Thromboxane A2 Generation Research Articles

Activation of the GP IIb-IIIa Complex Induced by Platelet Adhesion to Collagen Is Mediated by Both α2β1 Integrin and GP VI

alpha2beta1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha2beta1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha2beta1 integrin antibody. The combination of anti-alpha2beta1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha2beta1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha2beta1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha2beta1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.

The role of endothelin-1 as a mediator of the pressure response after air embolism in blood perfused lungs.

It is well known that lung embolism is associated with an increase in pulmonary vascular resistance. Since the mechanisms of pulmonary vascular reactions during embolism are still unclear, the aim of this study was to investigate the potential involvement of endothelin-1 (ET-1) and thromboxane A2 (TXA2) as mediators of the pulmonary artery pressure (PAP) increase after embolism using the selective ETA receptor antagonist LU135252 [1], the ETB receptor antagonist BQ788 [2], and the cyclooxygenase inhibitor diclofenac. Prospective experimental study in rabbits. Experimental laboratory in a university teaching hospital. 36 adult rabbits of either sex. The experiments were performed in 36 isolated and ventilated rabbit lungs which were perfused with a buffer solution containing 10% of autologous blood. Embolism was induced by the injection of 0.75 ml air into the pulmonary artery. PAP and lung weight, reflecting edema formation, were continuously recorded. Perfusate samples were drawn intermittently to determine TXA2 and ET-1 concentrations. Air injection resulted in an immediate increase in PAP up to 22.8 +/- 1.4 mm Hg at 2.5 min (control, n = 6), which was parallelled by an enhanced generation of TXA2. No relevant edema formation occurred during the observation period. Pretreatment with the ETA receptor antagonist LU135252 significantly reduced the pressure reaction after air embolism (p < 0.001) whereas the ETB receptor antagonist BQ788 (n = 6) was without marked effects. The administration of diclofenac (n = 6) did not alter the PAP increase 2.5 min after embolism, but significantly reduced the pressure reaction during the further observation period (p < 0.001). The application of LU135252 and diclofenac together (n = 6) also significantly reduced the PAP increase from 2.5 min during the total observation period (p < 0.001). The acute pressure reaction after air embolism is mainly mediated via ET-1 by an ETA receptor related mechanism. TXA2 seems to maintain this reaction for a longer time.

Acute coronary syndromes: unstable angina and non-Q-wave myocardial infarction.

Ischemic heart disease includes a wide spectrum of conditions, ranging from silent ischemia and exertion-induced angina, through unstable angina, to acute MI. Unstable angina occupies the center of this spectrum, causing disability and risk greater than that of chronic stable angina but less than that of acute MI1 (Fig 1⇓). Although non–Q-wave MI for many years was considered prognostically similar to unstable angina, recent longitudinal studies indicate that it is similar to Q-wave infarction2 3 (Fig 2⇓). Figure 1. Cumulative 6-month mortality from ischemic heart disease. Diagnosis on admission to hospital (n=21 761; 1985 to 1992). From Duke Cardiovascular Database. Reproduced with permission from Reference 1. Figure 2. Top, Cumulative 1-year combined death or MI among patients with Q-wave and non–Q-wave MI treated with fibrinolysis. Reproduced with permission from Reference 2. Bottom, Risk of subsequent cardiac events in stable convalescing patients after first non–Q-wave and Q-wave MI. Reproduced with permission from Reference 3. The concept of unstable angina has emerged from observations of frequent symptoms preceding acute MI, followed by prospective documentation that unstable symptoms frequently culminated in acute MI. The syndrome was rapidly accepted as a well-defined clinical entity as specific clinical manifestations, pathophysiological mechanisms, laboratory findings, and treatment became better characterized. Unstable angina is currently one of the leading causes of hospital admission for CAD, and non–Q-wave MI accounts for >30% of admissions for acute MI.1 4 Yet, the diagnosis of unstable angina remains clinical, based on symptom recognition. The physician caring for patients with unstable angina is in a privileged position of recognizing rapidly evolving CAD and being able to intervene to prevent irreversible left ventricular damage and progression of CAD. Unstable angina is classically described as a heterogeneous disease, referring to a wide spectrum of clinical manifestations from stable angina to MI, of disease …

