Metabolite Thromboxane B2 Research Articles

Impact of Short-Term Diesel Exhaust Exposure on Prothrombotic Markers in COPD: A Randomized, Double-blinded, Crossover Study.

Rationale: Growing evidence suggests that air pollution exposure is a major risk factor in chronic obstructive pulmonary disease (COPD) that is associated with an increased prothrombotic state and adverse cardiovascular outcomes. However, much of this work is based on observational data or human exposure studies involving younger participants. The biological causality and mechanism of air pollution-induced prothrombotic response in patients with COPD remain to be explored. Objective: The main aim of this work was to investigate the impact of short-term diesel exhaust (DE) exposure on circulating prothrombotic markers-fibrinogen and plasminogen activator inhibitor-1 (PAI-1)-and urinary eicosanoids in patients with COPD. Methods: Twenty-nine research participants were recruited in this randomized, double-blinded, crossover, controlled human exposure study to DE. Participants included former smokers with and without mild or moderate COPD (ES and COPD group) and healthy never-smokers without COPD (NS group). Each participant was exposed to DE (300 µg/m3 of PM2.5) and filtered air (FA) for 2 hours on different occasions, in randomized order, separated by a 4-week washout. Blood and urine samples were collected prior to and 24 hours after each exposure. Plasma fibrinogen and serum PAI-1 concentrations were quantified using ELISAs. Urinary eicosanoid concentrations were quantified using ultra- performance liquid chromatography coupled to tandem mass spectrometry. Linear mixed-effects models were used for statistical comparisons. Results: Participants with COPD showed an increase in plasma fibrinogen (effect estimate: 1.27 [1.06 to 1.53], p=0.01) after DE relative to FA, but no significant DE-associated change in serum PAI-1 (0.95 [0.87 to 1.04], p=0.26). In never-smokers and ex-smokers without COPD, fibrinogen (NS group: 1.10 [0.99 to 1.23], p=0.08; ES group: 0.86 [0.68 to 1.09], p=0.08] and PAI-1 (NS group: 1.12 [ 0.96 to 1.32], p=0.15; ES group: 0.90 [0.79 to 1.03], p=0.13) were not changed after DE exposure. COPD participants showed a DE-attributable increase in urinary thromboxane B2 (TXB2) metabolites concentrations as follows: 11-dehydro TXB2 (1.45 [1.02 to 2.08], p=0.04); 2,3-dinor-TXB2 (1.45 [1.05 to 2.00], p=0.03). Conclusions: Participants with COPD had increased plasma fibrinogen and urinary TXB2 metabolites after short-term DE exposure, suggesting they may be more susceptible to pollution-attributable prothrombotic response compared to healthy controls or ex-smokers without COPD. Clinical trial registration available at www.clinicaltrials.gov, ID: NCT02236039.

Adjustment for Renal Function Improves the Prognostic Performance of Urinary Thromboxane Metabolites.

Systemic thromboxane A2 generation, assessed by quantifying the concentration of stable thromboxane B2 metabolites (TXB2-M) in the urine adjusted for urinary creatinine, is strongly associated with mortality risk. We sought to define optimal TXB2-M cutpoints for aspirin users and nonusers and determine if adjusting TXB2-M for estimated glomerular filtration rate (eGFR) in addition to urinary creatinine improved mortality risk assessment. Urinary TXB2-M were measured by competitive ELISA in 1363 aspirin users and 1681 nonusers participating in the Framingham Heart Study. Cutpoints were determined for TXB2-M and TXB2-M/eGFR using log-rank statistics and used to assess mortality risk by Cox proportional hazard modeling and restricted mean survival time. Multivariable models were compared using the Akaike Information Criterion (AIC). A cohort of 105 aspirin users with heart failure was used for external validation. Optimized cutpoints of TXB2-M were 1291 and 5609 pg/mg creatinine and of TXB2-M/eGFR were 16.6 and 62.1 filtered prostanoid units (defined as pg·min/creatinine·mL·1.73 m2), for aspirin users and nonusers, respectively. TXB2-M/eGFR cutpoints provided more robust all-cause mortality risk discrimination than TXB2-M cutpoints, with a larger unadjusted hazard ratio (2.88 vs 2.16, AIC P < 0.0001) and greater differences in restricted mean survival time between exposure groups (1.46 vs 1.10 years), findings that were confirmed in the external validation cohort of aspirin users. TXB2-M/eGFR cutpoints also provided better cardiovascular/stroke mortality risk discrimination than TXB2-M cutpoints (unadjusted hazard ratio 3.31 vs 2.13, AIC P < 0.0001). Adjustment for eGFR strengthens the association of urinary TXB2-M with long-term mortality risk irrespective of aspirin use.

