Raneem O. Salem

Effects of alcohol on hemostasis.

Several epidemiologic studies have shown that moderate intake of alcohol is associated with a lower risk of cardiovascular disease (CVD), but the mechanism is not fully elucidated. One of the proposed mechanisms of the protective effect of moderate alcohol intake is its beneficial effect on hemostasis. The aim of this review is to summarize the effect of ethanol intake on platelet aggregation and activation, coagulation factors including von Willebrand factor (vWF), and the fibrinolytic system. With regard to the effect of alcohol on platelet function, evidence in the literature suggests both platelet activation and platelet inhibition by ethanol. A unifying hypothesis is that platelets are partially activated by ethanol, with partial degranulation allowing for continued circulation of platelets with impaired function. Evidence also exists showing that ethanol intake decreases fibrinogen, factor VII, and vWF levels. In addition, alcohol intake has been found to increase fibrinolysis by increasing tissue plasminogen activator activity. The effect of ethanol on platelets, coagulation factors, and the fibrinolytic system is likely to contribute to protection against CVD.

Fatty acid ethyl esters. Ethanol metabolites that reflect ethanol intake.

Fatty acid ethyl esters (FAEEs) are nonoxidative ethanol metabolites that have been implicated as mediators of alcohol-induced organ damage. FAEEs are detectable in the blood after ethanol ingestion, and on that basis represent markers of ethanol intake. FAEEs have also been quantitated in human liver and adipose tissue and have been shown to be postmortem markers of premortem ethanol intake. A substantial difference in FAEE concentration was found in liver and adipose tissue of patients with detectable blood ethanol at the time of autopsy vs those with no detectable blood ethanol, who were either chronic alcoholics or social drinkers. Most currently available diagnostic markers for chronic alcoholism have limited clinical utility. Data in this report demonstrate that the amount or type of FAEEs can be used to differentiate a chronic alcoholic from an episodic heavy drinker (binage drinker) at or near peak blood ethanol concentrations and approximately 24 hours after discontinuation of ethanol. Thus, FAEEs are markers of ethanol intake in blood and tissues and can be useful in distinguishing chronic alcoholics from binge drinkers.

Effects of a Pneumatic Tube System on Routine and Novel Hematology and Coagulation Parameters in Healthy Volunteers

CONTEXT Technologic advances affecting analyzers used in clinical laboratories have changed the methods used to obtain many laboratory measurements, and many novel parameters are now available. The effects of specimen transport through a pneumatic tube system on laboratory results obtained with such modern instruments are unclear. OBJECTIVE To determine the effects of sample transport through a pneumatic tube system on routine and novel hematology and coagulation parameters obtained on state-of-the-art analyzers. DESIGN Paired blood samples from 33 healthy volunteers were either hand delivered to the clinical laboratory or transported through a pneumatic tube system. RESULTS No statistically significant differences were observed for routine complete blood cell count and white cell differential parameters or markers of platelet activation, such as the mean platelet component, or of red cell fragmentation. When 2 donors who reported aspirin intake were excluded from the analysis, there was a statistically, but not clinically, significant impact of transport through the pneumatic tube system on the mean platelet component. There were no statistically significant differences for prothrombin time, activated partial thromboplastin time, waveform slopes for prothrombin time or activated partial thromboplastin time, fibrinogen, or fibrin monomers. CONCLUSIONS Although further study regarding the mean platelet component may be required, transport through a pneumatic tube system has no clinically significant effect on hematology and coagulation results obtained with certain modern instruments in blood samples from healthy volunteers.

A new automated screening assay for the diagnosis of von willebrand disease

A new, automated assay for von Willebrand factor (vWF) activity has recently become commercially available (HemosIL vWF activity assay, Instrumentation Laboratories, Lexington, MA). We prospectively studied 61 specimens from 58 patients undergoing laboratory testing for suspicion of von Willebrand disease with this new method, in comparison with the established ristocetin cofactor method. Assays for factor VIII and vWF antigen were also performed using an established method on an MDA-180 coagulation analyzer (bioMérieux, Durham, NC) and a new method on an ACL TOP coagulation analyzer (Instrumentation Laboratories). Blood types were determined. The results showed no significant difference between the assays for factor VIII (mean, 97% for MDA-180 and ACL TOP; P = .494) or vWF antigen (mean, MDA-180, 109%; ACL TOP, 111%; P = .766). The mean result for the ristocetin cofactor assay was 106% vs 93% with the automated vWF activity (P = .007). The automated activity assay was 100% sensitive and 86% specific for detecting vWF abnormalities and seems to be a suitable screening test. Abnormal results should be followed up with a ristocetin cofactor activity assay for confirmation. Further study is recommended to confirm these conclusions.

91 – Alcohol and Fatty Acid Ethyl Esters

This chapter discusses the relationship of alcohol and fatty acid ethyl esters. Fatty Acid Ethyl Esters (FAEE) are nonoxidative ethanol metabolites that are esterification products of fatty acids and ethanol. FAEE directly contribute to cell injury induced by ethanol abuse, but the exact mechanism by which FAEE mediate this toxic effect remains to be discovered. Most of the enzyme activities found to be catalytic for FAEE synthesis are esterification enzymes that link fatty acid or fatty acyl-CoA to ethanol. However, there are a number of reports that suggest that the hydrolysis of fatty acids from a more complex lipid molecule (such as phospholipid and triglyceride) is the rate limiting step in the formation of FAEE, and that the esterification step is presumably either nonrate-limiting or nonenzymatic. FAEE are of growing importance in the toxicity induced by excess ethanol intake. There is an increasing evidence that FAEE mediated cytotoxicity and FAEE are proving to be a robust marker of ethanol intake in a variety of cells and tissues, and in the blood. The use of FAEE to monitor ethanol intake or to identify individuals as chronic alcoholics should continue to strengthen as more clinical studies are completed.

