Effect of Armagnac fractions on human platelet aggregation in vitro and on rat arteriovenous shunt thrombosis in vivo probably not related only to polyphenols

Previous studies showed that alcohol-free extracts of Armagnac, an oak cask aged spirit rich in polyphenols, inhibit human platelet function in vitro and in vivo, in an experimental rat arteriovenous shunt thrombosis model and in human healthy volunteers. To identify active compounds, we fractionated a freeze-dried extract of a 10-year-old Armagnac using successively chloroform, diethyl ether and ethyl acetate. The 4 resulting fractions were tested on in vitro human platelet aggregation induced by ADP and in vivo on arteriovenous shunt thrombosis after 10 days oral treatment in rats. Active components were found mainly in fractions 1 and 3: at the highest concentration (2.4 10(-2) g/l), in vitro ADP-induced aggregation was inhibited by 62.7+/-2.1% and 51.2+/-3.8% for F1 and F3, respectively, vs 18.9+/-2.4% and 13.9+/-0.4% for fractions 2 and 4 and 33.6+/-1.5% for the crude extract. There was a significant decrease in thrombus weight with the crude extract and all fractions tested after 10 days treatment with 2.5 mg/kg/day orally, greatest with fraction 1. Characterisation of phenol content showed that fraction 1, the most biologically active, was essentially devoid of ellagic acid and ellagitannins, the polyphenols initially thought responsible for the effect, whereas fraction 2 which was mostly inactive, was the richest in polyphenols. CONCLUSION: The antiplatelet and antithrombotic activity of Armagnac seems mostly unrelated to polyphenols.     

A systematic review of the effects of acute psychological stress and physical activity on haemorheology, coagulation, fibrinolysis and platelet reactivity: Implications for the pathogenesis of acute coronary syndromes

Physical activity and psychological stress are two potential triggers for the onset of acute coronary syndromes (ACS). To examine the mechanisms underlying this association, we systematically reviewed the literature to determine the effects of acute psychological stress and physical activity on haemorheology and haemostasis. Studies examining the haemorheological and haemostatic response to an acute bout of physical activity (i.e. < 60 min) or laboratory psychological stress task were eligible for inclusion. The experimental evidence, although compromised by various methodological weaknesses, suggests that low and moderate intensity physical activity may be cardio-protective through beneficial effects on fibrinolytic system. High levels of physical activity, and psychological to a lesser extent, have been consistently associated with robust changes in haemorheology and haemostasis. Such findings imply that such activities may have the potential to trigger the onset of ACS, although in reality this may be limited sedentary individual and/or those with pre-existing vascular disease. In addition, the data also suggest that individuals may be at a greatest risk of stress-induced thrombogenesis in the period immediately following physical activity or psychological stress, rather than during the activity per se. In conclusion, psychological stress and physical activity may act as potential triggers for the onset of ACS via effects on haemostasis and haemorheology.     

Quantification of platelet composition in experimental venous thrombosis by real-time polymerase chain reaction

INTRODUCTION: Platelets play a key role in thrombus formation. Determination of the platelet component in a thrombus provides pathophysiological insights to the thrombotic event and aids in selecting an appropriate therapeutic intervention. In this study a sensitive and reliable method to characterize the cellular components of experimental thrombi was developed using real-time polymerase chain reaction (PCR). METHODS AND RESULTS: Vena cava thrombosis was induced by either oxidative injury to topical FeCl(2) (FeCl(2)-VT) or stenosis-limited blood flow and a hypotonic pressure stress (stasis-VT) in rats. High levels of platelets were identified in the thrombus containing vessels by real-time PCR analysis of target gene amplification using the 2(-DeltaDeltaCT) values by normalizing the data with gene expression in naive vessels and with a housekeeping gene, ribosomal protein L32. By this analysis, the levels of PF-4 (as a platelet marker) mRNA were significantly higher in FeCl(2)-VT (2(-DeltaDeltaCT)=7.8) than in stasis-VT (2(-DeltaDeltaCT)=4.2, p<0.05). Enhanced platelet enrichment in FeCl(2)-VT was also confirmed qualitatively by scanning electronic microscopic analysis. In addition, real-time PCR using a panel of genes representing vascular injury, inflammation and thrombosis showed marked induction (2(-DeltaDeltaCT)>5) in MCP-1, IL-1beta, iNOS and P-selectin mRNA expression in both models. CONCLUSIONS: These data demonstrate the utility of real-time PCR to quantitate platelets and other cell components in vascular thrombosis, which may facilitate the characterization and thus therapeutic intervention of a particular thrombotic event in both preclinical animal models and clinical conditions.     

