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Biomedical subjects

G A FitzGerald

Publications and source records attributed to G A FitzGerald.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of human 12-lipoxygenase genes.

Two human 12-lipoxygenase enzyme (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31)-related genes were characterized from 13 distinct clones isolated from three genomic bacteriophage and cosmid libraries. A complete gene (12-lipoxygenase gene 1) spanning approximately 17 kilobases and consisting of 14 exons with sequence matching the cloned platelet/human erythroleukemia (HEL) cell cDNA sequence was identified. Several consensus sites for transcription factors and two potential transcription initiation sites within the 5' flanking region, encompassing the putative promoter region, were identified. A segment of a second, probable pseudogene (12-lipoxygenase gene 2), which displays approximately 85% identity to gene 1 within exon sequences, was also characterized. The presence of two 12-lipoxygenase genes was also substantiated by Southern blot analysis of total human genomic DNA. Exon-intron boundaries for the 12-lipoxygenase genes were located in the identical corresponding positions to the previously cloned human 5-lipoxygenase and rabbit 15-lipoxygenase genes, indicating a highly related gene family. Three lipoxygenase genes (12-lipoxygenase genes 1 and 2, 15-lipoxygenase) were localized to human chromosome 17, whereas the most unrelated lipoxygenase (5-lipoxygenase) was mapped to chromosome 10 by PCR analysis of a human-hamster somatic hybrid DNA panel. 12-Lipoxygenase gene 1 expression could be detected in human erythroleukemia cells, platelets, and human umbilical vein endothelial cells with certainty by reverse transcription-PCR analysis. There was no detectable 12-lipoxygenase gene 2 expression in several tissues and cell lines.

Amino Acid Sequence↗
PubMed1992

Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction.

Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t-PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet-induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.

Blood Platelets↗
PubMed1992

Antiplatelet and anticoagulant drugs in coronary vascular disease.

The stimulation of platelets, activation of the coagulation cascade, release of platelet-derived vasoconstrictors, and endothelial dysfunction all contribute to the thrombotic vascular occlusion that results in myocardial infarction. Despite the importance of platelets in the initiation of this process, they are activated by multiple endogenous mediators. Thus, one might anticipate that redundancy in the system would confound the efficacy of antiplatelet drugs that were mediator-specific. The success of aspirin in clinical trials is likely to reflect the role of thromboxane A2 (TxA2) as an amplification signal for other platelet agonists. Activated platelets provide a substrate for assembly of the prothrombinase complex and both heparin and warfarin also reduce the mortality due to thrombotic vascular disease. The relative efficacy of these compounds versus aspirin and the safety of their combination, particularly in the setting of therapeutic thrombolysis, are under investigation. Novel antiplatelet agents, particularly those directed against the glycoprotein 11b/111a complex, are more potent than aspirin in animal models. Similarly, direct thrombin inhibitors seem superior to heparin. Whether such compounds can be administered safely in effective doses to humans is under study. It is hoped that the success of aspirin does not impede the clinical evaluation of theoretically more attractive antithrombotic drugs.

Animals↗
PubMed1992

Thromboxane synthesis and action within the kidney.

PGH2 and TxA2 exert their actions via tissue specific, receptor isoforms. PGH2/TxA2-dependent platelet aggregation and contraction of vascular and bronchial smooth muscle and of glomerular mesangial cells occur via receptors linked to activation of phospholipase C. Although PGH2/TxA2 appear to be of little importance in the maintenance of renal function under physiological circumstances, increased renal TxA2 biosynthesis has been documented in a variety of animal models of renal disease and in some clinical disorders (Table 2). The effects of this eicosanoid on renal tissues in vitro and of pharmacological manipulation of TxA2 synthesis and action in vivo suggest that such interventions will provide new drugs for the treatment of human kidney disease.

Animals↗
PubMed1992

Pharmacokinetics of tissue-type plasminogen activator during acute myocardial infarction in men. Effect of a prostacyclin analogue.

