A prospective randomized trial of aspirin-clopidogrel combination therapy and dose-adjusted warfarin on indices of thrombogenesis and platelet activation in atrial fibrillation

OBJECTIVES: This study was designed to investigate whether or not combination aspirin-clopidogrel therapy would reduce markers of thrombogenesis and platelet activation in atrial fibrillation (AF), in a manner similar to warfarin. BACKGROUND: Dose-adjusted warfarin is beneficial as thromboprophylaxis in AF, but potentially serious side effects and regular monitoring leave room for alternative therapies. METHODS; We randomized 70 patients with nonvalvular AF who were not on any antithrombotic therapy to either dose-adjusted warfarin (international normalized ratio 2 to 3) (Group I) or combination therapy with aspirin 75 mg and clopidogrel 75 mg (Group II). Plasma indices of thrombogenesis (fibrin D-dimer, prothrombin fragment 1+2) and platelet activation (beta-thromboglobulin [TG] and soluble P-selectin) were quantified, along with platelet aggregation responses to standard agonists, at baseline (pretreatment) and at six weeks posttreatment. RESULTS; Pretreatment levels of fibrin D-dimer (p = 0.001), beta-TG (p = 0.01) and soluble P-selectin (p = 0.03) were raised in patients with AF, whereas plasma prothrombin fragment 1+2 levels and platelet aggregation were not significantly different compared with controls. Dose-adjusted warfarin reduced plasma levels of fibrin D-dimer, prothrombin fragment 1+2 and beta-thromboglobulin levels at six weeks (all p < 0.001), enhanced plasma levels of soluble P-selectin (p < 0.001) and had no significant effect on platelet aggregation. Aspirin-clopidogrel combination therapy made no difference to the plasma markers of thrombogenesis or platelet activation (all p = NS), but the platelet aggregation responses to adenosine diphosphate (p < 0.001) and epinephrine (p = 0.02) were decreased. CONCLUSIONS: Aspirin-clopidogrel combination therapy failed to reduce plasma indices of thrombogenesis and platelet activation in AF, although some aspects of ex vivo platelet aggregation were altered. Anticoagulation with warfarin may be superior to combination aspirin-clopidogrel therapy as thromboprophylaxis in AF.     

A study of platelet activation in paroxysmal, persistent and permanent atrial fibrillation.

We hypothesized that the 'residual' thromboembolic risk in therapeutically anticoagulated patients undergoing cardioversion could potentially be related to abnormal haemorheology and platelet activation. To test this hypothesis, we firstly investigated the role of haemorheology and platelet activation in patients with paroxysmal and persistent atrial fibrillation (AF), who were compared with healthy controls and patients with permanent AF. Second, we compared these indices in patients with persistent AF, before and after successful cardioversion. We measured indices of haemorheology (haematocrit, plasma viscosity, and fibrinogen), fibrin D-dimer (an index of thrombogenesis and fibrin turnover) and platelet activation (as assessed by platelet aggregation and plasma levels of beta-thromboglobulin, and soluble P-selectin) in 29 patients with paroxysmal AF, 87 patients with permanent AF and 29 healthy controls in sinus rhythm. The effects of cardioversion were studied in 20 patients with persistent AF, who maintained sinus rhythm at 2 months follow-up. Plasma levels of beta-thromboglobulin (P = 0.03) and fibrin D-dimer (P = 0.001) were higher in patients with AF, when compared with controls; the highest levels were seen in those with permanent AF (Tukey's test, < 0.05). Plasma viscosity was significantly higher in the patients with paroxysmal AF compared with healthy controls (P = 0.02). Plasma soluble P-selectin levels and platelet aggregation responses to all four platelet agonists (adenosine diphosphate, collagen, epinephrine and thrombin) in patients with paroxysmal AF and permanent AF were similar to controls. Plasma fibrinogen, viscosity and other markers of platelet activation (including platelet aggregation) were not significantly different in patients with paroxysmal AF, during episodes of AF and sinus rhythm (P = not significant), although mean haematocrit was significantly higher during the episodes of AF compared with episodes of sinus rhythm (P = 0.03). Among the patients with persistent AF who remained in sinus rhythm at 2 months following successful cardioversion, there was a significant decrease in the plasma levels of soluble P-selectin at 2 weeks and 2 months, when compared with baseline (pre-cardioversion) levels (P < 0.001). Haemorheology and platelet aggregation response to agonists did not change significantly, except for a transient increase in platelet aggregation response to collagen at 2 weeks (P = 0.045). In conclusion. abnormal haemostatic and platelet activation in patients with permanent AF are not consistently observed in patients with paroxysmal and persistent AF. Abnormal haemorheology appears to play an important role in patients with paroxysmal AF, especially during the paroxysms of AF. Cardioversion of persistent AF to sinus rhythm appears to decrease the platelet activation, but whether this translates into a beneficial reduction in thromboembolic risk requires further study.     

