Thromboxane A2 Formation Research Articles

Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.

Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619.

According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.

Increased Renal Biosynthesis of Prostaglandin E2 and Thromboxane B2 in Human Congenital Obstructive Uropathy

Animal experiments have shown that after ureter obstruction hydronephrotic kidneys release increased amounts of prostaglandin E2 (PGE2) and thromboxane A2 (TxA2), suggesting that these prostanoids modify renal blood flow and excretory function in this model. To test the hypothesis that these mechanisms are also operative in congenital obstructive uropathy, we measured prostanoid excretion rates in 12 neonates and infants with congenital unilateral or bilateral hydronephrosis. Prostanoid determinations were performed by gas chromatography mass spectrometry. PGE2 and thromboxane B2 (TxB2) (non-enzymatic metabolite of TxA2) excretion exceeded the normal range in eight and 11 of 12 patients, respectively. Median PGE2 excretion was 22, range 4-572 ng/h/1.73 m2 (normal 3-16). Median TxB2 excretion was 22, range 3-188 ng/h/1.73 m2 (normal 3-7). No other renal prostanoids (prostaglandin F2 alpha, 6-keto-prostaglandin F1 alpha) or systemic prostanoid metabolites (PGE-M, 2,3-dinorthromboxane B2, 11-dehydro-thromboxane B2, 2,3-dinor-6-keto-prostaglandin F1 alpha) were consistently elevated. A second group of 12 neonates with congenital obstructive uropathy was followed sequentially. PGE2 and thromboxane B2 excretion rates increased even further after surgical decompression and gradually normalized during follow-up. There was a significant relationship between elevated FeNa and enhanced PGE2 and TxB2 excretion. These data suggest that endogenous renal formation of PGE2 and TxA2 is selectively stimulated in hydronephrotic kidneys in neonates and infants. PGE2 and TxA2 may be involved in modulating renal function in these infants.

Female Sex Hormones and Platelet/Endothelial Cell Interactions

The effects of estradiol and progesterone added to the growth medium of human umbilical vein endothelial cells for 72 h on the formation and release of prostacyclin were investigated. The influence on collagen-induced platelet aggregation and on the platelet formation of thromboxane A2 following aggregation, of the growth medium collected before and after thrombin stimulation of the endothelial cells, was studied simultaneously. Under basal conditions, endothelial cells grown with progesterone released significantly less prostacyclin into the growth medium than did controls (p less than 0.05). Following thrombin stimulation, endothelial cells grown with estradiol (p less than 0.05) or a combination of estradiol and progesterone (p less than 0.01) contained significantly less prostacyclin than controls. No significant effects on the platelet aggregation or platelet thromboxane formation could be found. This study indicates a lowering effect of both female sex hormones on the endothelial cell prostacyclin formation and release. This may be of significance for the increased risk of vascular disease in pregnant women and oral contraceptive users, but can hardly explain the consequences of the hormonal loss occurring at the menopause.

Inhibitory effects of endothelial cells and calcium channel blockers on platelet aggregation.

This study examined the effects of human umbilical vein endothelial cells (ECs) and calcium channel blockers (Ca2+ blockers), diltiazem, verapamil, and nicardipine, on platelet aggregation in vitro. ECs markedly inhibited the platelet aggregation induced by ADP, collagen, thromboxane A2 (TXA2), or thrombin. When platelets were incubated with ECs, the antiaggregatory activity reached a plateau within 5 to 10 min. As the number of ECs added to the platelet-rich plasma was increased, platelet aggregation declined progressively. The antiaggregatory activity of ECs was attenuated considerably by the addition of aspirin. All three Ca2+ blockers inhibited platelet aggregation in a dose-dependent manner. The combination of ECs and a Ca2+ blocker resulted in more potent inhibition of platelet aggregation than either alone, but the effects were not synergistic. Both ECs and Ca2+ blockers inhibited the synthesis of TXA2 during platelet aggregation. However, Ca2+ blockers did not significantly influence the production of prostaglandin I2 (PGI2) by ECs during incubation and aggregation. These results suggest that PGI2 is an important factor in endothelial antiaggregatory activity. Ca2+ blockers directly inhibit platelet aggregation by suppressing TXA2 formation, but do not appear to enhance the antiaggregatory activity of ECs.

