Pathology Patterns Reviews

Effects of Alcohol on Hemostasis

Raneem O. Salem, PhD, and Michael Laposata, MD, PhD

Key Words: Cardiovascular disease; Ethanol; Platelet aggregation; Fibrinogen; von Willebrand factor; Fibrinolysis; Tissue plasminogen activator; Tissue plasminogen activator inhibitor-1
DOI: 10.1309/113N8EUFXYUECCNA

Abstract
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 the protective effect of CVD.

The mechanisms by which alcohol affects the cardiovascular system, beneficially or detrimentally, have been the target of extensive research. Many epidemiologic studies have shown that moderate alcohol consumption (1-2 standard drinks per day or 10-20 g/d) can reduce the risk of heart disease and ischemic stroke by 20% to 60% and death of all causes by 10% to 20%.1 The underlying factors contributing to the protective or pathophysiologic effects related to alcohol consumption are not well understood. Alcohol consumption affects plasma lipid profiles and has been shown to raise high-density lipoprotein levels.2 A number of studies have indicated that ethanol also directly affects hemostasis via a number of mechanisms, including modulation of plasma coagulation factors, fibrinolysis, and platelet function.3-5 Thus, individuals without a history of alcoholism might benefit from moderate consumption of alcohol. Although the association between moderate drinking and a lower risk of coronary artery disease has been well documented, the mechanisms by which alcohol causes these effects remains just as elusive as those underlying alcohol-related diseases. Chronic alcohol consumption can predispose to bleeding. A study of patients in the United States and Sweden showed that the baseline incidence of acute upper gastrointestinal bleeding increased by 3-fold as alcohol consumption increased from 1 drink or fewer per week to more than 20 drinks per week.6 There is also a linear association between the consumption of ethanol and the risk for hemorrhagic stroke.7 In a study of a sample of Japanese men living in Hawaii, alcohol consumption reduced the incidence of myocardial infarction (MI) and cardiac death (ie, coronary thrombosis) but did not have the same protective effect against stable angina.8 This finding suggests that the effect of alcohol on hemostasis is an important, possibly predominant, mechanism in protecting against cardiovascular disease (CVD).
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This hypothesis is underscored by evidence that moderate drinkers lose this protective effect as soon as they stop drinking. This is not enough time for the lesions that characterize atherosclerosis to develop.8,9 This review summarizes studies on the effect of alcohol intake on platelets, coagulation factors, and fibrinolytic factors.

Effect of Ethanol on Platelet Aggregation

The benefit of moderate alcohol consumption in the prevention of CVD seems to be through the reduction of MI, not atherosclerosis. Because it is well established that coronary thrombosis is the occlusive event producing an acute MI, several studies have investigated the influence of alcohol on the various molecules and systems that are required for platelet activation and aggregation Table 1. Haut and Cowan,10 in 1974, were among the first to report the effect of alcohol on platelet activity. They demonstrated that alcohol added to human platelets in vitro, ingested by subjects, or infused intravenously into men markedly decreased platelet aggregation in response to thrombin, collagen, epinephrine, and adenosine-5'-diphosphate (ADP). Subsequent epidemiologic and clinical studies confirmed these results and showed that an increase in ethanol intake leads to a decrease in platelet aggregation in response to ADP and collagen.8-14 Renaud et al,15 in a cohort study, measured the effect of alcohol consumption on platelet aggregation in response to ADP and collagen in 1,600 men aged 49 to 66 years (part of the Caerphilly Prospective Heart Disease Study). Information

