 478 ± 10 vs. C, 421 ± 15 p = 0.01) % MVB/BW ratio (HF, 1.72 ± 0.8 vs. C, 0.8 ± 0.1, p < 0.001); glycemia (mg/dl, HF, 144 ± 4 vs. C, 127 ± 4, p = 0.01); triglyceridemia (mg/dl, HF, 166 ± 21 vs. C, 86 ± 9, p < 0,01) and systolic BP (mmHg, HF, 151 ± 2 vs. C, 127 ± 2, p < 0,001). In the HFMf group, Mf decreased MVB/BW ratio (1.38 ± 0.6, p < 0.05); glycemia, triglyceridemia, (mg/dl, 110 ± 11 vs. HF, p = 0.01; 65 ± 13 vs. HF, p = 0.01) and systolic BP (mmHg, 141 ± 1 vs. HF, p < 0.01). Moreover, HF diet increased the release of PR thromboxane (TX) B2, stable metabolite of the vasoconstrictor TXA2, (ng PR/mg tissue, HF, 121 ± 8 vs. C, 71 ± 6, p < 0,05); and prostaglandin (PG) F2alpha (ng/mg, HF, 166 ± 11 vs. C, 87 ± 9, p < 0,05). Mf decreased the release of (PG) F2alpha in the HFMf group (ng/mg, 81 ± 12 vs. HF, p < 0,01). Conclusions: In conclusion, metformin treatment reduces abdominal fat which plays a role in the pathogenesis of hypertension. A possible mechanism by which metformin lowers systolic BP in this experimental model could be the reduction of vasoconstrictor prostanoid release in the MVB, reducing peripheral resistance.

The improvement of walking abilities and endothelial function after the supervised training treadmill program (STTP) in patients with peripheral artery disease (PAD) is not related to prostacyclin and thromboxane release

BackgroundIn this prospective study we evaluated the relationship between thromboxane B2 (TXB2), prostacyclin (PGI2) and lactate concentrations, and the improvement of walking abilities and endothelial function in patients with peripheral artery disease (PAD) undergoing a supervised treadmill training program (STTP). MethodsA total of fifty-nine patients with stable intermittent claudication were included into a 12-week long STTP. Changes in blood pressure, biochemical parameters, ankle/brachial index (ABI), flow-mediated dilatation (FMD), maximal walking time (MWT) and pain-free walking time (PFWT) were assessed before and after STTP. Additional baseline and post-STTP measurements were taken for blood lactate, and TXB2 and PGI2 urinary derivatives before and after maximal exercise (ME). ResultsThe MWT improved significantly after STTP by 91% (p<0.0001) and PFWT by 97% (p<0.0001). Also, ABI values improved significantly after STTP in all patient groups and was more pronounced in those with longer MWT at baseline. FMD values increased by 45% (p<0.0001) after STTP. Urinary 11-dehydro-thromboxane B2 and 2,3-dinor-6-keto-PGF1α concentration tend to decrease after STTP and their ratio remained unchanged. Lactate levels did not change after the treadmill training program. Hs-CRP and fibrinogen concentration decreased significantly after STTP only in patients with longer MWT at baseline—fourth quartile. ConclusionsSTTP in patients with PAD showed significantly improved walking abilities and endothelial function. Lactate production, TXB2 release, and PGI2 release are not directly correlated with improvement of endothelial function and walking abilities. Patients with better-walking abilities at baseline derive greater clinical and metabolic benefits from STTP.

Low-dose aspirin plus low-molecular-weight heparin for the prevention of pre-eclampsia: yes, no or maybe.

Low-dose aspirin plus low-molecular-weight heparin for the prevention of pre-eclampsia: yes, no or maybe.

Effects of estrogen on cerebrovascular function: age-dependent shifts from beneficial to detrimental in small cerebral arteries of the rat.

