Cerebral platelet thromboembolism and thromboxane synthetase inhibition

Platelet aggregating sodium arachidonate was slowly infused into the internal carotid artery (1 mg, 100 microliters, 1 microliter/s) of nitrous oxide anesthetized rats. The electroencephalographic activity recorded by a Cerebral Function Monitor from the injected hemisphere was reduced within minutes. The somatosensory evoked responses to contralateral electrical stimulation of the whisker area were eliminated on the same side in most cases when measured five and fifteen minutes after the infusion. The brain was frozen in situ with liquid nitrogen after fifteen minutes. Regional tissue analysis showed ipsilateral derangement of the cerebral energy state and increased lactate levels. Pretreatment with the platelet antiaggregating thromboxane synthetase inhibitor OKY-1581 (Sodium-3-4-(3-pyridylmethyl)phenyl-2-methyl-acrylate), 30 mg/kg i.v., fifteen minutes before the sodium arachidonate infusion prevented cerebral energy failure and elimination of the sensory evoked responses.     

Effects of aspirin and alcohol on platelet thromboxane synthesis and vascular prostacyclin synthesis

We have studied interactions between aspirin and alcohol in vitro with regard to their effects on platelet thromboxane synthesis and vascular prostacyclin synthesis. Alcohol did not affect the inhibition by aspirin of platelet thromboxane synthesis. However, low concentrations of alcohol (0.05-0.15%, v/v) reversed the inhibition by aspirin of vascular prostacyclin synthesis. Since the effects on prostacyclin and thromboxane synthesis would favour bleeding rather than haemostasis, the observations reported herein may explain previous reports of a potentiation by alcohol of the aspirin-induced increase in bleeding time.     

Peripheral venous plasma aspirin concentrations and platelet aggregation inhibition produced by enteric-coated aspirin formulations

In a random cross-over design, six healthy consenting adult volunteers were given on separate occasions single doses of 300-650 mg of 3 different formulations of enteric-coated aspirin. Over various intervals for 48-54 h following dosage, plasma aspirin and salicylate concentrations were measured together with percentage inhibition of platelet aggregation activated by threshold concentrations of sodium arachidonate alone and combined with ADP and collagen. In all subjects each formulation delivered measurable quantities of aspirin to the peripheral circulation, the unchanged drug being detected at various times up to and including 28 h after dosage. Moreover, low aspirin concentrations were found to co-exist with unimpaired platelet aggregation. All 3 formulations yielded statistically significant (P less than 0.01) inhibition of platelet aggregation activated both by arachidonate and by the combination of aggregants when tested 24-29 and 48-54 h after dosage; there were no significant differences (P greater than 0.05) between the 3 formulations in this regard. Two different patterns of delivery of unchanged aspirin to the systemic circulation from these enteric-coated formulations were apparent. These patterns may be important when considering which aspirin formulation might be most appropriate in chronic use for an antiplatelet effect. None of the enteric-coated formulations used in this study may be optimal in this regard.     

Individual variation in platelet aggregability and serum thromboxane B2 concentrations after low-dose aspirin

The effects of low daily oral doses of aspirin (40 mg/day) on platelet aggregability and serum thromboxane B2 concentrations were studied in 19 poststroke patients. Although platelet aggregation was reduced significantly after 1 week, there was wide individual variation in the inhibition of platelet function in spite of marked decreases of serum thromboxane B2 concentrations by greater than 90% (from 224 +/- 58 to 8 +/- 8 ng/ml). There was no correlation between collagen-induced platelet aggregability and serum thromboxane B2 concentration before aspirin administration in the range 100-350 ng/ml, but after 1 week of repeated administration of aspirin, there was a correlation between platelet aggregability and serum thromboxane B2 concentrations of less than 25 ng/ml (r = 0.68, p less than 0.01). However, platelet inhibition was insufficient even in some patients with markedly decreased thromboxane B2 concentrations (less than 5 ng/ml). Our results suggest that individual variation of platelet aggregability in response to low-dose aspirin may be due to variation not only in the degree of inhibition of thromboxane A2 production but also in the relative dependence of platelet aggregation on extra-arachidonic pathways.     