Nonplatelet thromboxane generation is associated with impaired cardiovascular performance and mortality in heart failure.

Nonplatelet thromboxane generation, stimulated largely by oxidative stress, is a novel mortality risk factor in individuals with coronary artery disease. Though inversely associated with left ventricular ejection fraction (LVEF), a potential role in the pathobiology of heart failure (HF) remains poorly defined. Nonplatelet thromboxane generation and oxidative stress were assessed by measuring urine thromboxane-B2 metabolites (TXB2-M) and 8-isoPGF2α by ELISA in 105 subjects taking aspirin and undergoing right heart catheterization for evaluation of HF, valve disease, or after transplantation. Multivariable logistic regression and survival analyses were used to define associations of TXB2-M to invasive measures of cardiovascular performance and 4-year clinical outcomes. TXB2-M was elevated (>1,500 pg/mg creatinine) in 46% of subjects and correlated with HF severity by New York Heart Association (NYHA) functional class and brain natriuretic peptide level, modestly with LVEF, but not with HF etiology. There was no association of oxidative stress to HF type or etiology but a trend with NYHA functional class. Multiple invasive hemodynamic parameters independently associated with TXB2-M after adjustment for oxidative stress, age, sex, and race with pulmonary effective arterial elastance (Ea pulmonary), reflective of right ventricular afterload, being the most robust on hierarchical analysis. Similar to Ea pulmonary, elevated urinary TXB2-M is associated with increased risk of death (adjusted HR = 2.15, P = 0.037) and a combination of death, transplant, or mechanical support initiation (adjusted HR = 2.0, P = 0.042). Nonplatelet TXA2 thromboxane generation is independently associated with HF severity reflected by invasive measures of cardiovascular performance, particularly right ventricular afterload, and independently predicted long-term mortality risk.NEW & NOTEWORTHY Nonplatelet thromboxane generation in heart failure is independently associated with risk of death, transplant, or need for mechanical support. Measurement of urine thromboxane metabolites using a clinically available assay may be a useful surrogate for invasive measurement of cardiovascular hemodynamics and performance that could provide prognostic information and facilitate tailoring of therapy in patients with heart failure. Inhibiting thromboxane generation or its biological effects is a potential strategy for improving cardiovascular performance and outcomes in heart failure.

Association of Thromboxane Generation With Survival in Aspirin Users and Nonusers

BackgroundPersistent systemic thromboxane generation, predominantly from nonplatelet sources, in aspirin (ASA) users with cardiovascular disease (CVD) is a mortality risk factor. ObjectivesThis study sought to determine the mortality risk associated with systemic thromboxane generation in an unselected population irrespective of ASA use. MethodsStable thromboxane B2 metabolites (TXB2-M) were measured by enzyme-linked immunosorbent assay in banked urine from 3,044 participants (mean age 66 ± 9 years, 53.8% women) in the Framingham Heart Study. The association of TXB2-M to survival over a median observation period of 11.9 years (IQR: 10.6-12.7 years) was determined by multivariable modeling. ResultsIn 1,363 (44.8%) participants taking ASA at the index examination, median TXB2-M were lower than in ASA nonusers (1,147 pg/mg creatinine vs 4,179 pg/mg creatinine; P < 0.0001). TXB2-M were significantly associated with all-cause and cardiovascular mortality irrespective of ASA use (HR: 1.96 and 2.41, respectively; P < 0.0001 for both) for TXB2-M in the highest quartile based on ASA use compared with lower quartiles, and remained significant after adjustment for mortality risk factors for similarly aged individuals (HR: 1.49 and 1.82, respectively; P ≤ 0.005 for both). In 2,353 participants without CVD, TXB2-M were associated with cardiovascular mortality in ASA nonusers (adjusted HR: 3.04; 95% CI: 1.29-7.16) but not in ASA users, while ASA use was associated with all-cause mortality in those with low (adjusted HR: 1.46; 95% CI: 1.14-1.87) but not elevated TXB2-M. ConclusionsSystemic thromboxane generation is an independent risk factor for all-cause and cardiovascular mortality irrespective of ASA use, and its measurement may be useful for therapy modification, particularly in those without CVD.

