OBJECTIVE

We sought to determine whether platelet activity in patients with heart failure is related to an ischemic versus nonischemic etiologic condition, clinical disease severity, or adverse clinical outcomes.

BACKGROUND

Platelet activity may affect outcome in patients with heart failure. A prospective evaluation of the relation of baseline platelet function to etiologic condition, New York Heart Association (NYHA) class, and clinical outcomes has not been previously reported.

METHODS

Ninety-six consecutive outpatients with ambulatory heart failure with an ejection fraction <0.40 and NYHA Class II to IV symptoms who presented to the Duke Heart Failure Clinic and 14 healthy control subjects formed the study groups. Baseline characteristics and blood analyzed for thromboxane (Tx) B2, 6-keto PGF(1alpha), platelet contractile force, adenosine diphosphate/collagen shear-induced closure time, whole blood aggregation and CD41, CD31, CD62p, and CD51/CD61 by flow cytometry were determined. Survival status and hospitalizations were determined in the heart failure patient cohort.

RESULTS

The median age of patients was 65 years (22% female, 64% white). An ischemic etiologic condition was present in 61% of patients. The population had mild to moderate heart failure: NYHA class I (1%), II (41%), III (46%), and IV (12.5%) and severe ventricular dysfunction (median ejection fraction = 0.20). There were 39 clinical events (7 deaths, 3 cardiac transplants, 29 other first hospitalizations) in 305 median days of observation. Platelet activity, indicated by whole blood aggregation with 5 micromol adenosine diphosphate (P =.04) and Tx B2 (P =.01), was higher in patients with heart failure. Whole blood aggregation was greater than the 90th percentile in 22% of patients with heart failure versus 7% of control subjects. Platelet function did not differ for any of the markers between the ischemic and nonischemic groups and was not affected by antecedent aspirin. There was no relation of NYHA class or the occurrence of events to platelet activity.

CONCLUSIONS

Platelet activity is heightened in 22% of outpatients with stable heart failure symptoms and is not affected by antecedent aspirin therapy. The degree of platelet activation is similar in ischemic and nonischemic patients with heart failure and is not related to clinical disease severity. Current methods to assess platelet activation do not appear to predict outcome.

1. The pharmacological effects of cinnamophilin, a new lignan, isolated from Cinnamomum philippinense, was determined in vitro in human platelet, rat isolated aorta and guinea-pig isolated trachea and in vivo in mice and guinea-pigs. 2. Cinnamophilin inhibited dose-dependently human platelet-rich plasma (PRP) aggregation induced by arachidonic acid (AA), collagen and U-46619 with IC50 of 5.0 +/- 0.4, 5.6 +/- 0.6 and 3.0 +/- 0.4 microM, respectively. The second wave of ADP- or adrenaline-induced platelet aggregation was inhibited by cinnamophilin, while the first wave was only slightly inhibited by cinnamophilin above 30 microM. 3. Cinnamophilin was found to be a thromboxane A2 (TXA2) receptor blocking agent in human platelet, rat aorta and guinea-pig trachea as revealed by its competitive antagonism of U-46619-induced aggregation of human-PRP, contraction of rat aortic rings and guinea-pig tracheal rings with pA2 values of 7.3 +/- 0.2, 6.3 +/- 0.1 and 5.2 +/- 0.2, respectively. 4. [3H]-inositol monophosphate formation and the rise of intracellular Ca2+ caused by U-46619 in human platelet was suppressed by cinnamophilin (10 microM). 5. Cinnamophilin induced a dose-dependent inhibition of thromboxane B2 (TXB2) formation, while the prostaglandin E2 (PGE2) formation was increased. Cinnamophilin did not affect unstimulated platelet adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. When the platelets were challenged with AA, a dose-dependent rise in cyclic AMP was observed. Dazoxiben (a pure TX synthase inhibitor) and SQ 29548 (a pure TXA2 receptor antagonist) did not affect cyclic AMP levels in AA-treated platelets. 6. A high concentration of cinnamophilin (100 MicroM), failed to attenuate the contractile response of rat aorta to endothelin-l, angiotensin II, 5-hydroxytryptamine or noradrenaline. Contraction of tracheal rings induced by histamine, carbachol or KCl was also not inhibited by cinnamophilin (100 MicroM).7. Thirty min after intraperitoneal (i.p.) administration of cinnamophilin (100 microg kg-1), tail bleeding time of mice was prolonged more markedly than with indomethacin, dazoxiben or SQ 29548.8. Intravenous administration of AA (50 microg kg-1) to guinea-pig induced bronchoconstriction. Cinnamophilin(0.1 mg kg-1, i.v.) was administered 1 min before AA, the bronchoconstriction response to AA was abolished.9. It is concluded that cinnamophilin is a novel dual TX synthase inhibitor and TXA2 receptor antagonist and that it may be a useful tool for the investigation and treatment of diseases involving TXA2 disorders.