Indexes of hypercoagulability measured in peripheral blood reflect levels in intracardiac blood in patients with atrial fibrillation secondary to mitral stenosis

Systemic thromboembolism is a major complication in patients with mitral stenosis, especially in those who have atrial fibrillation (AF). It has been suggested that there may be increased regional left atrial coagulation activity in such patients, despite normal systemic coagulation activity on peripheral blood sampling. Our aim was to investigate whether there were significant differences between intracardiac versus peripheral indexes of hypercoagulability in 25 patients (5 men; mean age 60 years) with mitral stenosis who were undergoing percutaneous balloon mitral valvuloplasty and who were all in chronic AF. Two days after halting warfarin therapy, intracardiac (right and left atria) and peripheral (venous and arterial) blood samples from patients were obtained and compared with levels in matched healthy controls in sinus rhythm. Thrombogenicity was assessed by levels of fibrin D-dimer, fibrinogen, indexes of platelet activation (soluble P-selectin and beta thromboglobulin [betaTG]) and indexes of endothelial dysfunction (soluble thrombomodulin [sTM] and von Willebrand factor [vWF]). There were no statistically significant differences in the various markers between the femoral vein and artery, left and right atria, and between the femoral vein and both atria (all p = NS). Plasma fibrinogen, vWf (both p <0.005), and D-dimer (p = 0.011) were significantly higher and levels of sP-selectin and sTM were lower (both p <0.005) in patients when compared with controls. There was no significant difference in plasma betaTG levels. Our results suggest that there is no significant variation in indexes of thrombogenesis, platelet activation, and endothelial dysfunction between left atrium, right atrium, and the peripheral artery or vein. Peripheral samples therefore do reflect atrial coagulation, platelet, and endothelial activities.     

Increased markers of thrombogenesis in chronic atrial fibrillation: effects of warfarin treatment.

OBJECTIVE--To determine whether chronic atrial fibrillation is associated with abnormalities in plasma fibrinogen, von Willebrand factor (vWF) (a marker of endothelial disturbance), or fibrin D- dimer (a measure of fibrin turnover); and if so, whether such levels are related to haemodynamic disturbance (enlarged left atrium, poor left ventricular function) or existing treatment with warfarin or aspirin. To investigate the effects of introducing warfarin in patients with atrial fibrillation on fibrinogen and D- dimer levels. DESIGN--Cross sectional population sample controlled study and longitudinal study of patients undergoing anticoagulation. SETTING--District general hospital. SUBJECTS--87 patients (44 men and 43 women of mean (SEM) age 63.0 (1.0)) with chronic atrial fibrillation. At the time of the study, 37 were taking no antithrombotic medication (group 1), 31 were taking warfarin (including two on warfarin and aspirin) (group 2) and 19 were taking aspirin alone (group 3). They were compared with 158 population controls from a random population sample (the second Glasgow monitoring trends and determinants in cardiovascular disease study). As part of clinical treatment warfarin was introduced in 20 patients with chronic atrial fibrillation (14 men and six women of mean (SEM) (range) age 63.9 (2.35 (32-74) years). RESULTS--Plasma fibrinogen remained significantly increased in patients of group 1 (no antithrombotic medication) compared with that of the population controls (median difference 1.23 g/l; 95% confidence interval (CI) 0.88 to 1.62, P < 0.0001). There was also a significant increase in plasma D-dimer levels (median difference 77 ng/ml; 95% CI 38 to 122, P < 0.01) and vWF (median difference 63 IU/dl; 95% CI 38 to 89, P < 0.0001). There was no significant difference in plasma fibrinogen (median difference 0.14 g/l; 95% CI -0.44 to 0.77, P = 0.65) or vWF (median difference 3.5 IU/dl; 95% CI - 41 to 41, P = not significant in patients of group 2 (warfarin treatment) compared with that of patients in group 1. Levels of D-dimer were significantly lower in group 2 (median difference 90 ng/ml, 95% CI 39 to 150, P < 0.0001) than in group 1. There were no significant differences in plasma fibrinogen (median difference 0.08 g/l; 95% CI - 0.52 to 0.77, P = 0.73), D-dimer (median difference - 34 ng/ml; 95% CI - 114 to 21.0, P = 0.25), or vWF (median difference 2%; 95% CI - 35 to 41, P = not significant) levels between patients of groups 1 and 3. There were no significant correlations between the coagulation indices and left atrial volume or ventricular function. There was a significant positive correlation between plasma fibrin D-dimer and vWF levels in patients of groups 1 and 3 (r = 0.52, P < 0.001). There was a significant reduction in median plasma fibrin D-dimer levels at 2 months after the introduction of warfarin (181 ng/ml v 80 ng/ml, P < 0.001), but no effect on plasma fibrinogen. CONCLUSIONS--Increased median plasma fibrinogen and vWF levels were found in patients with chronic atrial fibrillation. Plasma D-dimer levels were also increased in patients with chronic atrial fibrillation not receiving warfarin, suggesting increased intravascular thrombogenesis in such patients. Introduction of warfarin normalised circulating fibrin D- dimer levels, suggesting that warfarin treatment was effective in preventing excessive fibrin turnover, consistent with the antithrombotic effects of warfarin. These results suggest three possible thrombotic markers to assess patients with atrial fibrillation who are at high risk of thrombogenesis; D-dimer also merits assessment as a measure of reduction in thrombotic risk in patients receiving warfarin.     

