BACKGROUND

Unstable angina is associated with enhanced lipid peroxidation and reduced antioxidant defenses. We have previously reported aspirin failure in the suppression of enhanced thromboxane (TX) biosynthesis in a subset of episodes of platelet activation in this setting. We tested the hypothesis that the in vivo formation of the F(2)-isoprostane 8-iso-prostaglandin (PG)F(2alpha), a bioactive product of arachidonic acid peroxidation, is enhanced in unstable angina and contributes to aspirin-insensitive TX biosynthesis.

RESULTS

Urine samples were obtained from patients with unstable angina (n=32), stable angina (n=32), or variant angina (n=4) and from 40 healthy subjects for the measurement of immunoreactive 8-iso-PGF(2alpha) and 11-dehydro-TXB(2). 8-Iso-PGF(2alpha) excretion was significantly higher in patients with unstable angina (339+/-122 pg/mg creatinine) than in matched patients with stable angina (236+/-83 pg/mg creatinine, P:=0.001) and control subjects (192+/-71 pg/mg creatinine, P:<0.0001). In patients with unstable angina, 8-iso-PGF(2alpha) was linearly correlated with 11-dehydro-TXB(2) excretion (rho=0.721, P:<0.0001) and inversely correlated with plasma vitamin E (rho=-0.710, P:=0. 004). Spontaneous myocardial ischemia in patients with variant angina or ischemia elicited by a stress test in patients with stable angina was not accompanied by any change in 8-iso-PGF(2alpha) excretion, thus excluding a role of ischemia per se in the induction of increased F(2)-isoprostane production.

CONCLUSIONS

These findings establish a putative biochemical link between increased oxidant stress and aspirin-insensitive TX biosynthesis in patients with unstable angina and provide a rationale for dose-finding studies of antioxidants in this setting.

BACKGROUND

F2 isoprostanes are stable, free radical-catalyzed products of arachidonic acid that reflect lipid peroxidation in vivo.

RESULTS

Specific assays were developed by use of mass spectrometry for the F2 isoprostanes iPF2alpha-III and iPF2alpha-VI and arachidonic acid (AA). Urinary excretion of the 2 F2 isoprostanes was significantly increased in hypercholesterolemic patients, whereas substrate AA in urine did not differ between the groups. iPF2alpha-III (pmol/mmol creatinine) was elevated (P<0.0005) in homozygous familial hypercholesterolemic (HFH) patients (85+/-5. 5; n=38) compared with age- and sex-matched normocholesterolemic control subjects (58+/-4.2; n=38), as were levels of iPF2alpha-VI (281+/-22 versus 175+/-13; P<0.0005). Serum cholesterol correlated with urinary iPF2alpha-III (r=0.41; P<0.02) and iPF2alpha-VI (r=0. 39; P<0.03) in HFH patients. Urinary excretion of iPF2alpha-III (81+/-10 versus 59+/-4; P<0.05) and iPF2alpha-VI (195+/-18 versus 149+/-20; P<0.05) was also increased in moderately hypercholesterolemic subjects (n=24) compared with their controls. Urinary excretion of iPF2alpha-III and iPF2alpha-VI was correlated (r=0.57; P<0.0001; n=106). LDL iPF2alpha-III levels (ng/mg arachidonate) were elevated (P<0.01) in HFH patients (0.32+/-0.08) compared with controls (0.09+/-0.02). The concentrations of iPF2-III in LDL and urine were significantly correlated (r=0.42; P<0.05) in HFH patients.

CONCLUSIONS

Asymptomatic patients with moderate and severe hypercholesterolemia have evidence of oxidant stress in vivo.

