Nurr1 haplotypes are associated with femoropopliteal restenosis/re-occlusion after percutaneous transluminal angioplasty.

Restenosis/re-occlusion remains a frequent complication in the first year after percutaneous transluminal angioplasty (PTA). In this study, association of nuclear receptor related 1 protein (Nurr1) haplotypes to the restenosis/re-occlusion rate after femoropopliteal PTA was investigated. Patients (n = 142) with disabling claudication or critical limb ischaemia, who had undergone technically successful femoropopliteal PTA, were prospectively followed up by vascular ultrasound imaging 12 months after the procedure. Nurr1 haplotypes 2 and 3 were associated significantly with the restenosis/re-occlusion rate (adjusted odds ratio 1.6, 95% confidence interval (CI) 1.1-2.3 and 2.0, 1.3-2.8, respectively) on univariate analysis.

Prevention of ischemic events in patients with peripheral arterial disease design, baseline characteristics and 2-year results an observational study.

AIM: Peripheral arterial disease (PAD) is associated with frequent cardiovascular ischemic events. We followed the survival of PAD patients and tested whether PAD remains an adverse prognostic indicator in spite of treatment according to the current European guidelines on cardiovascular disease prevention. METHODS: Eight hundred eleven patients with PAD and 778 control subjects, aged 65 (SD 9) years at inclusion, with a male/female ratio of 3/2 were treated according to the European guidelines on cardiovascular disease prevention and evaluated yearly for occurrence of death, non-fatal acute coronary syndrome, stroke or critical limb ischemia (major events) and revascularization procedures (minor events). At baseline, classical risk factors were significantly more prevalent in the PAD group and protective cardiovascular medication was prescribed to patients with PAD more frequently than to control subjects. RESULTS: In the PAD group, the 2-year Kaplan-Meier survival estimate was 96.7% (CI 95.4-97.9) vs. 98.2% (CI 97.2-99.1) in the control group, P=0.059. The groups differed in the 2-year major event-free survival: 93.5% (CI 92.7-95.3) in PAD vs. 97.1% (CI 95.9-98.4) in controls, P<0.017, as well as in event-free survival: 79.9% (CI 77.1-82.9) in PAD vs.96.4% (CI 95.0-97.9) in controls, P<0.001. CONCLUSION: Patients with PAD had a borderline higher risk of all-cause death and a significantly higher risk of major and minor non-fatal cardiovascular events compared to control subjects. However, treatment according to the European guidelines on cardiovascular disease prevention resulted in encouragingly low absolute mortality and morbidity. (ClinicalTrials.gov number NCT00761969.).

Design, synthesis and molecular modelling of 1-amidinopiperidine thrombin inhibitors.

Design, synthesis and biochemical evaluation of a series of novel non-covalent thrombin inhibitors with a 1-amidinopiperidine moiety are presented. Replacement of the planar benzamidine group in azaphenylalanine derivatives with 1-amidinopiperidine resulted in lower activity but higher selectivity for this type of compounds. The binding conformation of inhibitors in the active site of thrombin was revealed by molecular modelling studies.

Influence of CYP2C9 polymorphisms, demographic factors and concomitant drug therapy on warfarin metabolism and maintenance dose.

Warfarin is an anticoagulant drug with narrow therapeutic index and high interindividual variability in dose requirement. S-warfarin is metabolized mainly by polymorphic cytochrome P450 (CYP) 2C9. We systematically quantified the influence of CYP2C9 genotype, demographic factors and concomitant drug treatment on warfarin metabolism and maintenance dose. The mean warfarin doses were lower in carriers of one (2.71 mg/day, 59 patients) and two polymorphic alleles (1.64 mg/day, 11 patients) than in carriers of two wild-type alleles (4.88 mg/day, 118 patients). Multiple regression analysis demonstrated that CYP2C9 genotype, age, concomitant treatment with warfarin metabolism inducers and lean body weight contributed significantly to interindividual variability in warfarin dose requirement (adjusted R(2)=0.37). The same factors, except for age, significantly influenced S-warfarin clearance (adjusted R(2)=0.42). These results can serve as a starting point for designing prospective studies in patients in the initiation phase of genotype-based warfarin therapy.

Synthesis and in vitro evaluation of new azaphenylalanine derivatives as serine protease inhibitors.

New inhibitors of serine proteases with azaphenylalanine scaffold were synthesized and their activity was evaluated in vitro. We studied the effect of different substituents in the part of a molecule that binds in the distal pocket of the thrombin active site. Modifications generally led to decreased activity, however two derivatives are promising lead compounds as new thrombin and dual thrombin-factor Xa inhibitors.

Novel non-covalent azaphenylalanine thrombin inhibitors with an aminomethyl or amino group at the P1 position.

Design, synthesis and biological evaluation of a series of novel non-covalent azaphenylalanine thrombin inhibitors are presented. Replacement of the basic benzamidine moiety in azaphenylalanine derivatives by benzylamine (P1 part of a molecule) and the introduction of a N-cyclopentyl-N-methylamine moiety in the P2 part of a molecule resulted in the thrombin inhibitor LK-733 with greatly increased selectivity against trypsin and an in vitro Ki of 31 nM.

Classic risk factors, hypercoagulability and migraine in young women with cerebral lacunar infarctions.