The Effect of Red Blood Cells on the ADP-induced Aggregation of Human Platelets in Flow Through Tubes

The effect of red blood cells, rbc, and shear rate on the ADP-induced aggregation of platelets in whole blood, WB, flowing through polyethylene tubing was studied using a previously described technique (1). Effluent WB was collected into 0.5% glutaraldehyde and the red blood cells removed by centrifugation through Percoll. At 23 degrees C the rate of single platelet aggregation was upt to 9 x greater in WB than previously found in platelet-rich plasma (2) at mean tube shear rates G = 41.9, 335, and 1,920 s-1, and at both 0.2 and 1.0 microM ADP. At 0.2 microM ADP, the rate of aggregation was greatest at G = 41.9 s-1 over the first 1.7 s mean transit time through the flow tube, t, but decreased steadily with time. At G greater than or equal to 335 s-1 the rate of aggregation increased between t = 1.7 and 8.6 s; however, aggregate size decreased with increasing shear rate. At 1.0 microM ADP, the initial rate of single platelet aggregation was still highest at G = 41.9 s-1 where large aggregates up to several millimeters in diameter containing rbc formed by t = 43 s. At this ADP concentration, aggregate size was still limited at G greater than or equal to 335 s-1 but the rate of single platelet aggregation was markedly greater than at 0.2 microM ADP. By t = 43 s, no single platelets remained and rbc were not incorporated into aggregates. Although aggregate size increased slowly, large aggregates eventually formed. White blood cells were not significantly incorporated into aggregates at any shear rate or ADP concentration. Since the present technique did not induce platelet thromboxane A2 formation or cause cell lysis, these experiments provide evidence for a purely mechanical effect of rbc in augmenting platelet aggregation in WB.

Effects of Ethanol on Human Platelets Stimulated with Platelet‐Activating Factor, a Biologically Active Ether Phospholipid

The interaction between ethanol and 1-0-alkyl-2-acetyl-sn-glycerol-3-phosphocholine (platelet activating factor, PAF) was addressed using platelets obtained from normal nonalcoholic volunteers. Ethanol at concentrations of 20 to 100 mM inhibited PAF activation of human platelets. Ethanol inhibited prominently the second or arachidonic acid metabolite dependent wave of platelet aggregation, which occurs with human platelets in citrated plasma. It also inhibited serotonin release and thromboxane A2 formation associated with this secondary phase of aggregation. Ethanol did not readily inhibit the primary wave of PAF-induced aggregation. The incorporation of PAF into platelets or metabolism of PAF was not influenced by up to 100 mM ethanol. Since ethanol inhibited only the secondary response, a direct interaction between PAF, ethanol, and a platelet PAF receptor is unlikely. The effect of ethanol on PAF-induced platelet aggregation shows a selectivity similar to that demonstrated by other investigators for epinephrine and adenosine diphosphate.

Platelet Activation by Tetraprenol via Stimulation of Phospholipase A2 Action

Only tetraprenol (n = 4), among the (n)-polyprenols studied, induced activation of rabbit platelets. Tetraprenol-induced responses, including platelet aggregation, Ca2+ mobilization, inositol phosphate formation, and arachidonic acid release, were greatly inhibited by a thromboxane A2 (TXA2) receptor antagonist and a cyclooxygenase inhibitor, indicating an essential role for endogenously produced TXA2. The TXA2-mimetic agonist U46619 induced platelet aggregation, Ca2+ mobilization and phospholipase C action but did not induce arachidonic acid release. These results suggest that arachidonic acid is not released via phospholipase C but by phospholipase A2, and this is also supported by the finding that phospholipase C action was inhibited by depletion of extracellular Ca2+, while arachidonic acid release was not. Full arachidonic acid release was found to be induced by the synergistic action of U46619 and tetraprenol. Therefore, the initial, most essential response induced by tetraprenol is a small arachidonic acid release by phospholipase A2, which results in initial TXA2 formation. Further action of phospholipase C as well as Ca2+ mobilization and aggregation were induced by the initially formed TXA2 while further activation of phospholipase A2 required the synergistic action of tetraprenol and TXA2.

Role of thrombin and thromboxane A2 in reocclusion following coronary thrombolysis with tissue-type plasminogen activator.