Prevention of aspirin inhibition of platelet release reaction by the fatty acid precursor of platelet prostaglandins

Preincubating human platelet-rich citrated plasma with eicosatetraenoic acid (E-tetra), the immediate precursor of PGE₂ and F_2α, prevents the inhibitory effect of aspirin on the release reaction induced by epinephrine, ADP, thrombin or collagen. The methyl ester of E-tetra as well as a saline solution of arachidonyl C1 had the same effect as the free acid. In striking contrast to E-tetra, E-tri or its methyl ester, E-penta, stearic, oleic, linoleic and γ-linolenic acid failed to prevent the inhibitory effect of aspirin. Although E-tetra alone induces the release reaction, this phenomenon does not explain the action of E-tetra in blocking effects of aspirin. Thus, we tentatively postulate that E-tetra's unique protection of the platelet against the untoward effect of aspirin, may be attributable to the role it plays in platelet prostaglandin synthesis.     

Multi-parameter assessment of platelet inhibition and its stability during aspirin and clopidogrel therapy

Introduction

Poor response to antiplatelet drugs is associated with adverse outcomes. We assessed platelet inhibition and its stability and tested correlation and agreement between platelet function assays.

Methods

Peripheral blood from 58 patients on both aspirin and clopidogrel who underwent percutaneous coronary intervention (PCI) was collected at hospital discharge (visit-1) and at 30-90 days (visit-2). Platelet function was measured using light transmission aggregometry (LTA-AA and LTA-ADP), VerifyNow® (Aspirin; ARU and P2Y12; PRU), ex vivo TxB₂, urinary 11dhTxB₂, and VASP (PRI) assays. Data were analyzed as continuous, quartiles and binary. Patients were defined as aspirin poor responder (PR) with ARU ≥ 550, LTA-AA maximum ≥ 20%, TxB₂ ≥ 1 ng/mL or 11dhTxB₂ ≥ 1,500 pg/mg of creatinine and as clopidogrel PR with PRU ≥ 240, PRU ≥ 208, LTA-ADP maximum ≥ 40%, PRI ≥ 50%, or PRI ≥ 66%.

Results

Aspirin PR was 3-33% and clopidogrel PR was 10-35% in visit-1. LTA-AA, 11dhTxB_2, and all clopidogrel-response measures showed correlation and agreement between visit-1 and visit-2. The highest agreement between two visits was revealed by PRU ≥ 240 and PRI ≥ 66% (PRU-κ = 0.7, 95% CI = 0.47, 0.93; PRI-κ = 0.69, 95% CI = 0.42, 0.95, p-values < 0.001). Comparison of platelet function assays in a single visit (visit-1) revealed a poor correlation between LTA-AA and 11dhTxB₂ assays and no agreement among aspirin-response assays. The highest correlation and agreement were obtained between VerifyNow® P2Y12 and VASP assays (rho = 0.7, p-value < 0.001 and PRU ≥ 208-PRI-κ = 0.41-0.42, 95% CI = 0.13, 0.69, p-values < 0.001).

Conclusions

Platelet inhibition is stable during aspirin and clopidogrel treatment. Clopidogrel-response assays correlate and agree with each other better than aspirin-response assays.     

Effects of low-to-high doses of aspirin on platelet aggregability and metabolites of thromboxane A2 and prostacyclin.

