Major and potential prothrombotic genotypes in patients with venous thrombosis and in healthy subjects from Slovenia.

The objective of our study was to investigate the prevalence of the polymorphisms factor V Leiden (FVL), prothrombin G20210A (PT G20210A), methylenetetrahydrofolate reductase C677T (MTHFR C677T), plasminogen activator inhibitor type 1 -675 4G/5G (PAI-1 4G/5G) and factor XII -4 C/T (FXII -4 C/T) in 295 Slovenian patients with venous thrombosis (VT) and 223 healthy controls in order to establish their contribution to the risk for VT. The major genetic risk factor was FVL, while PT G20210A, MTHFR 677 C/T, PAI-1 4G/5G and FXII -4 C/T polymorphisms were not. However, PT G20210A increased the risk of recurrent VT, MTHFR C677T increased the risk in older patients, while the FXII -4 T allele suggested a possible protective effect in younger patients. The risk of VT increased with increasing number of genetic defects.

Hemostasis activation in thrombophilic subjects with or without a history of venous thrombosis.

Thrombophilia is considered to increase the risk of venous thrombosis (VT) due to hemostasis activation. To determine the level of hemostasis activation in thrombophilic subjects with or without a history of VT, hemostasis activation markers prothrombin fragment 1 and 2 (F1+2), thrombin-antithrombin complex (TAT), and cross-linked fibrin degradation products (D-dimer) were measured in 94 subjects with (patients) and 101 subjects without a history of VT (controls). A total of 34.8% of patients and 14.8% of controls (P= .002) had at least 1 thrombophilic defect (protein C deficiency, activated protein C [APC] resistance, presence of lupus anticoagulants, or prothrombin G20210A polymorphism). The subjects were divided into 4 subgroups: patients with (TF(+) patients) and without (TF(-) patients) thrombophilia, and controls with (TF(+) controls) and without (TF(-) controls) thrombophilia. Hemostasis activation was comparable between all patients and controls (TAT: 2.1 vs 2.6 microg/L; F1+2: 1.0 vs 0.9 nmol/L; D-dimer: 36 vs 37 microg/L, respectively) and between TF(+) and TF(- ) patients. However, TF(+) controls had a significantly higher prevalence of increased hemostasis activation markers compared with TF(-) controls (TAT>4.4 microg/L, 38.4 vs 7.3%; F1+2>1.1 nmol/L, 53.8 vs 22.0%; D-dimer >78 microg/L, 30.7 vs 8.8% of subjects, respectively; all P< .05). After stratification for thrombophilic defects, hemostasis activation was associated with APC resistance in controls and with protein C deficiency in patients. To conclude, thrombophilia was associated with hemostasis activation in controls. We assumed that, in patients, the differences in hemostasis activation between subjects with or without thrombophilia were blurred due to undetermined and unidentified thrombophilic defects.

Evidence of hypercoagulability and inflammation in young patients long after acute cerebral ischaemia.

INTRODUCTION: Young subjects with acute cerebral ischaemia - stroke or transient ischaemic attack - form an etiologically heterogeneous and often not clearly explained group of patients. The aim was to investigate possible disturbances in haemostasis and inflammation long after an acute cerebral ischaemic event. MATERIALS AND METHODS: Forty-four consecutive patients referred after having suffered from acute cerebral ischaemia before the age of 45 participated 1 to 9 years (median value 5 years) after the event. At the time of blood sampling 33 (75%) patients were receiving antithrombotic treatment. Forty-six apparently healthy subjects of the same age group served as controls. In all subjects global haemostasis parameters (overall haemostasis, coagulation and fibrinolytic potential), thrombophilia, several markers of haemostasis activation and inflammation were determined. RESULTS: Patients did not differ from controls in most of the conventional risk factors and the presence of most forms of thrombophilia, although in seven (17.5%) patients the weak presence of lupus anticoagulants was observed. Patients had significantly increased overall haemostasis and coagulation potential, increased soluble P-selectin and D-dimer, decreased overall fibrinolysis potential and increased fibrinogen and C-reactive protein compared to controls. The subgroups of patients receiving antiplatelet treatment, with thrombophilia and recurrent acute cerebral ischaemia, did not differ significantly from the other patients. CONCLUSIONS: In young patients long after acute cerebral ischaemia an imbalance in the haemostatic system and a minor, but significant degree of inflammation was detected. The mechanisms behind haemostatic imbalance seem to be enhanced thrombin generation, platelet activation and depressed fibrinolysis.

The influence of sequence variations in factor VII, gamma-glutamyl carboxylase and vitamin K epoxide reductase complex genes on warfarin dose requirement.

