Prothrombotic responses to exercise are little influenced by clopidogrel treatment.

AIMS: Adenosine diphosphate (ADP) is involved in shear-induced platelet activation, which may be important for platelet responses to stress. We therefore tested the hypothesis that ADP receptor antagonism by clopidogrel treatment would attenuate exercise-induced platelet activation. METHODS AND RESULTS: Fifteen healthy volunteers performed exhaustive exercise without and with clopidogrel pretreatment (75 mg/day; 7 days) in a randomised crossover study. Filtragometry readings (reflecting platelet aggregability in vivo) and 11-dehydro-thromboxane B(2) (TxM) in plasma were determined before and after exercise. Platelet and leukocyte activity, platelet-platelet (PPA), and platelet-leukocyte aggregates (PLAs) in vivo and their responsiveness to agonist stimulation in vitro were assessed by flow cytometry. Clopidogrel treatment inhibited ADP-induced platelet P-selectin expression by 72% (54-85%). Exercise increased platelet aggregation (filtragometry and PPAs), elevated plasma TxM, increased single platelet P-selectin expression, elevated circulating PLAs, and enhanced ADP and thrombin-stimulated P-selectin expression. Clopidogrel prolonged filtragometry readings and attenuated agonist stimulated P-selectin expression at rest, but did not influence TxM in plasma or urine or attenuate platelet or leukocyte responses to exercise. Clopidogrel treatment did not influence plasma CD40L (ligand) at rest or after exercise. CONCLUSION: Clopidogrel treatment attenuates platelet activity in vivo at rest, but exercise counteracts the platelet stabilizing effects of clopidogrel. The hypothesis that ADP is involved in stress-induced platelet activation was not supported.

A new assay for beta-thromboglobulin in urine.

Measurements of beta-thromboglobulin (beta TG) excretion in urine may be of value for "field" studies and due to problems with sampling artifacts for beta TG in plasma. Previous studies have used a radioimmunoassay designed for plasma without characterizing the "beta TG" immunoreactivity in urine. We describe modifications of the assay which increase its sensitivity and a sample work-up procedure using Sephadex G-25M columns separating high molecular weight (HMW) components (presumably intact beta TG) from low molecular weight (LMW) immunoreactivity (i.e. beta TG fragments and/or non-specific interferences). The sensitivity of the assay (with 2.5 ml sample) is less than 12 pg/ml HMW beta TG. Inter- and intraassay coefficients of variation were 7-10%. Only 33 (range 5-75)% of beta TG immunoreactivity in urine represented HMW beta TG. LMW immunoreactivity may be related to salt and other non-specific influences in the sample. Recoveries of beta TG were quite variable (9-100%) in unextracted urines, but high and reproducible (80 +/- 2%) in the HMW fraction. Thus, nonspecific interferences with beta TG measurements in certain urines are overcome by the separation step. Using Sephadex fractionation beta TG immunoreactivities in night urines (n = 15) were: 20 +/- 3 pg/ml in the HMW fraction, 70 +/- 8 pg/ml in the LMW fraction, and 85 +/- 10 pg/ml by direct assay. HMW beta TG increased in daytime samples (to 30 +/- 5 pg/ml; p less than 0.01), but no diurnal variation was seen in the LMW fraction or with the direct assay. Thus, selective analysis of HMW beta TG in urine circumvents problems with nonspecific immunoreactivity and apparent interferences with measurements of intact beta TG. The present more selective assay for HMW immunoreactivity increases the possibility of detecting physiological changes in beta TG release in vivo by urinary measurements.

Beta-thromboglobulin in urine and plasma: influence of coronary risk factors.

Blood platelet activation in vivo was evaluated by measuring beta-thromboglobulin in plasma and high molecular weight beta-thromboglobulin in urine in hypertensive smoking and nonsmoking middle-aged men (n=36) and in normotensive age-matched controls (n=40). We found no significant linear relationships between nocturnal or resting urinary high molecular weight beta-thromboglobulin and plasma beta-thromboglobulin in the combined hypertensive and normotensive groups. The excretion of high molecular weight beta-thromboglobulin correlated significantly with diastolic blood pressure when all subjects were pooled. After 60 minutes supine rest, nonsmokers had higher excretion of high molecular weight beta-thromboglobulin than smokers. Plasma beta-thromboglobulin levels tended to be higher in hypertensives. In multivariate analyses, resting high molecular weight beta-thromboglobulin excretion was positively related to diastolic blood pressure and negatively related to smoking, whereas plasma beta-thromboglobulin was positively related to diastolic blood pressure and inversely related to apolipoprotein A1 and B. We conclude that urinary high molecular weight beta-thromboglobulin and plasma beta-thromboglobulin are not closely related, but are complementary analyses, as there are methodological confounders for both variables.

Optimization of an enzyme immunoassay for 11-dehydro-thromboxane B(2) in urine: comparison with GC-MS.

The urinary excretion of stable metabolites of thromboxane A2, such as 11-dehydro-thromboxane B2, reflects platelet activity in vivo. Efficient sample purification is required before analysis of thromboxane metabolites, due to the presence of large amounts of interfering material in urine. Analysis by gas chromatography-mass spectrometry after extensive sample work-up procedures provides the most reliable data, but detection by enzyme immunoassay may be reliable if sample cleanup is adequate. We describe an improved immunoassay procedure for 11-dehydro-thromboxane B2, which is based on a simple one-step solid phase extraction, by using Bond-Elut Certify II columns, followed by enzyme immunoassay by using commercially available reagents. 11-Dehydro-thromboxane B2 exists in two forms, with different chemical and immunological characteristics, which are in pH-dependent equilibrium. We kept 11-dehydrothromboxane B2 in its open ring form throughout the assay, by incubating and handling samples at pH 8.6. The extraction step achieved a recovery of 83% (95% confidence interval 74-92%), the sensitivity of the enzyme immunoassay was doubled, and the reproducibility of the assay improved under these conditions. Intra- and interassay coefficients of variation were 3 and 13.8%, respectively. A single 500-mg dose of aspirin reduced the excretion of 11-dehydro-thromboxane B2 by 77+/-14%, suggesting good specificity. Comparison with gas chromatography-mass spectrometry in 28 urine samples showed excellent agreement between the two methods (r2 = 0.94; p<0.0001), and a regression line with a slope close to 1.0. The presently modified enzyme immunoassay for 11-dehydro-thromboxane B2 is suitable for clinical studies evaluating platelet function in vivo and has the advantage of being simpler and less expensive to use than gas chromatography-mass spectrometry.