BACKGROUND AND PURPOSE: The purpose of this study was to compare the effects of low-to-high doses of aspirin on platelet aggregability determined by different methods and on the metabolism of thromboxane A2 and prostacyclin. METHODS: We administered increasing doses (40, 320, and 1,280 mg/day) of aspirin to 19 poststroke patients and studied the differences in 1) the changes in platelet aggregability depending on the methods of evaluation and 2) the concentrations of prostaglandin metabolites in the blood and urine. RESULTS: Aggregation of platelet-rich plasma induced by a strong stimulus (10 microM ADP) was significantly reduced after 40 mg/day aspirin (p less than 0.005), and this reduction was similar to that after higher aspirin doses. In contrast, aggregation of platelet-rich plasma induced by weaker stimuli (1 and 5 microM ADP) decreased less significantly after 40 mg/day aspirin compared with that after higher aspirin doses. The serum thromboxane B2 generated after ex vivo incubation was reduced significantly (by 85%) after 40 mg/day aspirin and decreased further after 320 mg/day (by 96%) and 1,280 mg/day (by greater than 99%) of aspirin. The urinary 11-dehydro-thromboxane B2 concentration decreased less significantly after 40 mg/day aspirin (by 42%) compared with that after 320 mg/day (by 78%) and 1,280 mg/day (by 91%) aspirin doses. The urinary concentration of 2,3-dinor-6-keto-prostaglandin F1 alpha did not decrease after 40 mg/day aspirin but decreased significantly after higher doses of aspirin. CONCLUSIONS: These findings suggest that different doses of aspirin may be necessary to prevent thrombogenesis induced by different triggers of different strengths and that 40 mg/day aspirin is able to inhibit a large proportion of maximum thromboxane A2 release provoked acutely, with the prostaglandin I2 synthesis being little affected; however, higher doses of aspirin are required to attain further inhibition.     

Effect of thromboxane synthetase inhibition on platelet function and morphology during ovine pregnancy-induced hypertension

Arterial blood pressure, serum fibrin/fibrinogen degratory products, plasma thromboxane B2, in vitro platelet aggregation, and platelet ultrastructure were studied in ten gravid ewes during fast-triggered ovine pregnancy-induced hypertension and subsequent administration of the thromboxane synthetase inhibitors CGS13080 and CGS12970. During the hypertensive period, blood pressure (p less than 0.005) and plasma thromboxane B2 levels (p less than 0.005) were significantly altered. Collagen-induced in vitro platelet aggregation lag times increased (p less than 0.01), and percent aggregation (p less than 0.05), primary (p less than 0.01), and secondary (p less than 0.005) aggregatory slopes decreased. Collagen also failed to induce aggregation in some ewes. Primary slopes of ADP-induced in vitro platelet aggregation decreased (p less than 0.01) during hypertension. Degranulation and open canalicular tubule system swelling were observed in platelets which produced abnormal or no aggregation response. However, these ultrastructural abnormalities did not necessarily correspond to hypertensive periods. Thromboxane synthetase inhibitor administration lowered blood pressure (p less than 0.005) and plasma thromboxane B2 levels (p less than 0.005). Abnormalities in collagen and ADP-induced platelet aggregation curves were also corrected, and ultrastructural abnormalities were not detected. Marked elevations in plasma thromboxane levels during ovine pregnancy-induced hypertension may have had an "exhaustive" effect on thrombocytes which was reversed by thromboxane synthetase inhibition.     

Alterations of platelet aggregation kinetics with ultraviolet laser emission: the "stunned platelet" phenomenon.

