Platelet Thromboxane Formation Research Articles

Different forms of vitamin E and metabolite 13’-carboxychromanols inhibit cyclooxygenase-1 and its catalyzed thromboxane in platelets, and tocotrienols and 13’-carboxychromanols are competitive inhibitors of 5-lipoxygenase

Cyclooxygenase (COX-1 and COX-2)- and 5-lipoxygenase (5-LOX)-catalyzed biosynthesis of eicosanoids play important roles in inflammation and chronic diseases. The vitamin E family has four tocopherols and tocotrienols. We have shown that the metabolites of δ-tocopherol (δT) and δ-tocotrienol (δTE), i.e., δT-13’-carboxychromanol (COOH) and δTE-13’-COOH, respectively, inhibit COX-1/-2 and 5-LOX activity, but the nature of how they inhibit 5-LOX is not clear. Further, the impact of tocopherols and tocotrienols on COX-1/-2 or 5-LOX activity has not been fully delineated. In this study, we found that tocopherols and tocotrienols inhibited human recombinant COX-1 with IC50s of 1–12 µM, and suppressed COX-1-mediated formation of thromboxane in collagen-stimulated rat's platelets with IC50s of 8–50 µM. None of the vitamin E forms directly inhibited COX-2 activity. 13’-COOHs inhibited COX-1 and COX-2 enzyme activity with IC50s of 3–4 and 4–10 µM, respectively, blocked thromboxane formation in collagen- and ionophore-stimulated rats’ platelets with IC50s of 1.5-2.5 µM, and also inhibited COX-2-mediated prostaglandins in stimulated cells. Using enzyme kinetics, we observed that δT-13’-COOH, δTE-13’-COOH and δTE competitively inhibited 5-LOX activity with Ki of 1.6, 0.8 and 2.2 µM, respectively. These compounds decreased leukotriene B4 from stimulated neutrophil-like cells without affecting translocation of 5-LOX from cytosol to the nucleus. Our study reveals inhibitory effects of vitamin E forms and 13’-COOHs on COX-1 activity and thromboxane formation in platelets, and elucidates mechanisms underlying their inhibition of 5-LOX. These observations are useful for understanding the role of these compounds in disease prevention and therapy.

Garlic inhibitory effect on platelet activity induced by different agonists


 Platelets are essential elements of human blood. In addition to their normal role, platelets are involved in causing myocardial infarction, stroke and other thrombotic disorders. Platelet activation in vivo, probably involves a combination of agonists. Garlic has beneficial effects due to its ability to inhibit platelet aggregation and thromboxane formation. The aim of this work was to evaluate the ability of garlic extracts to inhibit platelet aggregation induced by different agonists and their mixtures in different donors. Significant differences were found in platelet aggregation in response to each agonist (P ≤ 0.05). The highest antiaggregatory effect was observed with arachidonic acid and the lowest effect with collagen-arachidonic acid mixture. Interaction effects between donor and agonist (or mixtures) were detected. The study showed the potential of aqueous garlic extracts to prevent platelet aggregation induced by different agonist.
 Highlights
 
 Platelets play a central role in the progression of atherosclerotic lesions.
 Blood from nine non-smoker healthy donors was used for in vitro platelet aggregation study.
 Significant differences were found in platelet aggregation in response to each agonist.
 Aqueous garlic extracts could prevent platelet aggregation induced by different agonist.

Synergistic inhibitory effect of capsaicin and dihydrocapsaicin on in-vitro platelet aggregation and thromboxane formation.

: Capsaicinoids, including capsaicin (CAP) and dihydrocapsaicin (DHC), the pungent principles of pepper fruits, individually inhibit in-vitro platelet aggregation. However, their effects, when present together, are not known. The aims of this study were to compare the effects of CAP and DHC alone, and in combination in the ratio that they are found in chilies (∼60% CAP : 40% DHC), on in-vitro platelet aggregation, platelet count and thromboxane B2 (TXB2) formation. The effects of 12.5 and 6.25 μmol/l CAP and DHC individually, and in combination (CAP : DHC, 60 : 40) on arachidonic acid-induced, ADP-induced and collagen-induced aggregation, were investigated. Platelet count was determined preincubation and postincubation with CAP and DHC and in combination. TXB2 formation from platelets treated with arachidonic acid in the absence and presence of CAP and DHC individually, and in combination, was measured. Compared with control, CAP and DHC (12.5 μmol/l) inhibited arachidonic acid-induced aggregation by 23.2 and 25.3%, respectively (both P < 0.01). In combination, CAP and DHC exhibited further inhibition in arachidonic acid-induced aggregation (CAP : DHC, 3.75 : 2.5 μmol/l, 36.5%, P = 0.01; 7.5 : 5 μmol/l, 57.5%, P < 0.001), compared with control. Incubation of platelets with caspaicinoids did not significantly affect the platelet count. In addition, the CAP : DHC (7.5 : 5 μmol/l) combination significantly inhibited (P < 0.001) TXB2 formation, compared with the individual capsaicinoids. Capsaicinoids had no effect ADP-induced or collagen-induced aggregation. The combination of CAP and DHC produces a significantly greater inhibitory effect on arachidonic acid-induced platelet aggregation and subsequent TXB2 formation, compared with the individual capsaicinoids.