Ticlopidine (T) and aspirin (ASA) are two antiplatelet drugs both capable of prolonging bleeding time (BT), with a different mechanism of action. A synergism in BT prolongation has been reported and is currently considered an argument for not recommending their combination. However, a profound suppression of platelet function might be a desirable counterpart of a marked prolongation of BT, with a possible use in selected clinical situations. We therefore studied ex vivo platelet function (aggregation by ADP 0.5-1-2.5 microM; adrenaline 0.75-2.5 microM; collagen 1.5-150 micrograms/ml; arachidonic acid 1 mM; PAF 1 microM; adrenaline 0.17 microM + ADP 0.62 microM; serum thromboxane [( TX]B2 generation) and BT (Mielke) in 6 patients with stable coronary artery disease receiving such combination. Patients underwent sequential laboratory evaluations at baseline, after 7 days of T 250 mg b.i.d., before and after the intravenous administration of ASA 500 mg, respectively, and, finally, after a minimum of 7 days of sole ASA oral administration (50 mg/day). The experimental design, therefore, allowed a comparison of T and ASA effects (2nd and 4th evaluation), and an assessment of the combination effect (3rd evaluation). Platelet aggregation in response to all doses of ADP was depressed more by T than by ASA. Conversely, responses to adrenaline, and arachidonate were affected more by ASA than by T. For all other agents, differences were not significant. T + ASA combination was more effective (p less than 0.05) than either treatment alone in depressing responses to high-dose collagen (% over control, mean +/- SEM: T: 95 +/- 3; ASA: 96 +/- 5; T + ASA: 89 +/- 4).(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to characterize the stimulus that activates the 5-lipoxygenase pathway in human peripheral monocytes (PM) during the process of contact activation. Incubation of PM, but not of polymorphonuclear leukocytes (PMN), in contact-activated, recalcified plasma induced a time-dependent release of leukotrienes (LT). The presence of platelets was required for the generation of cysteinyl-LT, but LTB4 formation also proceeded in their absence, although to a lesser extent. Plasmin, presumably generated via the intrinsic fibrinolytic pathway, was liable for the 5-lipoxygenase stimulation during contact activation inasmuch as (1) the 5-lipoxygenase pathway in PM was stimulated by contact-activated, recalcified, autologous or homologous plasma, but not by factor XII-deficient or prekallikrein-deficient plasma; (2) lysine analogs such as N alpha-acetyl-L-lysine, 6-aminohexanoic acid (6-AHA), or trans-4- (aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA), which inhibit plasmin(ogen) binding to PM plasmin(ogen) binding sites, concentration-dependently reduced the cysteinyl-LT release; (3) plasminogen activators such as urokinase or streptokinase concentration-dependently enhanced the cysteinyl-LT release up to 10 and 1,000 IU/mL, respectively, while higher concentrations were less effective leading to bell-shaped concentration-response curves; (4) plasmin inhibitors such as aprotinin or alpha 2-antiplasmin concentration-dependently inhibited the cysteinyl-LT release; and (5) preincubation of plasma with monoclonal antibodies directed against plasminogen and capable of preventing plasminogen activation blocked the contact-mediated 5-lipoxygenase stimulation. Moreover, incubation of PM with plasmin, but not with plasma kallikrein, in Hanks' balanced salt solution (HBSS)-bovine serum albumin (BSA) 0.4% triggered a concentration-dependent release of LTB4 up to 0.1 caseinolytic units (CU)/mL, with higher concentrations being less effective. By contrast, release of cyclooxygenase metabolites such as thromboxane (TX) B2 and prostaglandin (PG) E2 was not stimulated by plasmin, indicating specificity for the 5-lipoxygenase pathway. With plasmin as a hitherto unknown stimulus of the 5-lipoxygenase pathway in PM, a novel link between contact activation and inflammation has been established.

1. The aim of the present study was to characterize the subtypes of bradykinin (BK) receptors that evoke the relaxation and contraction induced by BK and to identify the main contracting and relaxing factors in isolated porcine basilar artery by measuring changes in isometric tension and a thromboxane (TX) metabolite. 2. Endothelial denudation completely abolished both responses. [Thi5,8, D-Phe7]-BK (a B2-receptor antagonist) inhibited the BK-induced relaxation and contraction, whereas des-Arg9, [Leu8]-BK (a B1-receptor antagonist) had no effect. 3. L-nitro-arginine (L-NA, a nitric oxide synthase inhibitor) completely inhibited BK-induced relaxation. Indomethacin (a cyclo-oxygenase inhibitor) completely and ONO-3708 (a TXA2/prostaglandin H2 receptor antagonist) partially inhibited BK-induced contraction, whereas OKY-046 (a TXA2 synthase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) did not. 4. In the presence of L-NA, the contractile response to BK was inhibited by indomethacin or ONO-3708 and was competitively antagonized by [Thi5,8, D-Phe7]-BK (pA2=7.50). In the presence of indomethacin, the relaxant response to BK was inhibited by L-NA and was competitively antagonized by [Thi5,8, D-Phe7]-BK (pA2=7.59). 5. TXA2 release was not induced by BK-stimulation. 6. These results suggest that the endothelium-dependent relaxation and contraction to BK in the porcine basilar artery is mediated via activation of endothelial B2-receptors. The main relaxing factor may be NO and the main contracting factor may be prostaglandin H2.