Continuing developments with the automated platelet count

The four main procedures for platelet counting are: manual phase contrast microscopy, impedance, optical light scatter/fluorescence and flow cytometry. Early methods to enumerate platelets were inaccurate and irreproducible. The manual count is still recognized as the gold standard or reference method, and until very recently the calibration of platelet counts by the manufacturers of automated cell counters and quality control material was performed by this method. However, it is time-consuming and results in high levels of imprecision. The introduction of automated full blood counters using impedance technology resulted in a dramatic improvement in precision. However, impedance counts still have limitations as cell size analysis cannot discriminate platelets from other similar-sized particles. More recently, light scatter or fluorescence methods have been introduced for automated platelet counting, but there are still occasional cases where an accurate platelet count remains a challenge. Thus, there has been interest in the development of an improved reference procedure to enable optimization of automated platelet counting. This method utilizes monoclonal antibodies to platelet cell surface antigens conjugated to a suitable fluorophore. This permits the possible implementation of a new reference method to calibrate cell counters, assign values to calibrators, and to obtain a direct platelet count on a variety of pathological samples. In future, analysers may introduce additional platelet parameters; a reliable method to quantify immature or reticulated platelets would be useful.     

Prolonged platelet activation in individuals with elevated blood pressure in response to a moderate exercise challenge

We examined the magnitude of 20-min moderate exercise-induced platelet activation in 50 volunteers with normal ( n =31) or elevated blood pressure (EBP; n =19). Blood was drawn before, immediately after, and 25 min after exercise. Antibody-staining for platelet activation markers, P-selectin, and fibrinogen receptors was done with and without adenosine diphosphate (ADP) stimulation in whole blood for flow cytometric analyses. Exercise led to increases in percent aggregated platelets and percent platelets expressing P-selectin or PAC-1 binding ( p s≤.001). This increase in percent platelets expressing P-selectin continued even after a 25-min rest only in the EBP group ( p ≤.01) accompanied by an increase in percent of aggregated platelets ( p ≤.05). Although ADP stimulation led to increased platelet activation at rest, it was attenuated following exercise, even among EBP individuals. A moderate exercise challenge induced prolonged platelet activation in individuals with EBP but attenuation in activation to further stimulation by an agonist. Findings suggest that a recovery period after physical stress appears critical in individuals with high BP regarding platelet activation and aggregation, which can lead to an acute coronary syndrome in vulnerable individuals.     

Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in patients with uterine cervix neoplasia

OBJECTIVE: Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities, in the platelets and serum, were examined in patients with uterine cervix neoplasia without treatment as well as in patients treated by conization or radiotherapy (RTX). DESIGN AND METHODS: The patients were divided based on the amount of time from the end of the treatments until the day of the blood sampling. Groups I (n=19) (conization) and III (n=11) (radiotherapy) (treated from one to five years earlier), groups II (n=19) (conization) and IV (n=16) (radiotherapy) (treated recently; up to three months earlier) and the non-treated group (cancer) (n=7). RESULTS: E-NPP and ADA in the platelets and E-NPP in the serum were decreased in all the treated groups in relation to the control and non-treated groups, while ADA in the serum was decreased only in the conization groups in relation to them. In group II, E-NPP and ADA, in the platelets, were increased in relation to group IV. CONCLUSION: The tendency of reduction for E-NPP and ADA indicates that they may act together to control nucleotide levels and it may also be speculated that surgery causes greater platelet activation contributing to the changes seen in the conization groups. In this sense, platelets seem to be more sensitive than serum.     