Platelets, a major constituent of thrombus, play a crucial role in the pathogenesis of acute ischemic coronary syndromes. The effect of ultraviolet laser emission on platelets within thrombi is unknown. The effects of increasing levels of laser energy on platelets in whole blood were investigated. Blood samples were obtained by aseptic venipuncture and anticoagulated with 3.8% sodium citrate. Samples were exposed to increased levels (0, 30, 45, 60 mJ/mm2; 25 Hz) of ultraviolet excimer laser fluence (308 nm wave-length) and then tested for ADP and collagen induced platelet aggregation, platelet concentration, and for platelet contractile force (PCF) development. Scanning electron microscopy was used to detect laser induced morphologic changes of platelets and by flow cytometric analysis to detect changes in expression of platelet surface antigens p-selectin (CD 62) and glycoprotein IIb/IIIa (CD 43). Exposure to excimer laser energy produced dose dependent suppression of platelet aggregation and force development ("stunned platelets"). ADP aggregation decreased from 8.0+/-1.1 Ohms (mean+/-SEM) to 3.7+/-0.8 Ohms (p<0.001) to 2.7+/-0.6 Ohms (p <0.001) and to 1.8+/-0.5 Ohms (p <0.001) as the laser energy increased from 0 to 30 to 45 to 60 mJ/mm2, respectively. Collagen induced aggregation decreased from 21.4+/-1.4 Ohms to 15.7+/-1.2 Ohms (p <0.001) to 11.7+/-1.1 Ohms (p <0.001) and to 9.9+/-1.0 Ohms (p <0.001), in response to the same incremental range of laser energy. Platelet contractile forces declined from 34,500+/-3700 to 27.800+/-2700 dynes as laser energy increased from 0 to 60 mJ/mm2 (p <0.03). Platelet concentration did not change with increasing laser energy. The expression of platelet surface antigen p-selectin (CD 62) remained stable through increasing levels of laser energy exposures while the percentage of CD 43 positive platelets significantly increased with exposure to laser energy, yet the level of expression did not exceed 0.5% of cells. Thus, aggregation kinetics are altered in platelets exposed to ultraviolet laser energy as manifested by decreased platelet aggregation and reduction in platelet force development capability. The response is dose dependent and most pronounced at higher energy levels such as 60 mJ/mm2.     

Effects of Y-20811, a long-lasting thromboxane synthetase inhibitor, on thromboxane production and platelet function

Effects of a new imidazole derivative, sodium 4-[alpha-hydroxy-5-(imidazolyl)-2-methylbenzyl]-3,5-dimethyl benzoate dihydrate (Y-20811), on thromboxane (TX) production and platelet aggregation were investigated. Y-20811 inhibited TX synthetase (IC50 = 2.2 X 10(-8) M) and platelet aggregation induced by arachidonic acid (AA) in human, guinea pig and rabbit platelets in vitro. Administered orally to rabbits, Y-20811 at a dose of 1 mg/kg decreased serum TXB2 concomitant with increasing 6-keto PGF1 alpha and at a dose of 3 mg/kg inhibited AA-induced platelet aggregation, in both cases for at least 48 hours. Y-20811 (0.3 mg/kg/day) administered to rabbits for 7 days decreased serum TXB2 levels by 50-90% during the medication, and these levels were restored to initial values 3 days after withdrawal of the drug. At a dose of 1 mg/kg Y-20811 protected rabbits against death induced by AA (2 mg/kg). These results indicate that Y-20811 is a selective and long-lasting TX synthetase inhibitor and an anti-aggregating agent useful in preventing thrombotic disorders.     

Magnitude and time course of platelet inhibition with extended release dipyridamole with or without aspirin in healthy Japanese volunteers. The AGgrenox versus Aspirin Therapy Evaluation (AGATE-Japan)

Randomized trials showed greater stroke prevention with extended release dipyridamole in combination with low dose aspirin than with either aspirin or dipyridamole alone. However, most studies with this formulation (Aggrenox) were carried out in Europe and North America. Considering potential inter-racial differences in drug response, we conducted a small randomized study in healthy Japanese volunteers to compare antiplatelet regimens with regard to the changes in the platelet biomarkers. Thirty healthy volunteers (18-40 years old, 15 male and 15 female) of Japanese descent were randomized to Aggrenox (n = 17) or aspirin 81 mg (n = 13 volunteers) for 30 days. Platelet function was assessed at baseline, and on days 15, and 30 by conventional aggregometry, whole blood flow cytometry, and cartridge-based analyzer. Both Aggrenox and aspirin provided sustained platelet inhibition at Day 15 and Day 30. Therapy with Aggrenox, however, was associated with more prominent and significant inhibition of collagen-induced aggregation (p = 0.08, Day 15), as well as prolongation of the closure time (p = 0.001, Day 30); diminished expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) (p = 0.02, Day 30), glycoprotein IIb (GPIIb) antigen (p = 0.001 and 0.024 for Day 15 and Day 30), and GPIIb/IIIa activity by PAC-1 antibody (p = 0.014 and 0.03), CD62 (P-selectin) (p = 0.03 for Day 15 and Day 30), as well as inhibition of protease activated receptors (PAR-1) associated with intact WEDE-15 (p = 0.002 and 0.003) and SPAN-12 (p = 0.002 and 0.04) thrombin receptors when compared with aspirin. The magnitude and durability of platelet response after Aggrenox in healthy Japanese is similar to those effects observed in Caucasians and African-Americans. A larger study to assess drug efficacy and safety in the Japanese post-stroke patients is warranted.     