Pharmacological studies of the contraction induced by 15-hydroperoxyarachidonic acid (15-HPAA) were done in the isolated canine basilar artery. The maximal contractile force produced by 15-HPAA was 1.5 times that of serotonin and was equivalent to that of prostaglandin (PG) F2 alpha or PGA1. At concentrations in which reductions of [14C]-6-keto-PGF1 alpha (the breakdown product of PGI2) occurred, both 15-HPAA and tranylcypromine contracted the artery, whereas indomethacin consistently relaxed it. In the case of indomethacin, as the drug inhibited the synthesis of [14C]-PGE2, F2 alpha and thromboxane(TX) B2 (the breakdown product of TXA2) as well, it has been considered that the balances between PGI2 and other vasoconstrictive PGs or TX may regulate the tone of the artery. Furthermore, it was shown that 15-HPAA enhanced the synthesis of lipoxygenase products from [14C]arachidonic acid. Experiments were done, therefore, to know whether these enhanced lipoxygenase products participated in the manifestation of contraction induced by 15-HPAA or not. As a result, although indomethacin did not affect the contraction, significant reductions of the contraction were shown by eicosatetraynoic acid. These results suggest that enhanced synthesis of lipoxygenase products may be involved in eliciting contractile responses of 15-HPAA.

The purpose of the present study was to examine the efficacy of U-74006F, a 21-aminosteroid, on lung dysfunction induced by endotoxaemia in awake sheep with lung lymph fistula and haemodynamic monitoring. We measured pulmonary haemodynamics, lung lymph balance, circulating leucocyte count, arterial blood gas tensions, and levels of thromboxane (Tx) B2 and 6-keto-prostaglandin (PG) F1 alpha in plasma and lung lymph. We performed two experiments. In experiment 1 (n = 6), we intravenously infused Escherichia coli lipopolysaccharide endotoxin (1 microgram/kg) over 30 min and observed the parameters over 5 h. In experiment 2 (n = 6), we pretreated sheep with an intravenous bolus of U-74006F (2 mg/kg) 30 min before the infusion of endotoxin in the same manner of experiment 1, and continuously infused U-74006F (0.5 mg/kg per h) over 5 h after the bolus during the experiment. The U-74006F significantly suppressed the early pulmonary hypertension, the late increase in pulmonary permeability and the elevations of TxB2 and 6-keto-PGF1 alpha levels in plasma and lung lymph during the early period following endotoxaemia, although the compound did not change the time course of leucocytopenia and hypoxaemia. These findings suggest that the administration of U-74006F attenuates the lung dysfunction induced by endotoxaemia in awake sheep.

The effects of the peptidoleukotrienes C4 (LTC4), D4 (LTD4), E4 (LTE4) and a series agonists and antagonists on arachidonic acid metabolism were characterized in minced guinea pig lung. In response to LTD4, guinea pig lung utilized and converted the endogenous arachidonic acid into a variety of cyclooxygenase metabolites [prostaglandins (PGs) E2, F2 alpha, 6-keto-F1 alpha and thromboxane (Tx) B2] and lipoxygenase metabolites (5,12-dihydroxy-6,8,11,14-eicosatetraneoic acid and 5,15-dihydroxy-5,9,11,13-eicosatetraneoic acid). Using radioimmunoassays, the stable, pharmacologically important metabolites of the cyclooxygenase pathway, 6-keto-PGF1 alpha and TxB2, were quantitated. LTD4, LTE4 and several synthetic agonists induced dose-dependent synthesis and release of TxB2 and 6-keto-PGF1 alpha. Agonist-induced synthesis and release of 6-keto-PGF1 alpha and TxB2 was time-dependent and was maximal after 10 to 12 min of incubation. The slow-reacting substance of anaphylaxis antagonist, FPL 55712, and the newly reported dithioacetal LTD4 receptor antagonists (SKF 102922 and SKF 102081) did not induce synthesis and release of TxB2 and 6-keto-PGF1 alpha in concentrations that blocked the agonist-induced smooth muscle contraction. Preincubation with these antagonists inhibited the synthesis and release of prostanoids induced by LTD4 in the guinea pig lung. Islet activating protein, which inactivates the inhibitory guanine nucleotide binding protein (Gi protein), partially inhibited the agonist-induced synthesis and release of prostanoids. Furthermore, the receptor binding affinities and/or the myotonic activities of the LTD4 agonists correlated linearly with the agonist-induced prostanoid synthesis and release effect.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

We aimed at investigating the relationship between Helicobacter pylori infection and in vivo lipid peroxidation and platelet activation, as reflected by urinary 8-iso-prostaglandin (PG)F(2alpha) and 11-dehydro-thromboxane (TX)B2, respectively, in otherwise healthy dyspeptic subjects.