Abstract 15635: Consumption of Aspirin and Naproxen by Platelet Donors and the Quality of Platelet Transfusion

Over-the-counter pain killers are amongst the most frequently consumed drugs world-wide. Individuals who self-report ingesting aspirin or aspirin containing drugs within the preceding 48-72 hours - depending on regional practice - are excluded from platelet donation, given the assumed platelet functional inhibition due to irreversible acetylation of platelet cyclooxygenase-1 (COX-1) by aspirin and the consequent suppression of thromboxane formation. Other nonsteroidal anti-inflammatory drugs, which bind COX-1 tightly enough to cause long acting platelet inhibition, such as naproxen, may also impact the quality of platelet preparations for transfusion but are not typically exclusionary. The prevalence of functionally relevant exposure of platelet donors to aspirin and naproxen is unknown. We used sensitive and specific mass spectrometry methods to quantitate the acetylation of platelet COX-1 by aspirin and platelet protein binding of naproxen in platelet transfusion units as indices of donor exposure to these drugs. Platelet thromboxane formation was assessed as a functional parameter. We screened single-donor apheresis platelet units transfused at the Hospital of the University of Pennsylvania. Over 30 % (71 out of 235) of the analyzed platelet units showed evidence of aspirin exposure ranging from residual COX-1 acetylation after an appropriate washout period to complete acetylation suggestive of recent aspirin exposure. Approximately 7% (16 out of 235) of the platelet units showed complete COX-1 acetylation and inhibition of thromboxane formation. More than 15% (22 out of 143) of analyzed platelet units showed high naproxen concentrations that were associated with depression of thromboxane formation. The prevalence of aspirin and naproxen consumption by platelet donors is as high as would be expected in the general population and results in transfusion of inhibited platelets with suppressed thromboxane formation.

Abstract 9050: Drug-Drug Interactions Between Aspirin and NSAIDs are Predicted by Kinetics of Cyclooxygenase-1 Inhibition

Background and aims : Therapy with NSAIDs may interfere with antiplatelet activity of aspirin. The mechanism of this drug-drug interaction has not yet been established on the level of enzyme kinetics and quantitative structure-activity relationship (QSAR). Methods: Time-dependent kinetics (log K) for the onset of COX-1 inhibition was measured for 17 common NSAIDs using arachidonic acid-induced COX-1 activity (human platelet microsomes, bioluminescence). IC50 values of all NSAIDs were determined by arachidonic acid-induced platelet thromboxane (TX) formation in platelet rich plasma, as well as the in vitro interaction with aspirin. Dragon and SYSTAT softwares served to develop QSAR models in which dependent parameters were log K and IC50 and independent parameters were structural/physico-chemical parameters (MoR32m, G2v, L3S, representing the shape, size and polarizibility). Results: All NSAIDs could be classified into a time-dependent (slow onset) or a time-independent (fast onset) mode of inhibition. Time-dependent NSAIDs included diclofenac, flurbiprofen, indomethacin, ketoprofen and ketorolac. These NSAIDs did not decrease TX inhibition by aspirin. In contrast, all other studied NSAIDs were time-independent inhibitors and prevented inhibiton by aspirin. The inhibition kinetics (log K) and IC50 were correlated with MoR32m, G2v and L3S (equations 1 and 2). eq.1: -log IC50 = 4.54 (±0.99) MoR32m - 64.62 (±12.58) G2v + 3.41 (±0.56) L3S + 8.59; r = 0.91; r2 = 0.84; s = 0.41; F = 22.81; PRESS = 5.00. eq.2: -log K = 2.35 (±0.69) MoR32m - 16.50 (±8.68) G2v + 1.18 (±0.39) L3S + 1.61; r = 0.74; r2 = 0.55; s = 0.29; F = 5.49, PRESS= 3.01; r, correlation coefficient; s, standard deviation; F, Fisher’s test; PRESS, sum of square differences. Conclusions: NSAIDs decrease the antiplatelet activity of aspirin. This is highly dependent on the inhibition kinetics of platelet COX-1, whereas the inhibition kinetics and IC50 values for TX inhibition further depends on MoR32m, G2v and L3S of a particular NSAID. Time-independent NSAIDs decrease the anti-platelet activity of aspirin. Therefore, time-dependent NSAIDs should be preferred in patients on antiplatelet treatment with aspirin. The QSAR models may predict the kinetics and enzyme inhibition by NSAIDs.