Platelet Adhesion to Native Type I Collagen Fibrils: ROLE OF GPVI IN DIVALENT CATION-DEPENDENT AND -INDEPENDENT ADHESION AND THROMBOXANE A2 GENERATION

Three glycoproteins (GPs), namely GPIa-IIa, GPVI, and GPIV, have been recently implicated in platelet-collagen adhesive interactions. We have employed antibodies to these GPs to investigate further their role in platelet adhesion to immobilized monomeric and polymeric fibrillar collagen under static conditions in the presence and the absence of Mg2+. In the presence of Mg2+, each antibody inhibited platelet adhesion to fibrillar collagen from 70 to 85%, especially during the early phase (<15 min), but the inhibitory effects diminished dramatically to 25% or less by 60 min. Combination of anti-GPVI with anti-GPIa-IIa antibodies completely inhibited platelet adhesion at 60 min. Anti-GPIV and anti-GPIa-IIa or anti-GPVI antibodies in combinations were more effective in inhibiting adhesion than was anti-GPIa-IIa or anti-GPVI alone. In the absence of Mg2+, anti-GPVI completely inhibited adhesion at 60 min, while anti-GPIV antibody inhibited adhesion by about 50% and minimal effects were seen with anti-GPIa-IIa, suggesting that GPIa-IIa does not play a significant role in the divalent cation-independent platelet adhesion to immobilized fibrillar collagen. Under either divalent cation-dependent or -independent conditions, platelets adhered to fibrillar collagen were able to secrete contents of both alpha-granules and dense granules and generate thromboxane A2 (TXA2), but platelets adhering to acid soluble monomeric collagen neither secreted their granular contents nor generated TXA2. Although anti-GPVI antibodies were not able to inhibit Mg2+-dependent adhesion, they completely inhibited TXA2 generation under both divalent cation-dependent and -independent conditions. With the other antibodies, TXA2 generation corresponded with the amount of adhesion observed. These results suggest that GPVI is directly associated with the TXA2 generating system during platelet-collagen interaction.

Contribution of thromboxane and endomembrane Ca2+-ATPases to variability in Ca2+ signalling of platelets from healthy volunteers

Inter-individual variability in Ca2+ signal generation was studied in platelets from 15 healthy volunteers. The possible involvement of variation in thromboxane A production and variation in sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) was investigated by using platelets isolated before and after intake of 500 mg aspirin, and by measuring the expression levels of two main SERCA isoforms (SERCA-2b and PL/IM 430-recognizable SERCA). Considerable difference in Ca2+ responses were detected after platelet stimulation with thrombin, collagen or the SERCA-2b inhibitor, thapsigargin (TG), with inter-individual coefficients of variance of 22-43% in the absence and 15-41% in the presence of aspirin. Differences in thromboxane A2 generation and SERCA expression contributed to this variability in various ways. In the absence of aspirin, the amount of formed thromboxane A2 partially explains the level of the Ca2+ response induced by TG. On the other hand, in the absence of thromboxane-dependent effects, the expression levels of SERCA-2b and SERCA PL/IM 430 were inversely related to the responses evoked by collagen and TG, respectively. None of these factors were related to the level of the thrombin-evoked Ca2+ signal.

Human group II 14 kDa phospholipase A2 activates human platelets.

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.

FMLP actions and its binding sites in isolated human coronary arteries.

The chemoattractant f-Met-Leu-Phe (FMLP) can modulate human coronary arterial tone without the involvement of peripheral leukocytes. We investigated the actions of FMLP and its cellular mechanism in human coronary arteries isolated 2-3 h after death. A single dose of FMLP (0.01-10 microM) produced transient contraction (or, followed by relaxation) responses in most human coronary rings examined. These responses to FMLP were in large part mediated by the generation of cyclooxygenase products, mainly thromboxane A2 (TXA2) and prostaglandin I2 (PGI2). Radiolabeled N-formyl hexapeptide. 125I-f-Nle-Leu-Phe-Nle-Tyr-Lys bound densely to intimal and adventitial sites that accumulated macrophages (CD68-positive) with a Kd of 14-29 nM and, further, weakly to the media with a Kd of 2.4-3.6 microM. Several cell types including macrophages, endothelial cells and smooth muscle cells were positively immunostained for both TXA2 synthase and PGI2 synthase. However, there was no significant relation between the magnitude of the responses to FMLP and dense macrophage accumulation in the intimal plaques or the adventitia. A reverse transcription-polymerase chain reaction showed predominant expression of FMLP receptor homologues, FPRH1 and FPRH2 mRNA, in human coronary medial tissues relative to that in leukocytes. In conclusion. FMLP produced transient tension changes in human coronary arteries, mainly via the generation of TXA2 and PGI2. This effect of FMLP did not appear to be mediated by the activation of densely accumulated intimal and/or adventitial macrophages, but by the activation of unidentified medial tissue cells which might have functional FMLP receptor homologues.