RESULTS

We measured urinary 8-iso-PGF2alpha and 11-dehydro-TXB2 excretion in 40 dyspeptic subjects with a positive 13C-urea breath test and 38 dyspeptic individuals with a negative test. Moreover, we investigated the effects of H pylori eradication on prostanoid metabolite excretion in 23 H pylori-positive subjects. We also measured prostanoid metabolite excretion before and after selective cyclooxygenase-2 inhibition with rofecoxib in 4 H pylori-positive subjects. Urinary 8-iso-PGF2alpha and 11-dehydro-TXB2 excretion was significantly higher in the H pylori-positive individuals than in controls. A significant direct correlation was found between the degree of positivity to the 13C-urea breath test and urinary 8-iso-PGF2alpha excretion. The latter was linearly correlated with urinary 11-dehydro-TXB2. Successful eradication of H pylori infection led to a significant reduction in both 8-iso-PGF(2alpha) and 11-dehydro-TXB2. Furthermore, their levels were unaffected after treatment with rofecoxib.

CONCLUSIONS

Our study provides evidence of enhanced in vivo lipid peroxidation and platelet activation in association with H pylori infection and suggests a novel mechanism by which an infectious agent could contribute to atherothrombosis.

The dynamics of production and excretion of prostanoids by histotropic larvae of Oesophagostomum dentatum and the role of prostanoids for the development of O. dentatum were studied under in vitro conditions. In control cultures fourth stage larvae (L4) were first seen on Day 7 of in vitro cultivation. Their numbers steadily increased until Day 21 of in vitro cultivation. Thereafter, the numbers of L4 no further increased. The continuous presence of inhibitors of prostanoid synthesis such as acetylsalicyclic acid (ASA, 5.6 mmole/liter) or indomethacin (INDO, 1.4 mmole/liter) in the culture medium blocked the development to L4 almost completely. When ASA was given first on Day 13 of in vitro cultivation L4 counts still increased during the following 4 days. However, the numbers of larvae which reached the L4 stage in these cultures was reduced to 65% of that in control cultures. Resumption of development to L4 was seen after the withdrawal of INDO in cultures treated with this drug until Day 13 of in vitro cultivation. Larvae and supernatants were collected separately on Days 7, 14, 21, or 28 of in vitro cultivation and larvae were homogenized. The presence of prostaglandin (PG) E2, PGD2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 was assayed radioimmunologically in homogenates and supernatants. In the homogenates only PGE2 and PGD2 were regularly detected. The total amount of PGE2 and PGD2 in the homogenates significantly increased with the increase in L4 numbers, whereas the homogenate protein-based values did not. In addition to PGD2 and PGE2, PGF2 alpha and TXB2 were found in the supernatants. PGE2 accounted for about 50% of the prostanoids in the supernatants on Day 7 of in vitro cultivation but was generally absent thereafter. After 14, 21, and 28 days of in vitro cultivation TXB2 was the most prominent single prostanoid in the supernatants. PGD2 was found in the supernatants only after 7 and 28 days of in vitro cultivation. PGF2 alpha was present in decreasing amounts in the supernatants throughout the cultivation period. In conclusion, histotropic larvae of O. dentatum produce and excrete mediators of the prostanoid type which may serve as endogenous hormone-like growth factors.

Particulate glucan (P) but not soluble glucan (F) has been shown to sensitize rats to endotoxins. This phenomenon is believed to be mediated by the reticuloendothelial system (RES). The effect of glucan-P and -F on the RES, and the response of glucan-treated rats to nonlethal doses of endotoxin were investigated. Rats were injected for 5 days with 10 mg/kg of glucan-P, -F or saline. Three days later rats were either (1) injected with colloidal carbon for clearance studies, (2) sacrificed for organ histology and determination of serum glucose, plasma thromboxane (Tx) B2, and plasma 6-keto-prostaglandin (PG) F1 alpha concentrations, or (3) challenged with a nonlethal dose of endotoxin. The latter were further subdivided into groups for either 30-day survival or for sacrifice at 30 min or 4 h post-endotoxin infusion to obtain blood samples for glucose, TxB2, and 6-keto-PGE1 alpha determinations. Glucan-P induced hepatosplenomegaly and granulomatous changes within the liver and spleen. The carbon clearance halftime was markedly decreased in these animals. In glucan-P-treated rats challenged with endotoxin, elevated concentrations of both plasma prostanoids were observed as well as alterations in serum glucose levels. These changes were less pronounced in glucan-F- or saline- treated rats. Following endotoxin challenge, only 40% of glucan-P-treated rats survived 30 days whereas 100% of both the glucan-F and saline-treated rats survived. We conclude that glucan-P, in contrast to glucan-F, significantly heightens RES function and that this effect likely accounts for the endotoxin sensitivity.

