Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.

Levels of prostaglandin (PG) E2 and thromboxane (TX) B2, the stable metabolite of TXA2, were measured by radioimmunoassay in cerebrospinal fluid (CSF) collected from the third ventricle and the cisterna magna of conscious cats. In the absence of fever, PGE2 was usually below the threshold of the assay (0.05-0.37 ng/ml), while TXB2 was measurable in the majority of cases and its concentration was greater in the third ventricle (about 0.7 ng/ml) than in the cisterna magna (about 0.2 ng/ml). At either site, TXB2 content rose if any manipulation was required for the collection of samples. PGE2 levels increased to measurable values (max 1.1-1.4 ng/ml) during fever produced by intrathecal or intravenous administration of leucocytic pyrogen. In contrast, TXB2 concentration rose to an average of 2.2-4 ng/ml only when pyrogen (bacterial or leukocytic) was given intrathecally. Moreover, TXB2 elevation, unlike PGE2 elevation, was limited to the uprise phase of the fever. Imidazole, given either intraperitoneally (50 mg/kg) or intrathecally (3 mg), attenuated the pyrogen fever and suppressed any rise in TXB2 levels. At the same time, the drug tended to increase the PGE2 content of the CSF. Evidence was also obtained suggesting that a fraction of PGE2 is bound to CSF protein, and this event may be important to the inactivation of the compound. These findings are consistent with the concept that PGE2 is involved in the sequence of events underlying pyrogen fever. A role for thromboxane A2 in this process remains to be established.

Increased concentrations of free arachidonic acid (AA) and its proinflammatory metabolites have been observed in psoriatic lesions. Replacement of arachidonic acid by alternative precursor polyunsaturated fatty acids (PUFA), especially eicosapentaenoic acid (EPA), which can be metabolized via the same enzymatic pathways as AA, might be a therapeutic option in psoriasis. However the results of studies evaluating the therapeutic benefit of dietary fish oil have been conflicting and not clearly dose-dependent. To overcome the slow kinetics and limited availability of oral supplementation, we have performed three studies to assess the efficacy and safety of an intravenously administered fish oil derived lipid emulsion on different forms of psoriasis. Patients received daily infusions of either an n-3 fatty acid-based lipid emulsion (Omegaven) or a conventional n-6 lipid emulsion (Lipoven) in different time and dose regimens. In addition to an overall assessment of the clinical course of psoriasis, EPA- and AA-derived neutrophil 5-lipoxygenase (LO)--products, thromboxane (TX) B2/B3, PAF and plasma free fatty acids were investigated. Treatment with n-3 fatty acids resulted in a considerably higher response rate than infusion of n-6 lipids. A more than 10-fold increase in neutrophil EPA-derived 5-LO product formation was noted in the n-3 group, accompanied by a rapid increase in plasma-free EPA within the first days. In conclusion, intravenous n-3-fatty acid administration causes reduction of psoriasis, which may be related to changes in inflammatory eicosanoid generation. The rapidity of the response to intravenous n-3 lipids exceeds by orders of magnitude the hitherto reported kinetics of improvement of psoriatic lesions upon use of oral supplementation.

Conscious cats were used to study the local release of prostaglandin (PG) E2 and thromboxane (Tx) B2 (the stable TxA2 by-product) from the preoptic-anterior hypothalamus (AH-POA) and the tuberal-posterior hypothalamus (PH-Tu) using a modified "push-pull" perfusion procedure. In the absence of fever, PGE2 release was steady from the 2nd h of perfusion onward, its rate at either site ranging between 0.08 and 0.12 pg/min. Local treatment with probenecid (1 mM) increased PGE2 release about threefold. Compared with PGE2, basal release of TxB2 was greater (0.15-0.43 pg/min) and, occasionally, tended to fall with time. Both compounds were found in higher amounts (2- to 10-fold increase) after locally injecting endotoxin, and the effect was greater in AH-POA than PH-Tu. Conversely, intravenous endotoxin (bolus) or interleukin 1 (IL-1) (bolus plus infusion) at doses causing a sustained fever selectively stimulated the formation of PGE2, but the response itself did not differ between AH-POA and PH-Tu. In either region, the degree of enhancement in PGE2 release correlated with the magnitude of the fever. Intravenous indomethacin (2 mg/kg) reversed both the fever and PGE2 elevation. These findings support an intermediary role for PGE2 in the central action of pyrogens and the ensuing fever. Blood-borne pyrogens may act at multiple sites in brain, which are tentatively identified with the circumventricular organs.