Platelet reactivity in overweight and obese patients undergoing cardiac surgery.

Body mass index (BMI) and specifically overweight and obesity have been associated with an increased platelet reactivity in different series of patients. This information is derived by different laboratory platelet function tests (PFTs) like mean platelet volume (MPV), platelet microparticles, thromboxane B2 metabolites, and others. Point-of-care PFT, which are often used in cardiac surgery, are rarely addressed. The present study aims to verify platelet reactivity using multiple-electrode aggregometry (MEA) as a function of BMI in cardiac surgery patients. One-hundred ninety-eight cardiac surgery patients free from the effects of drugs acting on the P2Y12 receptor and undergoing cardiac surgery received MEA-PFT immediately before surgery. Platelet reactivity was compared between normal weight and overweight-obese subjects. There were 99 underweight/normal (BMI <25), 60 overweight (BMI ≥25) and 39 obese (BMI ≥30) patients. Overweight-obese patients did not show higher platelet counts nor a clear platelet hyper-reactivity, when tested with MPV and MEA ADP test. At TRAPtest, the overweight/obese patients had a significantly (P=0.011) higher platelet reactivity (median 118, interquartile range 106-136) than controls (median 112, interquartile range 101-123) and a higher rate of platelet hyper-reactivity (odds ratio 2.19, 95% confidence interval 1.15-4.16, P=0.016) in a multivariable model. A minor association was found between the BMI and platelet reactivity at TRAPtest, with a higher degree of activity for increasing BMI. The BMI determines an increased thrombin-dependent platelet reactivity in cardiac surgery patients. Thrombin is extensively formed during cardiac surgery, and this may explain the lower postoperative bleeding observed in obese patients in previous studies.

Dysregulation of lipidomic profile and antiviral immunity in response to hyaluronan in patients with severe asthma

Author(s): Sokolowska, Milena; Chen, Li-Yuan; Liu, Yueqin; Martinez-Anton, Asuncion; Logun, Carolea; Alsaaty, Sara; Cuento, Rosemarie A; Cai, Rongman; Sun, Junfeng; Quehenberger, Oswald; Armando, Aaron M; Dennis, Edward A; Levine, Stewart J; Shelhamer, James H

Antiplatelet Effect Durability of a Novel, 24-Hour, Extended-Release Prescription Formulation of Acetylsalicylic Acid in Patients With Type 2 Diabetes Mellitus

High platelet reactivity and high platelet turnover have been implicated in incomplete platelet inhibition during immediate-release acetylsalicylic acid therapy in patients with type 2 diabetes mellitus (DM). An extended-release acetylsalicylic acid (ER-ASA; Durlaza) formulation was developed to provide 24-hour antithrombotic effects with once-daily dosing. The objective of the study was to evaluate the antiplatelet effects of ER-ASA in patients with DM. In this open-label, single-center study, patients with DM (n= 40) and multiple cardiovascular risk factors received ER-ASA 162.5mg/day for 14 ± 4days. Multiple platelet function tests, serum and urinary thromboxane B2 metabolites, prostacyclin metabolite, and high-sensitive C-reactive protein levels were assessed at 1, 12, 16, and 24hours post-dose. Patients with high platelet turnover and/or high platelet reactivity were treated with ER-ASA 325mg/day for 14 ± 4days, and laboratory analyses were repeated. All patients responded to ER-ASA 162.5mg/day as measured by arachidonic acid-induced aggregation, and there was no loss of the platelet inhibitory effect of ER-ASA 162.5mg/day over 24hours post-dose (p= not significant). The antiplatelet effect was sustained over 24hours for all platelet function measurements. Mean 1- to 24-hour serum thromboxane B2 levels were low with both doses and were lower with ER-ASA 325mg/day compared with 162.5mg/day therapy (p= 0.002). In conclusion, ER-ASA 162.5mg daily dose provided sustained antiplatelet effects over 24hours in patients with type 2 DM and multiple cardiovascular risk factors and had a favorable tolerability profile.