Interleukin-2 (IL-2), an agent known to activate neutrophils (PMN) with thromboxane (Tx)B2 release, produces pulmonary edema within 6 hours of intravenous infusion. This study tests the role of PMN in mediating the edema. Anesthetized rats received 10(6)U recombinant human IL-2 (n = 15) or vehicle (n = 14) as a constant intravenous infusion during a period of 1 hour. At this time there was leukopenia 3.63 +/- 0.43 (x10(3)/mm3) relative to vehicle-infused control rats 6.12 +/- 0.86 and a decline in PMN, 2.19 +/- 0.14 relative to control value of 3.33 +/- 0.05 (both p less than 0.05). After 6 hours edema, as measured by increase in the wet to dry weight (W/d) ratio, was present in the lungs (4.93 +/- 0.20 relative to control 4.06 +/- 0.10), heart (4.09 +/- 0.11 versus 3.76 +/- 0.08), liver (3.50 +/- 0.10 versus 3.18 +/- 0.10), and kidney (4.25 +/- 0.07 versus 4.00 +/- 0.07) (all p less than 0.05). There was increased lung permeability demonstrated by bronchoalveolar lavage fluid protein concentration of 1970 +/- 210 micrograms/mL relative to control 460 +/- 90 micrograms/mL (p less than 0.05). Interleukin-2 resulted in lung PMN sequestration of 53 +/- 7 PMN/10 high-power fields (HPF) relative to 23 +/- 2 PMN/10 HPF in controls (p less than 0.05) and increased plasma TxB2 levels to 1290 +/- 245 pg/mL relative to control 481 +/- 93 pg/mL (p less than 0.05). Pretreatment of other rats (n = 8) with selective anti-rat neutrophil antiserum 18 hours before the experiment led to a peripheral PMN count 10% of baseline and prevented edema in the lungs (W/d ratio 4.20 +/- 0.16) and heart (3.67 +/- 0.07) (both p less than 0.05) but not liver or kidney. Protein in lung lavage was reduced to 760 +/- 220 micrograms/mL (p less than 0.05). The protection afforded by leukopenia was associated with lack of PMN sequestration and prevention of the increase in plasma Tx levels (484 +/- 120 pg/mL, p less than 0.05). These data indicate that the rapid induction of lung and heart edema with a 1-hour infusion of IL-2 in the rat is mediated, in large part, by activated PMNs.

Essential thrombocythemia (ET) is characterized by abnormal megakaryopoiesis and enhanced thrombotic risk. Once-daily (od), low-dose aspirin is the recommended antithrombotic regimen, but accelerated platelet generation may reduce the duration of platelet cyclooxygenase (COX)-1 inhibition. We performed a multicenter, double-blind trial to investigate the efficacy of three aspirin regimens in optimizing platelet COX-1 inhibition while preserving COX-2-dependent vascular thromboresistance. Two-hundred-forty-five patients on chronic od low-dose aspirin were randomized (1:1:1) to receive 100 mg aspirin od, twice-daily, bid), or three-times daily (tid) for 2 weeks. Serum thromboxane B2 (sTXB2), a validated biomarker of platelet COX-1 activity, and urinary prostacyclin metabolite (PGIM) excretion were measured at randomization and after 2 weeks, as primary surrogate endpoints of efficacy and safety, respectively. Urinary TX metabolite (TXM) excretion, gastrointestinal tolerance, and ET-related symptoms were also investigated. Evaluable patients assigned to the bid and tid regimens showed substantially reduced inter-individual variability and lower median values of sTXB2: 19.3[9.7-40], 4 [2.1-6.7], and 2.5[1.4-5.65] ng/ml in the od (n=85), bid (n=79) and tid (n=79) arms, respectively. Urinary PGIM was comparable in the three arms. Urinary TXM was significantly reduced by 35% in both experimental arms. Patients in the tid arm reported a higher abdominal discomfort score. In conclusion, the currently recommended aspirin regimen of 75-100 od for cardiovascular prophylaxis appears largely inadequate in reducing platelet activation in the vast majority of ET patients. The antiplatelet response to low-dose aspirin can be markedly improved by shortening the dosing interval to 12 hours, with no improvement by further reducing it. (EudraCT 2016-002885-30).

S-5682 (Daflon-500 mg), a purified flavonoid fraction, consisting of 90% diosmin (a flavone derivative) and 10% hesperidin (a flavanone derivative), was administered to rats by intubation in the daily dose of 100 mg/d. 15 days after the start of treatment, polyurethane sponges were implanted in the subcutaneous connective tissue in the dorsolumbar region under rapid ether anaesthesia. Similar fragments of sponge were implanted in a group of control animals who received the vehicle (saccharose syrup) only, also by the oral route. The rats were sacrificed in fractions of 7 animals drawn from each of the two groups (control and treated) after 4, 8, 16 and 30 days (only 5 animals from each group on day 30) after implantation of the polyurethane sponges. The granulomas formed were removed, weighed and their prostaglandin (PG)E2, PGF2 alpha and thromboxane (Tx)B2 contents were determined. In addition a full cell count (polymorphs, lymphocytes, macrophages, plasmocytes and giant cells) was performed and the animals were histologically examined. The results show that treatment of the animals with S-5682 had the following effects: 1. A significant fall in the mean weight of the granulomas formed after 4 and 8 days was observed, reflecting inhibition of oedema formation during the early phase of the inflammatory reaction. 2. The synthesis of PGE2 (78.5% inhibition on day 4) and PGF2 alpha (45.2% on day 16) was inhibited. 3. There was very early inhibition of TxB2 synthesis (59.5% inhibition on day 4). 4. A later reduction in cell migration towards the inflammatory focus occurred which was statistically significant on day 16 (49.6% reduction in the total number of migrant cells). 5. Multiple histological aspects of the acute inflammatory reaction (diapedesis of polymorphs, lymphocytes, histiocytes and macrophages) and features of the chronic inflammatory reaction (newly formed microvascularisation of the granuloma tissue, perivascular oedema, presence of collagen fibres) were improved.

