Mojca Stegnar

High D-dimer levels predict cardiovascular events in patients with chronic atrial fibrillation during oral anticoagulant therapy

Atrial fibrillation (AF) is associated with hemostatic abnormalities and increased risk of thrombotic cardiovascular events even during oral anticoagulant therapy (OAT). The aim of our study was to evaluate the predictive value of hemostatic markers for the risk of major cardiovascular events during OAT. The study group comprised 113 patients with chronic AF (70.2 +/- 5.4 years old, 60% men), referred for OAT. Established clinical risk factors and levels of prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT), D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) antigen and activity, before and during OAT (after 3.9 +/- 0.7 months; INR 2.57 +/- 0.57) were determined. In all patients OAT significantly suppressed levels of F1+2 by 67%,TAT by 30% and D-dimer by 48% (all p <0.001). During an average follow-up of 44 months 22/111 (20%) patients suffered a combined cardiovascular event (stroke, myocardial infarction, peripheral vascular occlusion or vascular death). Patients with cardiovascular events were significantly older, had more frequent heart failure/systolic dysfunction and had significantly increased levels of D-dimer at entry (63 vs 39 ng/mL, p = 0.005) and during OAT (33 vs 18 ng/mL, p = 0.002), and of t-PA antigen at entry (14.3 vs 10.9 ng/mL, p = 0.02) and during OAT (15.0 vs 11.2 ng/mL, p = 0.05) (all values are medians). In multivariate Cox proportional hazard models, heart failure/systolic dysfunction (hazard ratio 2.91; 95% CI 1.17-7.26; p = 0.02), high levels of D-dimer on OAT (top vs. lower two quartiles) (hazard ratio 4.78, 95% CI 1.39-16.41; p = 0.01) and t-PA antigen levels (continuous variable) (hazard ratio 1.09; 95% CI 1.01-1.17; p = 0.02) were significantly associated with combined cardiovascular events. In conclusion, high levels of D-dimer and t-PA antigen during OAT are significant predictors of combined cardiovascular events in AF patients and, on this basis, could be useful additional markers of cardiovascular risk in such patients.

Impact of Adipose Tissue on Plasma Plasminogen Activator Inhibitor-1 in Dieting Obese Women

The increased incidence of cardiovascular diseases in obese subjects could be partially attributed to impaired fibrinolysis due to elevated plasma levels of tissue plasminogen activator inhibitor 1 (PAI-1). The associations between changes in plasma PAI-1, metabolic variables, and adipose tissue during weight loss and regain were studied in 52 healthy, premenopausal, obese women participating in a weight reduction program with a hypocaloric diet. PAI-1, insulin, triglyceride, leptin, and adipsin levels were determined at entry, after the first week, after completion of the program, and after 5 months of follow-up. In the 33 obese women who completed the program, decreases in PAI-1 antigen (-54%), PAI activity (-74%), and leptin (-51%), but not of adipsin, were observed. Changes in PAI-1 were associated with changes in body mass index (BMI), body fat, leptin, and insulin. The decreased level of PAI-1 remained low after follow-up in the 14 women who maintained their reduced weight but increased in the 16 women who regained weight. This increase in PAI-1 was correlated with an increase in body fat and leptin. On multivariate analysis, BMI was the major determinant of PAI-1 level. In conclusion, during weight reduction with a hypocaloric diet, the decrease in PAI-1 is more closely related to changes in adipose tissue than to changes in metabolic variables, suggesting a significant role for adipose tissue in regulating plasma levels of PAI-1.

A novel G/A and the 4G/5G polymorphism within the promoter of the plasminogen activator inhibitor-1 gene in patients with deep vein thrombosis.

