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G A FitzGerald

Publications and source records attributed to G A FitzGerald.

At least 109 records · Page 6Linked to original sources
PubMed1996

Oxidative stress and platelet activation in diabetes mellitus.

The role of oxidative stress and platelet activation in the development and evolution of diabetic vascular complications is unclear. They can both be the consequence of established vascular disease or a contributing factor to the evolution of atherosclerosis. Free radical generation may both lead to or result from platelet activation, suggesting that oxidative stress and platelet activation may be closely interrelated. Controversial results may partly reflect methodological constraints. Novel techniques for the measurement of in vivo indices of oxidant injury have been developed and will be used in conjunction with pharmacological probes to establish whether oxidative stress is enhanced in diabetes and whether this preceeds the onset of micro and macrovascular disease.

Animals↗
PubMed1996

8-Epi PGF2 alpha: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo.

1. Excessive free radical generation is thought to contribute to tissue injury in a broad spectrum of diseases. A particular constraint in addressing this hypothesis has been the inability to assess free radical generation in vivo and the lack of information on drugs or vitamins which act as effective antioxidants in vivo. 2. Traditional approaches have relied upon measures of substrate oxidizability or spin trapping of free radical adducts ex vivo. It is unknown how these measurements might relate, in a quantitative fashion, to the generation of reactive oxygen species in vivo. Isoeicosanoids are free radical catalyzed products of arachidonic acid. One of these compounds, 8-epi prostaglandin F2 alpha (8-epi PGF2 alpha) exhibits biological activity and may function as an autacoid. Specific analysis of this 8-epi PGF2 alpha isomer indicates that it is elevated in certain syndromes thought to be associated with oxidant stress. These include vascular reperfusion, paracetamol poisoning and liver cirrhosis. Apparently healthy individuals who smoke cigarettes or consume alcohol exhibit dose dependent increments in excretion of 8-epi PGF2 alpha. Excretion is depressed by antioxidant vitamins, although not by the nonspecific cyclooxygenase (COX) inhibitor, aspirin, even though 8-epi PGF2 alpha may be formed by either COX-1 or COX-2. 3. Specific analysis of this and other isoeicosanoids may afford an opportunity to evaluate the effects of antioxidant interventions in human diseases characterized by excessive lipid peroxidation in vivo.

Animals↗
PubMed1996

The human pharmacology of thrombin inhibition.

Heparin has an established place in the treatment of venous thrombosis. It is also effective in syndromes of arterial occlusion, such as unstable angina, although its benefit: risk ratio when combined with thrombolytic drugs in the treatment of myocardial infarction remains open to debate. Specific thrombin inhibitors have several theoretical advantages over heparin. However, they have failed to realize their promise in phase III trials, perhaps because of inadequacies in trial design. Our understanding of protease-activated receptors, such as that for thrombin, is modest and the comparative advantages of receptor antagonism versus specific thrombin inhibition are unknown. Increasing usage of low molecular weight heparins and glycoprotein IIb/IIIa antagonists provide a changing context for evaluation of these drugs. Problems in phase III clinical trials might be minimized by placing greater emphasis on dose finding in phase II.

Animals↗
PubMed1995

Cyclooxygenase-dependent formation of the isoprostane, 8-epi prostaglandin F2 alpha.

Isoprostanes are a family of prostaglandin (PG) isomers formed in an enzyme-independent manner. They circulate in plasma and are excreted in urine. One of them, 8-epi PGF2 alpha is a vasoconstrictor and mitogen, effects which are prevented by thromboxane antagonists. Given that 8-epi PGF2 alpha may be formed by cyclooxygenase (COX) (Corey, E. J., Shih, C., Shig, N-Y., and Shimoji, K. (1984) Tetrahedron Letts. 44, 5013-5016; Hecker, M., Ullrich, V., Fischer, C., and Meese, C.O. (1987) Eur J. Biochem. 169, 113-123) and that this might confound its use as an index of free radical generation, we sought to characterize the mechanism of its formation by human platelets. Activation of platelets by threshold concentrations of collagen, thrombin, and arachidonic acid resulted in formation of 8-epi PGF2 alpha coincident with that of the COX product, thromboxane, and the 12 lipoxygenase product, 12-hydroxyeicosatetraenoic acid, as detected by selected ion monitoring assays using gas chromatography-mass spectrometry. The effect appeared selective for 8-epi PGF2 alpha among the F2 isoprostanes. Pretreatment of platelets with aspirin or indomethacin abolished 8-epi PGF2 alpha formation. COX-independent activation of platelets by high doses of collagen or thrombin, by the phorbol ester, phorbol 12-myristate 13-acetate, or the prostaglandin endoperoxide analog, U 46619 was not associated with 8-epi PGF2 alpha formation. Confirmation of the nature of the material formed by platelet COX as 8-epi PGF2 alpha included its cochromatography over three highly resolving high performance liquid chromatography systems, identification by electron impact mass spectrometry, and its formation by partially purified COX. Inhibition of platelet thromboxane formation was associated with augmented 8-epi PGF2 alpha formation. A major component of 8-epi PGF2 alpha formed in serum by healthy volunteers was shown to be sensitive to inhibition by aspirin ex vivo. In addition to its generation by free radical catalyzed mechanisms, 8-epi PGF2 alpha may be formed as a PG by human platelets. Given that activation of platelet COX characterizes many of the human syndromes which are putatively associated with free radical generation, assessment of the contribution of this pathway is relevant to the use of 8-epi PGF2 alpha as an index of lipid peroxidation in vivo.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Formation of 8-iso-prostaglandin F2 alpha by human platelets.