Effects of bradykinin, histamine and serotonin on pulmonary vascular resistance and permeability.

The effects of bradykinin, histamine and serotonin on vascular resistance and microvascular permeability were investigated in isolated cell-free perfused rabbit lungs. The capillary filtration coefficient was determined from the slope of lung weight changes over periods of venous pressure elevation before application of bradykinin (n = 6), histamine (n = 6) and serotonin (n = 6), and 5, 20 and 50 min afterwards. To prevent rapid inactivation of bradykinin by the angiotensin-converting enzyme in the pulmonary circulation, the bradykinin effects were additionally studied in the presence of the angiotensin-converting enzyme inhibitor captopril (n = 6). Bolus application of each substance resulted in a short-lasting increase in the pulmonary vascular resistance (3.7-9.1 mmHg). Which was most pronounced in the bradykinin+captopril group. The capillary filtration coefficient was significantly increased after histamine application, and to an even greater extent after serotonin application, whereas bradykinin on its own, as well as bradykinin given in the presence of captopril, had no measurable influence on capillary filtration in the lung. As a result of the bradykinin challenge, there was an immediate massive generation of prostacyclin, which could not be further augmented by a application. Histamine injection entailed a delayed onset of prostacyclin generation after the second stimulation, whereas no prostacyclin increase was measured in the serotonin-treated lungs. Thromboxane A2 generation was exclusively observed after the first serotonin application. The data exemplify different pathophysiological characteristics of bradykinin, histamine and serotonin on lung barrier function. Histamine and serotonin induce oedema formation by enhancing microvascular permeability, whereas bradykinin does not.

Antithrombotic action of the kava pyrone (+)-kavain prepared from Piper methysticum on human platelets.

(+)-Kavain, a 4-methoxy-alpha-pyrone prepared from Piper methysticum Forst. (Piperaceae), was investigated regarding its assumed antithrombotic action on human platelets which was deduced from its ability to suppress arachidonic acid (AA)-induced aggregation, exocytosis of ATP, and inhibition of cyclooxygenase (COX) and thromboxane synthase (TXS) activity, the latter two effects being estimated from the generation of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2), respectively. Exogenously applied AA (100 mumol/l) provoked a 90% aggregation of platelets, the release of 14 pmol ATP, and the formation of either 220 pg TXA2 or 43 pg PGE2, each parameter being related to 10(6) platelets. An application of (+)-kavain 5 min before AA, dose-dependently diminished aggregation, ATP-release, and the synthesis of TXA2 and PGE2 with IC50 values of 78, 115, 71, and 86 mumol/l, respectively. The similarity of the IC50 values suggest an inhibition of COX by (+)-kavain as primary target, thus suppressing the generation of TXA2 which induces aggregation of platelets and exocytosis of ATP by its binding on TXA2-receptors.

8-Epi-PGF2alpha induces airflow obstruction and airway plasma exudation in vivo.

8-Epi-prostaglandin F2alpha (8-epi-PGF2alpha) is an F2-isoprostane formed mainly via noncyclooxygenase pathways in vivo. We investigated whether 8-epi-PGF2alpha has any effect on airflow obstruction and plasma exudation in vivo. Airflow obstruction was quantified by measuring lung resistance (RL) in anesthetized and ventilated guinea pigs, and plasma exudation was quantified by the Evans Blue dye method (20 mg/kg intravenously). Intratracheal instillation of 8-epi-PGF2alpha (1 nmol or 10 nmol) caused dose-related increases in RL. Furthermore, the higher dose of 8-epi-PGF2alpha produced Evans Blue dye extravasation in main bronchi and intrapulmonary airways. A prostanoid TP-receptor antagonist, BAY u3405 (1 mg/kg intravenously), abolished the airway effects of 8-epi-PGF2alpha (10 nmol). A thromboxane A2 (TxA2) synthase inhibitor, OKY-406 (30 mg/kg intravenously), significantly attenuated these effects of 8-epi-PGF2alpha (10 nmol). The level of TxB2, a stable TxA2 metabolite, increased in bronchoalveolar lavage fluid (BALF) after 8-epi-PGF2alpha instillation. We conclude that 8-epi-PGF2alpha causes airflow obstruction and plasma exudation in vivo. This effect may be mediated primarily via prostanoid TP-receptors, and a secondary generation of TxA2 may be involved in part of the airway responses in 8-epi-PGF2alpha in the guinea pig.