Reocclusion of the coronary artery occurs after thrombolytic therapy of acute myocardial infarction despite routine use of the anticoagulant heparin. However, heparin is inhibited by platelet activation, which is greatly enhanced in this setting. Consequently, it is unclear whether thrombin induces acute reocclusion. To address this possibility, we examined the effect of argatroban [MCI9038, (2R,4R)-4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8- quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid], a specific thrombin inhibitor, on the response to tissue-type plasminogen activator in a closed-chest canine model of coronary thrombosis. MCI9038 prolonged the thrombin time and shortened the time to reperfusion (28 +/- 2 min vs. 59 +/- 7 min in controls; mean +/- SEM, n = 5, P less than 0.01). At the highest dose, 2.5 mg/kg per hr, complete reocclusion was prevented in four of the five experimental animals, whereas reocclusion occurred in all five controls. However, reperfusion was complicated by cycles of decrease flow, which were abolished by the thromboxane A2 antagonist, GR32191. GR32191 at 1 mg/kg combined with MCI9038 at 0.5 mg/kg per hr prevented reocclusion, whereas, at these doses, either drug alone was without effect. In addition, thromboxane A2 biosynthesis, determined as excretion of its metabolite 2,3-dinor-thromboxane B2, was increased after reperfusion at all doses of MCI9038. These data demonstrate that thrombin impairs thrombolysis induced by tissue-type plasminogen activator in vivo and induces acute reocclusion. Furthermore, the response to thrombin inhibition may be impaired by continued formation of thromboxane A2.

Endogenous formation of prostanoids in neonates with persistent pulmonary hypertension.

Endogenous formation of thromboxane A2 and prostacyclin were evaluated in seven neonatates with persistent pulmonary hypertension by serial gas chromatographic mass spectrometric determination of their urinary metabolites dinor-thromboxane B2 and dinor-6-keto-prostaglandin F1 alpha, respectively. The patients were studied until their hypertension had resolved on clinical criteria. Urinary excretion of dinor-thromboxane B2 and dinor-6-keto-prostaglandin F1 alpha was increased when the persistent pulmonary hypertension was associated with group B streptococcal (n = 2) and pneumococcal (n = 1) sepsis. Based on urinary metabolite excretion, endogenous formation of thromboxane A2 and prostacyclin did not consistently differ from normal neonates in four patients with non-septic persistent pulmonary hypertension (hyaline membrane disease (n = 2), asphyxia, and meconium aspiration). These data suggest that thromboxane A2 is not a universal mediator of persistent pulmonary hypertension. It may, however, have a role in the pathophysiology of early onset group B streptococcal disease, and persistent pulmonary hypertension of other infectious aetiology. If these findings are confirmed by further studies, thromboxane synthetase inhibition or receptor antagonism may offer a potential therapeutic approach in neonates with persistent pulmonary hypertension associated with sepsis.

Significance of prostaglandin E2 in acute necrotising pancreatitis in rats.

Acute necrotising pancreatitis in rats was induced by injecting 5% sodium taurocholate into the pancreatic duct. Prostaglandin E2 (100 micrograms/kg subcutaneously twice) decreased the mortality rate from 100% to 60% (NS). When treatment with prostaglandin E2 was combined with simultaneous administration of either dazmegrel (UK 38,485, 50 mg/kg bodyweight) or Sibelium (Flunarizine R 14,950, 0.2 mg/kg body weight) a significant decrease in the mortality rate (p less than 0.05) was recorded. Dazmegrel is a selective thromboxane A2 synthetase inhibitor and prevents the formation of thromboxane A2. Flunarizine (a calcium entry blocker) decreases thromboxane A2 formation and also inhibits the effects of raised thromboxane A2 concentrations. As plasma thromboxane B2 (the stable metabolite of thromboxane A2) concentrations increase and the plasma prostaglandin E2 concentrations decrease in acute necrotising pancreatitis in rats, the results of the present study indicate that these prostaglandins play a role in the pathophysiology of the disease. It is suggested that restoration of the balance in prostanoid concentrations will have a beneficial effect on the course of acute necrotising pancreatitis.

Inhibitory effect of a selective thromboxane A2receptor antagonist, EP 092, on platelet aggregation in whole bloodex vivoandin vivo

