Thromboxane A2 biosynthesis in human disease

Thromboxane A2 (TxA2), the predominant cyclooxygenase product of human platelets, is a potent vasoconstrictor and platelet agonist. Although its biological properties are readily appreciable in vitro, it has been difficult to define its biological importance in vivo. To a large extent this reflected the problems associated with efforts to monitor biosynthesis of this eicosanoid and the lack of selective pharmacological probes that prevented the synthesis of TxA2 or antagonized its biological action in vivo. Recently the analysis of urinary metabolites of TxB2 has become simplified so that the methodology is readily applicable to clinical studies. This provides a noninvasive, time-integrated index of Tx biosynthesis. Although one cannot definitively establish a tissue of origin for metabolites measured in urine, indirect evidence suggests that urinary TxB2 derives primarily from the kidney whereas its dinor metabolite predominantly reflects platelet biosynthesis under physiological conditions. Although plasma concentrations of TxB2 are readily confounded by platelet activation ex vivo, the enzymatic metabolites formed from TxB2 have recently been identified and appear to bypass this problem. Combined analysis of long-lived (e.g., 11-dehydro-TxB2) and short-lived (e.g., 2,3-dinor-TxB2) metabolites in plasma promise to more accurately localize phasic increases in the biosynthesis of TxA2 and have been paralleled by the development of antagonists of the TxA2/prostaglandin endoperoxide receptor and their study of humans. The use of such specific probes in conditions characterized by abnormal biosynthesis of TxA2 promises to define the biological role of this mediator for humans.     

Prostaglandin endoperoxide/thromboxane A2 receptor desensitization. Cross-talk with adenylate cyclase in human platelets.

Platelet activation by the prostaglandin endoperoxide (PGH2)/thromboxane (Tx) A2 analog, U46619, involves stimulation of phospholipase (PL) C and an increase in intracellular calcium via distinct receptor subtypes. Agents which stimulate adenylate cyclase inhibit platelet function. We demonstrate that PGH2/TxA2 receptor desensitization is associated with enhanced stimulation of platelet cyclic AMP by the prostacyclin analog, iloprost and by forskolin. Sensitization of adenylate cyclase is mediated via the PGH2/TxA2 receptor subtype which activates PLC, as it is blocked by the specific antagonist, GR32191 (Takahara, K., Murray, R., FitzGerald, G. A., and Fitzgerald, D. J. (1990) J. Biol. Chem. 265, 6838-6844). This effect is not observed in platelets desensitized with thrombin or platelet activating factor and is not mediated by protein kinase C. Prior exposure of platelets to platelet activating factor results in much greater desensitization of PGH2/TxA2-induced aggregation (mean 64%) compared with calcium stimulation (mean 18%), consistent with selective heterologous desensitization of the PLC-linked PGH2/TxA2 receptor subtype. Platelet activation by PGH2/TxA2 is a tightly regulated process, involving both homologous desensitization of at least two receptor subtypes and sensitization of the platelet adenylase cyclase system.     

A pharmacological approach to thromboxane receptor antagonism

Thromboxane A2 (TxA2) appears to be an important mediator of ischemia and hypoxia. Despite its short half-life and the fact that it may not circulate in the blood until its values become quite high, TxA2 contributes to the pathogenesis of cardiopulmonary diseases (e.g., sudden death, myocardial ischemia, circulatory shock). It does so because it propagates its own formation by activating platelets and constricting blood vessels, thus activating more TxA2 and trapping it locally within an ischemic or hypoxic region. TxA2 concentrations in the extracellular fluid of lymph of ischemic regions may be much higher than that occurring in nonischemic, normally perfused regions. Specific and potent Tx receptor antagonists (TxRA) have recently become available for study. The TxRA are useful tools in the study of the pathophysiology of Tx-dependent disease processes and have been found to be effective in a variety of ischemic disorders including circulatory shock, myocardial ischemia, and sudden cardiopulmonary death. Moreover, inasmuch as early work indicates that these agents are both safe and effective in humans, Tx receptor antagonists may be employed as therapeutic agents in several cardiovascular disease states. Further investigation is necessary to clarify the role of TxRA as therapeutic agents.     