Plasma plasminogen activator inhibitor-1 (PAI-1) level was observed to be associated with sequence variations at the PAI-1 locus. Therefore, PAI-1 gene promoter was screened for possibly new polymorphisms and to investigate the contribution of these sequence variations to PAI-1 levels in patients with deep vein thrombosis (DVT). DNA was isolated from blood of 83 consecutive unrelated patients (42 +/- 11 years old) and from 50 apparently healthy subjects of similar age and gender distribution. Six fragments covering DNA sequence- 1523 base pairs (bp) upstream from the start of PAI-1 gene transcription to +90 bp in the first exon, were amplified by polymerase chain reaction and analyzed by single-strand conformation polymorphisms. Two polymorphisms were found: a previously described 4G/5G deletion/insertion polymorphism -675bp upstream from the start of transcription and a novel G/A single base substitution polymorphism further upstream at -844 bp. The two polymorphisms were in strong linkage disequilibrium. Significant differences between patients and controls were observed neither for the frequencies of the 4G/5G alleles (0.60/0.40 and 0.59/0.41, respectively) nor for the frequencies of the G/A alleles (0.33/0.67 and 0.41/0.59, respectively). The distribution of both polymorphisms was similar in idiopathic and secondary DVT as well as in first and recurrent DVT. In patients association between the 4G/5G genotypes and PAI activity was observed, with the highest values in the 4G/4G genotype (13.3 U/mL), median values in the 4G/5G genotype (9.8 U/mL) and the lowest values in the 5G/5G genotype (2.0 U/mL). Despite the lack of association between the G/A genotypes and plasma PAI-1 levels, electrophoretic mobility shift assay showed specific binding of a nuclear protein from human vascular endothelial cells extracts to both the G and the A variant, suggesting functional importance of this novel G/A polymorphism in regulating the expression of PAI-1 gene.

D-dimer, other markers of haemostasis activation and soluble adhesion molecules in patients with different clinical probabilities of deep vein thrombosis

Two automated turbidimetric D-dimer assays (BC D-dimer Plus, Dade Behring, Marburg, Germany and Auto-Dimer, Biopool, Umeå, Sweden) were compared to two enzyme-linked immunosorbent assays (ELISAs) (Enzygnost D-dimer micro, Dade Behring and Asserachrome D-dimer, Diagnostica Stago, Asnières, France) and two rapid D-dimer assays (SimpliRed, Agen Biomedical, Brisbane, Australia and Minutex, Biopool) in out-patients with suspected deep vein thrombosis (DVT). In addition, the performance of prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), and soluble adhesion molecules VCAM-1 and P-selectin for DVT diagnosis was assessed. One hundred and thirty-five consecutive out-patients with suspected DVT of the lower limb were included, and in 52 (39%), DVT was confirmed by compression ultrasound. All D-dimer assays investigated reliably excluded DVT in those patients without DVT irrespective of their pre-test clinical probability of DVT. One D-dimer ELISA (Dade Behring) gave the highest area under the receiver operating characteristic (ROC) curve compared to other assays, and therefore, this was the most accurate assay in differentiating patients with from patients without DVT. The diagnostic performance of one automated turbidimetric assay (Auto Dimer, Biopool) was similar to ELISA and its convenience close to rapid latex agglutination assays. Most patients with a high pre-test clinical probability of DVT had positive D-dimer regardless of the presence or absence of DVT, which decreased the specificity of the tests and made D-dimer determination less useful for this group of patients. Because the diagnostic accuracy [sensitivity, specificity, negative (NPV) and positive predictive value (PPV)] of F1+2, TAT, VCAM-1 and P-selectin was inferior to D-dimer assay, these assays could not substitute or supplement D-dimer testing in diagnosis of DVT. Levels of VCAM-1 and P-selectin were increased in patients with DVT and should therefore be investigated further to clarify their role in DVT.

Fibrinolytic response to venous occlusion in healthy subjects: Relationship to age, gender, body weight, blood lipids and insulin

The physiological variability of fibrinolytic response to 20 min upper arm venous occlusion was studied in 191 healthy women and men, 19-80 years old. It was observed that fibrinolytic response measured by the absolute amount of t-PA antigen after venous occlusion increased with increasing age (from 10.7 at 19-30 years to 25.0 ng/ml at 71-80 years), was higher in men than in women (20.9 vs 15.6 ng/ml), higher in obese than in slim subjects (29.5 vs 12.3 ng/ml) and higher in subjects with moderately elevated blood cholesterol (33.3 vs 18.2 ng/ml) and triglycerides (30.0 vs 24.4 ng/ml, all p < 0.05 or less) than in subjects with normal levels of these variables. Due to simultaneous increase in basal levels of t-PA antigen in all these cases, relative increases in t-PA antigen after venous occlusion were not altered. Fibrinolytic response measured by t-PA activity, but not with euglobulin clot lysis time, increased with age. PAI-1 antigen was not affected by venous occlusion, while PAI activity decreased to zero in most subjects (in 61-80%) regardless of age, gender or blood lipids. However, in obese subjects and especially in subjects with elevated insulin, fibrinolytic response was reduced as determined by residual PAI activity after venous occlusion (1.3 and 10.6 IU/ml, respectively) due to the increased basal level of PAI-1. It was concluded that age, gender, body weight, blood lipids and insulin significantly modulate fibrinolytic response to venous occlusion.

