Reduction of platelet cytosolic phospholipase A2 activity by atorvastatin and simvastatin: biochemical regulatory mechanisms.

UNLABELLED: Statins have demonstrated effects beyond reducing cholesterol level that may contribute to their clinical benefit, including effects on platelet biochemistry and function. OBJECTIVES: To explore and compare the antiplatelet effect of two lipophilic statins (atorvastatin and simvastatin) and one hydrophilic statin (pravastatin) concerning: a) collagen-induced platelet aggregation and thromboxane A2 (TXA2) synthesis; b) the additive effect of statins on TXA2 synthesis in platelets treated with a submaximally effective concentration of aspirin and c) the biochemical mechanisms involved. METHODS AND RESULTS: Washed human platelets were incubated with statins (1-20µM), and stimulated with collagen (1µg/ml) or arachidonic acid (AA) (200µM) and TXB2 was quantified by ELISA. Incubation with simvastatin or atorvastatin reduced (36.2% and 31.0%, respectively) collagen-induced TXB2 synthesis (p<0.05) and platelet aggregation (p<0.001), whereas pravastatin had no effects. Simultaneous incubation with a submaximally effective concentration of aspirin (1µM) and atorvastatin or simvastatin significantly increased the inhibition of TXB2 synthesis by aspirin by 4.4- and 4.1-fold, respectively. Statins did not affect AA-induced TXB2 synthesis, excluding an effect on COX-1/TXA2 synthase activities. Atorvastatin and simvastatin concentration-dependently inhibited the collagen-induced increase in cytosolic calcium and the kinetics of cPLA2 phosphorylation. Lipophilic statins reduced phosphorylation of both ERK1/2 and p38 MAPK, which regulate cPLA2 phosphorylation and calcium movement. CONCLUSION: We report for the first time a direct downregulation by atorvastatin and simvastatin of platelet cPLA2 activity through effects on calcium and MAPK, which reduce collagen-induced TXA2 synthesis. These mechanisms might contribute to their beneficial effects, even in aspirin-treated patients.

Association analysis of thromboxane A synthase 1 gene polymorphisms with aspirin intolerance in asthmatic patients.

AIM: Thromboxane A synthase (TBXAS1) converts prostaglandin H to thromboxane A, a potent constrictor of smooth respiratory muscle. Thus, functional alterations of the TBXAS1 gene may contribute to aspirin-intolerant asthma (AIA). MATERIALS & METHODS: We investigated the relationship between SNPs in the TBXAS1 gene and AIA. Asthmatics (n = 470) were categorized into AIA (20% or greater decreases in forced expiratory volume in 1 s [FEV(1)], or 15% to 19% decreases in FEV(1) with naso-ocular or cutaneous reactions) and aspirin-tolerant asthma (ATA). A total of 101 SNPs were genotyped. mRNA expression of the TBXAS1 gene by peripheral blood mononuclear cells and plasma thromboxane B2 (TXB2) concentrations were measured by reverse transcriptase (RT)-PCR and ELISA. RESULTS: Logistic regression analysis showed that the rare allele frequency of rs6962291 in intron 9 was significantly lower in the AIA group (n = 115) than in the ATA group (n = 270) (p(corr) = 0.04). The linear regression analysis revealed a strong association of rs6962291 with the aspirin challenge-induced FEV(1) fall (p = 0.003). RT-PCR revealed an exon-12-deleted splice variant. We measured TBXAS1 mRNA levels in peripheral blood mononuclear cells. The mRNA levels of the full-length wild-type and splice variant were significantly higher in the TT homozygotes than in the AA homozygotes of rs6962291 (1.00 ± 0.18 vs 0.57 ± 0.03 and 1.00 ± 0.18 vs 0.21 ± 0.05, p = 0.047 and 0.001, respectively). The plasma TXB2 level was significantly lower in rs6962291 AA carriers than in rs6962291 TT (p = 0.016) carriers. CONCLUSION: The rare allele of rs6962291 may play a protective role against aspirin hypersensitivity via a lower catalytic activity of the TBXAS1 gene, attributed to the increase of a nonfunctioning isoform of TBXAS1.