Abstract 14668: Prospective, Randomized Comparison of 500 mg and 250 mg Acetylsalicylic Acid I.v. and 300 mg p.o. in Patients With Acute Coronary Syndrome (ACUTE Trial)

Background: Current national and international guidelines recommend the use of oral and/or intravenous (IV) acetylsalicylic acid as an alternative mode of application in patients with acute coronary syndrome (ACS) who are unable to swallow. However, acetylsalicylic acid IV has not been investigated in clinical trials and is not available everywhere. Aim: The primary objective of the present open-label, randomized, verum-controlled, parallel-group, phase III study was to demonstrate superiority of 250 or 500 mg acetylsalicylic acid IV treatment over oral treatment with 300 mg acetylsalicylic acid tablet to inhibit thromboxane A2 release at 5 minutes after a single dose of study drug in subjects with ACS with and without ST elevation. Methods: Acetylsalicylic acid naive patients with ACS were randomized into three groups to receive a single dose of acetylsalicylic acid either 300 mg orally, 250 or 500 mg intravenously. ThromboxaneA2 release (measured as the stable serum metabolite thromboxane B2), platelet aggregation and prostacyclin levels were measured 5 and 20 minutes after study drug administration. Patients were followed for 30 days for clinical events. Results: A total of 270 patients were randomized. The results of the primary and secondary endpoints are given in the table. Conclusion: The administration of 250 or 500 mg acetylsalicylic acid intravenously compared to 300 mg orally is associated with a faster and more effective inhibition with respect to thromboxane generation and inhibition of platelet aggregation, without any increase in bleeding complications. Therefore, i.v. administration of acetylsalicylic acid should be preferred in the acute management of patients with ACS.

Metabolomic analysis revealed the role of DNA methylation in the balance of arachidonic acid metabolism and endothelial activation

Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.

The Effect of Cadmium on COX-1 and COX-2 Gene, Protein Expression, and Enzymatic Activity in THP-1 Macrophages.

The aim of this study was to examine the effects of cadmium in concentrations relevant to those detected in human serum on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) expression at mRNA, protein, and enzyme activity levels in THP-1 macrophages. Macrophages were incubated with various cadmium chloride (CdCl2) solutions for 48 h at final concentrations of 5 nM, 20 nM, 200 nM, and 2 μM CdCl2. The mRNA expression and protein levels of COXs were analyzed with RT-PCR and Western blotting, respectively. Prostaglandin E2 (PGE2) and stable metabolite of thromboxane B2 (TXB2) concentrations in culture media were determined using ELISA method. Our study demonstrates that cadmium at the highest tested concentrations modulates COX-1 and COX-2 at mRNA level in THP-1 macrophages; however, the lower tested cadmium concentrations appear to inhibit COX-1 protein expression. PGE2 and TXB2 production is not altered by all tested Cd concentrations; however, the significant stimulation of PGE2 and TXB2 production is observed when macrophages are exposed to both cadmium and COX-2 selective inhibitor, NS-398. The stimulatory effect of cadmium on COXs at mRNA level is not reflected at protein and enzymatic activity levels, suggesting the existence of some posttranscriptional, translational, and posttranslational events that result in silencing of those genes’ expression.

Comparison of Oral Aspirin Versus Topical Applied Methyl Salicylate for Platelet Inhibition