In the present study, interactions of age and estrogen in the modulation of cerebrovascular function were examined in small arteries <150 μM. The hypothesis tested was that age enhances deleterious effects of exogenous estrogen by augmenting constrictor prostanoid (CP)-potentiated reactivity of the female (F) cerebrovasculature. F Sprague-Dawley rats approximating key stages of "hormonal aging" in humans were studied: perimenopausal (mature multi-gravid, MA, cyclic, 5-6 mo of age) and postmenopausal (reproductively senescent, RS, acyclic 10-12 mo of age). Rats underwent bilateral ovariectomy and were given estrogen replacement therapy (E) or placebo (O) for 14-21 days. Vasopressin reactivity (VP, 10(-12)-10(-7) M) was measured in pressurized middle cerebral artery segments, alone or in the presence of COX-1- (SC560, 1 μM) or COX-2- (NS398, 10 μM) selective inhibitors. VP-stimulated release of prostacyclin (PGI2) and thromboxane (TXA2) were assessed by radioimmunoassay of 6-keto-PGF1α and TXB2 (stable metabolites). VP-induced vasoconstriction was attenuated in ovariectomized + estrogen-replaced, multigravid adult rats (5-6 mo; MAE) but potentiated in older ovariectomized + estrogen-replaced, reproductively senescent rats (12-14 mo; RSE). SC560 and NS398 reduced reactivity similarly in ovariectomized multigravid adult rats (5-6 mo; MAO) and ovariectomized reproductively senescent rat (12-14 mo; RSO). In MAE, reactivity to VP was reduced to a greater extent by SC560 than by NS398; however, in RSE, this effect was reversed. VP-stimulated PGI2 was increased by estrogen, yet reduced by age. VP-stimulated TXA2 was increased by estrogen and age in RSE but did not differ in MAO and RSO. Taken together, these data reveal that the vascular effects of estrogen are distinctly age-dependent in F rats. In younger MA, beneficial and protective effects of estrogen are evident (decreased vasoconstriction, increased dilator prostanoid function). Conversely, in older RS, detrimental effects of estrogen begin to be manifested (enhanced vasoconstriction and CP function). These findings may lead to age-specific estrogen replacement therapies that maximize beneficial and minimize detrimental effects of this hormone on small cerebral arteries that regulate blood flow.

Sudden Thrombosis in Coronary Artery Bypass Grafting Surgery

Sudden Thrombosis in Coronary Artery Bypass Grafting Surgery

Quinidine, but not eicosanoid antagonists or dexamethasone, protect the gut from platelet activating factor-induced vasoconstriction, edema and paralysis.

Intestinal circulatory disturbances, atony, edema and swelling are of great clinical relevance, but the related mechanisms and possible therapeutic options are poorly characterized, in part because of the difficulties to comprehensively analyze these conditions. To overcome these limitations we have developed a model of the isolated perfused rat small intestine where all of these symptoms can be studied simultaneously. Here we used this model to study the role of eicosanoids, steroids and quinidine in platelet-activating factor (PAF)-induced intestinal disorders. A vascular bolus of PAF (0.5 nmol) triggered release of thromboxane and peptidoleukotrienes into the vascular bed (peak concentration 35 nM and 0.8 nM) and reproduced all symptoms of intestinal failure: mesenteric vasoconstriction, translocation of fluid and macromolecules from the vasculature to the lumen and lymphatics, intestinal edema formation, loss of intestinal peristalsis and decreased galactose uptake. All effects of PAF were abolished by the PAF-receptor antagonist ABT491 (2.5 μM). The COX and LOX inhibitors ASA and AA861 (500 μM, 10 μM) did not exhibit barrier-protective effects and the eicosanoid antagonists SQ29548 and MK571 (10 μM, each) only moderately attenuated the loss of vascular fluid, the redistribution to the lumen and the transfer of FITC dextran to the lumen. The steroid dexamethasone (10 μM) showed no barrier-protective properties and failed to prevent edema formation. Quinidine (100 μM) inhibited the increase in arterial pressure, stabilized all the intestinal barriers, and reduced lymph production and the transfer of FITC dextran to the lymph. While quinidine by itself reduced peristalsis, it also obviated paralysis, preserved intestinal functions and prevented edema formation. We conclude that quinidine exerts multiple protective effects against vasoconstriction, edema formation and paralysis in the intestine. The therapeutic use of quinidine for intestinal ailments deserves further study.

Orai1‐induced store‐operated Ca2+ entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets

Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipaseC and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoproteinVI-mediated thromboxaneA2 release was strongly dependent on Orai1-mediated SOCE. The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.