Xanthohumol from hop cones (Humulus lupulus L.) prevents ADP-induced platelet reactivity.

Hop cones (Humulus lupulus L.), very rich source of phenolic compounds, possessing anticancer, antioxidant and anti-inflammatory activities, are considered as beneficial diet ingredients improving human health. In this study, the antiplatelet action of xanthohumol (XN), the principal flavonoid in hop cones, was investigated. XN significantly attenuated ADP-induced blood platelet aggregation (97.2 ± 35.7 AU for 6 µg/ml of XN vs. 120.4 ± 30.1 AU for 0.17% dimethyl sulfoxide (DMSO), p < 0.001) and significantly reduced the expression of fibrinogen receptor (activated form of GPIIbIIIa) on platelets' surface (47.6 ± 15.8 for 1.5 µg/ml XN, 44.6 ± 17.3% for 3 µg/ml XN vs. 54.5 ± 19.2% for control or 43.3 ± 18.4% for 6 µg/ml XN vs. 49.7 ± 19.4% for 0.17% DMSO, p < 0.05 or less). These findings suggest that the phenolic compounds originating from hops (XN) have a novel role as antiplatelet agents and can likely be used as dietary supplements in prophylactic approaches.

Patient-independent variables affecting the assessment of aspirin responsiveness by serum thromboxane measurement.

The serum TXB2 (sTXB2) assay reflects the pharmacodynamics of platelet inhibition by low-dose aspirin. However, different studies reported variable sTXB2 values. sTXB2 assay requires whole blood incubation at 37 °C as a condition for optimal thrombin generation, arachidonic acid release and its metabolism by platelet cyclooxygenase-1 to form TXA2. Access to 37 °C incubation may be variably delayed, and different methods to quantitate sTXB2 may contribute to variable results between different Centers. We investigated whether delaying 37 °C incubation and/or analytical issues affect sTXB2 concentrations, biasing the assessment of aspirin responsiveness. Sixty-eight samples from 54 volunteers, on- and off-aspirin, were incubated at 37 °C immediately after sampling (reference sample) or after 5, 10, 15, 20, 30 or 60 minutes at room temperature (RT); 8 samples remained at RT 60 minutes, without subsequent incubation; 314 sera were measured by enzyme immunoassay (EIA) and liquid chromatography-tandem mass-spectrometry (LC/MS-MS) methods. sTXB2 concentrations decreased exponentially as a function of the delay before 37 °C incubation, ranging from 94 ± 11 % at 5 minutes to 23 ± 22 % of the reference sample after 60 minutes at RT. There was high agreement between EIA and LC/MS-MS. Moreover, we simulated the influence of a 15- or 30-minute delayed incubation on 300 sTXB2 measurements from previously-studied, aspirin-treated patients. Delayed incubation reduced the percentage of aspirin 'non-responders' by 22 % to 52 %, depending on the response threshold. In conclusion, a variable delay in the 37 °C incubation of blood samples may affect the assessment of platelet cyclooxygenase-1 inhibition by aspirin and confound the characterization of the determinants of aspirin responsiveness.

Comparative impact on prostanoid biosynthesis of celecoxib and the novel nonsteroidal anti-inflammatory drug CG100649.

Nonsteroidal anti-inflammatory drugs (NSAIDs) elevate cardiovascular risk by disrupting cyclooxygenase-2 (COX-2)-dependent biosynthesis of prostacyclin (PGI(2)). CG100649 is a novel NSAID proposed to inhibit both COX-2 and carbonic anhydrase (CA)-I/-II. We compared its impact on prostanoid biosynthesis with that of celecoxib, an NSAID purposefully designed to selectively inhibit COX-2. In a controlled, double-blind randomized trial, single oral doses of 2 or 8 mg CG100649, 200 mg celecoxib, or placebo were well tolerated by healthy volunteers (n = 23). Both CG100649 and celecoxib had the effect of depressing urinary excretion of 2,3-dinor-6-keto-PGF(1α) (PGI-M); the effect of CG100649 was dose-dependent and more sustained (up to 240 h after the dose) than that of celecoxib. Neither CG100649 nor celecoxib significantly inhibited COX-1-dependent prostanoid formation. CA inhibition was not detected after administration of CG100649, despite its partitioning asymmetrically into erythrocytes. CG100649 and celecoxib are both relatively selective inhibitors of COX-2, but they differ in duration of action. Whether they have similar impact on cardiovascular events remains to be determined.