Acute, overwhelming sepsis or endotoxemia in experimental animals is associated with increased circulating levels of thromboxane (Tx)B2 (stable metabolite of TxA2) and 6-keto PGF1 alpha (stable metabolite of prostacyclin). The purpose of the present investigation was to determine the plasma prostanoid response to sepsis using an animal paradigm in which the septic process evolved more slowly than in previous similar studies. Bacterial peritonitis was induced in rats by cecal ligation (group B) or cecal ligation plus puncture with a 22-gauge needle (group C). Compared to sham-operated controls (group A), levels of immunoreactive 6-keto PGF1 alpha were significantly (p less than .05) elevated in group C rats at 6, 12, and 24 hr after surgery. At 48 hr after surgery, levels of this prostanoid were significantly (p less than .05) elevated in group B animals. In contrast, TxB2 levels were never significantly increased in septic (groups B and C) as compared to control (group A) rats. These data are consistent with results from several clinical studies and emphasize an important difference between the cecal ligation model and other experimental sepsis paradigms.

Carprofen is a nonsteroidal anti-inflammatory drug of the 2-arylpropionate subclass. It contains a single chiral centre and exists in two enantiomeric forms. In this study rac-carprofen, at two dosages, 0.7 and 4.0 mg/kg, and placebo were administered i.v. to six New Forest horses in a three period cross-over study. The concentration-time profiles were established for R(-) and S(+)-carprofen for plasma and both inflamed (exudate) and noninflamed (transudate) tissue cage fluids. R(-)-carprofen was the predominant enantiomer in all three fluids, as indicated by plasma area under the curve (AUC) values for R(-) and S(+)-carprofen of 117.4 and 22.6 microg h/mL (low dose carprofen) and 557.5 and 138.1 microg h/mL (high dose carprofen) respectively. Penetration of both enantiomers into exudate was slow and limited and passage into transudate was even lower. The pharmacodynamics of rac-carprofen was investigated at both the molecular level and in terms of the ability to suppress components of the tissue cage inflammatory response. Low dose carprofen produced only moderate and transient inhibition of serum thromboxane (Tx)B2 but failed to affect exudate prostaglandin (PG)E2 concentrations, whilst suppression of exudate leukotriene (LT)B4 and beta-glucuronidase was not significant. High dose carprofen produced greater and more persistent inhibition of serum TxB2 and virtually abolished exudate PGE2 synthesis. Some inhibition of LTB4 and beta-glucuronidase in exudate was also obtained. At both dosages rac-carprofen reduced the swelling produced by intradermal bradykinin injection but only high dose carprofen was anti-inflammatory as indicated by suppression of temperature rise over exudate tissue cages and neither dose affected leucocyte numbers in exudate. When considered in conjunction with previous data on carprofen, the present findings indicate that carprofen is not a selective inhibitor of cyclooxygenase (COX) isoenzymes, COX-1 and COX-2 in the horse, although it may show some preference for COX-2 inhibition. Because low dose carprofen, which is the clinically recommended dosage, produces minimal inhibition of COX, it is likely to achieve its therapeutic effects at least partially through other pathways, possibly including weak to moderate inhibition of 5-lipoxygenase and of enzyme release. The good safety margin of carprofen in clinical use might also be explained by weak COX inhibition and by other actions at the molecular level.