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

The time-dependent release of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), thromboxane (Tx) B2, and 6-keto-PGF1 alpha (6-keto) from brain was measured before, during, and after a 15-min interval of total ischemia (four-vessel occlusion) in halothane-anesthetized cats using the technique of cerebroventricular perfusion. Resting levels of PGE2, PGF2 alpha, 6-keto, and Tx were: 253 +/- 75, 953 +/- 300, 650 +/- 200, and 550 +/- 170 pg/ml, respectively. During the 15-min ischemia, all prostanoids rose significantly, yet the highest levels were not observed until the first 15-60 min of the reflow at which time levels of PGE2, PGF2 alpha, 6-keto, and Tx, as compared with the preischemic baseline, rose approximately 8, 3.4, 3, and 55-fold, respectively. Significantly, although all prostanoids showed increases relative to baseline, the ratios of PGF2 alpha/6-keto and PGE2/6-keto remained stable throughout the experiment in both groups of animals. In contrast, the Tx/6-keto ratio rose from approximately 1 to approximately 30 during the 60 min after reflow in untreated cats. Treatment with zomepirac sodium (5 mg/kg, i.v.), a cyclooxygenase inhibitor, resulted in highly significant reductions in the levels of all prostanoids during the preischemic period. In zomepirac sodium-treated animals, there were also highly significant reductions in the prostanoid response to ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostacyclin (PGI2) is an inhibitor of platelet function in vitro. We tested the hypothesis that PGI2 is formed in biologically active concentrations at the platelet-vascular interface in man and can be pharmacologically modulated to enhance its inhibitory properties. This became feasible when we developed a microquantitative technique that permits the measurement of eicosanoids in successive 40-microliters aliquots of whole blood emerging from a bleeding time wound. In 13 healthy volunteers the rate of production of thromboxane B2 (TXB2) gradually increased, reaching a maximum of 421 +/- 90 (mean +/- SEM) fg/microliters per s at 300 +/- 20 s. The hydration product of PGI2, 6-keto-PGF1 alpha, rose earlier and to a lesser degree, reaching a peak (68 +/- 34 fg/microliters per s) at 168 +/- 23 s. The generation of prostaglandins PGE2 and D2 resembled that of PGI2. Whereas the threshold concentration of PGI2 for an effect on platelets in vitro is approximately 30 fg/microliters, only less than 3 fg/microliters circulates under physiological conditions. By contrast, peak concentrations of 6-keto-PGF1 alpha obtained locally after vascular damage averaged 305 fg/microliters. Pharmacological regulation of PG endoperoxide metabolism at the platelet-vascular interface was demonstrated by administration of a TX synthase inhibitor. The rate of production of PGI2, PGE2, and PGD2 increased coincident with inhibition of TXA, as reflected by three indices; the concentration of TXB2 in bleeding time blood and serum, and excretion of the urinary metabolite, 2,3-dinor-TXB2. These studies indicate that PGI2 is formed locally in biologically effective concentrations at the site of vessel injury and provide direct evidence in support of transcellular metabolism of PG endoperoxides in man.

Toxoplasma gondii tachyzoites markedly alter the profile of eicosanoids released by human mononuclear phagocytes. Freshly isolated, 2-h adherent human monocytes release both cyclooxygenase (e.g., thromboxane [TX] B2, prostaglandin [PG] E2) and 5-lipoxygenase (e.g., leukotriene [LT] B4, LTC4) products of arachidonic acid metabolism after stimulation by the calcium ionophore A23187 or ingestion of opsonized zymosan particles or heat-killed T. gondii. However, after incubation with viable T. gondii, normal and chronic granulomatous disease monocytes release only the cyclooxygenase products TXB2 and PGE2 and fail to form LTB4, LTC4, or other 5-lipoxygenase products. Monocytes maintained in culture for 5 d lose this capacity to release TXB2 and PGE2 after incubation with T. gondii. T. gondii significantly inhibit calcium ionophore A23187-induced LTB4 release by monocyte-derived macrophages; heat-killed organisms do not affect this calcium ionophore A23187-induced release of LTB4. T. gondii-induced inhibition of LTB4 release by calcium ionophore A23187-stimulated monocyte-derived macrophage is reversed by interferon (IFN)-gamma treatment of the monolayers. LTB4 induced extensive damage to the cellular membranes and cytoplasmic contents of the organisms as observed by transmission electron microscopy. Exogenous LTB4 (10(-6) M) induced intracellular killing of ingested T. gondii by non-IFN-gamma-treated monocyte-derived macrophages. IFN-gamma-induced antitoxoplasma activity in monocyte-derived macrophages was inhibited by the selective 5-lipoxygenase inhibitor zileuton but not by the cyclooxygenase inhibitor indomethacin. These findings suggest a novel role for 5-lipoxygenase arachidonic acid products in human macrophage IFN-gamma-induced antitoxoplasma activity.