The PFA-100 system is a platelet function analyzer designed to measure platelet-related primary hemostasis. The instrument uses two disposable cartridges: a collagen/epinephrine (CEPI) and a collagen/ADP (CADP) cartridge. Previous experience has shown that CEPI cartridges detect qualitative platelet defects, including acetylsalicylic acid (ASA)-induced abnormalities, while CADP cartridges detect only thrombocytopathies and not ASA use. In this seven-center trial, 206 healthy subjects and 176 persons with various platelet-related defects, including 127 ASA users, were studied. The platelet function status was determined by a platelet function test panel. Comparisons were made as to how well the defects were identified by the PFA-100 system and by platelet aggregometry. The reference intervals for both cartridges, testing the 206 healthy subjects, were similar to values described in smaller studies in the literature [mean closure time (CT) 132 s for CEPI and 93 s for CADP]. The use of different lot numbers of cartridges or duplicate versus singleton testing revealed no differences. Compared with the platelet function status, the PFA-100 system had a clinical sensitivity of 94.9% and a specificity of 88.8%. For aggregometry, a sensitivity of 94.3% and a specificity of 88.3% were obtained. These values are based on all 382 specimens. A separate analysis of sensitivity by type of platelet defect, ASA use versus congenital thrombocytopathies, revealed for the PFA-100 system a 94.5% sensitivity in identifying ASA users and a 95.9% sensitivity in identifying the other defects. For aggregometry, the values were 100% for ASA users and 79.6% for congenital defects. Analysis of concordance between the PFA-100 system and aggregometry revealed no difference in clinical sensitivity and specificity between the systems (p>> 0.9999). The overall agreement was 87.5%, with a Kappa index of 0.751. The two tests are thus equivalent in their ability to identify normal and abnormal platelet defects. Testing 126 subjects who took 325 mg ASA revealed that the PFA-100 system (CEPI) was able to detect 71.7% of ASA-induced defects with a positive predictive value of 97.8%. The overall clinical accuracy of the system, calculated from the area under the ROC curve, was 0.977. The data suggest that the PFA-100 system is highly accurate in discriminating normal from abnormal platelet function. The ease of operation of the instrument makes it a useful tool to use in screening patients for platelet-related hemostasis defects.

Aspirin is currently the most cost-effective drug for the secondary prevention of cardiovascular disease, but treatment failures are relatively common. Several factors have been linked to these recurrent vascular events in patients prescribed aspirin, including smoking, drug interactions, nonadherence, comorbid conditions, and aspirin resistance. The term aspirin resistance has been used to describe not only an absence of the expected pharmacologic effects of aspirin on platelets but also poor clinical outcomes, such as recurrent vascular events, in patients treated with aspirin. Aspirin resistance is perhaps more precisely understood as the phenomenon of measurable, persisting platelet activation that occurs in patients prescribed a therapeutic dose of aspirin and may underlie an unknown proportion of aspirin treatment failures. Key challenges for future research are to standardize a definition of aspirin resistance and to compare whether different measures of platelet activation, either alone or in combination, independently predict cardiovascular events. These challenges must be met before researchers conduct studies to assess the clinical utility of testing on patient outcomes and cost-effective prescribing.

This study examined the association of single nucleotide polymorphisms in 4 candidate genes in a cohort of subjects with aspirin resistance. Aspirin resistance was significantly associated with genetic variation in the platelet surface adenosine 5-diphosphate receptor gene P2Y1.

Interactions between altered lipoproteins and platelets may be important in atherosclerosis lesion formation and thrombosis. The aims of this study were to compare the effects of oxidatively modified and native low density lipoproteins (LDL) and high density lipoproteins (HDL) on platelet responses in the presence and absence of other platelet agonists, and investigate the mechanism(s) by which lipoproteins influence platelet activation. We have shown that native and oxidatively modified lipoproteins differ importantly in their effects on platelets; oxidation renders lipoproteins more reactive to platelets. Native LDL promote aggregation of human platelets, enhance the mobilization of arachidonate from phospholipids, increase thromboxane B2 production, and decrease membrane fluidity. Oxidized LDL are more reactive than native LDL and alone cause aggregation. Native HDL inhibit platelet responses and increase membrane fluidity. Oxidized HDL promote aggregation and cause spontaneous aggregation. The enhanced platelet responses cannot be attributed to increased production of thromboxane A2 since cyclooxygenase inhibitors (aspirin, indomethacin) have little inhibitory effect. The data suggest that activation of platelets by lipoproteins results from changes in membrane fluidity. These observations shed new light on the potential role of altered lipoproteins in the pathogenesis of atherosclerosis and its complications.