Sequential alterations in haemorheology, endothelial dysfunction, platelet activation and thrombogenesis in relation to prognosis following acute stroke: The West Birmingham Stroke Project.

Abnormalities of haemorheology (plasma viscosity, fibrinogen), endothelial function [von Willebrand factor (vWf)], platelet activation (soluble P-selectin) and thrombogenesis [plasminogen activator inhibitor (PAI), and fibrin D-dimer] are common in cardiovascular disease. We investigated changes in these markers in 86 patients (58 males) presenting with acute stroke (all age < 75 years, with ictus < 12 h), and sequential changes at six time points (baseline on admission, 48 h, 1 week, 2 weeks, 3 months and 6 months following the onset of stroke). Baseline plasma viscosity, haematocrit, fibrinogen, vWf, PAI, soluble P-selectin and fibrin D-dimer levels were increased in the acute stroke patients compared with 35 age-matched and sex-matched controls. Following admission, there were significant increases in haematocrit at 2 weeks, vWf at 48 h and 1 week, fibrinogen at 1 week, PAI at 48 h and 1 and 2 weeks, soluble P-selectin at 48 h, and fibrin D-dimer at 48 h and 1 week following admission. Using univariate 'time to event' analysis, high (> median) mean age (log-rank test, P = 0.0262), diastolic blood pressure (P = 0.01), haematocrit (P = 0.0234), PAI-1 (P = 0.0066) and fibrin D-dimer levels (P = 0.0356) were associated with a shortened event-free survival. Using a multivariate Cox survival analysis, only PAI-1 levels remained an independent predictor of survival (P = 0.0349). We conclude that acute stroke patients have marked baseline abnormalities of haemorheology, endothelial disturbance, thrombogenesis, platelet activation and abnormal fibrinolysis, with further changes over the subsequent follow-up period. Abnormal thrombogenesis and fibrinolysis may significantly influence survival in patients with acute stroke. These changes may have potential implications for the pathogenesis of stroke and its complications, although the possibility remains that we are documenting an acute phase response that previous studies, which included stroke patients with a wide time range since ictus onset, have neglected to consider.     

Relation of Endothelium, Thrombogenesis, and Hemorheology in Systemic Hypertension to Ethnicity and Left Ventricular Hypertrophy

Although the arterial tree is exposed to increased pressure in hypertensive patients, paradoxically, the complications of hypertension (heart attacks, stroke) are mainly thrombotic rather than hemorrhagic. Patients with left ventricular (LV) hypertrophy are at high risk of the complications of hypertension. We performed a cross-sectional study of 178 patients attending a hypertension clinic in a city center teaching hospital, and measured plasma levels of the soluble adhesion molecule P-selectin (associated with platelet activity/function and atherosclerosis), the von Willebrand factor (vWf; a marker of endothelial dysfunction), fibrin D-dimer (an index of thrombogenesis), plasminogen activator inhibitor (PAI, an index of fibrinolysis), lipoprotein(a) (Lp(a), associated with thrombogenesis and atherogenesis) and hemorheological indexes (fibrinogen, hematocrit, plasma viscosity, hemoglobin) in patients with essential hypertension, in whom the LV mass and LV mass index were determined using echocardiography. The 178 patients (86 men, mean age 54 ± 15 years) were compared with 47 normotensive healthy controls (aged 56 ± 20 years). Hypertensive patients had higher P-selectin, PAI, vWf, fibrin D-dimer, Lp(a), plasma fibrinogen, and plasma viscosity when compared with controls. Black hypertensive patients had higher Lp(a) levels and LV septal and posterior wall thickness on echocardiography, but lower plasma PAI levels. Patients with LV hypertrophy (defined as a LV mass index>134 g/m² in men or>110 g/m² in women) had higher plasma fibrinogen compared with those without LV hypertrophy. Systolic blood pressures were significantly correlated to age, plasma viscosity, plasma fibrinogen, and vWf. Diastolic blood pressures were significantly correlated with age and plasma fibrinogen. Fibrinogen levels were correlated with LV mass, LV mass index, left atrial size, plasma viscosity, and vWf. Fibrin D-dimer levels were significantly correlated with vWf and fibrinogen levels. Thus, hypertensive patients have high plasma fibrinogen levels, thrombogenesis, and impaired fibrinolysis (as indicated by high D-dimer and PAI levels, respectively), platelet activation (raised soluble P-selectin), and endothelial dysfunction (high vWF). The high plasma fibrinogen levels were related to blood pressures, LV mass index (and LV hypertrophy), and left atrial size. These abnormalities in hemorheologic factors and markers of thrombogenesis and endothelial function may act synergistically to increase the risk of thrombogenesis and atherosclerosis in hypertensive patients.     