BACKGROUND

Even though the acetylation of platelet cyclooxygenase (COX)-1 at serine-529 is the direct mechanism of action of low-dose aspirin, its antiplatelet effect has been characterized using indirect indexes of COX-1 activity.

OBJECTIVE

We performed a clinical study with enteric-coated low-dose aspirin (EC-aspirin), in healthy subjects, to evaluate the effects on the extent and duration of platelet COX-1 acetylation, using a novel proteomic strategy for absolute protein quantification (termed AQUA), as compared with traditional pharmacokinetic and pharmacodynamic parameters.

METHODS

In a phase I, single-arm, open-label study of EC aspirin (100 mg day(-1) ) administered to 24 healthy subjects, we compared, over a 24 h-period on day 1 and 7, % platelet acetylated COX-1 (AceCOX-1) with traditional pharmacokinetic and pharmacodynamics [i.e. serum thromboxane (TX) B2 , platelet function by monitoring CEPI(collagen/epinephrine) closure time (CT) using whole-blood PFA-100 and urinary excretion of 11-dehydro-TXB2 ] parameters.

RESULTS

Acetylation of platelet COX-1 was measurable before detection of aspirin levels in the systemic circulation and increased in a cumulative fashion upon repeated dosing. After the last dose of EC-aspirin, %AceCOX-1, serum TXB2 and CEPI-CT values were maximally and persistently modified throughout 24 h; they averaged 76 ± 2%, 99.0 ± 0.4% and 271 ± 5 s, respectively. EC-aspirin caused 75% reduction in urinary 11-dehydro-TXB2 excretion. After chronic dosing with aspirin, the pharmacokinetics of acetylsalicylic acid was completely dissociated from pharmacodynamics.

CONCLUSIONS

The demonstrated feasibility of quantifying the extent and duration of platelet COX-1 acetylation will allow characterizing the genetic, pharmacokinetic and pharmacodynamic determinants of the inter-individual variability in the antiplatelet response to low-dose aspirin as well as identifying extra-platelet sites of drug action.

Injection of lambda-carrageenin into the pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B2 were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethylisopropylsilyl (DMiPS) ether or ME-methoxime-DMiPS ether derivatives, 6-keto-PGF1 alpha reached the maximum at 1 hr after carrageenin, then PGE2 and TXB2 showed peaks at 3 hr and waned off before 9 hr. The PGF/ alpha level was kept low, but PGD2, PGE1 and PGF1 alpha were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB2 levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE2, definitely play an important role in the exudation during the first 5 hr.

Thromboxane (TX) B2, 2,3-dinor-TXB2, 11-dehydro-TXB2, 6-oxoprostaglandin (PG)F1 alpha and 2,3-dinor-6-oxo-PGF1 alpha were measured in 24 h urine samples obtained from 30 apparently healthy chronic cigarette smokers and 37 closely matched non-smoking control subjects. Samples were analysed using a newly developed assay based on immunoaffinity chromatography and capillary column gas chromatography/electron capture negative ion chemical ionisation mass spectrometry. There were significant and comparable increases in the excretion rates of both 2,3-dinor-TXB2 and 11-dehydro-TXB2 in the smoking compared with the non-smoking group (2P less than 0.001). Excretion rates of 2,3-dinor-TXB2 were 418 +/- 35 and 265 +/- 26 pg/mg creatinine in the two groups, respectively. 11-Dehydro-TXB2 excretion rates were 440 +/- 54 and 221 +/- 18 pg/mg creatinine, respectively (mean +/- S.E.). There were significant (2P less than 0.05) positive correlations between average reported cigarette consumption and excretion of both thromboxane metabolites. There were small but significant (2P less than 0.02) increases in the excretion rates of both 6-oxo-PGF1 alpha and 2,3-dinor-6-oxo-PGF1 alpha in the smoking compared with the non-smoking group. There was no significant difference in the rates of excretion of TXB2 in the two groups. The effects of acute cigarette smoke exposure (five cigarettes in 2 h) was also studied in four normally non-smoking healthy volunteers. There was no significant change in the excretion rate of any of the eicosanoids measured during control and smoking periods (at least 2 weeks apart), indicating that increased TXA2 biosynthesis in chronic smokers is unlikely to be a consequence of acute platelet activation.