Fatty acid ethyl ester, a non-oxidative ethanol metabolite, reverses the inhibitory effect of ibuprofen on platelet aggregation.

Aspirin and certain non-aspirin non-steroidal anti-inflammatory drugs (NSAIDs) can produce an increased tendency to bleed by impairing thromboxane A2 (TXA2)-dependent platelet aggregation, especially in the presence of other risk factors for bleeding [1]. These substances reduce platelet aggregation by inhibiting platelet cyclooxygenase (COX), thereby blocking the formation of TXA2. Aspirin inhibits the COX enzyme through the irreversible acetylation of the hydroxyl group of serine 529 [2]. Its effects persist for the duration of the lifetime of the platelet in the circulation. In contrast, NSAIDs, such as indomethacin and ibuprofen, produce reversible COX inhibition. NSAIDs compete with the most commonly presented substrate, arachidonate, for the active site of the enzyme [3,4]. Approximately 25% of patients using aspirin and certain non-aspirin (e.g. ibuprofen) NSAIDs experience some kind of side effect. Themost serious adverse effect of NSAIDS is upper gastrointestinal bleeding [5]. The clinical risk for bleeding in patients taking aspirin or NSAIDs is enhanced by the concomitant use of ethanol. Some studies have shown that ethanol ingestion may amplify the effect of aspirin and ibuprofen on human platelets and increase risk of bleeding [6–8]. The ingestion of aspirin before ethanol intake also increases the blood ethanol concentration by the inhibition of gastric alcohol dehydrogenase (ADH) [9]. In this study, we evaluated the effect of fatty acid ethyl ester (FAEE), a non-oxidative ethanol metabolite, on platelet aggregation in the presence of aspirin or ibuprofen. Unexpectedly, we found that a physiological concentration of FAEE easily achieved with ethanol intake rapidly reverses the effect of ibuprofen, but not aspirin, on the platelet aggregation. Blood was collected in citrate anticoagulated tubes from volunteers. The volunteers had ingested either 400 mg of ibuprofen 2 h prior to blood collection or 325 mg aspirin the night before the collection. Platelet-rich plasma (PRP) was isolated and 500 lL aliquots were added to siliconized tubes containing different concentration of ethyl oleate (5, 10, or 25 lM), as a representative FAEE species. Ethyl oleate is elevated in concentration in chronic alcoholics [10]. All tubes were incubated for 1 min at 37 C. In another set of experiments, the tubes were incubated for different times (1, 5, 10, or 15 min). At the end of the incubation time, 50 lL of arachidonate (500 lg mL) (Sigma Chemical Co., St Louis, MO, USA) was added to all tubes. The tubes were then transferred to a platelet aggregation chromogenic kinetic system PACKS-4 (Helena Laboratories, Beaumont, TX, USA) to measure platelet aggregation. Platelet-rich plasmawas isolated and incubatedwith 5, 10, or 25 lMFAEE for 1 min (Fig. 1A). Platelets did not aggregate in response to arachidonate in samples where no FAEE was added. However, in samples where FAEE was added, a dose– response effect was observed, with 90% aggregation (a full response) reached at 4 min or less following the stimulation with arachidonate in the continued presence of 25 lM FAEE (Fig. 1A). Longer incubation times with FAEE (up to 15 min) showed the same results (Fig. 1B). We have detected up to 41 lM FAEE in the serum of patients presenting to our emergency department [11]. Other species of FAEE (ethyl palmitate, ethyl linolate, and ethyl arachidonate), like that shown in Fig. 1 with ethyl oleate, were able to restore normal platelet aggregation in response to arachidonate in samples isolated from subjects ingesting ibuprofen. Thus, FAEE restored normal platelet aggregation in response to arachidonate from subjects ingesting 400 mg ibuprofen in a doseand time-dependent manner. In addition, the presence of FAEE restored normal platelet aggregation in response to 10 lM epinephrine, which is also impaired in patients takingNSAIDs. Fatty acid ethyl ester (25 lM ethyl oleate) was also incubated with PRP obtained from three subjects who ingested aspirin (325 mg) 8–12 h prior to blood collection. Aspirin inhibited platelet aggregation in response to arachidonate in the presence and in the absence of FAEE. One possible explanation for the FAEE-induced reversal effect to normal platelet aggregation in subjects ingesting ibuprofen is that FAEE increases arachidonate release from phospholipids (R. O. Salem et al., unpublished data). The free arachidonate then effectively competes with ibuprofen Correspondence: Michael Laposata, Massachusetts General Hospital, Room 235 Gray Building, 55 Fruit Street, Boston, MA 02114, USA. Tel.: +1 617 726 8172; fax: +1 617 726 3256; e-mail: mlaposata@ partners.org