Aortic spontaneous echocardiographic contrast and hemostatic markers in patients with nonrheumatic atrial fibrillation

OBJECTIVES: To determine the relationship between spontaneous echocardiographic contrast (SEC) in the descending thoracic aorta and plasma levels of hemostatic markers in patients with nonrheumatic atrial fibrillation (AF). DESIGN AND SETTINGS: A cross-sectional study at a university hospital. PATIENTS AND MEASUREMENTS: In 91 consecutive patients (mean +/- SE age, 70 +/- 1 years; 68 men) with nonrheumatic AF who underwent transesophageal echocardiography, plasma levels of markers for platelet activity (platelet factor 4 [PF4] and beta-thromboglobulin [beta-TG]), thrombotic status (thrombin-antithrombin III complex [TAT]), and fibrinolytic status (D-dimer and plasmin-alpha(2)-plasmin inhibitor complex [PIC]) were determined. RESULTS: Forty-three patients who had aortic SEC (AoSEC) were older (72 years vs 68 years; p < 0.05) and had a higher prevalence of chronic AF (88% vs 52%; p < 0.05) than 48 patients without AoSEC. TAT, PIC, and D-dimer levels were significantly higher in patients with AoSEC than in those without AoSEC, whereas PF4 and beta-TG levels were not different between the two groups. Although the prevalence of cerebral embolism did not differ between the two groups (23% vs 29%), the prevalence of peripheral embolism was higher in patients with AoSEC than in those without AoSEC (10% vs 0%; p < 0.05). Multivariate analysis revealed mitral regurgitation (odds ratio, 7.53; p < 0.02), SEC in the left atrium (odds ratio, 2.14; p < 0.02), and aortic atherosclerosis (odds ratio, 1.87; p < 0.04) emerged as independent predictors of AoSEC. CONCLUSIONS: Patients with nonrheumatic AF who have AoSEC appear to have enhanced coagulation activity but not platelet activity. Intensive anticoagulation treatment might be required for these patients.     

A cross-sectional and diurnal study of thrombogenesis among patients with chronic atrial fibrillation

OBJECTIVES

First, we sought to determine whether there is diurnal variation in hemostatic factors related to thrombogenesis and hypercoagulability among patients with chronic atrial fibrillation (AF). Second, we sought to determine whether levels of soluble thrombomodulin (sTM), a marker of endothelial function, or soluble P-selectin (sP-sel), an index of platelet activation, are altered in patients with AF as compared with subjects in sinus rhythm.

BACKGROUND

Atrial fibrillation is associated with an increased risk of stroke and thromboembolism and is known to confer a hypercoagulable state, with abnormalities of thrombosis, platelet activation and endothelial cell function. Many cardiovascular events, such as acute myocardial infarction, have thrombosis as an underlying process, and they undergo diurnal variation.

METHODS

Fifty-two patients (45 men, mean [±SD] age 66 ± 6 years) with chronic AF, none of whom received antithrombotic therapy, were studied. Baseline levels of fibrinogen, sP-sel, sTM and von Willebrand factor (vWF) were compared to those levels in matched healthy control subjects in sinus rhythm. In a subgroup of 20 patients, five venous blood samples were collected through an indwelling cannula at 6-h intervals from 12 to 12 the following day and were analyzed for the same markers.

RESULTS

Patients with chronic AF had higher plasma sP-sel, sTM, vWF and fibrinogen levels as compared with control subjects in sinus rhythm. Significant correlations were found between fibrinogen and sP-sel in patients with AF (r = 0.567 [Spearman], p < 0.001) and in control subjects (r = 0.334, p = 0.016). There was no significant diurnal variation in plasma levels of sP-sel, sTM, vWF or fibrinogen over the 24-h study period (repeated measures analysis of variance, p = NS).