Prostaglandins are important modulators of the action of vasopressin. Others researchers have proposed that vasopressin stimulates prostaglandin synthesis, completing a negative feedback loop and thereby limiting vasopressin's antidiuretic effect. We have re-examined this question, using specific radioimmunoassay and thin-layer radiochromatography to determine prostaglandin synthesis by the toad bladder. Under control conditions, the bladder synthesizes prostaglandin (PG)E2 and thromboxane (TX)B2. There was no evidence for synthesis of PGE1 or PGF2 alpha by radioimmunoassay, or of other prostaglandins by radiochromatography. Furthermore, there was no evidence for metabolism of PGE2 by the bladder. Using a variety of protocols, in isolated epithelial cells as well as intact bladders, we were unable to detect any significant increase in PGE2 or TXB2 synthesis after stimulation with arginine vasopressin (AVP) or deamino-8-D-arginine vasopressin (DDAVP). Arachidonic acid, the specific precursor of prostaglandin synthesis, increased PGE2 synthesis twofold, and significantly inhibited AVP- and DDAVP-stimulated water flow by 60 and 75%, respectively. Naproxen and acetaminophen inhibited prostaglandin synthesis and enhanced water flow in response to AVP and DDAVP (44-54%). Our findings indicate that the toad bladder produces tow prostaglandins, PGE2 and TXB2, and that vasopressin does not alter their rate of synthesis. Because agents such as acetaminophen and naproxen inhibit prostaglandin synthesis and enhance vasopressin- and DDAVP-stimulated water flow, we suggest that it is the inhibitory effect of these agents on the hormone-independent rate of prostaglandin synthesis that is responsible for their enhancement of water flow. Furthermore, because AVP appears to increase prostaglandin synthesis by the intact kidney, we suggest that cells other than those of the collecting tubule are responsible for the increased prostaglandin production.

Platelet-activating factor (PAF), as well as PAF acetylhydrolase (PAF-AH) activity in the peripheral blood plasma of patients with psoriasis and palmoplantar pustolosis, was measured with a radioimmunoassay technique, and compared with leukotriene (LT) B4, LTC4, LTD4 and E4 (LTD4/E4), thromboxane (TX) B2 and prostaglandin (PG) E2 levels. In a normal healthy group (n = 12) PAF level was 25.9 +/- 6.5 pg/0.1 ml plasma (mean +/- standard error of the mean: SEM), and this was elevated in patients with psoriasis (68.1 +/- 11.8, n = 25, P < 0.01), without a change in the PAF-AH level. LTB4 showed a similar increase (115.0 +/- 21.6 pg/ml vs. 68.2 +/- 11.8 pg/ml, P < 0.05), while TXB2 and PGE2 showed insignificant (P>> 0.05) changes. LTC4 and LTD4/E4 were around the level of the limit of detection. Patients with palmoplantar pustulosis (n = 33) demonstrated similar, but milder and statistically insignificant, increases in PAF, LTB4, TXB2 and PGE2 levels. Modulation of the mediator levels before and after treatment was compared in 16 patients with psoriasis and 11 with palmoplantar pustulosis. PAF in psoriasis significantly decreased after treatment (70.9 +/- 17.1 to 25.1 +/- 5.5, P < 0.05) and this was moderately correlated (r = 0.298) with clinical improvement as indicated by the psoriasis area and severity index (38.5 +/- 7.5 to 10.9 +/- 4.2, P < 0.01). TXB2 (180.2 +/- 100.4 to 34.1 +/- 13.5), PGE2 (3.7 +/- 0.7 to 2.9 +/- 0.5) and LTB4 (120.1 +/- 31.1 to 84.2 +/- 8.2), in psoriasis, mildly decreased without statistical significance. Patients with palmoplantar pustulosis demonstrated a similar decrease in all mediators without statistical significance. The results obtained suggest a role of PAF in psoriasis. As the priming effects of PAF have been shown, for leucocytes and endothelial cells, to enhance their inflammatory response, we assume that PAF has roles in the acute phase of psoriatic and leucotactic inflammation.