Measurement of aspirin concentrations in portal and systemic blood in pigs: effect on platelet aggregation, thromboxane and prostacyclin production

Low doses of enteric-coated aspirin were administered orally to pigs. Plasma aspirin concentrations measured in blood obtained simultaneously from permanent catheters in a systemic artery and portal vein for 6 hours after dosage showed a large variation in the plasma aspirin concentration: time profile between pigs. After 50 mg single dose the ratio of the arterial: portal area under the plasma concentration versus time curve (AUC) was 0.63 +/- 0.08 (mean +/- SE, n = 6). In three pigs which received all three dosage regimens, the arterial: portal AUC ratios were 0.48 +/- 0.05 after 50 mg single dose, 0.52 +/- 0.02 after 100 mg single dose and 0.47 +/- 0.02 after 100 mg daily for 1 week. Platelet aggregation in response to sodium arachidonate (1.65 mM) was completely abolished after chronic aspirin administration of 100 mg daily. Thromboxane production (pg/10(6) platelets) induced by this stimulus decreased from 536 +/- 117 before aspirin to 57 +/- 14 after aspirin (mean +/- SE, n = 4; p = 0.03). Aortic prostacyclin synthesis, measured as 6-keto PGF1 alpha (ng/disc after 10 min incubation), was 1.66 +/- 0.28 (mean +/- SE, n = 4) in untreated pigs and 0.95 +/- 0.25 (n = 5) in treated pigs (p = 0.07). Results from this study support the idea that a difference between aspirin concentrations in the portal and systemic circulations can be achieved. Whether this can be translated into a clinically useful differential effect on the vessel wall compared to the platelet remains to be determined.     

The inhibitory effect of human endothelial cell monolayers on platelet reactions and its inhibition by aspirin

Primary cultures of human endothelial cells released an inhibitor of platelet aggregation when the cell monolayer was incubated with PRP, PPP or buffer. The amount released increased as the incubation time increased. The inhibitor also was released when the endothelial monolayer was incubated with platelets which had been treated with aspirin to block the formation of endoperoxides. The inhibitory effect also increased when PRP samples were exposed to collagen in the presence of endothelial cells. The inhibitory activity resembled that of PGI₂ (prostacyclin), and preincubation of the endothelial cells with aspirin inhibited the time-dependent increase observed in the controls. We suggest that the platelet inhibitory activity of endothelial cells is synthesized from an endogenous precursor and released to the surroundings. Synthesis of the inhibitor was increased by cyclic endoperoxides formed during platelet aggregation and was decreased by treatment of the endothelial monolayer with aspirin.     

Insights into the inhibition of platelet activation by omega-3 polyunsaturated fatty acids: beyond aspirin and clopidogrel

Objectives

We sought to examine the effects of escalating doses of omega-3 polyunsaturated fatty acid (PUFA) supplements on platelet function using light transmission aggregometry (LTA) and electrophoretic quasi-elastic light scattering technology (EQELS).

Background

PUFA may inhibit platelet function through fatty acid substitution in the platelet membrane by changing the surface charge density and causing decreased production of thromboxane A2. EQELS can measure platelet surface charge density and determine whether the platelet is in resting or activated state.

Methods

A total of 30volunteers were divided in 3 groups of 10 as follows: Group A, no antiplatelet agent; Group B, daily aspirin only, and Group C, daily aspirin and clopidogrel. All patients received escalating doses of omega-3PUFA from 1 to 8 g daily over 24 weeks. Platelet function was measured by template bleeding time, LTA, and EQELS at baseline and at 6, 12, 18 and 24 weeks.