about the amount of alcohol consumed and the frequency of drinking was obtained through the use of a detailed questionnaire. Blood samples were obtained from subjects who had fasted for at least 10 hours; platelet-rich plasma (PRP) was isolated for use in platelet aggregation studies. The results showed that platelet aggregation in response to ADP and collagen decreased as the frequency of alcohol consumption increased. These trends suggest that men who consume alcohol more frequently are more likely to manifest a lesser degree of platelet activity. On the other hand, there was increased platelet activation in response to thrombin at all levels of alcohol consumption. This increase in activity does not indicate the absence of an inhibitory effect of alcohol because studies in which blood samples are not obtained within 20 minutes of ethanol ingestion might miss the inhibitory effect of alcohol on platelet aggregation, especially in response to thrombin. This hypothesis is based on results from various studies, one of which reported that platelet aggregation in response to collagen, ADP, and thrombin was inhibited within 10 minutes of ethanol intake and then rebounded, especially in response to thrombin.16 Mikhailidis et al5 studied the effect of ethanol on platelet aggregation using blood collected from alcoholics admitted for detoxification. At baseline, platelets were hypocoagulable in response to ADP, collagen, and platelet activating factor. The effect of alcohol subsided 2 weeks after abstinence, and the platelets became hyperaggregable. This increase in platelet activity might contribute to the sudden death associated with withdrawal or binge drinking.4 Several studies have indicated that ethanol has a greater inhibitory effect in platelets enriched with saturated vs unsaturated fats.13,17,19 One of the earliest studies that investigated

Table 1 Literature Review of the Effect of Alcohol Consumption on Platelet Aggregation

Study Haut and Cowan,10 1974 Multiple studies8,9,11,12,14 Renaud et al,15 1992 Method Added ethanol to human platelets, also an in vivo study, with ethanol ingested or infused into men Effect of ethanol intake on platelet activity in healthy volunteers Blood samples were obtained from 1,600 men to perform platelet aggregation studies; the amounts of alcohol consumed were obtained from a detailed questionnaire Effect of alcohol consumption on platelet aggregation Results Alcohol decreased platelet aggregation in response to thrombin, ADP , collagen, and epinephrine Decreased platelet aggregation in response to ADP and collagen with increasing alcohol consumption Decreased aggregation with increasing frequency of alcohol consumption for ADP and collagen; increased platelet activation in response to thrombin Platelet aggregation in response to collagen, ADP , and thrombin was inhibited within 10 min of ethanol ingestion, then showed a rebound effect, especially in respond to thrombin At baseline, platelets were hyperaggregable in response to ADP , collagen, and platelet activating factor; 2 wk after alcohol abstinence, platelets became hyperaggregable Alcohol had a greater inhibitory effect in response to platelets enriched with saturated fat

Hillbom et al 198516

Mikhailidis et al,5 1986

Effect of ethanol on platelet aggregation using blood samples from alcoholics admitted for detoxification The effect of ethanol on platelets enriched with saturated vs unsaturated fat

,18 Multiple studies13,17

ADP, adenosine-5'-diphosphate.

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this relationship was performed on rats and demonstrated a significant inhibitory effect of alcohol on platelet function, but only in animals fed a diet high in saturated fatty acids.17 In studies involving cholesterol rather than saturated fatty acids, by using cholesterol-enriched platelets from rabbits fed a chow diet with added cholesterol, Gross et al20 showed that aggregation in response to ADP was not affected but that platelets became hypersensitive to collagen through a thromboxane A2 (TXA2)-dependent pathway. These enhanced collagen-induced responses were diminished on the administration of 6% ethanol in the drinking water of cholesterol-fed animals for 1 week by reduction of TXA2 formation.18