The protective effects of paclitaxel on platelet aggregation through the inhibition of thromboxane A2 synthase.

Paclitaxel is an anticancer drug used in the treatment of ovarian, breast, head and neck, lung, and prostate cancer. We investigated the antiplatelet activity of paclitaxel in vitro as well as a possible antiplatelet mechanism. Paclitaxel inhibited washed rabbit platelet aggregation induced by collagen in a concentration dependent manner, with an IC(50) of 59.7 +/- 3.5. However, it had little effect on platelet aggregation mediated by arachidonic acid, U46619, a thromboxane (TX) A(2) mimic, or thrombin, suggesting that paclitaxel may strongly inhibit collagen mediated signal transduction. In accordance with these findings, paclitaxel blocked collagen induced cytosolic calcium mobilization, arachidonic acid liberation, and serotonin secretion. In addition, it inhibited arachidonic acid mediated platelet aggregation by about 37% by interfering with TXA(2) synthase as measured by the formation of arachidonic acid mediated TXA(2) and prostaglandin D(2), as well as cyclooxygenase-1 and TXA(2) synthase activity assays. Taken together, these results point to a cellular mechanism for the antiplatelet activity of paclitaxel through the inhibition of TXA(2) synthase and cytosolic calcium mobilization. This may contribute to the beneficial effects of paclitaxel on the cardiovascular system.

Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome).

Studying consanguineous families with Ghosal hematodiaphyseal dysplasia syndrome (GHDD), a disorder of increased bone density, we identified mutations in TBXAS1, which encodes thromboxane synthase (TXAS). TXAS, an enzyme of the arachidonic acid cascade, produces thromboxane A(2) (TXA(2)). Platelets from subjects with GHDD showed a specific deficit in arachidonic acid-produced aggregation. We also found that TXAS and TXA(2) modulated expression of TNFSF11 and TNFRSF11B (encoding RANKL and osteoprotegerin (OPG), respectively) in primary cultured osteoblasts.

Isomers of trans fatty acids modify the activity of platelet 12-P lipoxygenase and cyclooxygenase/thromboxane synthase.

OBJECTIVES: Trans isomers of fatty acids (TFA) have been implicated in the initiation and progression of atherosclerosis. Previous studies have shown that trans-unsaturated fatty acids like their cis-unsaturated counterparts exert a modifying effect on platelet aggregation. The aim of this work was to determine the influence of TFA on the aforementioned enzymes: lipoxygenase and cyclooxygenase/thromboxane synthase. METHODS: Two C18 cis-trans fatty acid pairs: oleic/elaidic and linoleic/linolelaidic were chosen. Fasting blood was sampled from 30 healthy volunteers without any lipid abnormalities and not on medication for at least 7 days prior to sampling. Platelet-rich plasma (PRP) was prepared and the platelet count was adjusted to 3 x 10(8) cells x ml(-1). Fatty acids in hexane were added to a final concentration of 7 microM and evaporated to dryness under nitrogen, 500 microl of PRP was pipetted into each tube, thoroughly mixed and incubated at 37 degrees C for 60 min. 12-P LOX activity was measured with a spectrophotometric method and expressed per mg protein. Cyclooxygenase/thromboxane synthase activity was determined by TXB(2) production with an ELISA-based assay. Statistics were done with the Kruskal-Wallis test and Statistica software package. RESULTS: We have found that the activity of 12-P LOX was suppressed by all cis-trans fatty acids used by us. Cyclooxygenase/thromboxane synthase activity was significantly inhibited by polyunsaturated fatty acids only. Linolelaidic acid was more potent in comparison to its monounsaturated (elaidic acid) counterpart. CONCLUSIONS: We believe that the effects of fatty acids are demonstrated at the membrane level where fatty acids may produce a perturbation in specific lipid domains. TFA are able to interact with the platelet membrane and transmembrane proteins just as the cis isomers. By interacting with proteins exposed on the cytoplasmic membrane, TFA may modify the activity of receptors and other membrane proteins. In this way, changes on the membrane surface are propagated into the cell affecting the activity of cytoplasmic enzymes like 12-P LOX and cyclooxygenase/thromboxane synthase.

The antiplatelet activity of Danggijakyaksan by inhibition of phospholipase C.