Endogenous peroxynitrite modulates PGHS-1-dependent thromboxane A2 formation and aggregation in human platelets

Aggregation of activated platelets is considerably mediated by the autocrine action of thromboxane A2 (TxA2) which is formed in a prostaglandin endoperoxide H2 synthase-1 (PGHS-1 or COX-1)-dependent manner. The activity of PGHS-1 can be stimulated by peroxides, an effect termed "peroxide tone", that renders PGHS-1 the key regulatory enzyme in the formation of TxA2. Activated platelets release nitric oxide (*NO) and superoxide (O*2) but their interactions with the prostanoid pathway have been controversially discussed in platelet physiology and pathophysiology. The current study demonstrates that endogenously formed peroxynitrite at nanomolar concentrations, originating from the interaction of *NO and *O2, potently activated PGHS-1, which parallels TxA2 formation and aggregation in human platelets. Inhibition of the endogenous formation of either *NO or O*2 resulted in a concentration-dependent decline of PGHS-1 activity, TxA2 release, and aggregation. The concept of peroxynitrite as modulator of TxA2 formation and aggregation explains the interaction of *NO and O*2 with the PGHS pathway and suggests a mechanism by which antioxidants can regulate PGHS-1-dependent platelet aggregation. This may provide a molecular explanation for the clinically observed hyperreactivity of platelets in high-risk patients and serve as a basis for novel therapeutic interventions.     

Platelet and vascular smooth muscle thromboxane A2/prostaglandin H2 receptors

Thromboxane A2 (TxA2) and prostaglandin H2 (PGH2) aggregate platelets and contract vascular smooth muscle. Inasmuch as both compounds produce the same effects and presumably through the same receptor, their receptors have been referred to as TxA2/PGH2 receptors. Pharmacological studies of stable agonists and antagonists of the TxA2/PGH2 receptors have shown different rank order potencies for these compounds in platelets compared with blood vessels. These studies have provided evidence to support the hypothesis that the platelet TxA2/PGH2 receptor is different from the one found in vascular tissue. The vascular receptor has been named [TxA2/PGH2]tau and the platelet receptor has been named [TxA2/PGH2]alpha. In the past few years several radiolabeled antagonists and agonists have been developed and used in radioligand-binding studies, primarily in platelets. One of these ligands, 125I-labeled PTA-OH, a TxA2/PGH2 receptor antagonist, has been extensively used to characterize the human platelet TxA2/PGH2-binding site. It has been found to have a Kd of approximately 20 nM and a Bmax of 2500 binding sites/platelet. Through the combination of pharmacological and biochemical approaches, it should be possible to characterize platelet and vascular TxA2/PGH2 receptors.     

Edema from cyclooxygenase products of endogenous arachidonic acid in isolated lung

We infused A23187, a calcium ionophore, into the pulmonary circulation of dextran-salt-perfused isolated rabbit lungs to release endogenous arachidonic acid. This led to elevations in pulmonary arterial pressure and to pulmonary edema as measured by extravascular wet-to-dry weight ratios. The increase in pressure and edema was prevented by indomethacin, a cyclooxygenase enzyme inhibitor, and by 1-benzylimidazole, a selective inhibitor of thromboxane (Tx) A2 synthesis. Transvascular flux of 125I-albumin from vascular to extravascular spaces of the lung was not elevated by A23187 but was elevated by infusion of oleic acid, an agent known to produce permeability pulmonary edema. We confirmed that A23187 leads to elevations in cyclooxygenase products and that indomethacin and 1-benzylimidazole inhibit synthesis of all cyclooxygenase products and TxA2, respectively, by measuring perfusate levels of prostaglandin (PG) I2 as 6-ketoprostaglandin F1 alpha, PGE2, and PGF2 alpha and TxA2 as TxB2. We conclude that release of endogenous pulmonary arachidonic acid can lead to pulmonary edema from conversion of such arachidonic acid to cyclooxygenase products, most notably TxA2. This edema was most likely from a net hydrostatic accumulation of extravascular lung water with an unchanged permeability of the vascular space, since an index of permeability-surface area product (i.e., transvascular albumin flux) was not increased.     