Signal transduction in the platelet activation induced by IgG anti-streptokinase and anisoylated plasminogen-streptokinase activator complex

Streptokinase (SK) is one of the plasminogen activators currently used in therapeutics. SK antibodies may appear in the blood after thrombolytic therapy with SK or after ß-hemolytic streptococci infection. Such antibodies may both activate platelets and neutralize the ability of SK to convert plasminogen into plasmin. We previously demonstrated that platelet activation induced by the combination of IgG anti-SK and anisoylated plasminogen-SK activator complex (APSAC) is mediated by Fγ7RIIal receptor. However, the mechanism by which IgG anti-SK and APSAC (or SK) transduce an activating signal across the platelet plasma membrane remains unknown. We have demonstrated in the present study that the platelet aggregation induced by the combination of IgG anti-SK and APSAC is accompanied by an increase in inositol phosphate, Ca2+ mobilization and thromboxane (Tx) A2 generation. Neomycin, erbstatin and GF 109203X, which inhibit phospholipase C (PLC), protein tyrosine kinase (PTK) and protein kinase C (PKC) activities, respectively, abolished platelet aggregation induced by IgG anti-SK plus APSAC, indicating the pivotal roles of the PLC, PTK and PKC pathways in this immunological activation. In addition, TxA2 generation is also important since aspirin, a cyclo-oxygenase inhibitor and SQ 29548, a TxA2 receptor antagonist, showed significant inhibition of the platelet response. The contribution of released ADP was confirmed using apyrase, which significantly inhibited IgG anti-SK plus APSAC-induced platelet aggregation. Finally, WEB 2086, a platelet-activating factor (PAF) receptor antagonist, was not effective, indicating that PAF is not involved in this process. APSAC- or SK-induced platelet activation may limit the therapeutic effectiveness of the drug and may contribute to the pathogenesis of early reocclusion. The study of the mechanism leading to APSAC-induced platelet activation could be relevant for a better understanding of the physiopathology of immune complex disorder diseases and thrombolytic treatment failure.

Signal transduction in the platelet activation induced by IgG anti-streptokinase and anisoylated plasminogen-streptokinase activator complex

Streptokinase (SK) is one of the plasminogen activators currently used in therapeutics. SK antibodies may appear in the blood after thrombolytic therapy with SK or after-hemolytic streptococci infection. Such antibodies may both activate platelets and neutralize the ability of SK to convert plasminogen into plasmin. We previously demonstrated that platelet activation induced by the combination of IgG anti-SK and anisoylated plasminogen-SK activator complex (APSAC) is mediated by Fc gamma RIIa1 receptor. However, the mechanism by which IgG anti-SK and APSAC (or SK) transduce an activating signal across the platelet plasma membrane remains unknown. We have demonstrated in the present study that the platelet aggregation induced by the combination of IgG anti-SK and APSAC is accompanied by an increase in inositol phosphate, Ca2+ mobilization and thromboxane (Tx) A2 generation. Neomycin, erbstatin and GF 109203X, which inhibit phospholipase C (PLC), protein tyrosine kinase (PTK) and protein kinase C (PKC) activities, respectively, abolished platelet aggregation induced by IgG anti-SK plus APSAC, indicating the pivotal roles of the PLC, PTK and PKC pathways in this immunological activation. In addition, TxA2 generation is also important since aspirin, a cyclooxygenase inhibitor and SQ 29548, a TxA2 receptor antagonist, showed significant inhibition of the platelet response. The contribution of released ADP was confirmed using apyrase, which significantly inhibited IgG anti-SK plus APSAC-induced platelet aggregation. Finally, WEB 2086, a platelet-activating factor (PAF) receptor antagonist, was not effective, indicating that PAF is not involved in this process. APSAC- or SKinduced platelet activation may limit the therapeutic effectiveness of the drug and may contribute to the pathogenesis of early reocclusion. The study of the mechanism leading to APSAC-induced platelet activation could be relevant for a better understanding of the physiopathology of immune complex disorder diseases and thrombolytic treatment failure.