The degree of interpatient variability in the warfarin dose required to achieve the desired anticoagulant response can only partly be explained by polymorphisms in the CYP2C9 gene, suggesting that additional genetic factors such as polymorphisms in genes involved in blood coagulation may influence warfarin dose requirement. In total, 165 Caucasian outpatients on stable maintenance warfarin treatment previously genotyped for CYP2C9 were analysed for common polymorphisms in FVII, GGCX and VKORC1 genes. The -402G > A polymorphism and a variable number of repeats in intron 7 of FVII gene did not significantly influence warfarin dose. The mean warfarin doses increased with the number of (CAA) repeats in the GGCX gene, but the differences were significant only in the CYP2C9*1/*1 subgroup of patients (p = 0.032). Common polymorphism (6484C > T) in intron 1 of the VKORC1 gene led to lower warfarin dose requirement; the means were 5.70 (95% C.I. 4.95-6.45), 3.49 (3.07-3.90) and 2.11 (1.80-2.42) mg/day for 6484 CC, CT and TT genotypes, respectively (p < 0.001). In contrast, 9041G > A polymorphism in 3'UTR of theVKORC1 gene led to higher warfarin dose requirement; the means were 3.09 (2.58- 3.60), 4.26 (3.69-4.82) and 5.86 (4.53-7.19) mg/day for 9041 GG, GA and AA genotypes, respectively (p < 0.001). With a regression model we explained 60.0% of variability in warfarin dose, which was due to gene polymorphisms (CYP2C9, VKORC1), age and body-surface-area. When aiming for individualised warfarin therapy, at least VKORC1 polymorphisms should be included in predictive genotyping besides CYP2C9.

Endothelial function is impaired in patients with primary antiphospholipid syndrome.

INTRODUCTION: The aim of this study was to evaluate endothelial function in patients with primary antiphospholipid syndrome (PAPS). PATIENTS AND METHODS: Flow mediated (FMD) and glyceryl trinitrate (GTN) induced dilation of the right brachial artery were studied in 25 patients with PAPS and 25 controls matched by age, sex and conventional risk factors for atherosclerosis. Fibrinogen, D-dimer, adhesion molecules, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigens and activities were measured. RESULTS: Mean (SD) FMD was significantly lower in PAPS than in controls (8+/-5% vs. 15+/-6%, P<0.001); GTN-induced dilation did not differ between the groups. There was a correlation between the baseline diameter of the brachial artery and duration of the disease (-0.56, P<0.05) and between GTN induced dilation and duration of the disease (0.51, P<0.05). Concentrations of vascular cell adhesion molecule-1 (P<0.001), intracellular adhesion molecule-1 (P<0.001) and fibrinogen (P<0.05) were higher in patients than in controls but no differences were observed for D-dimer, t-PA and PAI-1 antigens and activities. There was correlation between concentration of vascular cell adhesion molecule-1 and FMD (-0.35, P<0.05) and between intracellular adhesion molecule-1 and FMD (-0.41, P<0.05). CONCLUSIONS: This study shows that endothelial function is impaired in patients with primary APS, possibly contributing to accelerated atherosclerosis and thromboembolic complications in these patients.

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.

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.

Fibrinolytic parameters and lipoprotein(a) in young women with myocardial infarction.

Impaired fibrinolysis and elevated lipoprotein(a) (Lp[a]) are possible nonclassic risk factors for myocardial infarction (MI) at young age. The fibrinolytic system in young women with MI has not been evaluated yet and the role of Lp(a) is still controversial. The authors determined fibrinolytic parameters and Lp(a) in premenopausal women (mean age 42+/-3 years, n = 22) 0.5 to 6 (mean 3.5) years after MI, who were all without severe classic risk factors and had an otherwise low risk for MI. Elevated levels of tissue type plasminogen activator (t-PA) (p< 0.05) were measured in comparison to 52 age-matched controls; no difference was found in plasminogen activator inhibitor, plasminogen, fibrinogen, euglobulin clotting time and D-dimers. Significantly more MI patients had Lp(a) levels greater than 300 mg/L compared to controls (36% vs 13.5%, p< 0.05). The combination of elevated Lp(a), mild hyperlipidemia, and nonsevere smoking was found in 62.5% of MI patients who had elevated levels of Lp(a), in 23% of all women with MI, and in none of the controls. Elevated t-PA is probably only a marker of increased risk of MI, whereas elevated Lp(a) probably has a causative role. A combination of elevated Lp(a), hyperlipidemia, and nonsevere smoking seems to be a high-risk profile, relatively common in young women with MI.

Enhanced thrombin generation in women with a history of oral contraception-related venous thrombosis.