Effect of Ethanol on Platelets

Studies using different models have indicated that ethanol can influence membrane-associated signaling systems. Studies have shown that ethanol can affect cyclic adenosine monophosphate (cAMP) synthesis or degradation (including activation of adenylate cyclase in membrane preparations), phospholipid breakdown, inositol-1,4,5triphosphate formation in hepatocytes, arachidonic acid metabolism, and calcium homeostasis.21,22 There is evidence that indicates ethanol inhibits platelet activation and that ethanol enhances platelet activation. Both sides of this argument follow. Evidence That Ethanol Inhibits Platelet Activation Different in vitro and in vivo studies have reported decreases in platelet aggregation in response to collagen, thrombin, and Ca2+ ionophore but not in response to arachidonic acid. Zhang et al23 studied the effect of physiologically relevant concentrations of alcohol (1-85-mmol/L concentrations) on platelet aggregation using whole blood samples and PRP from humans and rats. Ethanol (85-mmol/L concentration) inhibited collagen-induced platelet aggregation in whole blood samples and PRP. When ADP was used as an agonist, platelet aggregation was inhibited in whole blood samples but not in PRP in humans.23 The first step in formation of a plug is adhesion of platelets to collagen. Nguyen et al24 studied the effect of ethanol on platelet adhesion to collagen using platelets isolated from blood samples obtained from human donors. Their results indicated that ethanol had no effect on platelet adhesion to collagen or platelet secretion of [14C]-serotonin. However, alcohol inhibited the formation of TXA2 and decreased collagen-induced phosphorylation of phospholipase A2. Other studies have shown a decrease in arachidonate liberation from phospholipid membranes and, therefore, a decrease in the formation of TXA 2 with collagen stimulation.25,26 In these studies, washed human platelets
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were incubated with different ethanol concentrations (25250-mmol/L) and were activated with collagen. There was a dose-dependent inhibition of both aggregation and secretion in response to collagen. In addition, ethanol inhibited the release of arachidonic acid from phospholipids in platelets labeled with [ 3 H]arachidonic acid and incubated with ethanol; therefore, it inhibited the formation of TXA 2 . However, there was a partial restoration of aggregation and secretion with the addition of exogenous arachidonate. According to these results, ethanol affects mobilization of arachidonic acid by affecting phospholipase A2 rather than its conversion to TXA2. In these studies, normal levels of inositol phosphate and phosphatidic acid were detected, indicating that ethanol had no effect on the activation of phospholipase C by collagen.25 Another study, in which thrombin was used as the agonist instead of collagen showed the same results.27 This study showed an inhibition of thrombin-induced secretion of granules by ethanol. However, ethanol did not affect activation of phospholipase C or mobilization of intracellular calcium. Ethanol also had no effect on thrombin-induced shape change or formation of pseudopods, but ethanol inhibited granule centralization and fusion. The results from this study suggested that alcohol inhibits granule secretion through a Ca2+-mediated process, such as granule centralization, rather than through phospholipase C. Calcium mobilization has a major role in platelet activation and aggregation. Depletion of intracellular Ca2+ stores is linked to Ca2+ entry into platelets, and this entry is not dependent on membrane potential.28 The in vitro effect of ethanol on Ca2+ mobilization from the dense tubular system into the cytoplasm and on aggregation of platelets was recently studied. 29 Blood samples were obtained from healthy donors. Platelets were isolated, activated by thapsigargin (Ca2+-adenosinetriphosphatase inhibitor), and incubated with 85-, 170-, and 340-mmol/L concentrations of ethanol. Ethanol resulted in a dose-dependent inhibition of Ca2+ influx. A separate study showed a transient increase in cytosolic calcium levels following incubation with ethanol.21 When platelets are activated, certain markers such as Pselectin, glycoprotein (Gp) IIb/IIIa, and CD63 (lysosomal integral membrane protein [LIMP] or lysosomal-associated membrane protein [LAMP]-3) are exposed on their surfaces.30,31 McKenzie et al32 studied the effect of in vitro exposure to ethanol on platelet surface markers of activation. Blood samples were obtained from 10 healthy volunteers and divided into 3 groups. In the first group, 2.30 mg/dL of ethanol was added to the samples; in the second group only 1.15 mg/dL of ethanol was added; nothing was added to the third group, which served as a control for the other groups. The results showed a dose-dependent inhibition of P-selectin exposure and the appearance of LAMP-1
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(CD107a), GpIIb-IIIa, and CD 63 receptor expression in the samples to which ethanol was added. The final step in platelet aggregation is the binding of fibrinogen to GpIIb-IIIa on the surface of the activated platelet.33 GpIIb-IIIa inhibitors have been shown to inhibit platelet aggregation in response to all agonists, and they are used in patients at high risk for MI.34 Because ethanol was shown to inhibit GpIIb-IIIa receptor expression in vitro and inhibit platelet aggregation, ethanol was presumed to increase the antithrombotic effect of GpIIb-IIIa inhibitors. A study was conducted to test this hypothesis, in which blood samples were obtained from healthy volunteers and incubated with 4 mg/mL ethanol in the presence of LY309562, a GpIIb-IIIa inhibitor.35 Following incubation, platelet aggregation was
Table 2 Studies Showing That Ethanol Inhibits Platelet Activation
Study
Nguyen et al,24 1999