Results

Mean bleeding time increased in a dose-dependent manner with escalating omega-3 PUFA doses. LTA confirmed expected antiplatelet effects of aspirin and clopidogrel, but did not detect any additional antiplatelet effects of omega-3 PUFA. EQELS showed a significant increase in the negative resting platelet charge compared to baseline and an attenuated response to arachidonic acid mediated platelet activation. No bleeding events were observed.

Conclusions

In this pilot study we were able to successfully measure platelet surface charge variation as a measure of omega-3 PUFA effect on platelets. Our results suggest that omega-3 PUFA increase the total platelet surface charge and, therefore, attenuate platelet activation, even among patients taking aspirin or aspirin plus clopidogrel. Further studies are needed to determine the clinical significance of these measured effects and EQELS results.     

Optical platelet aggregation versus thromboxane metabolites in healthy individuals and patients with stable coronary artery disease after low-dose aspirin administration

IntroductionAspirin reduces cardiovascular events in patients with coronary artery disease (CAD), but studies report a highly variable response to aspirin, often referred to as ‘aspirin low-responsiveness’. We investigated whether 75 mg of daily non-enteric coated aspirin would completely inhibit the platelet cyclooxygenase-1 activity to a comparable extent in healthy individuals and stable CAD patients.MethodsWe assessed serum thromboxane B₂ (S-TxB₂), urinary 11-dehydro-TxB₂ (U-TxM) and arachidonic acid-induced optical platelet aggregometry (OPA) in 44 CAD patients on aspirin and in 22 healthy individuals before and after aspirin. OPA was performed in duplicate for four consecutive days during aspirin treatment after one week of treatment. Compliance was optimized by face-to-face interviews and pill counting and confirmed by S-TxB₂ measurements.ResultsAspirin inhibited S-TxB₂ > 99% in healthy individuals (median 1.1 ng/mL, interquartile range (IQR) = 0.8;1.9 after aspirin) and in patients, S-TxB₂ was reduced to a similar level (0.9 ng/mL (0.7;1.5)). Healthy individuals had a median U-TxM of 278.5 pg/mg creatinine (229.5;380.0) before aspirin and 68.5 pg/mg creatinine (59.0;99.7) on aspirin corresponding to an average 74% inhibition of the endogenous TxA₂ biosynthesis. In patients median U-TxM was 67.5 pg/mg creatinine (54.0;85.5). Seven study participants (11%) were aspirin low-responders according to OPA, but none had S-TxB₂ in the highest quartile.ConclusionsLow-dose aspirin suppressed S-TxB₂ to comparable levels in CAD patients and healthy individuals. Despite an almost complete inhibition of S-TxB₂, some participants were low-responders according to OPA. Thorough compliance control and use of thromboxane-specific assays are important when measuring platelet response to aspirin.     

Selective inhibition of thromboxane synthetase with dazoxiben - basis of its inhibitory effect on platelet adhesion

Platelet adhesion to rabbit aortic subendothelium or collagen-coated glass was quantitated in a rotating probe device by uptake of radio-labelled platelets. Under conditions in which aspirin had no effect, dazoxiben, a selective inhibitor of thromboxane synthetase, reduced platelet adhesion to aortic subendothelium by about 40% but did not affect adhesion to collagen-coated glass. Pre-treatment of aortic segments with 15-HPETE, a selective inhibitor of PGI2-synthetase, abolished the inhibitory effect of dazoxiben on adhesion. Concentrations of 6-oxo-PGF1 alpha in the perfusate were raised in the presence of dazoxiben alone, and following addition of thrombin (10 units/ml) there was a 2--3 fold increase in concentration. Perfusion of damaged aorta with platelets labelled with (14C)-arachidonic acid in the presence of thrombin and dazoxiben resulted in the appearance of (14C)-labelled-6-oxo-PGF1 alpha. Inhibition of thromboxane synthetase limits platelet adhesion probably by promoting vascular synthesis of PGI2 from endoperoxides liberated from adherent platelets, which subsequently promotes detachment of cells from the surface.