F2-isoprostanes are free radical catalyzed prostaglandin F2 isomers formed from arachidonic acid in an enzyme independent manner (1). Analogous families of other prostaglandin isomers have also been described. Detection of these compounds in vivo has been postulated to represent an approach to the quantitative assessment of free radical generation in humans (2). Additionally, the 8-iso analogues of PGF2 alpha and PGE2 have been shown to induce vasoconstriction, a response which is prevented by pharmacological antagonists of the thromboxane receptor (3). Consequently, it is conceivable that these particular isomers might exhibit an autacoidal function. We chose to explore the factors which regulate the biosynthesis of one of these compounds, 8-iso-PGF2 alpha, in vivo and by human platelets in vitro, to understand more clearly how the discovery of these compounds might be exploited to further our understanding of free radical catalyzed processes in vivo.

Adult↗
PubMed1994

Phosphorylation and regulated expression of the human thromboxane A2 receptor.

Recombinant forms of the human thromboxane A2 (TXA2) receptor composed of the carboxyl-terminal amino acid residues 220-343 were phosphorylated in vitro by both cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC), and these phosphorylations were competed for by synthetic peptides corresponding to the proposed carboxyl-terminal cytoplasmic tail and/or the third extracellular loop of the receptor, respectively. Exogenous addition of PKA or PKC to membrane preparations of human embryonic kidney 293 cells, transfected with the TXA2 receptor, typically reduced TXA2 receptor binding by 10 and 30%, respectively. In vivo inhibition of PKC or PKA in the transfected human embryonic kidney 293 cells increased TXA2 receptor binding to 121.4% (+/- 5.3%) and 110.4% (+/- 4.6%), respectively, relative to control cells. In vivo activation of PKC in the platelet-like human erythroleukemia (HEL) cells by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in an initial reduction followed by a time-dependent increase in TXA2 receptor ligand binding. Stimulation of HEL cells with the TXA2 receptor agonist [15-(alpha,2 beta(5Z)-3 alpha(E,3S)-4 alpha)]-7-[3- (3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7-oxabicyclo-[-2.2,1-]hept -2-yl]- 5-heptenoic acid or basic fibroblast growth factor, alone or together, resulted in a marked decrease in TXA2 receptor binding. Northern blot studies in HEL cells demonstrated that PMA stimulation induced the expression of the TXA2 receptor gene with mRNA levels peaking following PMA stimulation for 4-8 h. This induction is consistent with the presence of a phorbol ester response element in promoter I of the TXA2 receptor gene. Dexamethasone did not induce the expression of the receptor gene, despite the presence of a glucocorticoid response element in promoter II of the TXA2 receptor gene. In summary, our results indicate that the cellular responses to TXA2 are mediated both by phosphorylation of the TXA2 receptor by different protein kinases and by regulated expression of the TXA2 receptor gene.

Bridged Bicyclo Compounds↗
PubMed1993

Cloning and expression of a cDNA for the human prostaglandin E receptor EP1 subtype.

A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2 alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.

Amino Acid Sequence↗
PubMed1993

Effects of fish oil supplementation in the third trimester of pregnancy on prostacyclin and thromboxane production.