Oral acetylsalicylic acid (aspirin) is the primary antiplatelet therapy in the treatment of acute myocardial infarction and acute coronary syndrome. Methyl salicylate (MS; oil of wintergreen) is compounded into many over-the-counter antiinflammatory muscle preparations and has been shown to inhibit platelet aggregation locally and to be absorbed systemically. To assess the ability of topically applied MS to inhibit systemic platelet aggregation for patients who are unable to tolerate oral drug therapy. A randomized, prospective, blinded, crossover study was conducted in 9 healthy men, aged 30-46 years. All subjects ingested 162 mg of aspirin or applied 5 g of 30% MS preparation to their anterior thighs. There was a minimum 2-week washout period between study arms. Blood and urine were collected at baseline and at 6 hours. An aggregometer measured platelet aggregation over time against 5 standard concentrations of epinephrine, and a mean area under the curve (AUC) was calculated. Urinary metabolites of thromboxane B(2) were measured by a standard enzyme immunoassay. Differences in and between groups at baseline and 6 hours were tested by the Wilcoxon signed-rank test. Baseline platelet aggregation did not differ significantly between the 2 arms of the study (median AUC [% aggregation(*)min]; binominal confidence intervals): aspirin 183; 139 to 292 versus MS 197; 118 to 445 (p = 0.51). Both aspirin and MS produced statistically significant platelet inhibition; aspirin decreased the AUC from 183; 139 to 292 to 85; 48 to 128 (p = 0.008) and MS decreased the AUC from 197; 118 to 445 to 112; 88 to 306 (p = 0.011). No significant difference was detected between baseline and 6-hour thromboxane levels for either aspirin (p = 0.779) or MS (p = 0.327). Topical MS and oral aspirin both significantly decrease platelet aggregation in healthy human volunteers.

Assessment of aspirin resistance varies on a temporal basis in patients with ischaemic heart disease

Objective:Laboratory tests including optical platelet aggregometry (OPA), platelet function analyser (PFA-100), and thromboxane B2 (TXB2) metabolite levels have been used to define aspirin resistance. This study characterised the prevalence of...

Aspirin is insufficient in inhibition of platelet aggregation and thromboxane formation early after coronary artery bypass surgery

Aspirin administered early after coronary artery bypass grafting surgery (CABG) improves graft patency and patients survival. However, the antiplatelet effect of aspirin seems to be variable and aspirin resistance is currently still being discussed. The aim of the study was to assess aspirin efficacy in the early postoperative period. Forty patients undergoing elective CABG surgery (20 in on-pump and 20 in off-pump) were enrolled in the study. Functional and biochemical responses to aspirin were evaluated by arachidonic acid (ARA)-induced platelet aggregation and urine 11-dehydro Thromboxane B2 metabolite excretion. Samples were collected before surgery (baseline; > or =7 days after aspirin withdrawal) and on days 1, 2 and 5 after surgery. Median baseline ARA aggregability was 55%. On day 1, platelet aggregability decreased (12%, P < 0.001). On day 2, despite the aspirin administration, platelet aggregability exceeded the values from day 1 (38%, P < 0.001). Only on day 5, sufficient inhibition of platelet aggregation was achieved (8%, P < 0.001). Median preoperative urine concentration of 11-dehydroTxB2 was 106 ng/mmol of creatinine. On day 1, the concentration decreased only slightly and insignificantly (97 ng/ml, P = NS), similarly as on day 2 (86 ng/ml, P = NS). Only on day 5, significant decrease in concentration of thromboxane metabolite was achieved compared to preoperative values (46 ng/ml, P = 0.001). Aspirin did not sufficiently inhibit platelet aggregation and thromboxane formation in the early postoperative period. Thus, antiplatelet treatment strategy should be intensified or modified in patients early after bypass surgery.

Marked Interindividual Variability in the Response to Selective Inhibitors of Cyclooxygenase-2

Variability in response to drugs may influence both efficacy and safety. Cyclooxygenase (COX)-2 inhibitors pose a cardiovascular risk by potentially increasing the likelihood of thrombosis, hypertension, and atherogenesis. Differences between individuals in the response to COX-2 inhibitors would be expected to influence their susceptibility to cardiovascular complications. We examined the variability in degree and selectivity of COX-2 inhibition in humans in response to celecoxib and rofecoxib. Fifty healthy volunteers received placebo, rofecoxib (25 mg), and celecoxib (200 mg), randomized by order. COX-1 and COX-2 inhibition was determined using ex vivo and in vivo indices of enzymatic activity. A subset of 5 individuals underwent 5 replicate studies to estimate variability in drug response both within and between subjects. Despite the higher COX-2 selectivity of rofecoxib in vitro, the average selectivity attained by 25 mg rofecoxib and 200 mg celecoxib in vivo were not different. However, there was considerable variability at an individual level in the degree of COX-2 inhibition and selectivity attained by both drugs. Approximately one third of the variability was attributable to differences between individuals, suggesting the contribution of genetic sources of variance, such as candidate polymorphisms detected in COX-1 and CYP2C9. The actual degree of selectivity for inhibition of COX-2 achieved by the coxibs relates both to chemical properties of the drug and to factors within an individual that modulate drug response. These sources of variability might be exploited to identify patients uniquely susceptible to benefit or at developing risk of cardiovascular complications.