Effect of diallyl trisulfide on the pharmacokinetics of dipyridamole in rats

This study was aimed to evaluate the effect of diallyl trisulfide (DATS), a major component derived from garlic used to inhibit platelet thromboxane formation, on the pharmacokinetics of dipyridamole. Pharmacokinetic parameters of dipyridamole were determined in rats following intragastric (80 mg/kg suspension or 40 mg/kg solution) or intravenous (3 mg/kg) administration of dipyridamole with coadministration (20 mg/kg) and long-term pretreatment of DATS (10 or 20 mg/kg/day for 15 consecutive days). In addition, everted gut sac models were used to assess transepithelial transport of dipyridamole and the effect of DATS on the intestinal absorption of dipyridamole. After coadministration and long-term pretreatment of DATS, significantly lower Cmax and AUC(0-24 h) were observed for intragastric administration of dipyridamole, whereas little change was noted after intravenous dipyridamole administration. After adding DATS (10 and 50 μg/mL) in the everted gut sacs, absorption of dipyridamole was remarkably decreased in the ileum and jejunum (p < 0.01). In conclusion, DATS reduced the oral exposure of dipyridamole in rats likely by the modification of the dissolution rate and intestinal absorption of dipyridamole, indicating that combined use of DATS or DATS-containing supplements with dipyridamole may require caution as low plasma concentrations of dipyridamole may lead to a subtherapeutic effect of this agent.

Inhibition of platelet thromboxane formation and phosphoinositides breakdown by diisoeugenol.

Diisoeugenol inhibited the platelet aggregation and ATP release of rabbit platelets caused by ADP, arachidonic acid, platelet-activating factor (PAF), collagen and thrombin. Prolongation of the incubation time of platelets with diisoeugenol did not cause further inhibition and the aggregability of platelets could not be restored after washing. In human platelet-rich plasma, diisoeugenol inhibited the biphasic aggregation and ATP release induced by adrenaline and ADP in a concentration-dependent manner. Thromboxane B2 formation caused by arachidonic acid, collagen and thrombin was markedly inhibited by diisoeugenol in a concentration-dependent manner. Diisoeugenol also inhibited the formation of inositol monophosphate caused by collagen, PAF and thrombin. The cAMP level of washed platelets was not changed by diisoeugenol. It is concluded that the antiplatelet effect of diisoeugenol is due to the inhibition of thromboxane formation and phosphoinositides breakdown.

Differential effects of immunosuppressive drugs on COX-2 activity in vitro and in kidney transplant patients in vivo

It was hypothesized that calcineurin inhibitors suppress vascular cyclooxygenase (COX)-2 and exert a reciprocal influence on in vivo prostacyclin and thromboxane. This could contribute to cardiovascular morbidity in transplanted patients. The ability of immunosuppressives to suppress vascular COX-2 expression in vitro was studied in cultured human vascular smooth muscle cells. Blood and urine samples were collected from 28 renal transplant patients before and 2, 4 and 6 h after intake of immunosuppressives and from 11 controls. ELISA was used to measure (1) plasma 6-keto-PGF1alpha and TxB2; (2) urine excretion of PGI-M and TxB(2); (3) 6-keto-PGF1alpha in the whole-blood COX-2 assay; and (4) TxB2 in the whole-blood COX-1 assay. Platelet aggregation was measured optically. COX-2 in cultured vascular smooth muscle cells was suppressed by cyclosporine A (CsA); tacrolimus and rapamycin had no effect. Human renal arteries and vascular smooth muscle expressed calcineurin Abeta and Agamma isoforms. CsA had no effect on plasma 6-keto-PGF1alpha, whole-blood COX-2 activity or PGI-M urine excretion; after rapamycin intake, the former two increased. Plasma TxB2 did not change after drug intake. TxB2 in the COX-1 assay and urine excretion of TxB2 was significantly lower in tacrolimus- and rapamycin-treated patients compared to the CsA group. Platelet aggregation was increased significantly in the CsA group. Although CsA suppressed COX-2 in cultured vascular smooth muscle cells, systemic prostacyclin was not suppressed by either CsA or tacrolimus in vivo. Rapamycin and tacrolimus may actively suppress platelet and renal thromboxane formation. Differential changes in prostanoids may have implications for long-term cardiovascular hazard in patients treated with immunosuppressives.