Previously we reported that cultured endothelial cells (ECs) can promote or inhibit polymorphonuclear leukocyte (PMN) diapedesis and albumin permeability in vitro by altering monolayer intercellular integrity (an activity influenced by pretreatment with exogenous amines). Endothelial eicosanoid release was also seen to stimulate both PMN motility and diapedesis. We now demonstrate that these endothelial activities are related. Thromboxane (Tx) B2 pretreatment of ECs results in increased diapedesis and permeability across the monolayers whereas 6-keto-PGF1 alpha pretreatment has the opposite effects, demonstrating that these eicosanoids exert direct effects upon ECs, in addition to their direct effects upon PMNs as previously described. Norepinephrine (NE) or serotonin (5HT) pretreatment of ECs inhibits the release of TxB2 and 6-keto-PGF1 alpha, with the result that the stimulation of PMN motility by these EC metabolites is eliminated. In contrast, histamine increases the endothelial release of eicosanoids, resulting in a further increase in PMN motility. We conclude that histamine directly reduces EC monolayer integrity (by altering the endothelial cytoskeleton) and also increases eicosanoid release, actions which both enhance PMN motility and further reduce monolayer integrity. Conversely, NE and 5HT both increase intercellular integrity and decrease eicosanoid release, thereby decreasing PMN motility, diapedesis, and albumin permeability.

Conscious cats were used to study the effects of endotoxin and interleukin 1 (IL 1) on levels of prostaglandin (PG) E2 and thromboxane (TX) B2 (the stable TXA2 byproduct) in cerebrospinal fluid (CSF) from the third ventricle. Pyrogens were given intravenously or intraventricularly and prostanoids were measured by radioimmunoassay. PGE2 was normally less abundant than TXB2 (mean, 37 vs. 528 pg/ml), and its level increased severalfold during the sustained fever following intravenous endotoxin (bolus) or IL 1 (bolus plus infusion). PGE2 elevation preceded the fever and was maintained thereafter. Likewise, intraventricular pyrogens promoted PGE2 formation, and their effect was also manifest during the latent period of the fever. The PGE2 metabolite, 13,14-dihydro-15-keto-PGE2, was not measurable in CSF from either afebrile or febrile animals. Basal content of PGE2, on the other hand, was higher in animals pretreated with probenecid (30 mg/kg ip or iv; 50 or 100 micrograms ivt), confirming the importance of transport processes in removing prostanoids from brain. Unlike PGE2, TXB2 levels did not change during the fever to intravenous endotoxin. TXB2 rose instead in response to intraventricular endotoxin, although the elevation did not extend beyond fever uprise. Furthermore, a TXA2 analog (ONO-11113;2 or 4 micrograms ivt) had inconsistent effects on body temperature, while a TXA2 antagonist (ONO-11120;2 micrograms ivt) did not interfere with endotoxin fever. These findings strongly support a causative role for PGE2 in the onset and progression of pyrogen fever. No evidence of a similar role was obtained for TXA2.

Thrombotic complications of pulmonary circulation occur in patients with chronic obstructive pulmonary disease (COPD). In the present study, we sought to evaluate in vivo platelet activation through the measurement of 11/dehydro-thromboxane (Tx) B2 TxA2 major metabolite in the urine, in 29 patients with COPD, compared with 29 sex- and age-matched healthy subjects. The urinary excretion of 11-dehydro-TxB2 was significantly higher in patients with COPD than in control subjects: median (range), 753 (277-4,409) and 275 (129-612) pg/mg creatinine, respectively; p < 0.0001). Moreover, 11-dehydro-TxB2 excretion was inversely related with arterial oxygen tension (rho = -0.46; p = 0.0145). In five of the 29 patients a short-term therapeutic course with oxygen supplementation induced a significant decrease of urinary 11-dehydro-TxB2 excretion: median range, 941 (452-2,640) to 445 (166-1,560) pg/mg creatinine. Moreover, selective inhibition of platelet cyclooxygenase activity by low-dose aspirin was associated with more than 90% inhibition of thromboxane metabolite excretion, demonstrating its being of platelet origin. Plasma levels of prothrombin fragment F1 + 2 were higher in patients than in control subjects (2.6 +/- 1.5 versus 0.9 +/- 0.4 nM, p = 0.0001). No relation between 11-dehydro-TxB2 excretion and plasma F1 + 2 levels was found. We conclude that platelet TxA2 biosynthesis is enhanced in patients with COPD and may be influenced by arterial oxygen tension changes.