Herbal Medicine.

Herbal Medicine.

Inhibition of leukocyte eicosanoid generation and radical scavenging activity by gnaphalin, a lipophilic flavonol isolated from Helichrysum picardii.

The lipophilic aglycone 5,7-dihydroxy-3,8-dimethoxyflavone (gnaphalin) isolated from the aerial flowering parts of Helichrysum picardii Boiss. & Reuter (Asteraceae) was tested for interactions with the cyclo-oxygenase and 5-lipoxygenase pathways of arachidonate metabolism in stimulated rat peritoneal leukocytes, and for its effects on leukocyte granular enzyme release, cell viability and interactions with reactive oxygen species. Gnaphalin dose-dependently inhibited generation of the cyclo-oxygenase metabolite thromboxane B2 (IC50 = 39.9 +/- 3.9 microM), and of the 5-lipoxygenase metabolite leukotriene B4, although the potency was two-fold less (IC50 = 81.8 +/- 12.9 microM). At concentrations of 6 to 320 microM, gnaphalin did not affect secretion of the pro-inflammatory enzymes lysozyme, myeloperoxidase and beta-glucuronidase from the neutrophil secretory granules, and did not scavenge hydrogen peroxide or hypochlorous acid. However, gnaphalin effectively scavenged superoxide radicals generated in the hypoxanthine/xanthine oxidase system (IC50 = 40 microM) and by PMA-stimulated leukocytes (> 60% at 500 microM), directly inhibited xanthine oxidase (85% at 395 microM) and inhibited Fe(3+)-ascorbate-induced liposomal peroxidation (IC50 = 215 microM). Thus, like some other flavonoids found in medicinal herbs, gnaphalin possesses an array of potentially beneficial anti-eicosanoid and free-radical scavenging properties which may alongside other constituents contribute to the claimed medicinal properties of the plant from which it is derived.

Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein.

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet alpha-granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet-rich plasma with recombinant secreted APP (sAPP) isoforms dose-dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low-dose thrombin-induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either alpha- or beta-secretase cleavage. In fact, the N-terminal cysteine-rich region of APP (residues 18-194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist-induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA-induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP-inhibition of agonist-induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.

Apolipoprotein(a) enhances platelet responses to the thrombin receptor-activating peptide SFLLRN.

Elevated levels of lipoprotein(a) [Lp(a)] are correlated with an increased risk of atherosclerotic disease. We examined the effect of recombinant apolipoprotein(a) [r-apo(a)] and Lp(a) on responses of washed human platelets, prelabeled in the dense granules with [14C]serotonin and suspended in Tyrode's solution, to ADP and the thrombin receptor-activating peptide SFLLRN. No effect of the 17 kringle (K), 12K, or 6K r-apo(a) derivatives (at concentrations of 0.35 and 0.7 micromol/L) or Lp(a) (up to 0.1 micromol/L) on primary ADP-induced platelet aggregation was observed. In contrast, weak platelet responses stimulated by 7.5 micromol/L SFLLRN were significantly enhanced by the r-apo(a) derivatives; eg, 0.7 micromol/L 17K r-apo(a) increased aggregation from 15+/-4% to 58+/-6%, release of [14C]serotonin from 9+/-3% to 36+/-6%, and formation of thromboxane A2, measured as its stable metabolite thromboxane B2, from 7+/-1 to 29+/-5 ng/10(9) platelets (n=3; P<0.04 to 0.015). Significant enhancement of aggregation and release of granule contents was observed at a concentration of 17K r-apo(a) as low as 0.175 micromol/L. Purified Lp(a) (0.25 to 0.1 micromol/L) also enhanced SFLLRN-induced aggregation and release in a dose-dependent manner. Although plasminogen (0.7 and 1.5 micromol/L) and low density lipoprotein (0.025 to 0.1 micromol/L) both exhibited potentiating effects on SFLLRN-mediated platelet aggregation, the magnitude of the responses was less than that observed with either the r-apo(a) derivatives or Lp(a). The enhanced responses of platelets via the protease-activated receptor- thrombin receptor in the presence of Lp(a) may contribute to the increased risk of thromboembolic complications of atherosclerosis associated with this lipoprotein.