Relative effects of flurbiprofen on platelet 12-hydroxy-eicosatetraenoic acid and thromboxane A2 production: influence on collagen-induced platelet aggregation and adhesion

Flurbiprofen has been shown to inhibit cyclo-oxygenase metabolism of arachidonic acid to thromboxane A2 (TxA2), resulting in the inhibition of platelet aggregation. Recently, our laboratory reported that the "irreversible" phase of platelet aggregation and adhesion were regulated, in part, by the lipoxygenase metabolism of arachidonic acid to 12-hydroxy-eicosatetraenoic acid (12-HETE) in platelets, and that selective inhibition of one enzyme i.e. either cyclo-oxygenase or lipoxygenase, resulted in paradoxical effects on the metabolism of arachidonic acid and platelet response related to the other pathway. Therefore, we performed experiments to assess the relative effects of flurbiprofen on TxA2 and 12-HETE synthesis, and on collagen-induced platelet aggregation and platelet adhesion to collagen-coated surfaces. "Irreversible" collagen-induced platelet aggregation was only partially inhibited by pre-incubation with 1 x 10(-6) M flurbiprofen, while TxA2 production was elevated and 12-HETE production was maximally inhibited in these platelets. At this concentration of flurbiprofen (1 x 10(-6)M), collagen-induced platelet adhesion was also reduced by 50%. At higher concentrations of flurbiprofen, both platelet aggregation and adhesion were further reduced, with a corresponding inhibition of TxA2 production. Thus it appears that the lipoxygenase pathway of arachidonic acid metabolism in platelets is not only inhibited by flurbiprofen, but is more sensitive to inhibition by flurbiprofen than the cyclo-oxygenase pathway. This differential effect of flurbiprofen on arachidonic acid metabolism in the platelet is related to differential effects on platelet function.     

Directed vascular expression of the thromboxane A2 receptor results in intrauterine growth retardation

Thromboxane (Tx) A2 is a platelet agonist, smooth muscle cell constrictor, and mitogen. Urinary Tx metabolite (Tx-M) excretion is increased in syndromes of platelet activation and early in both normal pregnancies and in pregnancy-induced hypertension. A further increment occurs in patients presenting with severe preeclampsia, in whom Tx-M correlates with other indices of disease severity. TxA2 exerts its effects through a membrane receptor (TP), of which two isoforms (alpha and beta; refs. 5,6) have been cloned. Overexpression of TP in the vasculature under the control of the pre-proendothelin-1 promoter results in a murine model of intrauterine growth retardation (IUGR), which is rescued by timed suppression of Tx synthesis with indomethacin. IUGR is commonly associated with maternal diabetes or cigarette smoking, both conditions associated with increased TxA2 biosynthesis.     

The roles of LAT in platelet signaling induced by collagen, TxA2, or ADP

The work presented here demonstrates that platelets from mice lacking LAT (linker for the activation of T cells) show reversible aggregation in response to concentrations of collagen that cause TxA2/ADP-dependent irreversible aggregation of control platelets. The aggregation defect of the LAT-deficient platelets was shown to be the result of almost no TxA2 production and significantly diminished ADP secretion. In contrast, the LAT deficiency does not affect aggregation induced by high concentrations of collagen because that aggregation is not dependent on TxA2 and/or ADP. Even though ADP and TxA2 provide amplification signals for platelet activation in response to low concentrations of collagen, LAT-deficient platelets hyperaggregate to low levels of U46619, a TxA2 analog, or ADP. Though the mechanism(s) of costimulatory signals by collagen, ADP, and TxA2 remains unidentified, it is clear that LAT plays a positive role in collagen-induced, TxA2/ADP-dependent aggregation, and a negative role in TxA2 or ADP-induced platelet aggregation.     