We investigated the effects of the traditional Korean prescription, Danggijakyaksan (DJS) on antiplatelet activity in human platelet suspensions. The effect of oriental medicinal prescriptions, Danggijakyaksan consisting of 6 herbes of Paeoniae Radix (2 g), Poria Cocos (1.33 g), Angelicae Sinensis Radix (1 g), Cnidii Rhizoma (1 g), Atractylodis Macrocephalae Rhizoma (1.33 g) and Alismatis Rhizoma (1.66 g), was studied. In this study, the mechanism involved in the antiplatelet activity of DJS in human platelet suspensions was investigated. Danggijakyaksan did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and DJS (20 and 40 microg/mL) significantly inhibited [3H]arachidonic acid (AA) released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. Danggijakyaksan did not significantly affect nitrate production in collagen (10 microg/mL)-induced human platelet aggregation. On the other hand, various concentrations of DJS (10, 20, and 40 microg/mL) dose-dependently inhibited [3H]inositol monophosphate (IP) formation stimulated by collagen (10 microg/mL) in [3H]myoinositolloaded platelets at different incubation times (1, 2, 3, and 5 min). It is concluded that the antiplatelet activity of DJS may possibly be due to the inhibition of phospholipase C activity, leading to reduced phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists.

Effects of camonagrel, a selective inhibitor of platelet thromboxane synthase, on the platelet-subendothelium interaction.

The aim of this study was to compare the effects of a new thromboxane synthase inhibitor, camonagrel, on platelet aggregation and platelet-subendothelium interaction under flow conditions, in comparison with a standard thromboxane synthase inhibitor (dazoxiben) and a cyclooxygenase inhibitor (acetylsalicylic acid). With respect to platelet aggregation in whole blood, the 50% inhibitory concentrations (IC(50)) of camonagrel were between 318 and 797 micromol/l after induction with collagen and adenosine 5'-diphosphate, respectively. For inhibition of thromboxane B(2) synthesis, the IC(50) values were 868 +/- 68 micromol/l; prostaglandin E(2) was inhibited only by acetylsalicylic acid (IC(50) for camonagrel >2,000 micromol/l), and the leukocyte 6-keto-PGF(1alpha) level was increased by camonagrel. The greatest reduction in percentage subendothelial surface occupied by platelets (mainly in the thrombi) after blood perfusion was seen after incubation with camonagrel in the range of concentrations that inhibited collagen-induced platelet aggregation. In conclusion, camonagrel reduced platelet-subendothelium interaction under flow conditions, showing this effect in a range of concentrations lower than in inhibition of platelet aggregation.

Elevated expression of the genes encoding TNF-alpha and thromboxane synthase in leucocytes from patients with systemic sclerosis.

OBJECTIVE: To determine the expression of molecular markers of prostanoid/fatty acid signalling in leucocytes of patients with systemic sclerosis (SSc). METHODS: Gene expression in patient leucocytes was analysed using real-time fluorescence reverse transcriptase polymerase chain reaction for tumour necrosis factor alpha (TNF-alpha), thromboxane synthase (TXAS, CYP5A), prostacyclin synthase (CYP8A), monocyte chemoattractant protein-1 (MCP-1), peroxisome proliferator-activated receptors (PPAR) alpha, delta and gamma, low-density lipoprotein-associated lipoprotein lipase A(2) (LDL-PLA(2)), apolipoprotein E (apoE) and cholesterol 27-hydroxylase (CYP27). RESULTS: Both TNF-alpha and TXAS showed an increase in mean expression in the diseased group (6.3-fold and 5.6-fold respectively, P<0.0001). These two markers, along with CYP27, PPARgamma and apoE, provided predictive markers for the development of carotid artery disease within the SSc patient population. CONCLUSION: The elevated levels of TNF-alpha and thromboxane seen in SSc patient sera are paralleled by increases in the expression of the appropriate genes in leucocytes. This method will allow us to screen for a large number of candidate markers of disease in order to increase our understanding of the processes underlying the pathology of SSc.

1-Imidazolylcarbonyloxy-substituted tetrahydroquinolines and pyridines: synthesis and evaluation of P450 TxA2 inhibition.