evaluated using ADP, collagen, and a thrombin receptor agonist peptide (SFLLRN). Ethanol enhanced the inhibitory effect of LY309562 on platelet aggregation and secretion in response to all agonists used. LY309562 did not have a major effect on platelet aggregation when stimulated with ADP; neither did ethanol. Thus, most of the studies performed to investigate the effect of ethanol on platelet aggregation indicated an inhibitory effect of ethanol on platelets Table 2. The exact mechanism by which ethanol exerts its inhibitory effect on human platelets is unclear. Evidence That Ethanol Causes Activation of Platelets When platelets are activated, there is a decrease in the cAMP level.37 This decrease might be due to decreased adenyl

Study Design
Human platelets incubated with ethanol

Ethanol Dose (mmol/L)*

87

Measurement of Activation

Results

Comments

Rubin,25 1989

Benistant et al,27 1990

Rand et al,36 1989

Wakabayashi and Marumo,29 2002 McKenzie et al,32 2002

Ethanol had no effect on Ethanol, by affecting phosplatelet adhesion to phorylation of PLA2, collagen and granule might decrease its release activity and, therefore, Ethanol decreased formdecrease TXA2 ation of TXA2 following formation adhesion to collagen Ethanol delayed phosphorylation of PLA2 but had no effect on PLC phosphorylation Human platelets incubated 25-50 Aggregation and secretion Dose-dependent inhibition Ethanol mostly affected with ethanol for 1 min by collagen (0.5-10 g/mL) of aggregation and PLA2, rather than PLC before platelet stimulation Shape change secretion with collagen No effect on shape change Ethanol (150 mmol/L) inhibited release of AA from PL and decreased TXA2 synthesis No effect on Ca2+ level and mobilization Washed human platelets from 25-150 Release of 5-[14C]-serotonin Ethanol prevented granule Ethanol-inhibited AA healthy donors incubated Activation of PLC centralization and fusion release could be due with ethanol and activated Ethanol (150 mmol/L) to inhibition of PLA2 by thrombin (0.025 U/mL) inhibited serotonin release in response to thrombin Ethanol inhibited AA release in response to thrombin Ethanol had no effect on PLC Rabbit platelets incubated 0-4 mg/ Release of [14C]-serotonin PGI2 increased cAMP levels Ethanol potentiated the with ethanol in the presence mL cAMP levels Ethanol had no effect on effect of PGI2 on platelet of PGI2 cAMP levels or platelet aggregation but had no aggregation effect on platelet Ethanol increased inhibition aggregation in the of platelet aggregation in absence of PGI2 samples preincubated with PGI2 2+ Blood obtained from healthy 85-340 Ca mobilization by Ethanol as low as 85 Ethanol, by inhibiting Ca2+ donors to study the effect platelets preincubated mmol/L inhibited Ca2+ entry into platelets, of ethanol Ca2+ mobilization with Fura-2 (Sigma, influx) caused inhibition of St Louis, MO platelet aggregation Human platelets incubated 87 Expression of P-selectin Ethanol decreased Ethanol decreased with ethanol 2 min before and CD63 by flow P-selectinand CD63 expression of receptors stimulation with thrombin cytometry expression on surface of platelets (0-1 U/mL)

Adhesion to collagen [14C]serotonin release TXA2 formation PLA2 activity Phosphorylation of PLC

AA, arachidonate; cAMP, cyclic adenosine monophosphate; TXA2, thromboxane; PL, phospholipid; PLC, phospholipase C; PLA2, phospholipase A2; PGI2, prostacyclin * Unless otherwise indicated.