Oxidative stress-induced isoprostane formation may contribute to aspirin resistance in platelets

Recent studies suggest that aggregation of platelets from patients with coronary artery and cerebrovascular disease may be resistant to low-dose aspirin (ASA) treatment, which may promote plaque-associated thrombus formation. However, the underlying mechanisms of platelet ASA resistance are poorly understood. ASA is thought to inhibit platelet aggregation primarily by inactivating the cyclooxygenase (COX), thus decreasing the synthesis of the pro-aggregatory arachidonic acid metabolite thromboxane A(2) (TxA(2)). However, recent studies also identified a non-enzymatic, oxidation-dependent pathway for the synthesis of the arachidonic acid derivative isoprostanes, which exhibit potent vasoconstrictor and pro-aggregatory effects similar to that of TxA(2). Because the pathophysiological conditions that promote arteriosclerotic vascular diseases (e.g. hypercholesterolemia, diabetes, hyperhomocysteinemia) are thought to be associated with an increased formation of reactive oxygen species and increased plasma isoprostane levels, it can be hypothesized that increased COX-independent isoprostane formation in platelets contribute to ASA resistance.     

Role of eicosanoids in the cardiovascular system

Cardiovascular eicosanoids are of significance in relation to regulation of hemostasis and flow under healthy and pathological conditions. In healthy subjects, TxA2 and PGI2 participate in the maintenance of vascular integrity in relation to vascular injury. In this respect, vascular eicosanoids can be regarded as constituents of a balancing system which favours platelet deaggregation in intact vessels but platelet aggregation in a injured vessel. Degenerative arterial disease, like e.g. atherosclerosis, disturbs the balance and favours platelet activation and adhesion to vascular surfaces. This may promote the development of platelets thrombi in the absence of vascular injury and lead to thrombosis.     

Enhanced microvascular permeability of PMA-induced acute lung injury is not mediated by cyclooxygenase products.

Products of cyclooxygenase activity have been proposed to mediate the pulmonary hypertension and increased microvascular permeability associated with phorbol myristate acetate- (PMA) induced acute lung injury. Previously, we reported that thromboxane (Tx) does not mediate PMA-induced pulmonary hypertension in intact anesthetized dogs. In the present study, PMA was administered to isolated canine lungs perfused with autologous blood at constant flow to investigate a possible role for Tx in the PMA-induced increase in microvascular permeability. Changes in permeability were assessed by determining changes in the capillary filtration coefficient (Kfc). In lobes pretreated with papaverine to prevent PMA-induced increases in pulmonary vascular resistance, Kfc increased from a baseline value of 0.2 +/- 0.03 to 1.5 +/- 0.29 ml.min-1.cmH2O-1.100 g wet lobe wt-1 (P < 0.01) 30 min after PMA (5.8 x 10(-8) M, n = 10). Concomitantly, TxB2, the stable metabolite of TxA2, increased from 138 +/- 44 to 1,498 +/- 505 pg/ml (P < 0.05) in the blood. Both the selective Tx synthase inhibitor, OKY-046 (7 x 10(-4) M, n = 6), and the cyclooxygenase inhibitor, indomethacin (10(-4) M, n = 7), prevented the PMA-induced increase in TxB2, but neither compound attenuated the PMA-induced increase in Kfc. ONO-3708 (10(-6) M), a selective prostaglandin (PG) H2/TxA2 receptor antagonist, prevented the vasoconstriction resulting from administration of U-46619, a stable PGH2/TxA2 receptor agonist, but it did not prevent the PMA-induced increases in Kfc (n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased platelet thromboxane synthesis in renal glomerular diseases

Washed platelets were prepared from healthy children and adults, and patients with renal glomerular diseases, and incubated with [1-14C] arachidonate to measure the generation capacities of thromboxane (Tx) A2 and 12-hydroxyeicosatetraenoate (12-HETE). Tx generation capacity of platelets was significantly higher in patients with chronic glomerulonephritis, purpura nephritis and lupus nephritis than in healthy control subjects. There was no significant increase in minimal change nephrotic syndrome. 12-HETE showed a decreasing tendency in the glomerular diseases, which was restored to normal level by in vitro addition of indomethacin. Such increased Tx generation capacity of platelets may cause abnormal enhancement of platelet functions and conceivably constitute an aggravating factor of glomerular and microvascular damage in the affected kidney.     