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 resistance in patients with ischaemic heart disease (IHD) and investigated the concordance and repeatability of these tests. DESIGN, SETTING AND PATIENTS: Consecutive outpatients with stable IHD were enrolled. They were commenced on 150 mg aspirin daily (day 0) and had platelet function assessment (OPA and PFA-100) and quantitative analysis of serum/urine TXB2 at day > or =7 and then at a second visit approximately 2 weeks later. MAIN OUTCOME MEASURES: We assessed the prevalence of aspirin resistance by each method, concordance between methods of measuring response to aspirin and association between time points to assess the predictability of response over time. RESULTS: 172 patients (62.7 (SD 8.7) years, 83.1% male) were recruited. At visits 1 and 2, respectively, 1.7% and 4.7% were aspirin resistant by OPA, whereas 22.1% and 20.3% were aspirin resistant by PFA-100. There were poor associations between PFA-100 and OPA, and between TXB2 metabolites and platelet function tests. OPA and PFA-100 results were poorly associated between visits (kappa = 0.16 and kappa = 0.42, respectively) as were TXB2 metabolites, suggesting that aspirin resistance is not predictable over time. CONCLUSIONS: The prevalence of aspirin resistance is dependent on the method of testing. Response varies on a temporal basis, indicating that testing on a single occasion is inadequate to diagnose resistance or guide therapy in a clinical setting.

[Thrombogenic factors in the elderly. Evaluation of the plasma concentrations of thromboxane B2 and antithrombin III]. / Factores trombogénicos en el anciano. Valoración de las concentraciones plasmáticas de tromboxano B2 y antitrombina III.

The aim of this study is to prove the existence of a major tendency of platelet aggregation in elderly patients compared to medium-aged adults and, also, to detect whether it is affected by the presence of diabetes mellitus. Plasmatic concentrations of B2 thromboxane (TXB2) and antithrombin III (AT III) were determined in 73 elderly patients of both sexes; 56 without metabolic disease known (Group a) and 17 diabetic patients, 7 type I (Group bI) and 10 of type II (Group bII); and 12 healthy adults (control group). Medium plasmatic concentration of TXB2 was significantly higher (p less than 0.001) in Group a (55 +/- 14 ng/ml) compared to the control group (37 +/- 9 ng/ml) and there was no difference between Group bI (53 +/- 19 ng/ml) and bII (57 +/- 15 ng/ml). No variations were noted in ATIII concentration between the adults (27.4 +/- 2.3 mg/dl) and elderly patients (a = 29.6 +/- 4.4, bI = 29 +/- 2.6, bII = 31.2 +/- 5.9 mg/dl). In elderly patients, there appears to be a state of platelet pro-aggregation without influence of any risk factor, such as diabetes. This could explain the thrombogenic tendency of this age group.

Assessment of prostacyclin and thromboxane A2 release during reperfusion after global ischemia induced by crystalloid cardioplegia--comparison between warm and cold ischemia.