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cyclase activity or increased catabolism of cAMP by phosphodiesterase. Activation of protein kinase C (PKC) causes phosphorylation of phosphodiesterase and, thus, a decrease in the cAMP level in human platelets. Ethanol may affect the level of cAMP and PKC activity in platelets. DePetrillo and Swift38 studied the effect of moderate ethanol intake on the cAMP level. The results showed a decrease in the cAMP concentration below baseline in human platelets, with a nadir at 30 seconds following the addition of ethanol, which then returned to baseline levels. This decrease in the cAMP level in the presence of ethanol was lost when platelets were incubated with a phosphodiesterase inhibitor (1-(5-isoquinolinylsulfonyl)-2methylpiperazine) and when they were stimulated with PKCactivating phorbol esters. These results indicate that ethanol affects the cAMP level through activation of PKC, which in turn increases phosphodiesterase activity via phosphorylation. Conflicting reports exist on the effects of ethanol on the human platelet cAMP level. However, 2 reports indicated no effect of alcohol on the cAMP level.36,39 In these 2 studies, the cAMP level in platelets was measured at a longer incubation time and with a higher ethanol concentration than used in the study by DePetrillo and Swift.38 Ethanol has been shown to increase the activity of PKC in the brain.40 Several studies measured PKC activity levels in the presence of ethanol. Many of these studies were done in animals or in cultured cells, rarely in humans.41,42 Dietrich et al43 performed an in vivo study to investigate the effect of
Table 3 Studies Showing That Ethanol Causes Activation of Platelets
Study DePetrillo and Swift,38 1992 Study Design Ethanol Dose (mmol/L)* Obtained blood samples from blood transfusion services 2-32

alcohol on platelet PKC levels. This study showed an increase in PKC levels in platelets 60 minutes following ethanol intake (0.4 g/kg). PKC is activated by 1,2-diacylglycerol, which is formed by hydrolysis of phosphatidylinositol 4,5-biphosphate mediated by phospholipase C. Ethanol induces rapid but small increases in the level of inositol 1,4,5-triphosphate in human platelets owing to activation of phospholipase C. These changes might be responsible for the ethanol-induced calcium mobilization, which peaked to a maximum level within 5 seconds and decreased slowly during a 5-minute incubation period, leading to shape change.44 Thus, ethanol can cause activation of PKC indirectly by activating phospholipase C and the formation of 1,2-diacylglycerol. Taken together, these studies Table 3 support the hypothesis that ethanol initially causes a partial platelet activation within the first few minutes and that this partial activation produces platelets unable to fully contribute to platelet plug formation. This working hypothesis might explain the contradictory results of the various studies of the effects of ethanol on human platelets.

Effect of Ethanol on Coagulation Factors

Coagulation factors, specifically fibrinogen and factor VII, have been implicated as risk factors for atherosclerotic diseases. 45,46 Several cross-sectional studies also have

Measurement of Activation cAMP levels by HPLC

Results

Comments

Deitrich et al,43 1996 Rubin et al,44 1988

Healthy volunteers ingested ethanol; blood was obtained 60 min later Obtained blood samples from healthy donors

0.4 g/kg

300

Ethanol (8 or 32 mmol/L) caused Ethanol affected cAMP a decrease in cAMP levels for levels through activation of 30 s, which then recovered to PKC; therefore, there was basal level within 1 min activation of phosphodiesHigh ethanol concentration (512 terase by phosphorylation mmol/L) had no effect on cAMP levels Addition of phosphodiesterase inhibitor or PKC inhibitor blocked the effect of ethanol on cAMP levels PKC levels in Ethanol increased PKC levels 60 platelets min following ingestion, but ethanol decreased PKC in chronic alcoholics Ca2+ mobilization Ethanol caused shape change Ethanol caused activation of Aggregation studies within 30 s; addition of PLC and led to formation IP formation (32Pmore ethanol did not cause of IP3, mobilization of Ca2+, orthophosphate) further changes and activation of PKC, 32P phospholipids Ethanol (200 mmol/L) caused leading to shape change 2+ increase in Ca levels within 55 s, then returned to basal levels Ethanol (300 mmol/L) increased IP3 within 10 s Ethanol (300 mmol/L) increased PKC within 2 min

cAMP, cyclic adenosine monophosphate; HPLC, high-performance liquid chromatography; IP3, inositol triphosphate; PKC, protein kinase C. * Unless otherwise indicated.