The title compounds are derived from our model describing structural requirements for strong P450 TxA2 inhibition. In the present paper the syntheses of the 1-imidazolylcarbonyloxy-substituted tetrahydroquinolines 1, 3, and 4, tetrahydro-naphthalene 2 and 3-ethylpyridines 5 and 6 are described. Using our P450 TxA2 inhibition assay, 1-6 were tested for enzyme inhibitory activity. Compound 1 (5-(1-imidazolylcarbonyloxy)-5,6,7,8-tetrahydroquinoline) turned out to be the most active derivative showing a potency similar to the reference compound dazoxiben (IC50 values 1.6 and 1.1 microM).

Effects of cysteinyl-leukotriene receptor antagonist, thromboxane A2 receptor antagonist, and thromboxane A2 synthetase inhibitor on antigen-induced bronchoconstriction in patients with asthma.

BACKGROUND: Leukotriene (LT) and thromboxane A2 (TXA2) receptor antagonists have been used in the treatment of asthma. OBJECTIVES: We examined the effects of an LT receptor antagonist, TXA2 receptor antagonist, and TXA2 synthetase inhibitor on bronchoprovocation test (BPT) in patients with mild-to-moderate atopic asthma. METHODS: BPT was performed four times in each of six asthmatics. Development of the immediate asthmatic reaction (IAR) and late asthmatic reaction (LAR) was confirmed on the first BPT (BPT1). After a 7-day washout period, an LT receptor antagonist (pranlukast, 450 mg/d), TXA2 receptor antagonist (seratrodast, 80 mg/d), or TXA2 synthetase inhibitor (ozagrel, 800 mg/d) was administered orally over 7 days at random using a cross-over method (BPT2-4). Blood levels of LTB4, LTC4, LTD4, 11-dehydrothromboxane B2, eosinophil cationic protein, and histamine were measured at reaction phases of pre-BPT, IAR, and LAR. RESULTS: Administration of pranlukast suppressed IAR by 80.5% (p < 0.0001) and LAR by 54.6% (p = 0.0391). Ozagrel significantly suppressed IAR by 39.5% (p = 0.0413), but the fall in FEV1 was >20% (21.56+/-4.173%). Seratrodast did not suppress IAR or LAR. Blood levels of chemical mediators did not correlate with the suppressive effects of the tested drugs. CONCLUSIONS: The LT receptor antagonist was considered to be the most effective. LT might play a more important role in the pathogenesis of asthma than TXA2. Our data showed that measurement of blood levels of chemical mediators is not useful in identifying the pathogenic mechanisms of asthma.

Spectrofluorimetric quantification of malondialdehyde for evaluation of cyclooxygenase-1/thromboxane synthase inhibition.

The in vitro assay developed by Hartmann and Ledergerber (1995) utilizing the spectrofluorimetric quantification of malondialdehyde after reaction with thiobarbituric acid was modified and used for further investigations. The human whole blood was replaced by a platelet suspension of pig blood, and calcium ionophore A23187 was used instead of collagen for inducing the arachidonic acid cascade. The modified assay represents a simple, time and cost saving method for the evaluation of cyclooxygenase-1/thromboxane synthase inhibition. The reproducibility and comparability of results is given. Additional experiments allow classification of selective phospholipase A2, cyclooxygenase-1, and thromboxane synthase inhibitors. Further studies of malondialdehyde formation show that the cyclooxygenase and/or the thromboxane synthase are competitively inhibited by reaction products of the cyclooxygenase pathway by a negative feedback mechanism.

New tetrahydronaphthalene derivatives as combined thromboxane receptor antagonists and thromboxane synthase inhibitors.

A pyridine group was linked to the tetrahydronaphthalene moiety of the derivatives described in the preceding paper, to afford new combined thromboxane receptor (TP-receptor) antagonists and synthase inhibitors. The most interesting compound 2f inhibits TXA2 synthase with an IC50 value of 0.64 microM and the aggregation of human platelets with an IC50 value of 0.063 microM and shows a long duration of action in different species after oral administration.

Bleeding disorder due to platelet prostaglandin H synthase-1 (PGHS-1) deficiency.