Statin and fibrate treatment of combined hyperlipidemia: the effects on some novel risk factors.

The effects of cerivastatin and fenofibrate on proteins involved in haemostasis and on markers of inflammation were investigated in otherwise healthy middle-aged males with combined hyperlipidemia. Besides classical risk factors, other so-called novel risk factors for coronary artery disease are seen to be playing an increasingly important role in the development and progression of atherosclerosis. Thirty-eight males, aged 49 +/-5 years were randomised to 12 weeks treatment either with cerivastatin at a daily dose of 0.2 mg to 0.4 mg to achieve the LDL cholesterol goal of <3.0 mM, or with fenofibrate 250 mg daily. Fasting serum lipids, homocysteine, total and free tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor (PAI-1) and tissue plasminogen activator (t-PA) antigen and activity, C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were measured. No change in homocysteine level was observed in the cerivastatin group, while after fenofibrate administration it increased (p <0.0001). Total TFPI decreased significantly after cerivastatin (p = 0.002), but not after fenofibrate. Free TFPI did not decrease after either drug. Neither drug affected (t-PA) antigen and activity, while fenofibrate increased PAI-1 antigen (p <0.05) and activity (p <0.05). Cerivastatin decreased serum CRP values by 49.5% (p = 0.001), and fenofibrate by 29.8% (p = 0.03). The decreases of CRP in the two groups differed significantly (p = 0.04). IL-6 levels decreased significantly in the fenofibrate group (39%; p <0.0001), but not in the cerivastatin group (15%; p = 0.24) No significant decreases were observed for TNF-alpha. Cerivastatin had neutral effects on fibrinolysis, homocysteine or coagulation. On the other hand, fenofibrate increased PAI-1 antigen and activity and homocysteine, and did not affect coagulation. Both cerivastatin and fenofibrate reduced CRP levels, the decrease being significantly greater after cerivastatin. Fenofibrate also significantly decreased IL-6.

Prevalence of antiphospholipid antibodies in deep vein thrombosis and their relationship to blood coagulation and fibrinolysis.

Elevated levels of antiphospholipid antibodies are associated with an increased risk of thrombosis. To establish the prevalence of these antibodies in deep vein thrombosis (DVT), IgG and IgM antibodies to cardiolipin (aCL) and phosphatidylserine (aPS) were determined by enzyme-linked immunosorbent assay in 118 patients with DVT either during an acute episode (N = 53) or at least 2 months after acute DVT (N = 65). Most patients (76%) had proximal leg DVT and no one had evident autoimmune disorder. aCL and aPS values higher than 4 standard deviations above the mean value of the control group (147 blood donors) were considered increased. Increased IgG aCL were observed in 10% of DVT patients (controls: 5%, not significant), increased IgG aPS in 16% of DVT patients (controls: 5%, p less than 0.005) and both types in 4% of DVT patients (controls: 3%, not significant). In the subgroup of 41 patients with previous idiopathic DVT, prevalence of increased IgG aPS was the highest: 27% (p less than 0.001). Increased antibodies of IgM isotype were observed in 3% (aCL) and 2% (aPS) of all DVT patients (controls: 8% and 4%, respectively, not significant). Elevated IgG aCL or aPS were not associated with significant changes in platelet count, antithrombin III and protein C. However, in patients with increased IgG aPS deficient fibrinolysis due to high plasminogen activator inhibitor activity was observed before and after 20 min upper arm venous occlusion. DVT patients with increased IgG aPS might be exposed to a greater risk of rethrombosis due to deficient fibrinolysis than DVT patients without these antibodies.

Factor V Leiden, prothrombin 20210G --> A, methylenetetrahydrofolate reductase 677C --> T and plasminogen activator inhibitor 4G/5G polymorphism in women with pregnancy-related venous thromboembolism.