Thromboxane A2 potentiates thrombin-induced proliferation of coronary artery smooth muscle cells.

The activation of thrombin is the key event in clot formation after vascular injury. Thrombin itself, but also other clot-derived factors, such as thromboxane A2 (TXA2), are mitogenic for vascular smooth muscle cells. We have studied the possible interactions between thrombin and TXA2 in stimulation of coronary artery smooth muscle cell (SMC) proliferation. Thrombin (1 U/ml) caused a significant proliferatory response in SMC. U 46619, a stable TXA2 mimetic, had only a minor stimulating effect by its own but markedly potentiated the thrombin-induced mitogenesis. A possible mechanism for these potentiating effects is provided by the demonstration of a marked (6 fold) but transient (maximum after 20 min) increase in the expression of TXA2 receptor (TP receptor) mRNA in SMC by thrombin. Since a significant clot-related TXA2 generation was detected for at least 2 hours, the up-regulation of TP receptors by thrombin may represent a mechanism that is relevant for the in vivo situation of SMC proliferation after vessel injury.

Activation of Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) in human platelets.

Blood platelets respond to the physiological agonist thrombin with shape change, aggregation and release of granular contents (1). Thrombin also evokes the rapid release of arachidonic acid esterified to platelet membrane phospholipids and thereby initiates the synthesis of prostaglandin endoperoxides, thromboxane A2 (TXA2) and other metabolites. The release of arachidonic acid from thrombin-stimulated platelets can be attributed largely to the action of PLA2 that specifically hydrolyzes phospholipids containing arachidonic acid (2,3). Platelet TXA2 generation represents a major pathway mediating both physiological and pathological platelet activities (4,5) and therefore the modulation of the involved PLA2 in platelets may play an important role in both haemostasis and thrombosis.

Mechanisms of Bleeding and Thrombosis in Myeloproliferative Disorders

Arterial and venous thromboses and microcirculatory disturbances such as erythromelalgia and neurologic and visual symptoms are the thrombotic manifestations occurring in Polycythaemia Vera and Essential Thrombocythaemia. The increased in vivo thromboxane A2 generation existing in these patients and the selective sensitivity of erythromelalgia to aspirin suggest that platelet PGG/H synthase products may be involved in transducing the increased thrombotic risk. The relationship between Thromboxane A2 production and thrombotic accidents will be investigated by the European Collaboration on Low-Dose Aspirin in Polycythaemia Vera (ECLAP) which will test the efficacy of low-dose aspirin by a randomised clinical trial. The haemorrhagic diathesis of polycythaemic and thrombocythaemic subjects is generally mild and spontaneous bleeding usually manifests in patients with very high platelet count. Its mechanism may be related to quantitative as well as to qualitative platelet changes. Possible mechanisms linking the high grade thrombocytosis to bleeding are consumption of von Willebrand factor and clot fragility due to a mechanical effect of the high platelet count or to inhibition of fibrin polymerization by platelet Glycoprotein Ib.

The Bioactive Phospholipid, Lysophosphatidylcholine, Induces Cellular Effects via G-Protein-dependent Activation of Adenylyl Cyclase