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.

Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis.

Anti-inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug-drug interaction at the level of platelet cyclooxygenase-1 (COX-1). We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

We-P11:238 Effect of two different doses of aspirin on platelet aggregation and thromboxane formation in patients undergoing CABG

Objectifs Résumer les caractéristiques du tissu de comblement (« backfill ») chez des patients souffrant de spondyloarthrite (SpA) axiale et de dorsalgie non spécifique et chez des témoins sains et évaluer la valeur diagnostique du tissu de comblement (« backfill ») pour la SpA axiale.Méthodes Trois lecteurs en insu ont relevé le tissu de comblement (« backfill ») apparaissant sur des clichés IRM T1SE de 647 sujets : 297 patients atteints de spondylarthrite ankylosante (SA), 126 patients présentant une SpA axiale non radiographique (nr-axSpA), 147 patients souffrant de dorsalgie non spécifique et 77 témoins sains. Le tissu de comblement (« backfill ») a été évalué par la méthode SPARCC de score structural des articulations sacro-iliaques (SIJ Structural Score, SSS). Les évolutions du tissu de comblement (« backfill ») ont été évaluées sur les IRM de suivi de 157 patients. Nous avons résumé les caractéristiques du tissu de comblement (« backfill ») et calculé sa sensibilité et sa spécificité pour le diagnostic de la SpA axiale.La présence de tissu de comblement (« backfill ») a été constatée dans 78,8 % des cas de SA, 11,1 % des cas de nr-axSpA, 1,8 % des dorsalgies non spécifiques et chez aucun témoin sain. Il concernait plus fréquemment l’ilium et la moitié inférieure des articulations sacro-iliaques (ASI) dans la SpA axiale (les deux valeurs p < 0,05). Le score SSS du tissu de comblement (« backfill ») était beaucoup plus élevé dans la SpA axiale que dans les dorsalgies non spécifiques (les deux valeurs p < 0,01) et n’était pas corrélé aux caractéristiques démographiques ni aux indices BASDAI, BASFI et à la CRP (toutes les valeurs p > 0,05). Une seule corrélation positive a été observée entre le score du tissu de comblement (« backfill ») et la durée des symptômes dans la SA (r = 0,251, p < 0,01) et la nr-axSpA (r = 0,743, p < 0,01). Le tissu de comblement (« backfill ») n’a évolué que chez 8,9 % des patients sur une période moyenne de suivi de 1,09 an. Le tissu de comblement (« backfill ») présentait une forte spécificité (0,98) et une sensibilité modérée (0,59) pour le diagnostic de la SpA axiale.Nous avons résumé les caractéristiques du tissu de comblement (« backfill ») et conclu que sa présence sur les IRM T1SE constitue un signe spécifique de SpA axiale.

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a direct thrombin inhibitor with a suppressive effect on thromboxane formation in platelets

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a newly synthetic benzoxazinone derivative, has previously been shown to inhibit rabbit platelet aggregation caused by thrombin and arachidonic acid. In the present study, the mechanism for the antiplatelet effect of HPW-RX2 was further investigated. In human platelets, HPW-RX2 concentration-dependently inhibited platelet aggregation, ATP release, P-selectin expression, and intracellular calcium mobilization caused by thrombin. In contrast, HPW-RX2 had no significant effect on either SFLLRN- or GYPGKF-induced platelet aggregation, indicating that HPW-RX2 did not interfere with platelet thrombin receptors. Moreover, HPW-RX2 inhibited the amidolytic activity of thrombin and prolonged the fibrinogen clotting time. These results suggest that the inhibitory effect of HPW-RX2 on thrombin-induced platelet aggregation is via direct inhibition of thrombin proteolytic activity. Besides the inhibition on thrombin, HPW-RX2 also prevented platelet aggregation, ATP release, and increase in [Ca 2+] i caused by arachidonic acid and low concentration collagen. In a parallel manner, both arachidonic acid-induced thromboxane B 2 and prostaglandin D 2 formations were decreased in platelets treated with HPW-RX2. This indicates that HPW-RX2 is able to inhibit the arachidonic acid cascade at the cyclooxygenase level. This is the first report of a benzoxazinone derivative possessing both thrombin and cyclooxygenase inhibitory properties. The dual effect of HPW-RX2 might provide extra therapeutic benefits for treatment of arterial thrombosis.