The clinical use of interleukin-2 (IL-2) is limited by severe cardiopulmonary dysfunction. This study examines the mechanism of respiratory failure related to IL-2, using sheep with chronic lung lymph fistulae. Awake animals were infused with an intravenous (I.V.) bolus of IL-2 10(5) U/kg (n = 5) or its excipient (EXC) control (n = 3), every 8 hours for 4 to 5 days. Cardiopulmonary function was monitored daily for at least one 8-hour period. Within 2 hours after each IL-2 administration, mean pulmonary arterial pressure (MPAP) rose. On Day 1, the mean rise was from 13 to 26 mmHg (p less than 0.05), and on Day 5, to 29 mmHg (p less than 0.05). MPAP returned to baseline levels after 2-3 hours. Pulmonary arterial wedge pressure was unchanged from 4 mmHg. There were transient falls in arterial oxygen tension, from 88 to 77 mmHg on Day 1 and to 73 mmHg (p less than 0.05) on Day 5. Lung lymph flow (QL) rose from 2.4 to 6.8 ml/30 minutes (p less than 0.05) on Day 1, and from 4.7 to 10.2 ml/30 minutes (p less than 0.05) on Day 5, whereas the lymph/plasma protein ratio increased on Day 1 from 0.69 to 0.83 (p less than 0.05) and from 0.63 to 0.71 (p less than 0.05) on Day 5. This documents an increase in pulmonary microvascular permeability. Thromboxane (Tx)B2 levels increased transiently after each IL-2 injection in plasma from 195 to 340 pg/ml (p less than 0.05) and in lung lymph from 222 to 772 pg/ml (p less than 0.05) on Day 1, and to similar levels on Day 5. There was a progressive rise in cardiac output from 5.7 to 8.6 1/minute (p less than 0.05) during the 5 days of infusion. Systemic blood pressure did not change. Temperature rose from 39.1 to 41.2 C (p less than 0.05), and shaking chills were common. There was a progressive fall in leukocyte count, from 8.4 to 3.2 X 10(3)/mm3 (p less than 0.05) by Day 5, reflecting a 77% fall in lymphocytes. Lung lymph lymphocyte counts rose, and lymphocyte clearance increased.(ABSTRACT TRUNCATED AT 250 WORDS)

The left anterior descending coronary artery (LAD) of five dogs was ligated, and blood was withdrawn from the great cardiac vein, left marginal cardiac vein, femoral vein, and aorta. After ligation, immunoreactive 6-ketoprostaglandin (PG) F1 alpha rose from less than 0.1 to a mean value of 1.2 pmol/ml plasma in the great cardiac vein (GCV) and 0.88 pmol/ml in the left marginal vein, with no change in peripheral circulation. Immunoreactive thromboxane (TX) B2 remained below 0.075 pmol/ml throughout the experiments. LAD of 11 dogs was stenosed 60-80% with consequent cyclical reductions in blood flow. 6-Keto-PGF1 alpha in GCV rose in seven dogs (range 0.5-2.2 pmol/ml) and remained unchanged in four. No change was observed in peripheral plasma levels of 6-keto-PGF1 alpha. In these experimental conditions TXB2 remained below 0.075 pmol/ml. Lactate concentrations rose in both experimental conditions in GCV but not in peripheral circulation or in the left marginal vein. This study confirms a link between cardiac ischemia and increased coronary prostacyclin release, but we were unable to detect a similar correlation with TXB2 in plasma.

Lower torso ischemia leads during reperfusion to leukocyte (white blood cell)-dependent lung injury. This study tests the intermediary role of oxygen free radicals (OFRs) in mediating this event. Chronically instrumented anesthetized sheep underwent 2 hours of bilateral hindlimb ischemia. In untreated control animals (n = 7), 1 minute after tourniquet release, mean pulmonary artery pressure rose from 13 to 38 mm Hg (p less than 0.05), whereas pulmonary artery wedge pressure was unchanged from 4 mm Hg. The pulmonary hypertension was temporally related to an increase in plasma thromboxane (Tx) B2 levels from 211 to 735 pg/ml (p less than 0.05). At 30 minutes of reperfusion lung-lymph TxB2 levels rose from 400 to 1005 pg/ml (p less than 0.05). This coincided with an increase in lung-lymph flow from 4.3 to 8.3 ml/30 min (p less than 0.05), which remained elevated for 2 hours, an unchanged lymph/plasma protein ratio, and a rise in lymph protein clearance from 2.6 to 4.6 ml/30 min (p less than 0.05). These changes are consistent with increased lung microvascular permeability. White blood cell count fell during the first hour of reperfusion from 6853 to 3793/mm3 (p less than 0.05), and lung histologic findings showed marked leukosequestration relative to sham animals (n = 3). Pretreatment with the OFR scavengers, superoxide dismutase and catalase both conjugated to polyethylene glycol (n = 6) blunted the rise in mean pulmonary artery pressure to 19 mm Hg (P less than 0.05) and prevented the increase in plasma and lymph TxB2 lymph flow, and lymph protein clearance (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The pharmacodynamics and enantioselective pharmacokinetics of the arylpropionic acid non-steroidal anti-inflammatory drug, carprofen, were investigated in cats after administration of the racemic mixture (rac-carprofen) at dose rates ranging from 0.7 to 4.0 mg kg-1 intravenously and subcutaneously. A low dose of rac-carprofen (0.7 mg kg-1) partially inhibited the rise in skin temperature at a site of acute inflammation but had no effect on the ex vivo synthesis of serum thromboxane (Tx) B2. A higher dose (4.0 mg kg-1) inhibited oedematous swelling, although the response was statistically significant at only one time, and also reduced the ex vivo synthesis of serum TxB2 for 12 hours after intravenous injection or 24 hours after subcutaneous injection. The main features of carprofen pharmacokinetics were a low distribution volume, a relatively long elimination half-life, the predominance of the R(-) enantiomer and a bioavailability (after subcutaneous dosing) of 100 per cent and 92 per cent, respectively, after doses of 0.7 and 4.0 mg kg-1. On the basis of these data, it is suggested that a dose of 4.0 mg kg-1 by both intravenous and subcutaneous routes should be evaluated in clinical subjects.