The naturally occurring phospholipid, lysophosphatidylcholine (lyso-PC), regulates a broad range of cell processes, including gene transcription, mitogenesis, monocyte chemotaxis, smooth muscle relaxation, and platelet activation. Despite the growing list of cellular effects attributable to lyso-PC, the mechanism(s) by which it alters cell function have not been elucidated. In this report, we have examined the effects of exogenous lyso-PC on signal transduction processes within a variety of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Pretreatment of each of these cells with increasing concentrations of lyso-PC (25-150 microg/ml) was associated with a progressive increase in the cytosolic concentration of cAMP. The accumulation of cAMP in platelets correlated closely with the ability of lyso-PC to inhibit multiple platelet processes, including platelet aggregation, agonist-induced protein kinase C activation, thromboxane A2 generation, and the tyrosine phosphorylation of platelet proteins. In each of the cell types examined, the ability of lyso-PC to increase the cellular levels of cAMP was synergistically enhanced by pretreating the cells with the cAMP phosphodiesterase inhibitor, theophylline (5 mM), and was specifically inhibited by the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine. A role for the stimulatory G-protein, Gs, in the lyso-PC-induced activation of adenylyl cyclase was suggested by the ability of the GTPase inhibitor, guanylyl 5'-thiophosphate (0.2 mM), to inhibit the lyso-PC-stimulated increase in cAMP, and also by the ability of cholera toxin to inhibit increases in membrane GTPase activity in response to lyso-PC. The functional significance of lyso-PC-induced activation of adenylyl cyclase was investigated in MEG-01 cells. Treatment of these cells with either lyso-PC or dibutyryl cAMP for 36-40 h resulted in a 3-5-fold increase in the surface expression of the natural anticoagulant protein, thrombomodulin (TM). The ability of lyso-PC to increase TM expression was abolished by pretreating these cells with the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine, whereas the dibutyryl cAMP-induced increase in TM remained insensitive to adenylyl cyclase inhibition. These studies define an important role for the adenylyl cyclase signaling system in mediating cellular effects induced by lyso-PC.

AT1 and TxA2/PGH2 receptors maintain hypertension throughout 2K,1C Goldblatt hypertension in the rat.

Angiotension II (ANG II) increases the generation of vasoconstrictor prostaglandin endoperoxides (PGH2) and thromboxane A2 (TxA2). Two-kidney, one-clip (2K,1C) Goldblatt hypertensive rats have an increased plasma renin activity (PRA) and ANG II level during the early, but not the late, phases of hypertension. Therefore, the aim of these studies was to compare the antihypertensive efficacy of an ANG II type I (AT1) and a TxA2/PGH2 receptor antagonist during different phases of 2K,1C hypertension. Rats were maintained on a fixed sodium intake for 3 days before and throughout the period of drug administration. These studies assessed the antihypertensive response to administration of the AT1 receptor antagonist losartan (20 mg.kg-1.day-1), the TxA2/PGH2 receptor antagonist ifetroban (20 mg.kg-1.day-1) or vehicle given for 3 days to rats with early (2-4 wk postclip), intermediate (10-12 wk postclip), and late (36-42 wk post-clip) 2K,1C hypertension and to two control groups of rats corresponding in age to the early or intermediate and the late 2K,1C groups. The mean arterial pressure (MAP) was measured directly with indwelling arterial cannulas. The MAP of sham-operated rats was 109 +/- 5 mmHg. In the early phase of 2K,1C hypertension, the MAP was increased to 143 +/- 6 mmHg, and it was increased further to 162 +/- 5 mmHg during the intermediate and to 179 +/- 4 mmHg during the late phase. The PRA, compared with age-matched controls, was increased during early and intermediate, but not late phase 2K,1C hypertension. Neither drug lowered blood pressure in control rats. However, both drugs significantly reduced the blood pressure in the early, intermediate, and late phases of 2K, 1C hypertension. At the end of 3 days of administration, blood pressure in early 2K, 1C rats given losartan was reduced to levels of control rats, but remained slightly elevated in other groups and in those receiving ifetroban. In conclusion, AT1 and TxA2/PGH2 receptors maintain hypertension throughout the evolution of 2K, 1C hypertension in the rat, despite changes in PRA.

Evaluation of the thromboxane A2 synthetase inhibitor OKY-046 in a warm ischemia-reperfusion rat model.