Human polymorphonuclear leukocytes (PMN) activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), in the presence of cytochalasin B, are able to induce activation of coincubated autologous platelets "via" cathepsin G released from the azurophilic granules. However, thromboxane (Tx) B2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed, the amount of TxB2 found in supernatants of platelet/PMN suspensions challenged with 1 mumol/L fMLP was twofold to fourfold higher than that measured when platelets were stimulated by supernatants from fMLP-activated PMN. In the present report, we analyzed the possibility that PMN-induced TxB2 production in this system is the result of transcellular metabolism of arachidonic acid (AA) between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labeled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. Our results showed that: (1) 3H-TxB2 and 3H-12-HHT are synthesized when 3H-AA-labeled PMN are activated mixed to unlabeled platelets; (2) total radioactivity released by fMLP-stimulated PMN is increased in the presence of platelets, whereas the membrane content of unesterified 3H-AA is reduced; (3) platelet cyclooxygenase inhibition completely prevents 3H-TxB2 synthesis; and (4) inhibition of cathepsin G-induced platelet activation with the antiprotease eglin C blocks the formation of 3H-TxB2. These data show that in the experimental system used, platelets use PMN-derived unmetabolized AA to synthesize TxB2.

OBJECTIVE

The role of cyclooxygenase (COX)-2 was examined using a rat endotoxin shock model and the potency and selectivity of NS-398, a COX-2 selective inhibitor in vitro, for COX-2 activity was examined in vivo.

METHODS

Male Wistar rats (weighing 140-180 g) were used.

METHODS

Lipopolysaccharide (LPS, 1 mg/kg, i.v.) was administered to rats (LPS-treated rats) and expression of COX-1 mRNA and COX-2 mRNA in the aorta and peripheral blood leukocytes was examined by RT-PCR. COX activity was assessed by measuring the plasma 6-keto prostaglandin (PG) F1 alpha, PGE2 and thromboxane (TX)B2 30s after administration of arachidonic acid (AA, 3 mg/kg, i.v.), NS-398 (0.3-100 mg/kg, p.o.) or indomethacin (0.3-3 mg/kg, p.o.) was administered 1 h before the AA injection.

RESULTS

COX-2 mRNA was detectable in the aorta and peripheral blood leukocytes at least from 3 to 9 h after the LPS injection but not in non-LPS-treated rats. Plasma 6-keto PGF1 alpha, PGE2 and TXB2 levels after AA injection into LPS-treated rats were significantly enhanced compared to findings in non-LPS-treated rats. NS-398 showed significant inhibition of the increase in PGs in LPS-treated rats, the ED50 values being 0.35 mg/kg for 6-keto PGF1 alpha, 1.5 mg/kg for PGE2 and < 0.3 mg/kg for TXB2. NS-398 even at 100 mg/kg did not significantly suppress the increased PGs levels in non-LPS-treated rats. In contrast, indomethacin significantly inhibited plasma PGs levels after AA injection into LPS-treated rats and non-LPS-treated rats. The ED50 values in LPS-treated rats, determined by 6-keto PGF1 alpha, PGE2 and TXB2 production, were 1.0, 1.3 and 2.3 mg/kg and those in non-LPS-treated rats were 0.42, 0.24 and 0.93 mg/kg, respectively.

CONCLUSIONS

In a rat endotoxin shock model, expression of COX-2 plays a role in an increase in COX activity. NS-398 showed preferential inhibitory effects on COX-2 activity in vivo. This approach is useful to directly analyze the inhibitory activity of NSAIDs for COX-1 and COX-2 in vivo.

The possibility that isosorbide dinitrate (ISDN) inhibits platelet function in humans has been explored in vitro and in vivo. Incubation of citrated platelet-rich plasma from healthy subjects with scalar concentrations (1.25, 12.5 and 125 micrograms/ml) of ISDN for 5 and 10 minutes resulted in a decrease in platelet aggregation after ADP, adrenaline, and arachidonic acid at the highest drug concentration (mean decrease: 72% [p less than 0.01], 56% [p less than 0.05] and 62% [p less than 0.05], respectively, with the 10-minute incubation). Also, a significant reduction (30%) in generated thromboxane (TX)B2 levels was observed after arachidonic acid (p less than 0.01). ISDN was then infused at rate of 4 mg/hour for 30 minutes in 11 patients with angina and at a rate of 30 mg/hour for 20 minutes in 8. The smaller dose, which caused minor changes in arterial pressure and heart rate, was accompanied by a marked, significant decrease in ADP- and adrenaline-induced aggregation, with a nadir at 60 minutes from the infusion stop (decreases of 40% and 51% respectively). Circulating platelet aggregates also decreased, with a minimum (-41%, p less than 0.05) at the end of the infusion. The higher infusion rate, causing marked hemodynamic effects, was not accompanied by the occurrence of clear antiplatelet effects. Thus, ISDN can affect platelet function both in vitro and in vivo. The in vivo effect occurs at lower concentrations than in vitro but is blunted when a marked hemodynamic response occurs.