OBJECTIVE

High plasma cholesterol concentration and increased platelet activity are two major risk factors for atherosclerosis. Lovastatin, the lipophilic drug was shown to inhibit platelet aggregation whereas pravastatin, the hydrophilic drug had no such effect. Analysis of the effect of fluvastatin which is both a lipophilic and hydrophilic drug, on platelet aggregation was the goal of the present study.

METHODS

Fluvastatin 40 mg daily was administered to 25 patients with hypercholesterolaemia for up to 24 weeks. Normal subjects acted as controls. The influence of fluvastatin on plasma lipids and on platelet aggregation and fluidity was studied. The direct effect of fluvastatin on platelets was compared with that of other statins.

RESULTS

Fluvastatin therapy (40 mg day (-1) for a period of 4 weeks) in hypercholesterolaemic patients resulted in a 23% and 29% reduction in plasma levels of total cholesterol and LDL-cholesterol respectively. Platelet cholesterol/phospholipids molar ratio was reduced by 26% and platelet aggregation was significantly (P<0.02) reduced by 10% after 4 weeks of fluvastatin treatment. On continuing fluvastatin therapy for additional 20 weeks, no further decrement in plasma LDL cholesterol levels or in platelet cholesterol/phospholipid ratio were noted. However, platelet aggregation was further significantly (P<0.01) reduced by up to 15%. Incubation of platelets with increasing concentrations of fluvastatin or lovastatin, demonstrated a dose-dependent reduction in platelet aggregation, whereas pravastatin showed no effect. This inhibitory effect of fluvastatin or lovastatin on platelet aggregation (up to 34% or 22% respectively at a concentration of 1 microg statin ml (-1) was found both in platelet rich plasma and in washed platelet suspensions. Fluvastatin and lovastatin (but not pravastatin), seem to share similar platelet binding sites, as non labelled fluvastatin or lovastatin were able to displace [3H]-labeled-fluvastatin from its binding sites on platelets.

CONCLUSIONS

Fluvastatin therapy reduces platelet aggregation via a dual effect which involves its in vivo hypocholesterolaemic action on platelet cholesterol content, and also a direct effect of the drug binding to the platelets. The antiatherogenicity of fluvastatin may be related, in addition to its plasma cholesterol lowering ability, to its inhibitory effect on platelet activation.

OBJECTIVE

The aim of the study was to evaluate the relationship between daily aspirin use and urinary excretion of a stable thromboxane metabolite, 11-dehydrothromboxane B(2) (11-DTB2), in African American stroke patients.

METHODS

Subjects were a subgroup of those screened for the African American Antiplatelet Stroke Prevention Study. Subjects were within 4 months of noncardioembolic ischemic stroke and were not being treated with anticoagulants. Antithrombotic therapy at the time of urine collection varied according to the practice patterns of various attending physicians who treated the patients during their acute strokes. 11-DTB2 was measured by enzyme immunoassay in random urine samples 1 to 4 months after the stroke.

RESULTS

Eighty-seven of 92 patients enrolled were able to give a urine sample at the time of enrollment. There were 51 men and 36 women aged 36 to 87 (mean 62) years. On the basis of antithrombotic treatment before the sample collection, we divided patients into 4 groups: (1) 16 patients treated with no aspirin (no antithrombotic drugs [n=4] or ticlopidine [n=12]), (2) 21 patients treated with 81 to 325 mg aspirin per day (81 mg/d [n=2], 325 mg/d [n=19]), (3) 20 patients treated with 650 mg aspirin per day, and (4) 30 patients treated with 975 to 1300 mg aspirin per day (975 mg/d [n=2] and 1300 mg/d [n=28]). In patients taking daily aspirin at any dose, the median urinary 11-DTB2 was 783 pg/mg creatinine compared with 1386 pg/mg creatinine in patients not taking daily aspirin (P=0.01 by Wilcoxon rank sum test). In multivariate regression analysis, aspirin use remained significantly associated with lower urinary 11-DTB2 (P=0.008). There was no dose-response effect between the 3 aspirin dose groups and urinary 11-DTB2 (P=0.70).

CONCLUSIONS

In African American stroke patients, aspirin use is associated with significantly lower urinary 11-DTB2 independent of other vascular factors, and there does not appear to be a dose-response effect for aspirin doses of 325 to 1300 mg daily. The clinical significance of these finding remains to be determined.