BACKGROUND: Coronary reocclusion complicates the thrombolytic therapy of acute myocardial infarction despite the routine use of aspirin. This is consistent with experimental studies demonstrating that multiple agonists, in addition to thromboxane A2, mediate the platelet activation underlying reocclusion. Consequently, a more potent antiplatelet therapy with a broader spectrum of activity than aspirin may be required in this setting. Prostacyclin and its more stable analogue, iloprost, inhibit platelet aggregation to all known agonists and exert an additional effect over aspirin alone. Experiments in animal models have demonstrated, however, that iloprost increases the clearance of tissue-type plasminogen activator (t-PA) and impairs thrombolysis in vivo. This study examines whether a similar interaction occurs in humans. METHODS AND RESULTS: Twelve patients with acute myocardial infarction received t-PA intravenously, 60 mg in the first hour and a maintenance infusion of 13.3 mg/hr for 3 hours. Patients were assigned in a double-blind fashion to iloprost (2 ng/kg/min) or placebo following the initial 90 minutes of the maintenance infusion of t-PA. Iloprost decreased mean arterial blood pressure (-10 +/- 2.9 mm Hg, p less than 0.05) but did not alter heart rate. Steady-state plasma iloprost concentration was 591 +/- 64 pmol/l. At this concentration, iloprost markedly inhibited platelet aggregation in vitro, particularly in the presence of aspirin. Steady-state clearance of t-PA was unchanged by iloprost (454 +/- 65 versus 443 +/- 136 ml/min in controls, p = NS). Furthermore, neither elimination kinetics nor plasma protein binding of t-PA was altered by iloprost. CONCLUSIONS: At plasma levels that exert a potent antiplatelet effect, iloprost did not alter the pharmacokinetics of t-PA in men. Prostacyclin analogues may prove useful as an adjunct to plasminogen activators, particularly in patients at high risk for thrombotic reocclusion.

Acute Disease↗
PubMed1992

Thromboxane synthase inhibition in allergen challenged sheep lung: effect on eicosanoid synthesis.

We determined the effect of a novel inhibitor of thromboxane synthase, DP1904, on the baseline and allergen stimulated release of eicosanoids into the bronchoalveolar lavage fluid of sheep. DP1904 was effective in reducing the baseline and allergen stimulated production of TXB2. Inhibition of thromboxane synthase was associated with an increase in other prostaglandin endoperoxide metabolites, PGE2 and PGD2. Diversion of endoperoxide metabolism occurs in sheep lung tissue in vivo.

Allergens↗
PubMed1991

Suppression of thromboxane A2 but not of systemic prostacyclin by controlled-release aspirin.

BACKGROUND: The antithrombotic efficacy of aspirin is attributed to its inhibition of the enzyme prostaglandin G/H synthase, which is necessary for the formation of thromboxane A2 in platelets. Thromboxane A2 is a potent vasoconstrictor and platelet agonist. However, the formation of prostacyclin by vascular endothelium also requires prostaglandin G/H synthase, and prostacyclin exerts opposite effects on platelet function and vascular tone. We wanted to see whether controlled-release aspirin would affect the formation of thromboxane A2 but not prostacyclin by reducing the aspirin concentration that reaches the posthepatic circulation. METHODS: A controlled-release formulation containing 75 mg of aspirin, designed to release 10 mg per hour, was developed to inhibit prostaglandin G/H synthase in platelets in the prehepatic circulation. The effects of the controlled-release preparation on plasma levels of aspirin and salicylate, serum levels of thromboxane B2, and urinary dinor metabolites of prostacyclin and thromboxane B2 (measured by gas chromatography-mass spectrometry) were compared with those of conventional immediate-release aspirin in normal volunteers. Prostacyclin release was stimulated by intravenous bradykinin. RESULTS: Steady-state inhibition of serum thromboxane B2 required two to four days and appeared slower with 75 mg of controlled-release than with the same amount of immediate-release aspirin. Maximal inhibition was achieved rapidly by adding a single loading dose of 162.5 mg of immediate-release aspirin to the regimen. Over a 28-day period, suppression of thromboxane A2 with this regimen was comparable to that with immediate-release aspirin taken either as 162.5 mg daily or as 325 mg on alternate days, despite the minimal systemic bioavailability of controlled-release aspirin. Bleeding time was prolonged to a similar degree with each of the three regimens. The five- to sixfold increase in the prostacyclin metabolite induced by bradykinin was depressed by pretreatment for four days with 75 mg of immediate-release aspirin, but not by 75 mg of controlled-release aspirin. CONCLUSIONS: Maximal inhibition of platelet thromboxane A2 production was sustained during long-term dosing with controlled-release aspirin, whereas basal prostacyclin biosynthesis fell only slightly and systemic synthesis of prostacyclin stimulated by bradykinin was preserved. Controlled-release aspirin may facilitate determination of the clinical importance of preserving prostacyclin during platelet inhibition in humans.