Biological effect of increased maintenance dose of clopidogrel in cardiovascular outpatients and influence of the cytochrome P450 2C19*2 allele on clopidogrel responsiveness

INTRODUCTION: The first aim of this study is to evaluate the biological effect of doubling the maintenance dose of clopidogrel in pre-defined clopidogrel "low responders", compared to the biological effect of the standard dose in "responders". The second aim is to test the influence of the CYP 2C19*2 allele on clopidogrel responsiveness. MATERIALS AND METHODS: The platelet reactivity index (PRI), based on the phosphorylation status of the vasodilatator phosphoprotein, was determined in 81 consecutive cardiovascular outpatients who had been taking clopidogrel 75 mg/day for at least 15 days (visit 1). Patients with PRI>or=50% ("low responders") were then given clopidogrel 150 mg/day. All the patients were again evaluated 15 days later (visit 2) and were genotyped for the CYP 2C19*2 allele. RESULTS: At visit 1, PRI values ranged from 12.6% to 80.4%. In "low responders" (n=45), the mean PRI fell from 62.0+/-6.7% to 49.4+/-11.3% (P<0.001) after 15 days of clopidogrel 150 mg/day, while no significant change was observed in the other patients ("responders"), who remained on the standard dose (mean PRI: 37.7+/-10.4% and 39.9+/-10.8%, P=0.22, in visit 1 and 2, respectively). The CYP 2C19*2 allele was not associated with clopidogrel responsiveness. CONCLUSIONS: Increasing the maintenance dose of clopidogrel from 75 to 150 mg/day for 15 days in "low responders" is associated with a relative 20%-increase in its biological effect, independently of the CYP2C19 genotype, but without reaching the levels observed in "responders". The CYP 2C19*2 allele is not associated with clopidogrel responsiveness in our population of cardiovascular outpatients.     

Lack of biological relevance of platelet cyclooxygenase-2 dependent thromboxane A2 production

INTRODUCTION: There is emerging evidence of a considerable variability of the impact of aspirin on clinical outcome and laboratory findings. Persistent TxA2 production seems to be the most likely reason. Aim of this study was to determine whether the mechanism responsible for TxA2 persistent production is, at least partially, dependent upon aspirin-insensitive platelet COX-2 enzymatic pathway. METHODS AND RESULTS: In 100 consecutive patients, under chronic aspirin anti-platelet treatment (100-160 mg/day) selected on the basis of detectable plasma salicylate levels, serum and Arachidonic Acid (AA)-induced platelet TxA2 production, immunoblot analysis of platelet COX-1/COX-2 expression and COX-2 activity were studied. Immunoblot revealed COX-2 expression in 46% patients, in an amount that was markedly lower than COX-1. In 10 COX-2 positive patients with TxA2 levels over the median, AA-induced TxA2 production performed in vitro in the presence of the COX-2 inhibitor CAY10404 and aspirin demonstrated that COX-2 dependent TxA2 production is less than 2%. CONCLUSION: Our data demonstrate that the inter-individual variability of platelet sensitivity to aspirin is due to a reduced efficacy of aspirin on platelet COX-1 despite ascertained patient compliance. We suggest that serum TxA2 assay might be performed in future clinical studies to improve our knowledge on the residual TxA2 production in aspirin-treated patients.     

Transitioning patients from cangrelor to clopidogrel: pharmacodynamic evidence of a competitive effect

BACKGROUND: Cangrelor is a direct, parenteral, and reversible inhibitor of the platelet P2Y12 receptor currently undergoing Phase III testing. As many individuals treated acutely with cangrelor will often be treated long-term with a thienopyridine, it is important to determine the effects of concurrent cangrelor and clopidogrel administration. METHODS AND RESULTS: Ten healthy volunteers received a 600 mg oral loading dose of clopidogrel and then underwent serial platelet function monitoring for 6 h. Two weeks later these same individuals received a 600 mg clopidogrel loading dose simultaneously with a cangrelor IV bolus (30 microg/kg) and a 2-hour infusion (4 microg/kg/min). A separate group of ten volunteers received a 600 mg clopidogrel loading dose after administration of a cangrelor bolus and a 1-hour infusion. The effects on ADP-induced platelet activation and aggregation were evaluated by flow cytometry, whole-blood electrical impedance, and light-transmittance aggregometry. Cangrelor and clopidogrel alone achieved the expected levels of platelet inhibition. However, the sustained platelet inhibition anticipated for clopidogrel treatment did not occur when cangrelor was initiated simultaneously. No such effect was found when clopidogrel was started upon completion of the cangrelor infusion. CONCLUSION: To achieve sustained platelet P2Y12 inhibition in patients treated with cangrelor, clopidogrel administration should be started when the cangrelor infusion is terminated.