Is cardiopulmonary bypass a reason for aspirin resistance after coronary artery bypass grafting?

'Off-pump' coronary artery bypass grafting (OPCAB) is an alternative to conventional coronary artery bypass grafting (CABG) using cardiopulmonary bypass (CPB). While midterm results after OPCAB have become available, systematic studies of changes in platelet function after OPCAB are still missing. Since we have previously shown that oral aspirin treatment (100mg) does not achieve sufficient platelet inhibition in the majority of patients operated on with CPB, we hypothesized that bypass surgery without CPB (off-pump coronary artery bypass, OPCAB) causes less impairment of platelet inhibition by aspirin. The aim of this study was to investigate platelet function and the antiplatelet effect of aspirin after off-pump coronary artery bypass grafting in comparison with conventional on-pump surgery. We compared platelet function (in vitro aggregation and thromboxane formation) before and at days 1 and 5 after coronary artery bypass grafting, performed with (n=15) or without (n=14) CPB. Oral aspirin treatment (100mg/d) was started at day 1 after surgery. After a 5 day oral treatment with aspirin, platelet aggregation was inhibited significantly in OPCAB-patients to 55.7+/-16.3% of control before surgery (P<0.05), whereas aggregation remained unchanged after CPB (105.8+/-26.9% of control before surgery; P>0.05). Since aspirin primarily inhibits platelet thromboxane formation, thromoboxane was determined after in vitro aggregation. According to platelet aggregation, thromboxane formation was only inhibited significantly after OPCAB (29.2+/-13.0% of control before surgery, P<0.05), but not after CPB (74.5+/-21.4% of control before surgery, P>0.05). This resistance to aspirin after CPB may be caused by an increased release of new platelets which are competent to form thromboxane, since the number of platelets decreased from 237+/-11x10(3)/microl before CPB to 174+/-13x10(3)/microl at day 1 after surgery and increased significantly the following days reaching 303+/-17x10(3)/microl at day 5. Platelet counts of patients operated on without CPB showed no significant changes (236+/-16x10(3)/microl before OPCAB, 220+/-16x10(3)/microl at day 1 and 266+/-31x10(3)/microl at day 5 after surgery). The antiplatelet effect of aspirin is largely impaired after CPB, but not after CABG without CPB. Hence, increased platelet turnover after CPB seems to contribute to aspirin resistance, since an increased number of platelets might be competent to form thromboxane within the dosing intervals.

The antithrombotic profile of aspirin. Aspirin resistance, or simply failure?

The antithrombotic profile of aspirin. Aspirin resistance, or simply failure?

Influence of Dietary Linoleic Acid Intake with Different Fat Intakes on Arachidonic Acid Concentrations in Plasma and Platelet Lipids and Eicosanoid Biosynthesis in Female Volunteers

Background/Aim: N-6 fatty acids are considered to promote diseases prevalent in industrialized countries and characterized by an increased eicosanoid biosynthesis from arachidonic acid (AA). We investigated the impact of the linoleic acid (LA) intake on AA levels in humans. Methods: Six healthy female volunteers (age range 23–34 years) were given liquid formula diets (LFD) devoid of AA for 6 weeks, providing a constant intake of zero energy% (LFD 0: protein 15%, carbohydrates 85%) or 20 energy% (LFD 20: protein 15%, carbohydrates 55%, fat 30%) LA, for 3 weeks each. Fatty acids of plasma cholesteryl esters and platelet lipids were determined each week, and the prostaglandin biosynthesis was measured in 24-hour urine samples. Results: LFD 0 increased (+31% of initial value) and LFD 20 lowered (–30% of initial value) the percentage of AA in plasma cholesteryl esters and platelet lipids. Moreover, absence of dietary AA lowered the percentages of AA in plasma (–31% week 0 vs. week 6) and platelet (–11%) lipids, indicating a low transformation of LA. LFD 0 reduced urinary metabolite levels of prostaglandins D, E, and F in 24-hour urine samples (–48%, p < 0.001) within 24 h, but did not significantly affect platelet aggregation (–10%) and thromboxane formation (–25%). LFD 20 significantly lowered platelet aggregation (–25%) and thromboxane formation (–43%). The prostaglandin metabolite levels increased during the first 10 days, declined thereafter, and were lower than the preexperimental values at the end of the 3-week period. Conclusions: The results show that dietary LA does not increase the AA levels in plasma or platelet lipids and does not persistently contribute to prostaglandin biosynthesis which is increased by AA intake with Western diets.