CONCLUSIONS

There is no circadian or diurnal variation in the hypercoagulable state seen in AF, as assessed by plasma fibrinogen and markers of platelet (sP-sel) and endothelial function (vWF and sTM). The persistent hypercoagulable state, together with the loss of diurnal variation in various hemostatic markers, in chronic AF may contribute to the high risk of stroke and thromboembolic complications in these patients.     

Plasma adenosine levels and platelet activation in patients with atrial fibrillation

Platelet activation is observed in patients with atrial fibrillation (AF). P-selectin, which is expressed on platelet activation, plays an important role in the formation of thromboemobli. Because adenosine is known to attenuate platelet activation, we evaluated adenosine levels and 2 indicators of platelet activation, i.e., expression of P-selectin on platelets and plasma levels of beta-thromboglobulin, in 28 patients with AF (20 men and 8 women, age range 64+/-2 years) with sex- and age-matched (+/-2 years) subjects with sinus rhythm. The incidence of risk factors for stroke except for coronary heart disease and in echocardiographic parameters did not differ between the 2 groups. Plasma adenosine levels were lower (p <0.05) in patients with AF than in controls (mean [interquartile range] 13.4 [19.3-9.3] vs 19.1 [30.8-11.9] nmol/L). The expression of P-selectin on platelets (6.8% [13.6-3.4] vs 4.0% [8.8-1.8]) and plasma levels of beta-thromboglobulin were higher (p <0.05) in patients with AF. Flow cytometric analysis revealed that an antagonist of adenosine receptors, 8-sulfophenyltheophylline, increased the expression of P-selectin on platelets in a dose-dependent manner in the in vitro study. These results suggest that decreased plasma levels of adenosine were associated with platelet activation in patients with AF. Substitution of adenosine may provide a strategy for preventing platelet activation in these patients.     

Relationship between flow dynamics in the left atrium and hemostatic abnormalities in patients with nonvalvular atrial fibrillation

To investigate the relationship between left atrial (LA) flow dynamics and hemostatic markers in nonvalvular atrial fibrillation (AF), 45 patients with nonvalvular AF who had not received anticoagulants were evaluated by transesophageal echocardiography. We determined the LA appendage flow and the presence of LA spontaneous echo contrast (SEC) or thrombus. We measured plasma levels of thrombin-antithrombin III complex (TAT), fibrinopeptide A, D-dimer, beta-thromboglobulin, and platelet factor 4 in peripheral blood as hemostatic markers. The patients were divided into a low-velocity group (n = 19; sum of peak emptying and filling LA appendage flow velocities < 40 cm/s) and a high-velocity group (n = 26; > or = 40 cm/s). The maximum LA diameter was significantly greater and the LA expansion fraction was significantly smaller in the low-velocity group than in the high-velocity group. LA SEC or thrombus was observed in 11 patients (58%) in the low-velocity group, but not in any patients in the high-velocity group (p < 0.001). The plasma levels of TAT, fibrinopeptide A, D-dimer, beta-thromboglobulin, and platelet factor 4 were significantly higher in the low-velocity group than in the high-velocity group. The plasma levels of TAT, fibrinopeptide A, beta-thromboglobulin, and platelet factor 4 were significantly higher in 8 patients without LA SEC or thrombus in the low-velocity group than in 26 patients in the high-velocity group. Patients with nonvalvular AF accompanied by an enlarged and dysfunctioning LA and a decreased LA appendage flow velocity had increased intravascular coagulation-fibrinolysis activity and platelet activation. These abnormalities may be closely related to the thrombogenetic state in patients with nonvalvular AF.     

The value of proteomics for the diagnosis of a platelet-related bleeding disorder

Familial bleeding problems are frequently difficult to diagnose because currently used clinical tests cannot identify intracellular molecular defects of platelets. Using platelet proteomics, a comprehensive analytical tool, we diagnosed a family with severe bleeding problems of unknown origin with Quebec Platelet Disorder. Prior to proteomic analysis, we determined platelet counts, presence of glycoprotein (GP) Ib and GPIIb/IIIa, platelet aggregation, dense granule content and release, plasma levels of fibrinogen, Factor XIII and fibrin degradation products in four family members. Abnormalities were detected in platelet aggregation studies, which revealed variably reduced responses to ADP, collagen and epinephrine with concomitantly decreased ATP/serotonin secretion. In addition, D-dimer levels were significantly elevated 72 hours after in vitro thrombin stimulation of platelet-rich plasma. Together with the autosomal dominant inheritance and the delayed onset of bleeding in two of the four patients these results did not support any known platelet disorder. Therefore, the proteome of platelet lysates separated by one-dimensional SDS-PAGE was analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Platelet proteomics showed reduced amounts of alpha-granule proteins multimerin, fibrinogen and thrombospondin-1 in patient compared to control samples suggestive of Quebec Platelet Disorder. The diagnosis of Quebec Platelet Disorder was confirmed by urokinase-specific Western blots. Urokinase causes the degradation of alpha-granule proteins in this disorder. Diagnosis of rare bleeding disorders has important implications for prophylactic and acute treatment of bleeding patients. This is the first report using proteomics to identify a familial platelet defect.     