Adolescent↗
PubMed1991

Mechanisms of platelet activation: thromboxane A2 as an amplifying signal for other agonists.

Thromboxane (Tx) A2 is a product of cyclooxygenase catalyzed metabolism of arachidonic acid. It is formed via prostaglandin (PG) endoperoxide intermediates (PGG2 and PGH2) by a specific synthase. PGH2 appears to exert the same biologic effects as TxA2. The cDNA for a TxA2 receptor has been cloned from a human placental library. Although pharmacologic and biochemical studies suggest the presence of multiple isoforms, this remains to be confirmed at the molecular level. A hydropathy plot of the deduced amino acid sequence of the available clone suggests that it has 7 transmembrane spanning domains, typical of a G protein linked receptor. Pharmacologic studies imply that Tx receptors in platelets are linked to phospholipase C activation via pertussis toxin insensitive G proteins. Candidates include the 42 kD Gq and the 60 kD Ge. TxA2 acts as an amplifying signal for platelet agonists and the response to this eicosanoid is tightly regulated. Mechanisms include rapid hydrolysis of the agonist to the inactive TxB2, autoinactivation of Tx synthase, rapid homologous TxA2 receptor desensitization due to receptor-G protein uncoupling, coincidental sensitization to counterregulatory Gs linked receptor systems and stimulation of prostacyclin formation by TxA2. Due to its role as an amplification signal in platelet activation, inhibition of Tx synthesis and action is an effective mechanism for preventing platelet-dependent vascular occlusion. Aspirin is of proven efficacy in this regard. Tx synthase inhibitors and antagonists are under clinical investigation.

Humans↗
PubMed1991

Eicosanoid forming enzyme mRNA in human tissues. Analysis by quantitative polymerase chain reaction.

The key enzymes in the formation of eicosanoids, including leukocyte 5-lipoxygenase (5LX), platelet 12-lipoxygenase (12LX), reticulocyte 15-lipoxygenase (15LX), prostaglandin G/H synthase cyclooxygenase, and leukotriene A4 (LTA) hydrolase have been studied extensively in recent years. Little is known, however, about the regulation of these enzymes at the gene level. We have developed a quantitative polymerase chain reaction (PCR) assay to quantify the mRNAs for these five enzymes, as well as for cytoplasmic beta-actin (bACT) mRNA. Human erythroleukemia (HEL) cells, which display megakaryocytic/erythroid characteristics, were selected as a source of RNA to characterize the assay. These cells expressed mRNA for bACT, LTA, cyclooxygenase, and 12LX (in decreasing order). mRNA for 5LX and 15LX was undetectable. Bronchoalveolar lavage fluid cells obtained from asthmatic patients, primarily alveolar macrophages, contained mRNA for bACT, LTA, 5LX, cyclooxygenase, and 15LX (in decreasing order). Treatment of HEL cells with phorbol 12-myristate 13-acetate or steroid administration to asthmatic patients apparently selectively regulated certain of these target genes. The utility of this assay in quantifying mRNA for the various target genes in blood cells, including platelets from patients with chronic myelogenous leukemia, has also been demonstrated. Studies on the regulation of genes for enzymes involved in the leukotriene and prostaglandin biosynthetic pathways, especially when only small tissue samples are available, will be facilitated with this approach.