The pathophysiology of ischemia-reperfusion renal injury is mediated, in part, by the generation of the vasoconstricting prostanoid thromboxane A2 (TXA2). This study was undertaken to evaluate the renoprotective effects, as well as the optimal timing and dosage, of a selective thromboxane synthetase inhibitor, OKY-046, in a unilateral nephrectomized, 60 min ischemia, 72 hr reperfusion, rodent model. Forty-one rats were subjected to right nephrectomy only (group A), or right nephrectomy with 60 min of left renal ischemia and treatment with inactive vehicle only (group B), or 2 mg/kg or 4 mg/kg of OKY-046 administered intravenously before (groups C and D) or after (groups E and F) pedicle clamping. Outcome variables included animal survival; change in kidney weight; 0, 24, and 72 hr plasma creatinine (CR); urea nitrogen (BUN); thromboxane B2 (TXB2) and 6-keto prostaglandin F(1alpha) (6 kPGF(2alpha)) levels; creatinine clearance (CRCL); and histologic evidence of renal injury. Animal survival and postperfusion kidney weight were not significantly different among the groups. However, renal functional parameters were significantly improved with the 2 mg/kg dose of OKY-046 administered after renal ischemia. (group B 72 hr Cr= 8.01 +/- 1.1 mg% vs. group E=3.99 +/- 1.5 mg%, and group B 72 hr BUN=241.3 +/- 32.8 mg% vs. group E=52.6 +/- 22.5 mg%). The CRCL was also improved in group E vs. group B, although these results did not reach statistical significance (group B=0.069 ml/min vs. group E=0.194 ml/ min). The 24 hr TXB2 levels were significantly increased in group B (0 hr=754.1 +/- 219.4 pg/ml vs. 24 hr=2055.9 +/- 550.0 pg/ml), and pre- or posttreatment with OKY-046 abrogated this increase (group C 0 hr=517.1 +/- 80.9 pg/ml vs. 24 hr=384.7 +/- 251.5 pg/ml, and group E 0 hr=781.6 +/- 390.4 pg/ml vs. 24 hr=183.0 +/- 81.4 pg/ml). The 24 hr 6 kPGF(1alpha) levels decreased in all groups, whereas 72 hr 6 kPGF(1alpha) levels increased above baseline in groups A, C, and E, but not in group B. These data demonstrate the beneficial effects of thromboxane A2 synthesis inhibition in the setting of ischemia-reperfusion injury and suggest that this renoprotection correlates with late vasodilatory prostanoid synthesis.

Does thromboxane mediate the fetal ACTH response to acidemia?

Intravenous mineral acid infusions into fetal sheep stimulate increases in plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations that correlate to the induced changes in arterial pH (pHa). We have recently demonstrated that ACTH and cortisol responses to mineral acid infusion in adult sheep are mediated by thromboxane A2 (TxA2). We designed the present experiments to test the hypothesis that fetal ACTH and cortisol responses are also mediated by TxA2. We infused chronically instrumented fetal sheep with 1 N HCl (0.5 ml/min i.v.) for 60 min, with or without pretreatment with the cyclooxygenase inhibitor flunixin-N-methylglucamine. HCl infusion significantly decreased pHa and significantly increased the arterial partial pressure of O2 (PaO2) and CO2 (PaCO2). Flunixin pretreatment significantly decreased fetal plasma thromboxane B2 (TxB2) concentrations but did not significantly alter the blood gas and pH response to HCl. TxB2 is a stable metabolite of TxA2 and was measured as an index of TxA2 generation. HCl increased fetal heart rate only in the flunixin group. Plasma ACTH and cortisol concentrations were increased significantly in both groups; flunixin did not significantly alter the responses. HCl infusion did not significantly alter plasma TxB2 concentrations. We conclude that the fetal ACTH and cortisol responses to HCl infusion are not mediated by TxA2 or other prostanoids whose synthesis depends on cyclooxygenase activity.

Altered Prostaglandin Metabolism Induced by Angiotensin-Converting Enzyme Inhibitors in Broncho-Alveolar Lavage Fluid of the Guinea Pig

The aim of this study was to investigate if prostaglandin (PG) metabolism is altered by angiotensin-converting enzyme (ACE) inhibitors as determined in the broncho-alveolar lavage fluid (BALF) of the guinea pig. Enalapril or imidapril was orally administered once a day for 2 weeks to Hartley male guinea pigs. Twenty-four hours after the last treatment, BALF was collected and the concentrations of PGI2, thromboxane A2 (TXA2) and PGE2 were measured by enzyme immunoassay. Enalapril significantly (P<0.05) increased the TXA2 content, which was inhibited by indomethacin treatment and significantly (P<0.05) decreased the PGI2 content. Imidapril, however, did not affect TXA2 or PGI2 generation. These findings suggest that altered PG metabolism may be associated with coughing as a side effect of enalapril.