Arachidonate metabolites and the control of glomerular function

The glomerulus is a dynamic structure capable of regulating the glomerular filtration rate (GFR) by mesangial contraction, thereby decreasing Kf. The mesangium contracts in response to angiotensin II (AII) and arginine vasopressin (AVP), both of which are potent stimuli of vasodilatory prostaglandin (PG) production. We studied interactions among these opposing factors in glomeruli. Normal rat glomeruli synthesized PGF2 alpha greater than PGE2 greater than 6-keto-PGF1 alpha = thromboxane (Tx) B2. Rat glomerular epithelial and mesangial cells, although capable of producing these four cyclooxy-genase end products, responded to AVP and AII stimulation with a preferential increase of PGE2, which suggests an intraglomerular feedback system between constrictor and dilator factors. Whole glomeruli, when incubated in AII, decreased in size, with a maximum decrement of surface area at 10(-10) M AII. In these glomerular contraction studies, preincubation with either arachidonate or PGE2 decreased the contractile response to AII, whereas PG inhibition enhanced the glomerular contractile response. Stable endoperoxide analogs also contracted glomeruli. In the acute phase of nephrotoxic serum nephritis (NSN) there were marked increments in glomerular production of TxA2, which correlated temporally with decrements of GFR and filtration fraction. Inhibition of TxA2 synthesis normalized GFR and filtration fraction 1-3 h after induction of NSN. These studies suggest not only an important physiological feedback role of vasodilatory PGs, as modulators of AII-induced glomerular contraction, but also a direct mesangial contractile effect of the arachidonate metabolite TxA2.     

Daltroban blocks thromboxane responses in the pulmonary vascular bed of the cat.

The influence of daltroban (BM13.505; SK&F 96148), a thromboxane (Tx) A2-receptor-blocking agent, on responses to the TxA2 mimics U-46619 and U-44069 was investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. Daltroban (5 mg/kg iv) had no significant effect on mean baseline vascular pressures but significantly decreased responses to the TxA2 mimics without altering responses to prostaglandin (PG) F2 alpha or PGD2 or the PGD2 metabolite 9 alpha, 11 beta-PGF2. Dose-response curves for U-46619 and U-44069 were shifted to the right in a parallel manner, and daltroban had no significant effect on responses to norepinephrine, serotonin, angiotensin II, BAY K 8644, endothelin-(ET) 1, ET-2, or platelet-activating factor (PAF). After administration of daltroban, responses to U-46619 returned to 50% of control in 90 min and responses to the PG and TxA2 precursor arachidonic acid were decreased significantly. These results suggest that daltroban selectively antagonizes TxA2-receptor-mediated responses in a competitive and reversible manner. These data provide support for the hypothesis that discrete TxA2 receptors unrelated to receptors stimulated by PGF2 alpha, PGD2, or 9 alpha, 11 beta-PGF2 are present in the pulmonary vascular bed of the cat. The present data suggest that pulmonary vasoconstrictor responses to PAF and ET peptides are not dependent on activation of TxA2 receptors in the cat.     

Characterization of thromboxane receptor blocking effects of SQ 29548 in the feline pulmonary vascular bed.

The effects of SQ 29548, a thromboxane (Tx) A2 receptor blocking agent, on responses to the TxA2 mimic U46619 were investigated in the pulmonary vascular bed of the intact-chest cat under constant-flow conditions. The administration of SQ 29548 in doses of 0.25-1 mg/kg iv reduced vasoconstrictor responses to U-46619; however, responses to prostaglandins (PG) F2 alpha and D2 and to serotonin were also decreased. After administration of SQ 29548 in doses of 0.05-0.1 mg/kg iv, responses to U-46619 and U-44069 were reduced significantly, and the dose-response curves for these TxA2 mimics were shifted to the right in a parallel manner at a time when responses to PGF2 alpha and PGD2 were not altered. The low doses of the TxA2 receptor blocking agent significantly reduced responses to the PG and TxA2 precursor arachidonic acid but were without significant effect on vasoconstrictor responses to serotonin; histamine; norepinephrine; angiotensin II; the major PGD2 metabolite 9 alpha,11 beta-PGF2; BAY K 8644, an agent that enhances calcium entry; and endothelin-1. The present data show that at low doses SQ 29548 selectively blocks TxA2 receptor-mediated responses in a competitive and reversible manner in the pulmonary vascular bed. These data suggest that responses to arachidonic acid are mediated in large part by the formation of TxA2 and provide evidence in support of the hypothesis that a discrete TxA2 receptor unrelated to PGF2 alpha or PGD2 receptors is present in undefined resistance vessel elements in the feline pulmonary vascular bed.     