Actins↗
PubMed1991

Symptomatic patent ductus arteriosus following prophylactic indomethacin. A clinical and biochemical appraisal.

Seven of 36 premature infants with birth weight less than 1,250 g who had been randomly assigned to either high-frequency ventilatory ventilation or conventional mechanical ventilation developed symptomatic patent ductus arteriosus (PDA) after receiving prophylactic indomethacin, 0.2 mg/kg, i.v. 24 h after birth. Infants who developed symptomatic PDA were more likely to be white and male and have more severe pulmonary insufficiency than infants who did not develop symptomatic PDA. Serum indomethacin levels were similar in both groups of patients. There was no difference in the degree of suppression or subsequent recovery of endogenous eicosanoid biosynthesis between infants who did and did not develop symptomatic PDA. Compared to conventional mechanical ventilation, high-frequency oscillatory ventilation had no significant effect on endogenous eicosanoid biosynthesis. The susceptibility of white male infants to symptomatic PDA following prophylactic indomethacin may represent the influence of maturational factors on ductus patency. Cyclooxygenase products do not appear to be involved in mediating ductus patency when symptomatic PDA occurs immediately following administration of prophylactic indomethacin.

Dinoprostone↗
PubMed1991

Combined administration of aspirin and a specific thrombin inhibitor in man.

BACKGROUND: Heparin is of limited value as an antithrombotic drug in the presence of platelet activation and residual thrombus. Greater anticoagulant activity can be achieved in vivo with more specific thrombin inhibitors. Heparin may also increase the risk of bleeding by an effect on platelets that is independent of its thrombin inhibitory activity. METHODS AND RESULTS: The pharmacodynamic and pharmacokinetic effects of a novel thrombin inhibitor, argatroban, were examined alone and in combination with aspirin in normal male volunteers. Argatroban induced a dose-dependent prolongation of the thrombin time and the activated partial thromboplastin time (aPTT). aPTT had returned to its pretreatment value 1 hour after stopping the infusion of argatroban. Six male subjects received an infusion of 1 micrograms/kg/min argatroban after the administration of two doses of 162.5 mg aspirin or a matching placebo. At this dose, aspirin decreased serum thromboxane B2 by a mean of 99% and prolonged the bleeding time (230 +/- 52 versus 320 +/- 113 seconds, p less than 0.01). Argatroban given alone increased thrombin time by 454 +/- 18% and aPTT by 160 +/- 3%. Steady-state plasma concentrations were achieved at 1 hour and declined exponentially with an elimination half-life of 24 +/- 4 minutes. Neither the anticoagulant effects nor the plasma concentrations of argatroban were altered by aspirin. Furthermore, argatroban did not increase the bleeding time when given alone and did not further prolong the bleeding time when combined with aspirin. CONCLUSION: The combination of aspirin and argatroban may prove to be an effective therapeutic strategy in the prevention of coronary thrombosis.

Adult↗
PubMed1991

Suppression of eicosanoid biosynthesis during coronary angioplasty by fish oil and aspirin.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) is an acute, localized stimulus to platelet and vascular function. Periprocedural cardiovascular complications are reduced by moderate-dose aspirin (ASA), presumably due to inhibition of thromboxane (TX) A2. METHODS AND RESULTS: Excretion of TXA2 and prostacyclin (PGI2) metabolites in urine increased during PTCA. Pretreatment for 3 days with either moderate- (325 mg/day) or low-dose (80 mg/day) ASA inhibited the increase in both eicosanoids. Pretreatment for 3 weeks with fish oil (10 g/day) only partially suppressed TXA2. Formation of trienoic eicosanoids and accumulation of omega-3 fatty acids in platelet membranes confirmed fish oil ingestion. Although basal PGI2 was not inhibited, the PTCA-related increment was suppressed. CONCLUSIONS: PTCA results in an acute, transient alteration of eicosanoid biosynthesis consistent with accelerated platelet-vascular interactions. Pretreatment for 3 days with moderate or low doses of ASA suppresses TXA to a similar extent during PTCA, and their effects on acute cardiovascular complications of this procedure are likely to be comparable. It is unlikely that even prolonged pretreatment with fish oil can substitute for the platelet inhibitory action of ASA during PTCA. Suppression of PGI2 may contribute to the residual acute periprocedural complication rate in patients taking ASA.