Current concepts of atherogenesis, based on animal models, suggest a role for platelets in the development of atherosclerotic lesions, possibly through the release of alpha granule constituents. Platelets may also contribute to the development of vascular spasm through thromboxane A2 production. Platelet activation in the coronary circulation in patients with coronary artery disease (CAD) should occur if these hypotheses apply clinically. We measured aortic and coronary sinus plasma levels of the platelet alpha granule constituent beta-thromboglobulin (B-TG) and thromboxane B2 (TX B2) by radioimmunoassay in 15 patients with severe atherosclerotic CAD, seven patients with angiographically normal coronaries, and five patients undergoing evaluation for coronary artery spasm (CAS). Compared with the controls, CAD patients had significantly greater transmyocardial release of B-TG (11.1 +/- 8.1 ng/ml, mean +/- SEM vs 62.5 17.2, p less than 0.05 by rank sum test); TX B2 gradients showed a similar trend but the difference was not statistically significant (-0.08 +/- 0.03 ng/ml vs 0.22 +/- 0.02, 0.05 less than p less than 0.10). Three of the five patients studied developed CAS which was associated with acute elevation in coronary sinus TX B2; the two non-CAS patients with drug provocation had undetectable coronary sinus TX B2. We conclude that abnormal platelet activation takes place in the coronary circulation of CAD patients, and that production of acute myocardial ischemia by CAS occurs with increased coronary sinus TX B2.

Polymorphonuclear leukocyte (PMN) sequestration within the pulmonary microvasculature is known to occur in association with ischemia/reoxygenation (I/R). This sequestration is dependent on eicosanoids and reactive oxygen species. PMN sequestration within the lungs suggests that pulmonary microvascular endothelial cells (MECs) may in part regulate the I/R response. Simulating I/R, we examined the effect of hypoxia/reoxygenation (H/R) on pulmonary MECs in vitro, with and without PMNs. Significant cellular injury, assessed by 51Cr release, occurred upon reoxygenation of MECs (P < .01). Addition of PMNs to the H/R-injured monolayers did not increase MEC injury. Reoxygenation of MECs also resulted in increased thromboxane (Tx) B2 production compared to controls (P < .01). Inhibition of Tx secretion by aspirin reduced H/R-induced PMN adhesion to MECs (P < .01). Furthermore, H/R-induced increases in PMN-MEC adhesion were prevented by allopurinol and superoxide dismutase (P < .01). These data suggest that the pulmonary response to H/R is mediated by MEC generation of reactive oxygen radical species and Tx, which promotes increased PMN adhesion.

One hundred NZB/W F1 female mice were studied to compare the effects of a thromboxane synthase inhibitor (TSI), a stable prostacyclin analog (iloprost) and prostaglandin E1 (PGE1) in the evolution of the nephritis. At 10 weeks of age mice were randomly assigned to cohorts of 20 to receive either no treatment, vehicle control, PGE1, iloprost or TSI. Proteinuria, mortality, systemic blood pressure, renal immune complex deposition, urinary TX B2 and 6 keto PGF1 alpha levels were measured. Mice receiving PGE1 and iloprost had a significant delay in the onset of proteinuria and reduction in mortality at 40 weeks. The TSI treatment had no apparent effect on proteinuria or mortality. The amelioration of the nephritis was not associated with an alteration in immune complex deposition in survivors at 40 weeks. Although PGE1 and iloprost lessened the age related increase in urinary TX B2, increased the urinary 6 keto PGF1 alpha levels and the ratio of 6 keto PGF1 alpha to TX B2; so did the TSI. The PGE1 treated mice did experience a marked and persistent reduction in blood pressure but this was not observed in the iloprost- or the TSI-treated mice. All drugs tested reduced the age-related increase in thromboxane B2 but only the PGE1 and iloprost had a significant effect on the evolution of the nephritis.

Both the iris and the retina of the rabbit released prostaglandins (PG) E2, F2 alpha, 6-keto-F1 alpha and thromboxane (Tx) B2, when incubated in vitro. PGE2 was the major cyclooxygenase product formed by each tissue. The kinetics of PGE2 release by the iris and the retina were similar: high initial output followed by a decline to a steady-state value. The production of PGE2 was inhibited by indomethacin and stimulated by ionophore A23187. The iris and the retina converted exogenous arachidonic acid into 12- and 15-hydroxy-eicosatetraenoic acid (HETE): inhibition by eicosatetraynoic acid (ETYA) indicated the involvement of lipoxygenase enzymes. This lipoxygenase activity was important relatively to cyclooxygenase in the retina, but was only a minor pathway in the iris. Leukotriene (LT) B4 was released by the iris and the retina in amounts smaller than PGE2, but compatible with a biological activity: ionophore A23187 stimulated LTB4 production in both tissues. Our data support the hypothesis that PGE2 and LTB4 could play a role in the initiation of ocular inflammation.