The role of PAR4 in thrombin-induced thromboxane production in human platelets.

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [(3) H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca(2+)](i) and activation of phospholipase A(2). These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

The public health potential of aspirin.

The public health potential of aspirin.

Towards a definition of aspirin resistance: a typological approach

'Aspirin resistance' is a poorly defined term to describe the inability of aspirin to protect individuals from thrombotic complications and there are conflicting reports on incidence rates and clinical relevance of this phenomenon. Using collagen (1 w g/ml)-induced platelet aggregation and thromboxane formation (measured as thromboxane B 2 ) in citrated platelet-rich plasma, this study demonstrates that aspirin resistance can be classified into three distinct types. In aspirin responders, both, collagen-induced platelet aggregation and thromboxane formation was completely (>95%) inhibited by oral aspirin treatment (100 mg/day). In type I resistance (pharmacokinetic type), oral treatment with aspirin was ineffective but addition of aspirin (100 w M) in vitro resulted in a complete inhibition of collagen-induced platelet aggregation and thromboxane formation. In type II resistance (pharmacodynamic type), neither oral treatment with aspirin nor addition of aspirin in vitro inhibited collagen-induced platelet aggregation and thromboxane formation. In type III resistance (pseudo-resistance), platelet aggregation was induced by a low concentration of collagen (1 w g/ml) despite of a complete inhibition of thromboxane formation by oral aspirin treatment. This typology of aspirin resistance should help to clarify the mechanisms, the actual rate, and the possible clinical consequences of this phenomenon.

Aspirin resistance after coronary artery bypass grafting.

The antiplatelet effect of aspirin may be compromised by an enhanced platelet turnover, generating an increased fraction of platelets that is able to form thromboxane within the dosing intervals (usually 1 day). This may be of particular relevance for patients undergoing CABG after cardiopulmonary bypass, when postoperative platelet turnover is increased. The present work examines whether the antiplatelet effect of aspirin, which is based on the inhibition of platelet thromboxane production, may be impaired by an increased platelet turnover. Determination of thromboxane in collagen-stimulated platelet-rich plasma after CABG showed a remarkable enhancement of the capacity for thromboxane formation, despite the fact that aspirin was administered at an antiplatelet dose (100 mg once daily) that largely suppressed thromboxane synthesis in healthy control subjects. Aspirin is widely used to reduce graft thrombosis after coronary artery bypass grafting (CABG).1 However, aspirin appears to not always be an effective inhibitor of platelet function because considerable individual variations in the antiplatelet effect of aspirin have been reported in patients assigned to CABG.2 ASPIRIN RESISTANCE AFTER CORONARY ARTERY BYPASS GRAFTING

Thromboxane-dependent platelet activation in vivo precedes arterial thrombosis in thrombocythaemia: a rationale for the use of low-dose aspirin as an antithrombotic agent.

The clinical course of patients with polycythaemia vera (PV) and essential thrombocythaemia (ET) is frequently complicated by arterial thrombotic events. The pathogenesis is not clearly understood but attributed to abnormalities in platelet function. An increase in platelet thromboxane formation has been described in the majority of asymptomatic patients with thrombocythaemia, probably reflecting spontaneous platelet activation in vivo. In the present study we prospectively investigated whether an increase in platelet thromboxane formation actually precedes arterial microvascular thrombosis. In addition, we studied the effect of selective inhibition of platelet thromboxane formation on clinical outcome by reinstitution of low-dose aspirin (50 mg/ d). Six ET patients and one PV patient participated in this study. Within 10 d after withdrawal of aspirin, three patients developed arterial microvascular thrombosis of extremities (erythromelalgia), which was preceded by a 3-30-fold increase in urinary thromboxane excretion as compared with patients who remained asymptomatic. The increased urinary thromboxane excretion and clinical signs could be inhibited by a platelet-specific aspirin regimen of 50 mg/d without affecting vascular cyclooxygenase, indicating that platelets were the main source of the increased thromboxane generation. These data suggest that in symptomatic patients an enhanced formation of thromboxane by platelets, reflecting platelet activation in vivo, precedes the development of arterial microvascular thrombosis. These data provide a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythaemia.