B4 Synthesis Research Articles

Polymorphonuclear Leukocyte-Platelet Interaction: Role of P-Selectin in Thromboxane B2 and Leukotriene C4 Cooperative Synthesis

In PMN/platelet suspensions stimulated by fMLP giant mixed aggregates are formed and TxB2 and LTC4 are synthesized as the result of the cooperation in the arachidonic acid (AA) metabolism during cell/cell contact. PMN-derived cathepsin G induced the expression of P-selectin on platelet surface. GE12, an antibody against P-selectin, significantly reduced mixed cell aggregates. GE12 did not affect platelet aggregation induced by PMN-derived supernatants, indicating that the inhibitory effect of GE12 on mixed cell aggregation depends on inhibition of PMN/platelet adhesion. GE12 significantly reduced TxB2 and LTC4 production in PMN/platelet mixed cell suspensions stimulated by fMLP. As previously reported, synthesis of 3H-TxB2 in 3H-AA-labeled PMN/unlabeled platelets indicates that platelets utilize 3H-AA from PMN. 3H-LTC4 production in unlabeled PMN/3H-AA-labeled platelets indicates that bidirectional routes are utilized in this system for LTC4 synthesis. GE12 significantly reduced 3H-TxB2 and 3H-LTC4 synthesis. These results show that cathepsin G released by activated PMN induces the expression of P-selectin on platelet membrane: this adhesive glycoprotein modulates cell-cell contact and transcellular metabolism of AA.

Influence of aspirin on human megakaryocyte prostaglandin synthesis.

To evaluate whether currently popular aspirin regimens have an effect on the prostaglandin synthesis in human megakaryocytes we measured thromboxane B2 (TXB2) synthesis in response to thrombin stimulation in human megakaryocytes ex vivo. Human megakaryocytes were purified by Counterflow Centrifugal Elutriation from bone marrow punctures, taken from volunteers before and 2 hours after ingestion of one dose of 500 mg (n = 4), 80 mg (n = 4) or 40 mg (n = 2) aspirin. Subsequently, megakaryocytes were purified before and 12 h after ingestion of 80 mg (n = 3) aspirin twice daily for 1 week and 12 h after 500 mg (n = 3) aspirin. On average, 140 +/- 102 x 10(3) (mean +/- 1 SD) megakaryocytes were recovered. We found that aspirin inhibits megakaryocyte cyclooxygenase in a dose-dependent manner. Two hours after 500 mg of aspirin, TXB2 synthesis in megakaryocytes was inhibited by 96.8 +/- 2%, whereas one dose of 80 and 40 mg aspirin showed an inhibition of 79.4 +/- 13.7% and 80 +/- 6.2% respectively. However, the inhibition of TXB2 synthesis seems not to be long-lasting since, 12 h after the ingestion of aspirin, an increase of megakaryocyte TXB2 production could be observed which reached significance after the 500 mg aspirin dosage (p < 0.048). We conclude that human megakaryocyte cyclooxygenase is sensitive to aspirin inhibition and that low doses of aspirin (40 and 80 mg) enter the systemic circulation and are able to inhibit megakaryocyte cyclooxygenase, but this inhibition is incomplete and megakaryocyte cyclooxygenase seems to recover within 12 h after ingestion of aspirin.

Rapid and Selective Inhibition of Platelet Aggregation and Thromboxane Formation by Intravenous Low Dose Aspirin in Man

1. One of the major problems in the clinical use of low dose aspirin for the prevention of vascular occlusion is that it takes about 3-5 days to become effective, a time too long for patients with unstable angina or coronary thrombolysis. Intravenous aspirin may be expected to exert a more rapid effect, but its influence on endothelial prostacyclin synthesis is uncertain. 2. In a single-blind, randomized, prospective study, we compared the effects of a single intravenous low dose (50 mg) or high dose (500 mg) of aspirin or placebo infused over a 60 min period on platelet aggregation, platelet thromboxane A2 production and whole-body prostanoid synthesis in 10 healthy male subjects by gas chromatography-tandem mass spectrometry. 3. Before the study, blood flow rates in the basilic and subclavian veins were determined by sonographic colour velocity imaging; the infusion rate for low dose aspirin was calculated to avoid biologically effective plasma levels of aspirin in the systemic circulation. 4. Platelet aggregation induced by 1 mmol/l arachidonic acid was similarly inhibited by > 85% within 30 min after the start of the infusion of high dose or low dose aspirin, respectively, and remained suppressed for 24 h. Platelet thromboxane A2 release declined gradually after low dose aspirin, reaching a minimum of 93% inhibition after 60 min. High dose aspirin suppressed platelet thromboxane A2 release to below the detection limit after 10 min. 5. Urinary excretion of the major urinary metabolite of thromboxane A2 (2,3-dinor-thromboxane B2) was equally suppressed by both dosages of aspirin [no significant difference between high dose (-83.2%) and low dose (-67.4%)].(ABSTRACT TRUNCATED AT 250 WORDS)

An Efficient Formal Synthesis of Aflatoxin B2 by Use of the Kikuchi Rearrangement Reaction

An Efficient Formal Synthesis of Aflatoxin B2 by Use of the Kikuchi Rearrangement Reaction

Stereoselective cyclooxygenase inhibition in cellular models by the enantiomers of ketoprofen.

The pharmacological activity of rac-ketoprofen and its enantiomers was investigated in vitro using different cellular models. The effect of these compounds on arachidonic acid metabolism was assessed by measuring the inhibition of prostanoid generation under the action of several agonists. Thus, we have evaluated the inhibition of (1) thromboxane B2 synthesis in rabbit platelets and human polymorphonuclear leukocytes (PMNs), (2) prostaglandin E2 synthesis in three cultured cells, namely human umbilical vein endothelial cells (HUVEC), human keratinocytes, and mouse macrophage-like P388D1 cells. The IC50 values found for (+)-(S)-ketoprofen were in the range between 0.1 nM and 0.8 microM, being slightly lower in all models than those found for rac-ketoprofen (0.4 nM-3 microM). On the other hand (-)-(R)-ketoprofen showed inhibition of cyclooxygenase only at concentrations two or three orders of magnitude higher than those required for the (+)-(S) enantiomer. These results, obtained with cell types of relevance for inflammatory processes and with compounds of high optical purity, demonstrate that the prostanoid biosynthesis inhibition caused by the drug rac-ketoprofen is exclusively due to its dextrorotatory enantiomer.

Prostaglandin cyclooxygenase products but not thromboxane A2 are involved in the pathogenesis of ewthromelalgia in thrombocythaemia

Fluid of artificial blisters from erythromelalgic skin areas in primary thrombocythaemia contained a high amount of prostaglandin-E-like activity. Dazoxiben did not alleviate the erythromelalgia in patients with primary thrombocythaemia despite complete inhibition of platelet malondialdehyde and thromboxane B2 synthesis and no inhibition of prostaglandin-E-like material. During a 10-day dazoxiben treatment period, persistent erythromelalgia was associated with a significant shortened mean platelet life span of 3.2 days. During subsequent treatment with low dose acetylsalicylic acid daily complete relief of erythromelalgia was associated with inhibition of platelet prostaglandin endoperoxide production and correction of platelet mean life span to normal, 7.9 days. These observations indicate that prostaglandin E2, or another prostaglandin endoperoxide metabolite, is involved in the pathogenesisof erythromelalgia. The presented study does not give one single clue as to the origin (platelet, vessel wall or other) of the prostanoid, but very likely originates from platelets because a very low dose of acetylsalicylic acid (250 to 500 mg every other day), which irreversibly inhibits platelet cyclooxygenase, is highly effective in the prevention of erythromelalgia in thrombocythaemia.

Effect of dipyridamole with or without aspirin on urine protein excretion in patients with membranous glomerulonephritis.

The antiproteinuric effect of the antiplatelet agent dipyridamole has been assessed after inhibition of thromboxane B2 (TxB2) synthesis in 8 patients with confirmed membranous glomerulonephritis. There were three study periods, each of 30 days, and 45 days apart, namely a washout period, treatment with dipyridamole 300 mg/d, and dipyridamole 225 mg/d plus aspirin 150 mg/d. On Days 1 and 30 of each study period serum and urine creatinine, 24-h excretion of protein, creatinine clearance, platelet aggregometry on whole blood and serum TxB2 were measured. Treatment with dipyridamole alone or with aspirin produced significant inhibition of platelet aggregation and a fall in 24-h protein excretion; the latter amounted to 54% with dipyridamole alone and 56% with dipyridamole plus aspirin (NS). Dipyridamole plus aspirin caused an 82% reduction in serum TxB2.

Altered prostaglandin synthesis and impaired sodium conservation in the kidneys of old rats.

1. The aim of this investigation was to study the role of prostaglandins in the impaired Na+ conservation of the ageing kidney. 2. We measured the urinary excretion of thromboxane B2, 6-keto-prostaglandin F1 alpha and prostaglandin E2 in young (3-4 months) and old (20-21 months) rats after 12, 24 and 36 h of Na+ deprivation. In a separate protocol, we measured prostanoid synthesis by isolated glomeruli, cortical homogenates, medullary slices and papillary slices from young and old rats in basal conditions and after 15 days of dietary Na+ deprivation. 3. In the acute study, urinary excretion of 6-keto-prostaglandin F1 alpha and prostaglandin E2 decreased in young but not in old rats. Urinary excretion of prostaglandin E2 was lower in old rats, but did not vary significantly with Na+ deprivation. 4. In old rats, thromboxane B2 synthesis was increased in all the portions of the kidney except the medulla. Production of 6-keto-prostaglandin F1 alpha was elevated in glomeruli and tended to increase in the cortex. Prostaglandin E2 synthesis was also elevated in the cortex. Thromboxane B2 synthesis tended to increase in the medulla and was enhanced in the papilla. After Na+ deprivation, only glomerular prostaglandin E2 synthesis increased in young rats. In old rats, cortical and papillary synthesis of 6-keto-prostaglandin F1 alpha increased, whereas prostaglandin E2 synthesis did not change. 5. The results suggest increased thromboxane synthesis in the ageing kidney. Increased prostacyclin and prostaglandin E2 synthesis may be an attempt to counteract enhanced thromboxane production.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that Mos protein may not act directly on cyclin.

Using affinity-purified antiserum we have examined cyclin B2 levels in Xenopus oocytes at various stages of oogenesis. We found that cyclin B2 is detected from stage 2 to stage 6 as two bands, one of which is phosphorylated, and that cyclin B2 mass increases about 28-fold between stage 2 and stage 6. To examine the effect of Mos protein on cyclin phosphorylation, we microinjected synthetic Xenopus c-mos (c-mosxe) RNA into stages 4, 5, and 6 Xenopus oocytes. In stage 6 oocytes, maturation was induced by c-mosxe RNA, and, as is the case with progesterone treatment, all cyclin B2 was shifted to the phosphorylated form. However, c-mosxe RNA injected into stage 4 or 5 oocytes did not induce maturation or cause a shift in the relative proportion of the two cyclin B2 bands. These data suggest that Mos does not act directly to phosphorylate cyclin B2, causing the band shift during maturation. Cyclin B2 synthesis increases about 2-fold during maturation, in concert with total protein synthesis. Data from experiments on cyclin B2 stability indicate that the half-life of cyclin B2 is about 85 hr in stage 6. This suggests that if Mos protein has a direct effect on cyclin stability, it does so only at a later stage in oocyte maturation, but not at the onset.

Colorectal leukotriene B4 synthesis in vitro in inflammatory bowel disease: inhibition by the selective 5-lipoxygenase inhibitor BWA4C.

The in vitro synthesis of leukotriene B4 (LTB4) was evaluated in colorectal biopsy specimens and resection tissue from patients with inflammatory bowel disease. The in vitro formation of LTB4 from biopsy tissues stimulated with calcium ionophore A23187 correlated with the degree of mucosal inflammation assessed at sigmoidoscopy, and with neutrophil infiltration measured as myeloperoxidase activity. Biopsy specimens from patients taking prednisolone formed less LTB4 than those from patients not on prednisolone, with comparable levels of inflammation seen at sigmoidoscopy. The formation of LTB4 was reduced dose-dependently by the acetohydroxamic acid 5-lipoxygenase inhibitor BWA4C, with no significant inhibition of prostaglandin E2 or thromboxane B2 synthesis. In inflamed colonic resection tissue from colitic patients, the IC50 for inhibition of LTB4 formation by BWA4C was 0.03 mumol/l, compared with an IC50 of 0.8 mumol/l for NDGA. Thus, BWA4C is a potent and selective inhibitor of LTB4 synthesis in colonic tissue from patients with ulcerative colitis. Acetohydroxamic acid 5-lipoxygenase inhibitors, exemplified by BWA4C, may be useful to evaluate the clinical importance of LTB4 in ulcerative colitis, and offer a novel therapy for the disease.

Intravenous Acetylsalicylic Acid – Dose-Related Effects on Platelet Function and Fibrinolysis in Healthy Males

Low-dose acetylsalicylic acid (ASA) has been shown to be beneficial in patients with acute myocardial infarction and unstable angina pectoris. Oral administration of ASA is difficult in the acute phase of these syndromes. In this study we evaluated the effect of 25 mg, 50 mg or 100 mg of ASA given as an intravenous bolus injection on platelet function and fibrinolysis in healthy males and related this to plasma concentrations of ASA. No adverse effects were found. A complete inhibition of serum thromboxane B2 synthesis was demonstrated 5 min after injection of 100 mg ASA intravenously. ASA disappeared from the circulation within 60 min after bolus injection and at this time thromboxane B2 synthesis was inhibited dose-dependently by 71%, 90% and 100% for doses of 25 mg, 50 mg and 100 mg, respectively. Inhibition of thromboxane B2 synthesis after 100 mg of intravenous ASA was still 96.5% at 24 h and 93.4% at 48 h after the injection. The bleeding time measured at 30 min after ASA administration was significantly prolonged on the average by 70 s, 144 s and 211 s after 25 mg, 50 mg and 100 mg of ASA, respectively. Minor, but significant changes were found in tissue plasminogen activator antigen and in plasminogen activator inhibitor within the first hour after injection of low dose ASA, but similar changes were found after injection of saline. No change in tissue plasminogen activator activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

CALCIUM-CHANNEL BLOCKING AGENTS VERAPAMIL AND DILTIAZEM ARE INHIBITORS OF VASOPRESSIN-INDUCED HUMAN PLATELET ACTIVATION

1. This study investigated the influences of calcium-channel blocking agents verapamil and diltiazem on platelet responses induced by arginine vasopressin (AVP) and lysine vasopressin (LVP). 2. The substances inhibited platelet aggregation induced by both low and high AVP concentrations, LVP and adrenaline plus AVP. IC50 values of each drug are lower than those determined for ADP- and collagen-elicited aggregation. Verapamil and diltiazem also decreased AVP-induced thromboxane B2 synthesis. 3. Other series of experiments showed that the addition of ethyleneglycol-bis-(beta-amino-ethyl ether) N, N'-tetra-acetic acid to platelet-rich plasma samples also prevented the platelet response to vasopressin polypeptides. 4. Our data provide evidence that the effects of verapamil and diltiazem on vasopressin-induced platelet responses may be directly related to inhibition of extracellular calcium entry.

A study of the effects of p,p′-DDE and other related chlorinated hydrocarbons on inhibition of platelet aggregation

A series of chlorinated hydrocarbons of interest in environmental toxicology were investigated concerning their effects on human platelet aggregation. Most potent in inhibiting platelet aggregation were p,p'-DDE and Arochlor 1242. Aggregation induced by arachidonic acid (1 mM) was more sensitive to inhibition by p,p'-DDE than was aggregation induced by ADP (10 microM). The former was completely inhibited by p,p'-DDE at a concentration of 1 x 10(-4) M, whereas there was only a 31% inhibition of the latter. Addition of Ca2+ (1 mM) blocked the inhibitory effect of p,p'-DDE on aggregation induced by both arachidonic acid and ADP. Calmodulin (1 microgram/ml) blocked the inhibitory effect of p,p'-DDE on aggregation induced by arachidonic acid but not that induced by ADP. The calmodulin inhibitory drugs trifluoperazine and calmidazolium had no effect at all or only a weak effect (-14%), respectively, on platelet aggregation. Increasing the concentrations of p,p'-DDE and Arochlor 1242 caused a delay in the onset of aggregation induced by the addition of arachidonic acid. The synthesis of thromboxane B2 and other prostaglandins in platelet membranes was dose-dependently reduced by p,p'-DDE. The structurally closely related isomers o,p'-DDE and p,p'-DDT did not significantly inhibit arachidonic acid-induced platelet aggregation or thromboxane B2 synthesis. It is concluded that p,p'-DDE and Arochlor 1242 inhibited platelet aggregation by inhibiting platelet cyclooxygenase activity.

Erythrocytes Metabolically Enhance Collagen-Induced Platelet Responsiveness Via Increased Thromboxane Production, Adenosine Diphosphate Release, and Recruitment

Erythrocytes promoted platelet reactivity in a plasma medium, as demonstrated in an in vitro system that independently evaluated the biochemistry of platelet activation and recruitment. The prothrombotic erythrocyte effects were metabolically regulated, as evidenced by lack of activity of ATP-depleted or glutaraldehyde-fixed erythrocytes. They occurred in the absence of cell lysis as verified by lactate dehydrogenase assays, and had an absolute requirement for platelet activation. The presence of erythrocytes induced a twofold increase in platelet thromboxane B2 (TXB2) synthesis upon collagen stimulation, indicating that erythrocytes modulated platelet eicosanoid formation. Cell-free releasates from stimulated platelet-erythrocyte suspensions, which exhibited increased recruiting capacity, contained 6.9-fold more ADP and 4.9-fold more ATP than releasates from stimulated platelets alone. Following aspirin ingestion, TXB2 formation was blocked, but erythrocyte promotion of platelet reactivity persisted at those doses of collagen that reinduced platelet activation. Moreover, when platelet mixtures consisted of as little as 10% obtained before aspirin plus 90% obtained post-aspirin ingestion, significant erythrocyte enhancement of platelet reactivity occurred, even at low agonist concentrations. These erythrocyte effects would decrease the therapeutic potential of inhibition of platelet cyclooxygenase by aspirin. The erythrocyte-induced modulation of platelet biochemistry and function emphasizes the importance of cell-cell interactions in stimulus-response coupling.

Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment

Erythrocytes promoted platelet reactivity in a plasma medium, as demonstrated in an in vitro system that independently evaluated the biochemistry of platelet activation and recruitment. The prothrombotic erythrocyte effects were metabolically regulated, as evidenced by lack of activity of ATP-depleted or glutaraldehyde-fixed erythrocytes. They occurred in the absence of cell lysis as verified by lactate dehydrogenase assays, and had an absolute requirement for platelet activation. The presence of erythrocytes induced a twofold increase in platelet thromboxane B2 (TXB2) synthesis upon collagen stimulation, indicating that erythrocytes modulated platelet eicosanoid formation. Cell-free releasates from stimulated platelet-erythrocyte suspensions, which exhibited increased recruiting capacity, contained 6.9-fold more ADP and 4.9-fold more ATP than releasates from stimulated platelets alone. Following aspirin ingestion, TXB2 formation was blocked, but erythrocyte promotion of platelet reactivity persisted at those doses of collagen that reinduced platelet activation. Moreover, when platelet mixtures consisted of as little as 10% obtained before aspirin plus 90% obtained post-aspirin ingestion, significant erythrocyte enhancement of platelet reactivity occurred, even at low agonist concentrations. These erythrocyte effects would decrease the therapeutic potential of inhibition of platelet cyclooxygenase by aspirin. The erythrocyte- induced modulation of platelet biochemistry and function emphasizes the importance of cell-cell interactions in stimulus-response coupling.

Mesangial cell immune injury

We assessed the renal hemodynamic changes occurring acutely after glomerular mesangial cell immune injury and the effects of thromboxane receptor antagonism on these changes. A single intravenous proteinuric dose of a monoclonal antibody raised against the rat thymocyte antigen Thy 1.1 (ER4), which is also expressed in rat mesangial cells, induced acute decrements in glomerular filtration rate and in renal blood flow in male Munich-Wistar rats. One hour after administration of 4 to 6 mg/kg of ER4 antibody, glomerular filtration rate and renal blood flow decreased by 80 and 36%, respectively. These decrements were associated with significant increments in basal thromboxane B2 synthesis in isolated glomeruli and no changes in glomerular prostaglandin E2 synthesis. Pretreatment of animals with the thromboxane receptor antagonist SQ-29,548 (2 mg/kg) significantly ameliorated decrements in glomerular filtration rate and renal blood flow. Pretreatment with a structurally dissimilar thromboxane receptor antagonist, L-670,596 (3 mg/kg) had no effect. Both antagonists at the doses employed abolished the decrements in renal blood flow induced by systemic administration of the thromboxane mimetic U-46619. Whereas the SQ-29,548 antagonist had no effect on glomerular leukotriene B4 and 12-hydroxyeicosatetraenoic acid synthesis, the L-670,596 thromboxane receptor antagonist significantly inhibited glomerular synthesis of these eicosanoids in immunologically injured glomeruli. These observations indicate that in mesangial cell immune injury the protective effect of thromboxane A2 receptor antagonism on glomerular filtration rate and renal blood flow is not solely due to inhibition of the vasoconstrictor effects of thromboxane A2. An effect on the synthesis of arachidonate lipoxygenation products may also play a role.

Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.

Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase.

Phenothiazines inhibit collagen-induced thromboxane B2 synthesis and increase forskolin anti-aggregating effects in human platelets

1. 1. The aim of the present study was to investigate the effects of phenothiazine compounds chlorpromazine and trifluoperazine on human platelet response to collagen and sodium arachidonate. 2. 2. The results demonstrated that phenothiazines inhibit collagen-induced platelet aggregation and thromboxane B2 synthesis in a dose-dependent way and increase the antiaggregating properties of forskolin. 3. 3. Phenothiazines at high concentrations decreased the platelet aggregation in response to arachidonate, without a significant reduction of thromboxane B2 production. 4. 4. The data provides evidence that phenothiazine effects could involve calmodulin-dependent enzymatic activities.

High dose eicosapentaenoic acid ethyl ester: effects on lipids and neutrophil leukotriene production in normal volunteers.

1. A 93% pure ethyl ester of eicosapentaenoic acid was investigated for tolerability and biochemical effects on neutrophil leukotriene synthesis and plasma lipoproteins when given in high dose. Six healthy volunteers received 6 g eicosapentaenoic acid ethyl ester daily for 6 weeks, followed by a 4 week wash-out and then 18 g daily for 6 weeks. 2. There was inhibition of neutrophil leukotriene B4 and 5-hydroxyeicosatetraenoic acid synthesis, with no significant differences between low and high dose. 3. There was a dose dependent increase in leukotriene B5 and 5-hydroxyeicosatetraenoic acid acid synthesis. 4. Plasma triglycerides were reduced maximally on 6 g daily, with no greater suppression at 18 g daily. 5. Plasma cholesterol was only suppressed significantly at 18 g daily. 6. The 6 g daily dose was well tolerated but the 18 g daily dose produced diarrhoea and steatorrhoea.

ChemInform Abstract: The Regiospecific p‐Deiodination of 2,4‐Diiodophenols. A New Synthesis of Aflatoxin B2.

AbstractTosylation of the phenol (I) and subsequent oxidative cyclization yield the furobenzofuran derivative (III) which is detosylated to form the phenol (IV).