Defective platelet prostaglandin H synthase (PGHS) activity has been recognized as a cause of bleeding disorders, but the defect has not been characterized. We evaluated three female patients aged 37, 48 and 55 who presented with a mild bleeding disorder due to platelet dysfunction. None of the patients had underlying diseases or reported use of aspirin or other nonsteroidal anti-inflammatory drugs. Coagulation screening tests and platelet count were normal in each patient. Platelet aggregation in response to adenosine diphosphate (ADP), collagen and epinephrine were subnormal, characterized by an abnormal second-wave aggregation and propensity for disaggregation. Arachidonate-induced platelet aggregation was defective, whereas PGH2-induced aggregation was normal. Platelet thromboxane A2 (TXA2) production in response to arachidonic acid was reduced in all three patients, i.e. 11.7, 4.6 and 4.4 ng TXB2/3 x 10(8) plt respectively (normal range was 49-81 ng/3 x 10(8) plt), whereas they were normal in response to exogenous PGH2, i.e. 71.4, 56.6 and 48.9 ng/3 x 10(8) respectively (normal range 49-85 ng/3 x 10(8) plt). These results are consistent with a deficiency of platelet PGHS activity. The level of the constitutive platelet PGHS-1 and TXA2 synthase (TXAS) proteins were determined on platelet microsomal fractions by Western blot analysis using affinity-purified polyclonal antibodies highly specific for human PGHS-1 and TXAS, respectively. In two patients the 70 kD PGHS-1 protein was undetectable, whereas it was normal in the third patient. The 60 kD TXAS band was normal in all three patients. These findings indicate that human platelet PGHS-1 deficiency is due to two types of enzyme defects: type 1 defect is manifested by an undetectable PGHS-1 protein in platelets whereas the type 2 defect is manifested by a normal quantity of PGHS-1 protein which has an impaired catalytic activity.

Novel antiasthmatic agents with dual activities of thromboxane A2 synthetase inhibition and bronchodilation. VI. Indazole derivatives.

Synthesis and pharmacological evaluation of novel indazole derivatives are described. These compounds were found to exhibit both thromboxane A2 (TXA2) synthetase-inhibitory and bronchodilatory activities. This observation supports the idea that the partial structure of the 3-pyridyl and phenyl groups with a methylene insertion is an important component for well-balanced activities.

Development of a screening assay for the in vitro evaluation of thromboxane A2 synthase inhibitors.

Inhibitors of thromboxane A2 (TxA2) synthase are regarded as potentially useful agents in the treatment of cardiovascular diseases and in the prevention of tumour cell metastases. We report here a novel in vitro assay for the evaluation of TxA2 synthase inhibitors. For the determination of inhibitory activity, malondialdehyde (MDA) formation by TxA2 synthase in whole blood was utilized. After reaction with thiobarbituric acid MDA was quantified spectrofluorimetrically. The blank value was obtained by incubation with a selective TxA2 synthase inhibitor. For the screening of compounds the simple MDA assay represents an alternative to the rather expensive and time consuming radioimmunoassay, HPLC and TLC methods. Only for compounds which have been shown to be good inhibitors in the MDA assay should a radioimmunoassay for selective inhibition of TxA2 synthase be performed.

6,6-Disubstituted Hex-5-enoic acid derivatives as combined thromboxane A2 receptor antagonists and synthetase inhibitors.

A series of omega-disubstituted alkenoic acid derivatives were designed and synthesized as antithrombotic inhibitors of thromboxane A2 synthetase and thromboxane A2 receptor antagonists. Hexenoic acid derivatives with a 3-pyridyl group and a 4-(2-benzenesulfonamidoethyl)phenyl substituent were found to be optimal with regard to the dual mode of action. The most potent compound, (E)-6-(4-(2-(((4-chlorophenyl)sulfonyl)amino)ethyl)phenyl)-6-(3-pyridyl) hex-5-enoic acid (36), inhibits thromboxane A2 synthetase in gel-filtered human platelets with an IC50 value of 4.5 +/- 0.5 nM (n = 4), whereas an inhibitory effect on cyclooxygenase is seen only at a much higher concentration (IC50: 240 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that 36 blocks the prostaglandin H2/thromboxane A2 receptor with an IC50 of 19 +/- 5 nM (n = 5) and is therefore 85-fold more potent than another combined thromboxane A2 synthetase inhibitor/receptor antagonist, Ridogrel (4). Compound 36 inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an EC50 of 1 microM (Ridogrel: 16 microM) and 100 nM, respectively, and was selected for further development.