Intravillous Eicosanoid Compartmentalization and Regulation of Placental Blood Flow

To determine the roles of the eicosanoids thromboxane and prostacyclin, and their compartmentalization, in the regulation of placental blood flow. First, the sites of production of thromboxane and prostacyclin were determined within the placental villus using immunohistochemical staining for thromboxane and prostacyclin synthetase. Second, the production of both eicosanoids was studied in cultured trophoblasts and compared with that in the villous core by measuring the metabolites thromboxane B2 and 6-keto-prostaglandin F 1 alpha. Finally, eicosanoid production was assessed in intact villi after stimulation by an acute change in oxygen content, 5% to 95%. Immunohistochemical staining showed that thromboxane production was primarily within the trophoblasts, whereas prostacyclin production was localized to the endothelial cells within the villi. In culture, we found preferential production of prostacyclin by the villous core cells and increased production of thromboxane by trophoblasts. Perifusion of intact villi demonstrated increased production of thromboxane by trophoblasts in response to an increase in oxygen content. Prostacyclin levels were too low to be detected. Placental blood flow appears to be regulated by compartmentalized eicosanoids, with thromboxane affecting primarily the maternal side of the placental circulation and prostacyclin affecting primarily the fetal side.

β-Carotene attenuates peroxide-induced vasoconstriction in the human placenta

Placental lipid peroxides and thromboxane are abnormally increased in preeclampsia. Peroxides stimulate thromboxane to increase placental vasoconstriction. Antioxidants, such as beta-carotene, control lipid peroxidation. Reduced levels of beta-carotene (0.25 mumol/L) have been found in preeclamptic women. We hypothesized that normal (0.5 mumol/L) or elevated (1.0 mumol/L) levels of beta-carotene would attenuate peroxide-induced vasoconstriction in the human placenta, whereas low levels would not. Isolated human placental cotyledons (n = 8) were sequentially perfused with control Krebs-Ringer-bicarbonate buffer, 200 mumol/L t-butyl hydroperoxide, and then with progressively increasing concentrations of beta-carotene (0.25, 0.5, and 1 mumol/L) plus t-butyl hydroperoxide. Fetal perfusion pressure was monitored continuously, and maternal and fetal effluent samples were collected for each treatment. Samples were analyzed for lipid peroxides and for thromboxane and prostacyclin by their stable metabolites thromboxane B2 (TXB2) and 6-keto-prostaglandin (PG)F1 alpha. Compared with control, t-butyl hydroperoxide significantly increased perfusion pressure, vascular resistance, and the secretion rates of lipid peroxides, TXB2 and 6-keto-PGF1 alpha. Perfusion with normal (0.5 mumol/L) or increased (1 mumol/L) levels of beta-carotene significantly or completely inhibited t-butyl hydroperoxide-induced vasoconstriction and the increases in lipid peroxide and TXB2 secretion, but did not inhibit peroxide-induced increases in 6-keto-PGF1 alpha secretion. Reduced levels of beta-carotene (0.25 mumol/L) did not inhibit t-butyl hydroperoxide-induced vasoconstriction or the increases in lipid peroxide secretion. beta-carotene at levels found in normal pregnant women (0.5 mumol/L) or at levels achieved with beta-carotene supplementation (1 mumol/L) inhibited peroxide-induced vasoconstriction and lipid peroxide and thromboxane secretion. Levels of beta-carotene found in preeclamptic women (0.25 mumol/L) did not inhibit peroxide-induced vasoconstriction or lipid peroxide secretion.