Prothrombin fragment F1 + 2 and thrombin-antithrombin III complex are useful markers of the hypercoagulable state in atrial fibrillation.

It is well known that atrial fibrillation (AF) is one of the most important diseases that predispose patients to thrombosis. We have attempted to identify patients with AF in the hypercoagulable state by measuring molecular markers such as thrombin-antithrombin III complex (TAT) and prothrombin fragment 1 + 2 (PTF) and determining the effect of antithrombotic therapy on these markers; 83 patients with AF were studied. Increased levels of plasma TAT and PTF were more frequently observed in patients with AF and associated mitral stenosis than in patients with AF alone. In cases of AF without mitral stenosis, plasma levels of TAT and PTF were significantly lower in those patients receiving antithrombotic agents (aspirin or warfarin) than in those receiving no antithrombotic agents. Furthermore, plasma levels of PTF were significantly lower in patients given warfarin than in those receiving aspirin. These results suggest that (1) patients with AF and mitral stenosis who are not given warfarin are in an extremely hypercoagulable state and (2) some patients with AF without mitral stenosis who are not given antithrombotic agents are also moderately hypercoagulable. In vivo activation of blood coagulation was more effectively controlled in patients receiving warfarin than in those taking aspirin.     

Effects of anticoagulation intensity on hemostatic markers in patients with non-valvular atrial fibrillation.

BACKGROUND: Elevation of hemostatic markers may account for the increased risk of ischemic stroke in patients with non-valvular atrial fibrillation (NVAF). The aim of this study was to determine the effects of anticoagulation intensity on hemostatic markers in patients with NVAF. METHODS AND RESULTS: In 509 patients with NVAF, comprising 263 patients treated with warfarin and 246 patients without warfarin, the hemostatic markers of prothrombin fragment F1.2 (F1.2), fibrin D-dimer, platelet factor 4 (PF4), and beta-thromboglobulin were determined and compared with those in 111 patients with sinus rhythm. F1.2 was inversely related with anticoagulation intensity and D-dimer increased with age. All hemostatic markers, except F1.2, were greater in patients with NVAF than in patients with sinus rhythm. F1.2 and D-dimer were significantly lower in patients with international normalized ratio (INR) > or =1.5 than in NVAF patients without warfarin and were not different between NVAF patients with INR of 1.5-1.9 and with INR > or =2.0. CONCLUSIONS: Low intensity of anticoagulation (INR 1.5-1.9) suppresses the elevated concentration of F1.2 and D-dimer in patients with NVAF, and might be favorable in Japanese patients with NVAF in view of the balance between prevention of thromboembolism and the adverse effect by warfarin (ie, bleeding).     

Thoracic aortic plaque enhances hypercoagulability in patients with nonrheumatic atrial fibrillation.

BACKGROUND: Patients with atrial fibrillation (AF) are at risk for thromboembolism, and coexistent cardiovascular diseases could affect their prothrombotic profiles. The relationship between plasma hemostatic markers and aortic atherosclerosis was determined in patients with AF or in sinus rhythm (SR). METHODS AND RESULTS: Sixty patients with nonrheumatic AF and 46 patients in SR who underwent transesophageal echocardiography and did not receive anticoagulant therapy constituted the study group. Markers for platelet activity (platelet factor 4 and beta-thromboglobulin), thrombotic status (thrombin-antithrombin III complex and prothrombin fragment 1+2 (F1+2)) and fibrinolytic status (plasmin-alpha2-plasmin inhibitor complex (PIC) and D-dimer) were determined. Levels of F1+2, PIC and D-dimer were higher in AF patients with severe atheroma than in those without severe atheroma (p<0.05). In patients in SR, hemostatic markers were not significantly increased even if they had severe aortic atherosclerosis. AF (Odds ratio (OR) 4.06, p=0.04) and age>or=75 years (OR 3.98, p=0.02) were independently predictive of elevated D-dimer levels and severe atheroma was predictive of elevated F1+2 levels (OR 5.52, p=0.04). CONCLUSIONS: Elderly patients with AF and severe aortic atherosclerosis might be in a prothrombotic state, and could benefit from intensive antithrombotic therapy.     