Amino-terminal topology of thromboxane synthase in the endoplasmic reticulum.

The membrane topology of the NH2-terminal portion of human thromboxane synthase (TXS), a member of the cytochrome P450 superfamily, has been investigated. By sequence alignment, the first 6 residues of the mature TXS polypeptide are likely to form a distinctive "tail" structure not found in many other mammalian cytochromes P450 in the endoplasmic reticulum membrane. Peptides with either the ultimate 10 or 15 residues of the NH2 terminus of TXS were synthesized and used to produce site-directed antibodies. The resulting peptide antibodies were highly specific and recognized human TXS, as shown by binding assays and Western blot analysis. Binding of the peptide antibodies to recombinant TXS in transfected COS-1 and to endogenous TXS in THP-1 cells was analyzed by immunocytochemistry. Selective permeabilization of the plasma membrane to immunoglobulin was achieved with streptolysin O; general permeabilization, including the endoplasmic reticulum membrane, was accomplished with Triton X-100. Permeabilization of the plasma membrane was sufficient to produce binding of both peptide antibodies to their epitopes, indicating that the epitopes for both of the peptide antibodies were exposed on the cytoplasmic side of the endoplasmic reticulum membrane. The results with the peptide antibodies provide direct experimental evidence supporting the topological model for membrane-bound cytochrome P450 proposed by Nelson and Strobel (Nelson, D. R., and Strobel, H. W. (1988) J. Biol. Chem. 263, 6038-6050), in which the NH2 terminus is oriented toward the cytoplasmic side of the endoplasmic reticulum membrane.

Toradol, an NSAID used for renal colic, decreases renal perfusion and ureteral pressure in a canine model of unilateral ureteral obstruction.

Toradol is a new parenteral, nonsteroidal anti-inflammatory drug which is efficacious in treating renal coli. In the present experiments, Toradol was administered to both control dogs and dogs with unilateral ureteral obstruction. In control dogs, Toradol had no effect on RBF or GFR, despite inhibition of renal prostaglandin synthesis (measured as urinary prostaglandin release). In contrast, RBF fell acutely by 35% (p < 0.001) within 15 minutes of Toradol administration in the setting of ureteral obstruction; contralateral RBF was unaffected. Ipsilateral ureteral pressure also fell. Changes in RBF and ureteral pressure, together with the known effects of NSAIDs on pain pathways, may contribute to the pain relief observed clinically with Toradol. However, the abrupt changes in renal hemodynamics brought on by Toradol to the obstructed kidney may compromise renal reserve, and Toradol should be used cautiously in treating renal colic.

Changes in plasma and urinary 11-dehydrothromboxane B2 in healthy subjects produced by oral CS-518, a novel thromboxane synthase inhibitor.

When 50 mg CS-518, a novel thromboxane (TX) A2 synthase inhibitor, was orally administered to healthy male volunteers, the plasma concentration of CS-518 peaked after 0.5 h and then decreased with a half-life of 0.44 h. There was no significant change in the plasma concentration of circulating TXB2, whereas that of circulating 11-dehydrothromboxane B2 (11-dhTXB2), an enzymatic metabolite of TXB2, was significantly decreased from 0.5 h to 24 h after administration; the maximal decrease to about 25% of the pre-dose value was found at 6 h. After CS-518 100 mg b.d. for 4.5 days, plasma 11-dhTXB2 was suppressed to the same extent as after the single dose of 50 mg from 6 h after the initial dose throughout the administration period. The urinary excretion of 11-dhTXB2 corrected for the creatinine level was significantly decreased by 70-84% throughout the treatment. These results suggest that CS-518 causes long-lasting inhibition of TXA2 synthase despite its rapid elimination from plasma, and that circulating 11-dhTXB2 in plasma and its urinary excretion can serve as a quantitative index of TXA2 synthase inhibition in vivo by CS-518.