Mechanisms involved in agonist-induced hyperaggregability of platelets from normal pregnancy

There is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy. Platelet activation occurs in pregnancy with a risk of the development of preeclampsia. In this study, platelet behavior was studied during 28-40 weeks of gestation in a group of women who remained normotensive and a group of nonpregnant female controls. Platelet aggregation and ATP release stimulated by agonists (i.e. collagen and adenosine 5'-diphosphate) were markedly enhanced in washed platelets from pregnant subjects. Furthermore, the collagen-evoked increase in intracellular Ca(2+) ([Ca(2+)](i)) mobilization in fura-2-AM-loaded platelets was also enhanced in pregnant subjects. Moreover, the binding activity of fluorescein isothiocyanate-triflavin toward the platelet glycoprotein IIb/IIIa complex did not significantly differ between the nonpregnant and pregnant groups. In addition, the amount of thromboxane A(2) (TxA(2)) formation from pregnant subjects was significantly greater than that from nonpregnant subjects in both resting and collagen-activated platelets. On the other hand, prostaglandin E(2) formation in the presence of imidazole in either resting or arachidonic acid (100 microM)-treated platelets did not significantly differ between these two groups. The levels of cyclic AMP formation in both resting and prostaglandin E(1) (10 microM)-treated platelets from pregnant subjects were significantly lower than those in nonpregnant subjects. Nitric oxide production was measured by a chemiluminescence detection method in this study. The extent of nitrate production in either resting or collagen-stimulated platelets from pregnant subjects did not significantly differ from that of platelets from the nonpregnant group. We conclude that the agonist-induced hyperaggregability of platelets from normal pregnancy may be due, at least partly, to an increase in TxA(2) formation and a lowering of the level of cyclic AMP formation, which leads to increased [Ca(2+)](i) mobilization and finally to enhanced platelet aggregation and ATP release.     

Cyclooxygenase inhibition prevents PMA-induced increases in lung vascular permeability

The effect of cyclooxygenase inhibition in phorbol myristate acetate (PMA)-induced acute lung injury was studied in isolated constant-flow blood-perfused rabbit lungs. PMA caused a 51% increase in pulmonary arterial pressure (localized in the arterial and middle segments as measured by vascular occlusion pressures), a 71% increase in microvascular permeability (measured by the microvascular fluid filtration coefficient, Kf), and a nearly threefold increase in perfusate thromboxane (Tx) B2 levels. Cyclooxygenase inhibition with three chemically dissimilar inhibitors, indomethacin (10(-7) and 10(-6) M), meclofenamate (10(-6) M), and ibuprofen (10(-5) M), prevented the Kf increase without affecting the pulmonary arterial pressure increase or resistance distribution changes after PMA administration. The specific role of TxA2 was investigated by pretreatment with OKY-046, a specific Tx synthase inhibitor, or infusion of SQ 29548, a TxA2 receptor antagonist; both compounds failed to protect against either the PMA-induced permeability or the vascular resistance increase. These results indicate that cyclooxygenase-mediated products of arachidonic acid other than TxA2 mediate the PMA-induced permeability increase but not the hypertension.     

Modulation of Platelet thromboxane A2 and arterial prostacyclin by dietary vitamin E

Platelets from vitamin E-deficient and vitamin E-supplemented rats generate the same amount of thromboxane A2 (TxA2) when they are incubated with unesterified arachidonic acid. Platelets from vitamin E-deficient rats produced more TxA2 than platelets from vitamin E-supplemented rats when the platelets are challenged with collagen. Arterial tissue from vitamin E-deficient rats generates less prostacyclin (PGI2) than arterial tissue from vitamin E- supplemented rats. The vitamin E effect with arterial tissue is observed when the tissue is incubated with and without added unesterified arachidonic acid. These data show that arterial prostacyclin synthesis is diminished in vitamin E-deficient rats. Vitamin E, in vivo, inhibits platelet aggregation both by lowering platelet TxA2 and by raising arterial PGI2.