Platelet surface CD62P and CD63, mean platelet volume, and soluble/platelet P-selectin as indexes of platelet function in atrial fibrillation: a comparison of "healthy control subjects" and "disease control subjects" in sinus rhythm.

OBJECTIVES: The aim of this work was to comprehensively study the role of platelets in atrial fibrillation (AF), in relation to the underlying cardiovascular diseases and type of AF, and to analyze the effect of antithrombotic treatment on different aspects of platelet activation. BACKGROUND: Platelet activation is present in nonvalvular AF, but there is debate whether this is due to AF itself and/or to underlying cardiovascular diseases. METHODS: A total of 121 AF patients were compared with 65 "healthy control subjects" and 78 "disease control subjects" in sinus rhythm. Platelet activation was assessed using 4 different aspects of platelet pathophysiology: 1) platelet surface expression of CD62P (P-selectin) and CD63 (a lysosomal glycoprotein) (by flow cytometry); 2) mean platelet volume (MPV) (by flow cytometry); 3) plasma levels of soluble P-selectin (sP-selectin, enzyme-linked immunoadsorbent assay); and 4) total amount of P-selectin per platelet (pP-selectin) ("platelet lysis" assay). RESULTS: Both AF patients and "disease control subjects" had higher levels of CD62P (p < 0.001), CD63 (p < 0.001), and sP-selectin (p < 0.001) compared with "healthy control subjects," with no difference between AF patients and "disease control subjects." Patients with permanent AF had higher levels of sP-selectin (p = 0.014) and MPV (p = 0.025) compared with those with paroxysmal AF. The presence of AF independently affected the levels of CD62P expression, while "high-risk" AF patients (CHADS score > or =2) had higher levels of CD62P compared with those with "low risk." Introducing warfarin resulted in a reduction of pP-selectin (p = 0.013). CONCLUSIONS: There is a degree of excess of platelet activation in AF compared with "healthy control subjects," but no significant difference between AF patients and "disease control subjects" in sinus rhythm. Platelet activation may differ according to the subtype of AF, but this is not in excess of the underlying comorbidities that lead to AF. Platelet activation in AF may be due to underlying cardiovascular diseases, rather than due to AF per se.     

Platelet activity in mitral valve prolapse: a study of platelet aggregation, malondialdehyde production, and plasma beta-thromboglobulin

Platelet aggregation, malondialdehyde (MDA) production, and recovery after aspirin (ASA) administration and plasma levels of beta-thromboglobulin (BTG) were determined in 40 asymptomatic patients with mitral valve prolapse (MVP) and 17 normal subjects. Platelet aggregation was similar in patients and controls, although two patients presented spontaneous aggregation. Production of MDA and plasma levels of BTG were higher in MVP than in controls; however, recovery after ASA was similar in both groups. The results further indicate that platelet hyperactivity is present in a significant number of asymptomatic patients with MVP.     

Laboratory assessment of anti-thrombotic therapy in heart failure,atrial fibrillation and coronary artery disease: insights using thrombelastography and a micro-titre plate assay of thrombogenesis and fibrinolysis

As heart failure, coronary artery disease and atrial fibrillation all bring a risk of thrombosis, anti-thrombotic therapy is recommended. Despite such treatment, major cardiovascular events such as myocardial infarction and stroke still occur, implying inadequate suppression of thrombus formation. Accordingly, identification of patients whose haemostasis remains unimpaired by treatment is valuable. We compared indices for assessing thrombogenesis and fibrinolysis by two different techniques in patients on different anti-thrombotic agents, i.e. aspirin or warfarin. We determined fibrin clot formation and fibrinolysis by a microplate assay and thromboelastography, and platelet marker soluble P selectin in 181 patients with acute or chronic heart failure, coronary artery disease who were taking either aspirin or warfarin. Five thromboelastograph indices and four microplate assay indices were different on aspirin versus warfarin (p < 0.05). In multivariate regression analysis, only microplate assay indices rate of clot formation and rate of clot dissolution were independently related to aspirin or warfarin use (p ≤ 0.001). Five microplate assay indices, but no thrombelastograph index, were different (p < 0.001) in aspirin users. Three microplate assay indices were different (p ≤ 0.002) in warfarin users. The microplate assay indices of lag time and rate of clot formation were abnormal in chronic heart failure patients on aspirin, suggesting increased risk of thrombosis despite anti-platelet use. Soluble P selectin was lower in patients on aspirin (p = 0.0175) but failed to correlate with any other index of haemostasis. The microplate assay shows promise as a tool for dissecting thrombogenesis and fibrinolysis in cardiovascular disease, and the impact of antithrombotic therapy. Prospective studies are required to determine a role in predicting thrombotic risk.     