OBJECTIVE

The goal of this study was to test the hypothesis that NCX-4016 may have broader anti-inflammatory and antithrombotic effects as well as better gastric tolerability than aspirin in humans.

BACKGROUND

NCX-4016 is an aspirin derivative containing a nitric oxide-releasing moiety that prevents platelet activation and modulates tissue factor (TF) expression and cytokine release from lipopolysaccharide (LPS)-stimulated monocytes.

METHODS

This was a blind-observer, placebo-controlled, parallel-group study in which 48 healthy subjects were randomized to receive NCX-4016 800 mg twice a day, NCX-4016 800 mg twice a day plus aspirin 325 mg, aspirin 325 mg, or placebo for 21 days.

RESULTS

Similar to aspirin alone, NCX-4016 effectively inhibited platelet aggregation induced by 0.6 mmol/ arachidonic acid, clot-stimulated thromboxane (TX) B2 generation in whole blood, and urinary excretion of 11-dehydro-TXB2. Unlike aspirin alone, the administration of NCX-4016 significantly inhibited TF expression in monocytes stimulated ex vivo with 10 micromol/l LPS (determined by flow-cytometry analysis of TF on CD14 positive cells). NCX-4016 also inhibited the rapid TF expression induced in monocytes by a proteinase activated receptor agonist (thrombin receptor activator protein, 2 micromol/l) as well as LPS-induced expression of CD11b . Ex vivo, release of MCP-1 and interleukin-6 were significantly inhibited by NCX-4016, but not by aspirin. NCX-4016 was not associated with gastric damage, and significantly reduced gastric injury when co-administered with aspirin, although both drugs reduced gastric PGE2 production to the same extent.

CONCLUSIONS

NCX-4016 is equally effective as aspirin in inhibiting cyclooxygenase activity. However, NCX-4016 causes less gastric damage and prevents monocyte activation. Larger multicenter trials are warranted to establish clinical efficacy and safety of NCX-4016.

BACKGROUND

We have established that thromboxane B2 (TX) blood levels increase across the pulmonary circulation after total cardiopulmonary bypass (CPB) but not after partial CPB. In the present study, we used the same model and examined the parameters of pulmonary injury after total or partial CPB.

RESULTS

Fourteen anesthetized sheep were placed on total CPB (n = 7), without ventilation and with occlusion of the pulmonary artery, or partial CPB (n = 7), with ventilation and an open pulmonary artery. After 90 minutes, the sheep were separated from CPB, and the pulmonary artery was perfused normally. After 30 minutes, we elevated left atrial pressure in all sheep. Plasma TX, plasma leukotriene B4, platelet counts, white blood cell counts, and plasma protein concentration were measured before CPB and every 15 minutes after CPB for 1 hour. The right and left atrial blood samples were obtained simultaneously. Pulmonary arterial pressure, left atrial pressure, and pulmonary arterial flow were measured. Pulmonary vascular resistance (PVR) was calculated for 30 minutes after CPB. Lung lymph protein, TX, leukotriene B4, and flow were measured before CPB and every 30 minutes after CPB for 1 hour. Pulmonary biopsies and bronchoalveolar lavage fluid were obtained before CPB and at the end of the experiment. After total CPB, levels of TX across the pulmonary circulation increased significantly, but leukotriene B4 remained constant. Platelets and white blood cells were consumed across the pulmonary circuit after total CPB but not after partial CPB. PVR increased by 170%, lymph flow increased by 233%, lung water content increased by 15%, and the ratio of lymph to plasma protein decreased by 20% after total CPB, but similar changes did not occur after partial CPB.

CONCLUSIONS

During total CPB, the lungs are totally dependent on oxygen supply provided by nonpulsatile bronchial arterial flow. Lung injury seen with restoration of pulmonary artery flow and ventilation may be the result of an inflammatory response associated with TX elevation after a period of relative pulmonary ischemia. Pulmonary injury was not seen after less severe pulmonary arterial flow deprivation, with maintenance of ventilation (partial CPB). Although the specific cause is undetermined from these data, the occurrence of elevated TX levels and lung damage after total CPB is clearly established.