OBJECTIVE To establish the prevalence of inherited and acquired risk factors for development of venous thromboembolism (VTE) in pregnancy and the puerperium. STUDY DESIGN In a retrospective study, 30 women with a history of objectively confirmed venous thromboembolism during pregnancy or the puerperium were studied. Fifty-six women with normal pregnancies were included as controls. Antithrombin, protein C, protein S, lupus anticoagulants, homocysteine, factor V Leiden mutation, prothrombin 20210G-->A polymorphism, methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism and the -675 (4G/5G) polymorphism in plasminogen activator inhibitor 1 gene were analysed. RESULTS At least one thrombophilic defect was observed in 16 (53.3%) cases and in 12 (21.4%) controls (P=0.003); factor V Leiden in 8 (26.7%) cases and 3 (5.7%) controls (P=0.009); prothrombin 20210G-->A polymorphism in 8 (26.7%) cases and 4 (7.5%) controls (P=0.021) and antithrombin deficiency in 4 (13.3%) cases and in 1 (1.8%) control (P=0.029). Other inherited and acquired risk factors were similarly distributed among cases and controls. CONCLUSION Women with pregnancy-related venous thromboembolism have an increased prevalence of inheritable thrombophilic defects predisposing them to an increased risk of thrombosis.

Influence of degraded phosphatidylserine on binding of antiphospholipid antibodies

BACKGROUND Antiphospholipid antibodies (aPL) show great heterogeneity. Different phospholipids, with or without protein cofactor(s), and phospholipid binding proteins alone have been proposed as the target molecules for aPL. In order to determine the influence of phospholipid degradation products on the binding of aPL, sera from 6 patients with the antiphospholipid syndrome were studied. METHODS Fresh and aged phosphatidylserine and cardiolipin were used as coating reagents in solid-phase immunoassay procedures. Antibody reactivity was tested by enzyme-linked immunosorbent assay in the sera and in eluates from columns packed with polystyrene scrapings coated with either cardiolipin or phoshatidylserine. RESULTS Three reaction patterns of affinity-purified antibodies were seen: (1) reactivity with phosphatidylserine but not with cardiolipin or degraded phosphatidylserine, (2) reactivity with cardiolipin and degraded phosphatidlyserine, and (3) reactivity with all three phospholipid antigens. CONCLUSIONS Striking differences in the antiphospholipid antibody reactivity with cardiolipin, phosphatidylserine and degraded phosphatidylserine in the presence of serum proteins were observed among patients with venous thromboembolism. The analyses showed that the degradation of phosphatidylserine influences the binding of aPL in in vitro assays.

Markers of hemostatic system activation during treatment of deep vein thrombosis with subcutaneous unfractionated or low-molecular weight heparin

Prothrombin fragments (F1+2), thrombin-antithrombin complexes (TAT) and D-dimers, markers of hemostatic system activation, were measured in 59 consecutive patients with deep vein thrombosis (DVT). Patients were randomly treated either with subcutaneous unfractionated heparin (UH) administered in two to three subcutaneous doses adjusted to activated partial thromboplastin time (APTT) or with low-molecular weight heparin (LMWH) (dalteparin) administered in a fixed dose of 200 IU/kg body weight in one subcutaneous injection daily. Before treatment, F1+2, TAT and D-dimer were above the cut-off level in 27/59 (46%), 34/59 (58%) and all (100%) patients, respectively. Significant associations were observed between F1+2 and TAT (r=.66, P<.001), TAT and D-dimer (r=.36, P<.005) and F1+2 and D-dimer (r=.30, P<.050). On the third day of treatment, F1+2 and TAT significantly decreased to reference values in almost all patients (in 64/66 determinations of both F1+2 and TAT) and remained low on the seventh day of treatment. Compared to pretreatment values, a nonsignificant decrease of D-dimer was noted in both groups, but all values remained above the cut-off value. When markers of hemostatic system activation in the UH and LMWH groups were compared, no significant differences were observed. It was concluded that subcutaneous UH in an APTT-adjusted dose and subcutaneous LMWH in a once-daily weight-adjusted dose controlled these markers of hemostatic system activation in a similar manner.