1. The inhibitory effect of a selective prostaglandin H2 (PGH2)/thromboxane A2 receptor antagonist, EP 092, on platelet aggregatory responses in whole blood ex vivo (guinea-pig: Rhesus monkey) and intravascular aggregation in vivo (rabbit) has been investigated. 2. Collagen (0.1-10.0 micrograms ml-1) caused a concentration-dependent decrease in single platelet count in samples of both guinea-pig and Rhesus monkey citrated whole blood incubated ex vivo. EP 092 administered to guinea-pigs by intravenous (0.1-3.0 mg kg-1) or oral (1.0-10.0 mg kg-1) routes significantly inhibited the platelet responses to collagen (ED50 values 1.3 +/- 0.2 and 1.4 +/- 0.2 mg kg-1 respectively). Similar potency against collagen-induced whole blood aggregation was observed in Rhesus monkey blood samples following EP 092 given orally (ED50 0.9 +/- 0.3 mg kg-1). 3. The duration of action of EP 092 against collagen aggregatory responses ex vivo in both guinea-pigs and Rhesus monkeys was between 3 and 6 h following oral administration at 3.0 mg kg-1. 4. The inhibitory activity demonstrated by EP 092 against collagen-induced aggregation of Rhesus monkey whole blood ex vivo was not accompanied by any significant reduction in thromboxane A2 formation except at the highest dose tested (10 mg kg-1). 5. The intravascular aggregatory response induced by collagen or thrombin in the anaesthetized rabbit was significantly inhibited by an intravenous infusion of EP 092 (10 mg kg-1). EP 092 appeared less potent and its effect was of shorter duration in this preparation compared with its inhibitory effect on ex vivo aggregation, being evident immediately after infusion of drug but not after a further 30 min. 6. It is concluded that collagen-induced platelet aggregatory response in guinea-pig and Rhesus monkey whole blood ex vivo and rabbit in vivo exhibit a thromboxane-dependent component which can be inhibited in a dose-related fashion by pretreatment with the thromboxane antagonist EP 092. In the rabbit, moreover, the data support the possibility of a role for thromboxane in the intravascular aggregatory response to thrombin.

Mechanism of Potentiation by Manganese Ion of Aggregation of Porcine Pancreatic Elastase-Treated Human Platelets

The effect of manganese ion (Mn2+) on the aggregation of porcine pancreatic elastase-treated platelets (ETP) induced by fibrinogen (Fbg) was studied. Mn2+ enhanced the aggregation of ETP on addition of Fbg specifically and dose-dependently. This effect of Mn2+ was not associated with the formation of thromboxane A2, and was not affected by pretreatment of ETP with acetylsalicylic acid in the presence of Mn2+. Moreover, it was not dependent on extracellular adenosine diphosphate, as shown by removal of extracellular adenosine diphosphate by pretreatment of ETP with creatine phosphate/creatine phosphokinase. Studies on the binding of 125I-Fbg to ETP showed that Mn2+ increased the Kd value of binding but did not affect the number of Fbg binding sites. These results indicate that Mn2+ specifically and dose-dependently potentiated Fbg-induced aggregation of ETP and that this effect of Mn2+ may be due to an increase in the affinity of binding of Fbg to the glycoprotein IIb-IIIa complex on the membranes of ETP.

Thromboxane synthase inhibition enhances action of converting enzyme inhibitors.

Mean arterial blood pressure was measured over a 24-hour period from the femoral artery of conscious, unrestrained spontaneously hypertensive rats. Oral administration of the angiotensin converting enzyme inhibitor CGS 16617 significantly lowered mean arterial pressure. In contrast, both the thromboxane synthase inhibitor CGS 12970 and the thromboxane receptor antagonist BM 13505 lacked an antihypertensive action in the spontaneously hypertensive rat. When administered concurrently, the thromboxane synthase inhibitor CGS 12970 potentiated the antihypertensive action of the angiotensin converting enzyme inhibitor CGS 16617. This effect was not observed with the thromboxane receptor antagonist BM 13505. In addition to CGS 16617, CGS 12970 also potentiated the hypotensive effect of two structurally dissimilar angiotensin converting enzyme inhibitors, benazapril HCL and captopril. Indomethacin blocked the thromboxane synthase inhibition-induced potentiation of the antihypertensive action of angiotensin converting enzyme inhibitors. The thromboxane synthase inhibitor CGS 12970 had no effect on the hypotension induced by hydralazine, indicating that the hypotension is not a nonspecific action related to the fall in blood pressure. These results may suggest that converting enzyme inhibition augments the levels and actions of a hormone that stimulates prostaglandin formation. It is well established that thromboxane synthase inhibitors eliminate the formation of the vasoconstrictor thromboxane A2 and allow reorientation of eicosanoid production toward the formation of vasodilating prostaglandins, which could enhance the antihypertensive action of angiotensin converting enzyme inhibitors.

The renal cytochrome P450 system generates novel arachidonic acid metabolites.