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suggested an association between factor VIII and von Willebrand factor (vWF) with atherosclerotic heart disease, but other studies have found no association with heart disease.47,48 Thus, a reduction in the risk of CVD can be related to the prevention of clotting or thrombosis, especially because 90% of patients with fatal MI have associated coronary thrombosis. Several studies have shown an effect of alcohol consumption on coagulation factors. The MONICA study, which was a cross-sectional study of the prevalence and population distributions of major CVD, investigated the effect of alcohol consumption on the plasma fibrinogen concentration in 4,940 healthy volunteers.49 The results showed a U-shaped relationship between alcohol consumption and fibrinogen concentration that was less significant in men older than 45 years and absent in young women. In all age and sex strata, nondrinkers had the highest fibrinogen levels, whereas subjects who consumed 40 to 59 g of ethanol daily had the lowest fibrinogen levels. A recent analysis of the Framingham Offspring study investigated the relation between alcohol consumption and fibrinogen, factor VII, and vWF levels in plasma.50 In this study, 1,456 men and 1,767 women were interviewed and asked to complete a questionnaire about the average amount of alcohol consumed, as beer, wine, and liquor. Fibrinogen and vWF levels were lowest among men and women who reported more than 7 drinks per week. The factor VII concentration was lowest in subjects who consumed the highest amount of alcohol. The type of alcohol consumed had no effect on the coagulation factor concentrations in either sex. The similarity of the results among the different types of alcohol indicated that the effect of alcohol consumption on fibrinogen, vWF, and factor VII levels is caused mainly by ethanol and not by other substances found in alcoholic beverages.
Table 4 Studies Showing the Effect of Ethanol on Coagulation Factors
Study Method

In a cross-sectional cohort study, Wannamethee et al,51 studied the relationships between alcohol intake and hemostatic markers in 3,158 healthy men. An increase in alcohol consumption from less than 1 drink per day to more than 5 drinks per day caused a significant increase in factor IX levels with no effect on factor VII and factor VIII levels. On the other hand, the increase in alcohol intake led to a decrease in plasma fibrinogen and vWF concentrations. The results of this study indicated that alcohol intake is inversely associated with a risk of CVD, as are the concentrations of fibrinogen and vWF. In another study, alcohol intake (4 glasses of beer per day) caused a 12% decrease in fibrinogen levels compared with abstainers.52 Some studies have measured the influence of moderate alcohol consumption on blood coagulation in patients with existing CVD. Gorinstein et al 53 showed a significant decrease in factor VII levels compared with baseline in 28 patients with CVD after they consumed 20 g of beer daily for 30 days. However, there were no statistically significant changes in fibrinogen or prothrombin time levels.53 Taken together, most of the clinical and epidemiologic experiments to study the effect of alcohol on fibrinogen concentration have found lower fibrinogen levels in drinkers compared with abstainers, whereas few studies have found no difference in fibrinogen concentrations between the 2 groups.14,53,54 The relation between ethanol ingestion and levels of factor VII and vWF are not clear because different epidemiologic and experimental studies have shown contradictory results of the effect of alcohol consumption on these levels.3,54,55 The studies reviewed Table 4 indicate that alcohol consumption has a complex association with hemostatic parameters. More studies are needed to clarify this relationship and reveal the mechanism by which alcohol produces an effect on coagulation factors and thereby explains the observed decrease in risk for CVD.

Results U-shaped relationship that was less significant in men <45 y and absent in young women Fibrinogen and vWF levels were lowest in subjects who reported >7 drinks per wk; factor VII lowest in subjects who consumed the highest amount of ethanol Fibrinogen and vWF levels decreased with higher ethanol intake; factor IX levels increased with higher alcohol consumption; factor VII and VIII levels did not change with ethanol intake Fibrinogen levels decreased by 12% in beer drinkers compared with nondrinkers Factor VII levels decreased in patients with CVD after the consumption of 20 g of beer per day for 30 d; no significant changes in fibrinogen levels or PT

Krobot et al,49 Cross-sectional study of the effect of alcohol consumption 1992 on fibrinogen level in 4,940 healthy volunteers Mukamal et al,50 Relation between alcohol consumption and fibrinogen, 2001 factor VII, and vWF levels in plasma from 1,456 men and 1,767 women Wannamethee Cross-sectional study of the effect of alcohol consumption et al,51 2003 on fibrinogen, vWF , and factors VII, VIII, and IX levels in 3,158 healthy volunteers Sierksma et al,52 Randomized study of the effect of consuming 4 glasses of 2002 beer per day by healthy volunteers Gorinstein et al,53 The influence of moderate alcohol consumption on blood 1997 coagulation in patients with CVD

CVD, cardiovascular disease; PT, prothrombin time; vWF, von Willebrand factor.