Angioplasty, Balloon, Coronary↗
PubMed1991

Systemic lysis protects against the effects of platelet activation during coronary thrombolysis.

Systemic lysis may protect against the platelet activation and ongoing thrombosis associated with coronary thrombolysis. To address this hypothesis, we compared urokinase and tissue-type plasminogen activator (t-PA) given intravenously in a chronic, canine model of coronary thrombosis. T-PA 10 micrograms/kg per min induced reperfusion in 55 +/- 7 min but complete reocclusion occurred in 9/10 animals. Reocclusion was prevented by combining t-PA with 7E3, an antibody to the platelet glycoprotein IIb/IIIa which abolished ex vivo platelet aggregation. A similar time to reperfusion was seen with urokinase 750-1,000 U/kg per min. In contrast to t-PA, complete reocclusion occurred in only 1/20 cases (P less than 0.001 vs. t-PA), despite evidence of continued platelet activation in vivo and platelet aggregation ex vivo. Furthermore, this did not reflect a difference in the clearance of the two plasminogen activators. However, plasma fibrinogen was undetectable after urokinase in contrast with t-PA. Furthermore, in animals treated with prourokinase 20 micrograms/kg per min, reocclusion (4/7) correlated with the degree of systemic lysis. To determine whether platelet activation modified the response to urokinase, it was combined with 7E3. 7E3 0.8 mg/kg reduced the time to reperfusion with t-PA (30 +/- 5, n = 6; P = 0.025), but not with urokinase (56 +/- 8 vs. 62 +/- 6, P = ns). Systemic lysis protects against the propensity of continued thrombosis during coronary thrombolysis to delay reperfusion and induce reocclusion. This may modify the requirement for adjunctive antiplatelet therapy.

Animals↗
PubMed1991

Presystemic acetylation of platelets by aspirin: reduction in rate of drug delivery to improve biochemical selectivity for thromboxane A2.

The utility of aspirin in the treatment of vascular occlusive disease has been ascribed to its blockade of platelet thromboxane A2 (TxA2), a potent inhibitor of vascular smooth muscle contraction and platelet aggregation. Coincident with the inhibition of TxA2 by aspirin at currently used dose regimens is a decrease in formation of prostacyclin (PGI2), an eicosanoid with opposing effects to TxA2 in vivo. The coincident inhibition of PGI2 could potentially limit the therapeutic efficacy of aspirin. This study addressed the issue as to whether a reduction in the rate of drug delivery could enhance the biochemical selectivity of aspirin toward inhibition of TxA2. Intubation studies were performed in parallel groups of healthy volunteers in which a 50-mg aspirin dose was administered as either a bolus or a 5 or 10 mg/hr infusion via a nasogastric tube. A control group received buffer solution. Systemic plasma levels of aspirin, major urinary metabolites of TxA2 and PGI2 and serum thromboxane B2 levels ex vivo were evaluated. Relative to the group receiving bolus aspirin, the slower rates of aspirin delivery increased the effective selectivity of the administered aspirin for TxA2. To investigate further this apparent selectivity, controlled release (CR) dose forms of aspirin were prepared and evaluated in a preliminary dose-ranging study in healthy subjects. The doses studied were 50 and 75 mg CR and a 75-mg aspirin solution. The CR aspirin dose forms decreased the maximal plasma concentration of aspirin relative to a bolus dose. The pharmacodynamic effects of the CR formulations were assessed via measurement of ex vivo serum thromboxane B2 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