The generation of prostaglandins and other oxygenated metabolites of arachidonic acid (AA) is a complex process initiated by the release of ester-ified AA from cellular lipids. Once liberated from membrane lipids by diverse stimuli (peptide hormones, neurotransmitters and mechanical disruption), the free AA is rapidly metabolized. Metabolism of AA involves three pathways: (a) cyclooxygenase, leading to the formation of prostaglandins, thromboxane A2 (TxA2), and prostacyclin (PGI2); (b) lipoxygenases, leading to the formation of hydroxy- and dihydroxyeicosatetraenoic acids (HETEs and diHETEs) and leukotrienes; (c) cytochrome P450-dependent monoxy-genase system which metabolizes AA by an NADPH-dependent mechanism to a variety of oxygenated products such as HETEs, epoxyeicosatrienoic acids or epoxides (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids or diols (DHTs) as well as ω and ω-1 hydroxylated acids (1–4). The pattern of AA metabolism in the kidney is distinct and AA metabolites participate in integrated renal function. Among the structures that metabolize AA via cyclooxygenase are collecting tubules (5), glomeruli (6), medullary interstitium (7) and blood vessels (8). Other structures such as the convoluted tubules have low or negligible cyclooxygenase activity (8). Lipoxygenase activity is mainly associated with leukocytes or platelets (9,10) and was reported to be present in isolated glomeruli (11).

Manipulation of cytochrome P-450 dependent renal thromboxane synthase activity in spontaneously hypertensive rats.

Thromboxane synthase is a cytochrome P-450-like enzyme requiring an iron-centered oxygen attack of the prostaglandin endoperoxide substrate (PGH2) for subsequent thromboxane A2 (TxA2) formation. The activity and levels of P-450 enzymes can be manipulated by decreasing heme availability. Stannous chloride (SnCl2) selectively induces renal heme oxygenase activity, depleting heme and decreasing hemoprotein synthesis. We therefore manipulated the renal cytochrome P-450 system to influence thromboxane synthase activity, as measured by the conversion of 14C-PGH2 to thromboxane B2 (TxB2) in renal cortical microsomes from spontaneously hypertensive rats (SHR). Seven-week-old SHR were treated subcutaneously with SnCl2 (1, 10 and 15 mg/100 g body weight) for 4 consecutive days, and cortical microsomal heme oxygenase activity, heme content, P-450 content, thromboxane synthase activity and systolic blood pressure were measured. Heme oxygenase activity was significantly increased from 1058 +/- 62 nmol/mg protein in controls to 3125 +/- 918, 5057 +/- 690--and 4236 +/- 581 nmol/mg protein in SHR treated with 1, 10 and 15 mg/100 g body weight SnCl2, respectively. The increase in heme oxygenase activity was associated with corresponding decreases in heme content (0.29 mumol/mg protein, for control to 0.12 mumol/mg protein for SHR treated with SnCl2, 10 mg/100 g body weight) and cytochrome P-450 content (0.18 +/- 0.1 nmol/mg protein for control to 0.06 +/- 0.01 nmol/mg protein for SHR treated with SnCl2 10 mg/100 g body weight). The reduction in heme and P-450 content was associated with a reduction in thromboxane synthase activity, i.e., decreases of 38, 35 and 47% from control levels at doses of 1, 10 and 15 mg/100 g body weight.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostanoid production in varicose veins: evidence for decreased prostacyclin with increased thromboxane A2 and prostaglandin E2 formation.

In this study, formation of arachidonic acid-derived prostanoids was investigated in saphenous veins of varicosed and nonvaricosed patients, all undergoing saphenectomy respectively for varicosis or in preparation for coronary bypass operation. Venous production of prostacyclin (PGI2), thromboxane A2 (TXA2) and prostaglandin E2 (PGE2) was assessed by bioassay and/or radioimmunologic assays as appropriate. Fragments of saphenous veins from varicosed patients produced significantly less PGI2 and more TXA2 and PGE2 than those from the control patients. Addition of arachidonic acid to incubation mixtures dose dependently increased release of these prostanoids, but the levels of PGI2 produced were consistently lower in veins from varicosed patients. No differences were found in varicosed patients between various segments of the same vein, no matter whether macroscopically affected or unaffected. These results demonstrate that the cyclooxygenase pathway in the venous wall of subjects with varicosis is shifted toward lesser formation of PGI2 and higher production of proaggregatory and proinflammatory prostanoids such as PGE2 and TXA2. These biochemical changes may be relevant to inflammation and thrombogenesis in varicosis.