The metabolites of prostacyclin (PGI2) and thromboxane A2 (TxA2), 6-keto-PGF1 alpha and thromboxane B2 (TxB2), were investigated during reperfusion (RP) following warm (37 degrees C, 60 min, n = 9) or cold (15 degrees C, 120 min, n = 11) ischemia induced by cold (4 degrees C) or normothermic (30 degrees C) K+ cardioplegia (CP) in isolated canine hearts subjected to global ischemia and RP. 6-Keto-PGF1 alpha flux was significantly higher (p less than 0.025) in the warm group at 1, 5, and 10 min of RP (4,202 +/- 1,412, 2,475 +/- 1,875, and 1,255 +/- 633 pg/g.min, mean +/- SD) compared to those in the cold group (1,504 +/- 1,245, 434 +/- 641, and 370 +/- 329 pg/g.min). TxB2 flux was small in amount compared to 6-keto-PGF1 alpha in both groups. Regarding the coronary hemodynamics, the cold group alone showed statistically significant relationships of coronary sinus blood flow to TxB2 level and TxB2/6-keto-PGF1 alpha ratio in coronary sinus blood. Also, coronary vascular resistance showed linear relations to these two parameters of the metabolites. In a supplementary experiment only with cold ischemia for 180 min, 6-keto-PGF1 alpha was released at each coronary flush-out by CP and the incremental amount showed a gradual increase during ischemia. These results indicated that significant production and release of PGI2 occurred during ischemia and RP following CP arrest and these related to the degree of myocardial damage while the response of TxA2 seemed less significant. The role of PGI2 release during RP following cardioplegic arrest was discussed.

Re-examination of the assay for plasma prostanoids by solid-phase extraction, and radioimmunoassay.

A method for the routine assay of plasma thromboxane and prostacyclin metabolites, thromboxane B2 and 6 keto-prostaglandin F1 alpha (employing solid phase extraction to remove the metabolites from interfering substances) and using commercially available antisera for their radioimmunoassay has been investigated. Modifications of previously published procedures have been explored, including the use of non-explosive solvents in the extraction procedure, and inclusion of a non-cross reacting internal standard, prostaglandin D2, to estimate recovery through the extraction procedures. Both the original and modified procedures can produce high blank values, which apparently result from the solid-phase extraction. These high backgrounds are constant for a given lot of solid phase extraction cartridges and can be corrected by subtraction. The modified method is linear with volume of plasma assayed to as little as one ml, and all cross-reacting material in normal human plasma was found to co-chromatograph with authentic standards on reverse-phase high performance liquid chromatography. Values for normal adult plasmas were found to be 0.44 +/- .15 pmol/ml 6 keto PGF1 alpha (mean +/- SD) and .103 +/- .07 for thromboxane B2. The method reported provides a convenient, reproducible way to assay these important plasma prostanoids.

An objective assessment of the interaction of heparin and its fractions with human platelets.

We have shown that heparin and heparin fractions cause in vitro platelet aggregation in a large portion of a normal population. Furthermore, this aggregation occurs in a concentration-dependent manner and is not related to the anti-Xa activity of heparin or its fractions. In addition, it appears that at least part of the mechanism by which heparin induces aggregation is through the production of thromboxane. However, this is not the sole mechanism, since approximately 20% aggregation still occurs when thromboxane production is totally inhibited or the thromboxane receptor is completely blocked. Furthermore, although protamine (at the concentrations used) completely neutralizes the anticoagulant activity of heparin, it does not always completely inhibit the platelet aggregating activity of heparin. Finally, we have shown that heparin alone promotes thromboxane production and PF4 release in a whole blood system. Additional studies are needed to characterize further the mechanisms of heparin-induced platelet aggregation.

Assessment of platelet function in patients with Raynaud's syndrome.

Platelet function was studied in 11 patients with Raynaud's syndrome and 11 healthy controls. Platelets obtained from patients with Raynaud's syndrome were significantly more responsive to adrenaline, produced more thromboxane A2, and were resistant to prostaglandin inhibitors (prostacyclin and prostaglandin E1) of platelet aggregation. Platelets from control subjects and patients with Raynaud's syndrome were more resistant to prostaglandin inhibitors when reactions were carried out at 27 degrees C rather than at 37 degrees C. Patients with Raynaud's syndrome also had significantly increased plasma concentrations of beta-thromboglobulin, fibrinogen, and circulating platelet aggregates. In an attempt to elicit local platelet responses, the forearms of control subjects and patients with Raynaud's syndrome were cooled in water tanks and platelet function tests performed before and after cooling. No significant difference in the results was observed. The potential role of platelets in the pathogenesis of Raynaud's syndrome is discussed.