To investigate the role of vasodilator prostanoids in the CO2-induced relaxation of cerebral arterioles, the present study examined the effect of exogenous prostaglandin (PG) E2 and nonprostanoid vasodilators, adenosine and sodium nitroprusside, on the indomethacin-impaired pial arteriolar response to CO2 in newborn piglets. Reactivity of pial arterioles (52-131 microns diam) was determined using a closed cranial window with intravital microscopy. Cortical prostanoid synthesis was assessed by analyzing for select prostanoids in cerebrospinal fluid sampled from under the cranial window. Inhalation of 7% CO2 caused an elevation of cortical 6-keto-PGF1 alpha and thromboxane (Tx) B2 and increased pial arteriolar diameter by 34 +/- 5%. Two cyclooxygenase inhibitors, indomethacin (5 mg/kg i.v.) and ibuprofen (30 mg/kg i.v.), abolished the CO2-induced elevation of cortical prostanoids. Indomethacin, but not ibuprofen, blocked the CO2-induced increase in pial arteriolar diameter. The indomethacin-impaired vasodilator response to CO2 was restored when PGE2 (0.1-1 microM) was applied topically to the cortical surface. Adenosine (1-100 microM) and sodium nitroprusside (0.5 microM) only partially restored the vasodilator response to CO2. The data suggest that vasodilator prostanoids facilitate cerebrovascular relaxation to CO2 and may play a permissive role in the relaxation response of vascular smooth muscle. The fact that adenosine (adenosine 3',5'-cyclic monophosphate-mediated dilator) and sodium nitroprusside (guanosine 3',5'-cyclic monophosphate-mediated dilator), were partially effective suggests a role for those intracellular signaling pathways. We speculate that receptor activation of intracellular pathways may alter Ca2+ sensitivity of the contractile apparatus in such a way that the relaxation response to CO2 can occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet density profiles, intraplatelet nucleotides, intraplatelet beta thromboglobulin (beta TG), plasma beta TG levels, intraplatelet cyclic AMP (cAMP) levels, platelet release reaction, platelet thromboxane (TX)B2 production and plasma fibrinogen levels were investigated in 24 newly diagnosed, non-insulin-dependent diabetic patients and 12 comparable controls. These variables were measured at diagnosis, after a 3-6 week dietary run-in period, and again after 6 months on treatment with either metformin or gliclazide therapy. With dietary restriction of refined carbohydrate and oral hypoglycaemic therapy, there was a reduction in platelet density (p less than 0.05), intraplatelet nucleotides (p less than 0.001), intraplatelet beta TG (p less than 0.001), plasma beta TG (p less than 0.001) and there was an increase in intraplatelet cAMP levels (p less than 0.05). Although these platelet variables returned towards normal, only the platelet density mean returned to within the normal range. There was no significant change in the platelet TXB2 production and plasma fibrinogen levels with treatment. Metformin and gliclazide were equally effective in the glycaemic control of non-insulin-dependent diabetes, and there was no difference between the platelet variables measured in the two groups. We would therefore suggest that improvement of glycaemic control, rather than any specific effect of the oral hypoglycaemic agent employed, is the most important factor in returning these parameters towards normality.

In a model of closed head injury in rats, a calibrated weight drop device was allowed to fall onto the skull's convexity over the left hemisphere 1 to 2 mm lateral from the midline. Prostaglandin (PG) levels were determined in the frontal cortex region remote from the site of injury where no macroscopic damage could be seen. Differential patterns of temporal changes were evident for PGE2, PGD2, thromboxane (TX) B2, and 6-keto-PGF1 alpha in the contused hemisphere, but no changes were found in the contralateral hemisphere. The major changes in PG levels were increased levels of PGD2 and 6-keto-PGF1 alpha that persisted from 18 hours until 10 days after injury. The ratio between TXB2 and 6-keto-PGF1 alpha, which reflects the vascular tone, increased during the early postinjury period (15 minutes and 1 hour) and decreased later, up to 10 days. Thus, a sustained imbalance in favor of the vasodilator is apparent; this may suggest an improved blood supply to the region. Both PGD2 and PGI2 have protective effects in the brain. We suggest that their endogenous increase may be part of a repair mechanism at the periphery of the injured zone.