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Effect of Ethanol on the Fibrinolytic System

Reduction in the risk of CVD can be related to the prevention and control of clotting and thrombosis. Fibrinolysis controls the formation and degradation of thrombi and, therefore, has a major role in CVD and the development of MI. Multiple studies have led to the suggestion that moderate alcohol intake might mediate additional cardioprotection by promoting fibrinolysis through changes in the activity, level, or interaction of one or more factors of the fibrinolytic system. Ridker et al56 assessed the effect of ethanol ingestion on the plasma concentration of endogenous tissue-type plasminogen activator (t-PA) in 631 healthy men aged 40 to 84 years with no history of CVD (participants in the Physicians Health Study). The study was a randomized, double-blind, placebo-controlled trial of aspirin and carotene. Alcohol consumption data were obtained through a questionnaire, and subjects were grouped into daily, weekly, monthly, and rarely-or-never consumers of alcohol. The results indicate a positive association between the alcohol consumption pattern and the plasma concentration of endogenous t-PA antigen. The highest level of t-PA was found among daily consumers of alcohol who reported daily consumption of 2 or more drinks per day. The lowest levels of t-PA were found in the rarely-or-never group. Other studies, such as the Northwick Park Heart Study, indicated higher fibrinolytic activity among drinkers than nondrinkers by measuring the clot-lysis time as a marker of fibrinolytic activity.57 Plasminogen activator inhibitor-1 (PAI-1) is an efficient inhibitor of t-PAs that convert plasminogen to its active form, plasmin, and, on that basis, are thought to influence fibrinolysis. Some studies have shown a positive relationship between PAI-1 and CVD.58-60 Epidemiologic studies have indicated that alcohol intake is associated with increased PAI-1 levels, which lead to decreased fibrinolysis.3,61,62 However, these studies did not assess the effect of different ethanol doses on PAI-1 levels. Djousse et al,63 in a cross-sectional study, studied the effect of alcohol doses on PAI-1 levels in 1,862 subjects as part of the National Heart, Lung, and Blood Institute Family Heart Study. Moderate ethanol intake (14.9 g/d or approximately 1 drink per day) had no effect on PAI-1 levels in male and female subjects. On the other hand, subjects who consumed 15 g or more of ethanol daily had higher PAI-1 compared with subjects with a lower ethanol intake. The results of this study, which showed no increase in PAI-1 levels with moderate ethanol intake, are compatible with a protective effect of moderate alcohol intake on CVD by another mechanism. Results of other studies showed an
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increase in PAI-1 levels with high alcohol consumption, resulting in increased mortality in heavy alcohol drinkers and binge drinkers.3,62,64,65 The hemostatic system has a strong circadian pattern. Increased platelet aggregativeness and decreased fibrinolytic activity occur in the morning. This circadian variation might explain the high incidence of MI and ischemic stroke in the morning.66,67 Numminen et al68 studied the effect of alcohol intake on the fibrinolytic activity in relation to circadian periodicity. Twelve healthy men aged 20 to 39 years were assigned to ingest ethanol (1.5 g/kg of body weight) in the morning or in the evening. The PAI-1 level was increased significantly, peaking at 3 hours following ethanol intake regardless of the time of intake (ie, morning or evening), and then it began to decrease rapidly. These results suggest that ethanol ingestion caused a transient decrease in fibrinolysis. In this study, the t-PA activity level, another important parameter reflecting fibrinolytic activity, was not measured. Another study, by Hendriks et al,61 investigated the short-term effect of moderate alcohol consumption with an evening meal on the fibrinolytic system in middle-aged men. There was a significant increase in t-PA antigen and PAI-1 activity and a decrease in t-PA activity following the ingestion of 3 types of alcoholic beverages. However, during the night, PAI-1 activity returned to control values, whereas the t-PA antigen level remained elevated, resulting in increased t-PA activity in the morning. A third study, by van de Wiel et al,64 measured the effect of low ethanol intake (2 glasses of wine) and high ethanol intake (6 glasses of wine) on PAI-1 and t-PA levels and clot-lysis time. Five hours after ingestion of a high dose of ethanol, PAI-1 antigen and activity, t-PA antigen, and clot-lysis time were increased, whereas t-PA activity was decreased. The prolonged clot-lysis time persisted until the next morning, indicating an inhibition of fibrinolysis. This study showed that high alcohol intake in the evening leads to inhibition of fibrinolysis, which might accelerate a thrombotic coronary event. Taken together, these results indicate that moderate alcohol consumption increases fibrinolytic activity, especially in the morning, when there is higher risk for clot formation and development of MI. Theses studies Table 5 support the hypothesis that moderate alcohol consumption has a cardioprotective effect through improved antithrombotic and fibrinolysis profiles.