INTRODUCTION: In women who suffer venous thrombosis (VT) during oral contraceptive (OC) use, a transient risk factor (OC) is removed during the acute event, while most co-existing forms of thrombophilia persist and presumably continue to maintain hypercoagulability. The aim of this study was to establish if hypercoagulability persists long after OC-related VT and if it could be attributed to thrombophilia. MATERIALS AND METHODS: 60 women (age 33.0 ± 8.5 years) were investigated 5 - 64 (median 33) months after OC-related VT (patients) and compared to 63 apparently healthy women (controls). All women were tested for thrombophilia, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, P-selectin and C-reactive protein. Thrombin generation was measured by Technothrombin® TGA assay. Overall haemostasis potential (OHP) assay with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) as supplementary parameters were measured by repeated fibrin formation and degradation registration. RESULTS: In patients increased endogenous thrombin potential (4,205 ± 440 nM x min vs 4,015 ± 421 nM x min, p=0.017), increased OCP (22.6 ± 4.6 Abs-sum vs 20.8 ± 4.1 Abs-sum, p=0.025), shorter APTT (30.9 ± 3.8s vs 33.4 ± 3.6s, p<0.001) and lower antithrombin activity (99, 93-105% vs 104, 100-109%, p<0.05) were observed. Thrombophilia was observed in 22/60 (36%) patients and in 5/63 (7.9%, p<0.001) controls. The only significant difference between thrombophilic and non-thrombophilic patients was higher soluble P-selectin in the former subgroup (22, 20-33 µg/L vs 17, 12-22 µg/L, p=0.012). CONCLUSIONS: In women with a history of OC-related VT persistent hypercoagulability was observed, which, however was not augmented by the presence of thrombophilia.

The influence of co-treatment with carbamazepine, amiodarone and statins on warfarin metabolism and maintenance dose.

AIMS: Warfarin is a frequently used anticoagulant drug with narrow therapeutic index and high interindividual variability in the dose requirement. We have previously shown that warfarin dose is influenced by cytochrome P450 (CYP) 2C9 genotype, age, body weight and co-treatment with drugs that interfere with warfarin metabolism. As, in many patients, drug co-treatment cannot be avoided, we investigated the effect of co-treatment with carbamazepine, amiodarone and statins on warfarin metabolism and maintenance dose. METHODS: Caucasian patients on stable maintenance warfarin therapy with CYP2C9*1/*1 genotype (n=82) were included in the study. Plasma concentrations of (S)- and (R)-warfarin as well as warfarin hydroxylated metabolites were determined using HPLC assay and corresponding clearances of (S)- and (R)-warfarin were calculated. RESULTS: Patients co-treated with carbamazepine (n=5) had significantly higher plasma 10-hydroxywarfarin concentrations than patients not taking any interacting drugs (n=54) (median: 0.327 microg/ml vs 0.030 microg/ml, p=0.003). (S)- and (R)-warfarin clearances were also higher in the carbamazepine co-treated group (p=0.003), as were warfarin dose requirements (median: 9.00 mg/day vs 3.86 mg/day, p=0.003). Under the conditions of this study, patients co-treated with amiodarone (n=6) did not differ significantly regarding any measured characteristic from patients with no interacting drug treatment, while patients co-treated with simvastatin or lovastatin (n=17) had lower 10-hydroxywarfarin concentration (p=0.02). CONCLUSIONS: We confirmed important interaction between carbamazepine and warfarin metabolism which can be of major clinical importance. If treatment with carbamazepine cannot be avoided, patients taking warfarin should be frequently monitored, especially when initiating or stopping carbamazepine therapy.

Intensity of long-term treatment with warfarin is influenced by seasonal variations.

In a prospective study, which lasted 1 year and was performed on a large cohort of 800 patients treated at an outpatient anticoagulant clinic, we investigated possible seasonal variations in the intensity of the effect of warfarin treatment. For every season--winter, spring, summer and autumn--the mean prothrombin time reported as an international normalised ratio (INR) and the percentages of INR below 2.0 and above 4.0 were calculated. Significant seasonal variations in mean INR measurements were found, with the lowest values in summer and the highest values in autumn (F = 14.2, p < 0.0001). In addition, a trend toward a higher percentage of INR below 2.0 in summer and above 4.0 in autumn was observed. No significant differences were found between older (>65 years) and younger (<65 years) patients, although there was a trend toward more pronounced variations in younger patients. Consideration of seasonal variations might result in more accurate and safe guidance of warfarin treatment.

Vascular bed specific alterations in coagulation and fibrinolytic parameters in young women following myocardial infarction, lacunar cerebral infarction and deep vein thrombosis.

The possible existence of distinctive, vascular bed specific alterations of coagulation and fibrinolytic parameters associated with three different types of thrombosis was investigated in young women (n = 68, <45 years at onset of the event) following myocardial infarction (MI) (n = 22), lacunar cerebral infarction (LACI) (n = 16), idiopathic deep vein thrombosis (VT) (n = 14) and venous thrombosis due to oral contraceptive use (n = 16) in the stable period after the acute thrombotic event. Coagulation and fibrinolytic parameters, as well as classical metabolic variables, were measured and compared with 52 age-matched, healthy controls. In MI women we observed elevated tissue type plasminogen activator (t-PA) antigen levels, which correlated significantly with parameters of the plurimetabolic syndrome. In LACI women we found elevated fibrinogen, which correlated with D-dimer, systolic blood pressure, smoking, and sedimentation rate. Prolonged euglobulin clot lysis time, elevated t-PA antigen, PAI-1 antigen and activity, which all correlated with parameters of the plurimetabolic syndrome, were found in women with idiopathic VT, who were also clearly obese but not in women in whom oral contraceptives were the triggering factor for VT. Our results showed not parallel, but different profiles of alterations in fibrinolytic and coagulation parameters in line with the prediction of a vascular bed specific thrombosis process.