Prostacyclin and thromboxane A2 formation by atherosclerotic carotid artery: Comparison with normal aorta, saphenous vein, and platelets

Prostacyclin (PGI2) and thromboxane A2 (TxA2) formation by whole-tissue segments of nine carotid endarterectomy specimens (CES), five normal aortic specimens (NAS), six saphenous vein specimens (SVS), and four platelet samples were determined by incubation with 10 μmol/L 1-14C-radiolabeled prostaglandin endoperoxide H2 (PGH2), and in other experiments with and without 10 μmol/L of CGS 13080, a TxA2 synthase inhibitor. PGI2 formation (expressed as picomoles 6-keto-PGF1α/2-min incubation per sample) by nonatheromatous proximal intima of CES (307 ± 23, mean ± standard error) and distal intima of CES (260 ± 22) was not statistically different; however, it was greater than atheromatous transitional plaque (159 ± 13 pmol) (p < 0.01) and ulceration regions (140 ± 15 pmol) (p < 0.01) of CES, NAS (204 ± 16 pmol) (p < 0.01), and SVS (165 ± 9 pmol) (p < 0.01). TxA2 formation (expressed as picomoles TxB2/2-min incubation per sample) by CES ulceration (51 ± 2 pmol) was low but greater than proximal (17 ± 2 pmol) (p < 0.01), distal (19 ± 3 pmol) (p < 0.01), and transitional (23 ± 3 pmol) (p < 0.01) regions. TxA2 formation by NAS and SVS was not detected (less than 10 pmol). CGS 13080 inhibited TxA2 formation by CES below the limits of detection. Incubation of 1.9 × 105 intact platelets with 10 μmol/L of PGH2 formed a quantity of TxA2 equal to that of CES ulceration. Platelet homogenization reduced TxA2 formation (p < 0.01), demonstrating the sensitivity of TxA2 synthase activity to experimental conditions and the requirement for whole-tissue incubation with and without CGS 13080 to confirm its presence. Diminished PGI2 and minimal, but detectable, TxA2 formation by ulcerated regions of CES may partially explain platelet adherence and thrombogenicity at these foci because of imbalance between vasoregulatory eicosanoids.

Interrelation of platelet aggregation, release reaction and thromboxane A2 production

Aggregation of platelets, stimulated by different agonists, was inhibited by omitting sample stirring or by preincubation of platelets with a monoclonal antibody against glycoproteins IIb-IIIa or with a pentapeptide containing the sequence Arg-Gly-Asp-Ser. In platelets stimulated by collagen, ADP and epinephrine, the inhibition of aggregation paralleled a reduction of both release reaction and thromboxane A2 formation. When thrombin was the stimulus, ATP release and thromboxane A2 production were unaffected (or only slightly modified) by the inhibition of platelet aggregation. These data add further evidence to the hypothesis that aggregation supports the activation of platelets stimulated by weak agonists.

Potentiation of myocardial salvage by tissue type plasminogen activator in combination with a thromboxane synthetase inhibitor in ischemic cat myocardium.

We studied the effects of a thrombolytic agent (t-PA) and a thromboxane synthetase inhibitor (CGS-13080) in a model of myocardial ischemia and reperfusion. Occlusion of the left anterior descending coronary artery for 2 hours followed by 4 hours of reperfusion in anesthetized cats results in a large washout of creatine kinase into the blood (32 +/- 7 IU/mg protein) and an area of necrotic tissue comprising 52 +/- 5% of the area at risk and 9 +/- 0.6% of the left ventricle. Intravenous administration of t-PA (500 IU/kg.min) for 30 minutes alone at reperfusion or infusion of CGS-13080 (500 micrograms/kg.hr) had no effect on washout of creatine kinase or extent of necrotic tissue development. Administration of the same doses of both t-PA and CGS-13080 together markedly attenuated creatine kinase release to 10 +/- 2 IU/mg protein (p less than 0.01) and reduced the area of necrotic tissue to 9 +/- 2% of the area at risk and only 1.3 +/- 0.3% of the left ventricle (p less than 0.001). No significant sustained effects of these agents were observed on mean arterial blood pressure, heart rate, or the pressure rate index in these experiments. Thus, t-PA and CGS-13080 exert synergistic effects in preserving myocardial integrity in cats subjected to acute myocardial ischemia followed by reperfusion. The mechanism of this beneficial effect does not appear to be via reduced myocardial oxygen demand, increased myocardial oxygen supply, or enhanced inhibition of thromboxane A2 formation. The mechanism of this anti-ischemic effect is not clear but may involve a metabolic or a cytoprotective effect.