Evaluation of platelet function in aspirin treated patients with CAD

Background: Studies of platelet function in the acute coronary syndrome (ACS) have revealed both increased and unchanged platelet activation. To obtain a better understanding of platelet function in coronary artery disease in the setting of aspirin therapy, we performed platelet functional testing in patients with ACS and compared results to patients without CAD. Methods: We measured platelet aggregation and activation in response to ADP and epinephrine in 80 age and gender matched hospitalized patients (40 with ACS, 40 with non-cardiac chest pain (NCCP)). All subjects received ASA (81–325 mg). Platelet aggregation was performed using standard light transmission in platelet-rich plasma and activation was measured via flow cytometric analyses. We also studied platelet function under high shear rates measured by the platelet function analyzer (PFA-100). Results: ASA effect was found to be present in all subjects by blunted platelet aggregation in response to arachidonic acid. Patients with ACS showed significantly higher levels of platelet aggregation to epinephrine compared to patients with NCCP (p = 0.001). Other measures of platelet function including ADP aggregation, Pselectin, activated glycoprotein IIb/IIIa expression, and PFA-100 were unchanged between the two groups. Conclusions: We have found conflicting results of platelet functional testing in ACS. Specifically aspirin therapy in patients with ACS is effective in suppressing the platelet release response and is effective in the partial suppression of platelet aggregation; however, it appears that ACS patients have increased platelet aggregation to adrenergic stimuli when compared to age and gender matched controls without CAD despite the use of aspirin. In some studies, because ACS patients have an accentuated response to adrenergic stimuli this might be interpreted as aspirin resistance. Our study suggests that depending on the assay used to determine aspirin resistance, not all patients with this label are resistant to the biological effects of aspirin but they may have higher than normal baseline platelet sensitivity to adrenergic stimuli. This research was supported by grant K23HL67066 from the National Institute of Health.     

Platelet Aggregation in Primary Raynaud's Phenomenon and the Effect of Enalapril Administration

Platelet aggregation using a single platelet counting technique in whole blood, was determined on 18 patients with primary Raynaud's phenomenon and 17 age-matched controls. Platelet aggregation in the Raynaud's patients was also assessed during a double-blind, crossover trial to investigate the efficacy of the angiotensin converting enzyme (ACE) inhibitor, enalapril. There were no differences in platelet aggregation to collagen, arachidonic acid, ADP or PAF, or in plasma levels of beta-thromboglobulin (BTG), platelet factor 4 (PF4) or thromboxane B(2) (TxB(2)) between the Raynaud's group and the normal controls. Similarly, there were no differences in these parameters in the Raynaud's group during treatment with enalapril when compared to placebo. It is concluded that patients with primary Raynaud's phenomenon have no evidence of abnormal platelet aggregation or increased platelet activation, and that platelet aggregation is not affected by enalapril.     

Enhanced Fcgamma receptor I, alphaMbeta2 integrin receptor expression by monocytes and neutrophils in rheumatoid arthritis: interaction with platelets

OBJECTIVE: To investigate platelet and leukocyte activation and interaction in patients with rheumatoid arthritis (RA) and the effect of methotrexate (MTX) or anti-tumor necrosis factor-a (TNF-a) treatment on these variables. METHODS: Four-color flow cytometry analysis was performed for quantitative measurement of platelet (P-selectin, PAC-1) and leukocyte (CD11b, CD64) activation markers and estimation of percentage of leukocyte-platelet complexes in whole blood in 20 patients with RA before and after 6 weeks of therapy and in 20 controls. In addition, measures of soluble P-selectin (sP-selectin), beta-thromboglobulin, fibrinogen, prothrombin fragment 1+2, D-dimer, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin 6 (IL-6), and TNF-a and tender and swollen joint counts were carried out. RESULTS: Before therapy, PAC-1 binding, expression of CD11b and CD64 on monocytes and neutrophils, circulating levels of monocyte (CD11b+ or CD64+)-platelet complexes, monocyte-PAC-1+ platelet complexes, CRP, ESR, IL-6, TNF-a, fibrinogen, D-dimer and sP-selectin were significantly higher in RA patients compared to controls. The anti-TNF-a therapy significantly reduced levels of monocyte-PAC-1+ platelet complexes, sP-selectin, CRP, ESR, IL-6, TNF-a, fibrinogen, and D-dimer and tender and swollen joint counts. CD64 expression on monocytes was significantly decreased by MTX therapy. PAC-1 binding was not inhibited by MTX or anti-TNF-a. CONCLUSION: Increased platelet and leukocyte activation and increased formation of leukocyte-platelet complexes in patients with RA suggest a status of simultaneous activation of the immune and hemostatic systems.