OBJECTIVE: Disturbance in thromboxane and prostacyclin biosynthesis has been observed in preeclampsia. We studied whether fish oil supplementation in late pregnancy interferes with maternal and fetal production of thromboxane A2 and prostacyclin I2. STUDY DESIGN: Forty-seven women in the thirtieth week of pregnancy were randomly assigned in a ratio of 2:1:1 to receive fish oil (2.7 gm of n-3 fatty acid per day [Pikasol], or either olive oil or no oil supplementation as controls. Metabolites of thromboxane A2 and A3 and of prostacyclin I2 and I3 were quantified by mass spectrometry methods in serum and urine, respectively. Maternal serum and urine were sampled at baseline, in the thirty-third and thirty-seventh weeks of pregnancy. Fetal serum was sampled at delivery. RESULTS: At the thirty-seventh week the mean concentrations of the eicosapentaenoic-derived metabolites, thromboxane B3 and prostacyclin I3, was twofold to threefold higher (p < 0.001) in the group receiving fish oil compared with combined control groups. There were no significant effects of fish oil on the prostacyclin I2 metabolite, although there was a trend toward a reduction in thromboxane B2 in this group. In umbilical cord blood the mean concentration of thromboxane B2 was lowest in the group receiving fish oil (p = 0.03). CONCLUSIONS: Fish oil was metabolized to the eicosapentaenoic acid-derived eicosanoids thromboxane A3 and prostacyclin I3 in pregnant women. Correspondingly, analog products of arachidonic acid tended to be depressed. It remains to be established whether these biochemical effects will prove beneficial in the prevention or treatment of preeclampsia and intrauterine growth retardation.

Adult↗
PubMed1993

Interruption of vascular thrombus formation and vascular lesion formation by dietary n-3 fatty acids in fish oil in nonhuman primates.

BACKGROUND: Because of discrepant claims regarding the relative biological effects of n-3 fatty acids (n-3FAs), we have concurrently measured the effects of dietary n-3FAs on blood and vascular lipid composition, hemostatic function, blood thrombotic responses, vascular thrombus formation, and vascular lesion formation in baboons. METHODS AND RESULTS: Dietary n-3FAs displaced n-6FAs in plasma, platelets, blood vessels, and corresponding urinary eicosanoid metabolites (p < 0.01 in all cases) within weeks after initiation of a semipurified diet containing 1 g/kg per day n-3FA-ethyl ester concentrate (composed of two thirds eicosapentanoic acid and one third docosahexanoic acid). Coincidentally, platelet hemostatic function became minimally impaired (template bleeding times prolonged from 4.3 +/- 0.5 minutes to 7.6 +/- 1.3 minutes, p = 0.039); concentrations of collagen producing half-maximal platelet aggregation increased (from 6.4 +/- 2.1 to 8.5 +/- 2.5 micrograms/mL, p = 0.045); and tissue factor expression by endotoxin-stimulated blood monocytes fell (from 6.5 +/- 1.2 to 1.7 +/- 0.14 mU/10(6) cells, p < 0.005). Dietary n-3FAs decreased deposition of platelets onto thrombogenic segments of Dacron vascular graft incorporated into chronic exteriorized femoral arteriovenous (AV) shunts, a thrombotic process resistant to the effects of both aspirin and heparin (111In-labeled platelet deposition decreased from 14.1 +/- 1.4 x 10(9) platelets/5-cm segment at 40-60 minutes with occlusion to 7.5 +/- 0.8 x 10(9) platelets/5-cm segment without occlusion; p < 0.001). Platelet deposition onto segments of endarterectomized homologous normal aorta in the AV shunts of n-3FA-treated animals was similarly reduced (from 4.4 +/- 0.9 to 1.8 +/- 0.4 x 10(9) platelets; p < 0.01). Dietary n-3FAs interrupted vascular thrombus formation at sites of surgical carotid endarterectomy (platelet deposition, 1.5 +/- 0.4 versus 4.4 +/- 1.0 x 10(9) platelets in untreated controls; p < 0.001). Moreover, endarterectomized aortic segments (EASs) from n-3FA-treated donors exhibited little capacity to induce thrombus formation when tested in the AV shunts of control recipient animals (0.24 +/- 0.10 versus 4.4 +/- 0.90 x 10(9) platelets). However, in the converse crossover experiments, EASs from control animals actively accumulated platelets when studied in the AV shunts of n-3FA-treated animals (1.8 +/- 0.4 x 10(9) platelets; p < 0.01 versus n-3FA-treated EASs in shunts of normal animals). Dietary n-3FAs also abolished vascular lesion formation at sites of carotid endarterectomy 6 weeks after surgery (cross-sectional area of neointima 0.048 +/- 0.031 mm2 compared with 0.428 +/- 0.104 mm2 in control arteries; p = 0.010). CONCLUSIONS: In nonhuman primates, dietary n-3FAs in high doses eliminate both vascular thrombus formation and vascular lesion formation after mechanical vascular injury while largely sparing hemostatic function and modestly reducing blood thrombotic responses. These effects are attributed to selective n-3FA-dependent alterations in cellular membrane functions.

Animals↗
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↗