Summary
Ethanol or its metabolites inhibit platelet aggregation in response to collagen and ADP. On the other hand, ethanol decreases the intracellular cAMP level and
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Table 5 Studies Showing the Effect of Ethanol on Proteins Associated With Fibrinolysis
Study Ridker et al,56 1994 Meade et al,57 1979 Djousse et al,63 2000 Numminen et al,68 2000 Hendriks et al,61 1994 Method Measured the effect of ethanol on t-PA plasma levels in 631 patients with CVD Compared fibrinolytic activity among drinkers and nondrinkers by measuring the clot-lysis time Measured the effect of different ethanol doses on PAI-1 levels in 1,862 people The effect of alcohol intake on the fibrinolytic activity in relation to circadian periodicity; 12 men ingested ethanol in the morning or in the evening Investigated the short-term effect of moderate alcohol intake with an evening meal on the fibrinolytic system in middle-aged men Results Highest levels of t-PA found in patients who consumed 2 drinks per day Higher fibrinolytic activity among drinkers than nondrinkers PAI-1 levels increased with high ethanol intake (>15 g/d) and remained the same with moderate intake PAI-1 levels increased following ethanol ingestion, peaking at 3 h, regardless of the time of ingestion, then started to decrease after 3 h; t-PA levels were not measured t-PA antigen and PAI-1 activity increased, and t-PA activity decreased following ingestion of ethanol; during the night, PAI-1 activity returned to control values, but t-PA antigen values remained elevated; overall alcohol consumption increased fibrinolytic activity, especially in the morning PAI-1, t-PA antigen, and clot-lysis time increased with high dose of ethanol; and t-PA activity decreased; overall high ethanol intake in the evening inhibited fibrinolysis until morning

van de Wiel et al,64 2001

Studied the effect of low (2 glasses) and high (6 glasses) of wine in the evening on PAI-1, t-PA, and clot lysis

CVD, cardiovascular disease; PAI-1, plasminogen activator inhibitor-1; t-PA, tissue-type plasminogen activator.

increases the intracellular inositol-1,4,5-triphosphate concentration, leading to an increase in intracellular Ca2+ level and, therefore, platelet activation. One working hypothesis is that ethanol or its metabolites partially activate platelets and that these circulating, incompletely granulated platelets are not fully functional and poorly promote hemostasis. High levels of coagulation factors, especially fibrinogen, have been implicated as risk factors for thrombosis. Some studies have shown that ethanol induces a decrease in fibrinogen, vWF, and factor VII levels. A smaller group of studies have shown no effect of ethanol on these factors. Alcohol consumption also affects the fibrinolytic system. Moderate ethanol intake causes an increase in the tPA level with no change in the PAI-1 concentration, which increases fibrinolysis. High ethanol intake, on the other hand, apparently leads to a decrease in fibrinolysis by increasing the PAI-1 concentration, which elevates the risk for a thrombotic event. Ethanol has multiple effects on hemostasis, affecting platelets, coagulation factors, and the fibrinolytic system. Although conflicting results exist in the literature, growing evidence is pointing to the following effects of ethanol or its metabolites: partial platelet activation generating poorly functioning platelets, decreased levels of fibrinogen and selected other factors, and increased fibrinolysis.
From the Division of Laboratory Medicine, Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston. Address reprint requests to Dr Laposata: MGH Clinical Laboratories, Gray 235, 55 Fruit St, Boston, MA 02114.
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