OBJECTIVE: Lacunar cerebral infarctions (LACI) in young women is a rare condition which pathogenesis is still not fully recognized. We explored the presence of classic risk factors, hypercoagulability and migraine in young women with LACI. METHODS: Charts of 192 consecutive premenopausal women suffering cerebrovascular insult [125 (65%) haemorhagic, 58 (30%) ischaemic and 9 (5%) unclassified] during a period of 5 years were reviewed. Sixteen out of 58 (27%) patients with ischaemic stroke were identified to have LACI and included in a study. RESULTS: Ten and seven out of 16 LACI women had at least one classical risk factor (hypertension, hyperlipidaemia, smoking or oral contraceptives) or migraine, respectively. LACI patients had slight hypercoagulable state indicated by shorter thrombin and thromboplastin times, higher fibrinogen and higher t-PA antigen than 47 age matched controls (all P < 0.05). In addition in LACI patients with migraine the trend toward more pronounced hypercoagulable state in comparison to LACI patients without migraine was found. The combination of migraine, at least one classic risk factor and hypercoagulability was present in 5/16 (31.25%) of patients. CONCLUSIONS: The combination of slightly to moderately expressed classic risk factors, hypercoagulability and migraine might be a risk profile for LACI in young women. Further studies are needed to clarify risk profile, rather than isolated risk factors, for LACI in a specific group as young women are.

Subcutaneous abdominal, but not femoral fat expression of plasminogen activator inhibitor-1 (PAI-1) is related to plasma PAI-1 levels and insulin resistance and decreases after weight loss.

AIMS/HYPOTHESIS: Abdominal fat produces plasminogen activator inhibitor-1 (PAI-1) and could contribute to increased plasma PAI-1 values in human obesity associated with insulin resistance. Femoral fat, which is not associated with insulin resistance, is thought to be metabolically different from the abdominal fat. This study aimed to assess PAI-1 expression in these two fat territories in obese and lean subjects and to determine if concomitant changes of plasma and adipose tissue PAI-1 values occur after weight reduction. METHODS: In 24 obese and 16 lean subjects, PAI-1 expression in abdominal and femoral subcutaneous fat, plasma PAI-1, insulin, triglyceride concentrations and insulin resistance were determined at the start of the study and in obese subjects after a 3-month weight reduction programme as well. RESULTS: PAI-1 mRNA content in the abdominal subcutaneous fat was higher in obese than in lean subjects and positively correlated with plasma PAI-1 values (p < 0.01) and markers of insulin resistance (p < 0.05). In 18 obese subjects, re-examined after successful dieting, PAI-1 mRNA content decreased in the abdominal subcutaneous fat along with plasma PAI-1. However, the absolute changes of these two variables were not associated. In contrast, PAI-1 mRNA content in the femoral subcutaneous fat did not differ between lean and obese subjects, was not associated with plasma PAI-1 values or with markers of insulin resistance, and did not change after weight loss. CONCLUSION/INTERPRETATION: Only the abdominal, but not the femoral subcutaneous fat PAI-1 expression is a potential contributor to increases in plasma PAI-1 in obesity. Both plasma and abdominal subcutaneous fat PAI-1 values decreased significantly after weight reduction, although their absolute changes were not associated.

Novel thrombin inhibitors with azaphenylalanine scaffold.

In this paper the synthesis and antithrombotic activity of a series of novel thrombin inhibitors with azaphenylalanine scaffold are described. By systematic structural modifications for this series we have identified optimal groups for achieving nanomolar potency, that led to potent inhibitors of thrombin with Ki values up to 11 nM.

Impact of weight reduction on early carotid atherosclerosis in obese premenopausal women.

OBJECTIVE: To investigate the extent of carotid atherosclerosis and the effect of weight loss on carotid intima-media thickness (IMT) in obese premenopausal women. RESEARCH METHODS AND PROCEDURES: In 43 obese premenopausal women who participated in a 3-month weight reduction program with a hypocaloric diet, IMT was measured by B-mode high-resolution ultrasound at entry and after 5 months of follow-up. Blood samples were analyzed at entry, after intervention, and after 5 months of follow-up. Nineteen lean women served as control subjects. RESULTS: At entry, common carotid IMT (0.72 vs. 0.59 mm), carotid bulb IMT (0.90 vs. 0.71 mm), and overall mean IMT (0.81 vs. 0.65 mm) were greater in obese women than in lean women (all p < 0.01). After dietary intervention decreases in blood pressure, low density lipoprotein to high density lipoprotein cholesterol ratio, triglycerides, fibrinogen, plasminogen activator inhibitor-1, and an increase in tissue-type plasminogen activator activity levels were observed. These effects persisted after follow-up in 14 women who maintained reduced weight. Reduction in carotid bulb IMT (to 0.81 mm, p < 0.01) and overall mean IMT (to 0.79 mm, p < 0.05) was observed in this subgroup. No significant change of carotid IMT was detected in eight women who regained weight. Changes in IMT were associated independently and significantly with changes in body mass index, low density lipoprotein to high density lipoprotein cholesterol ratio, and plasminogen activator inhibitor-1 antigen. DISCUSSION: Obese premenopausal women had greater IMT than did age-matched lean controls. It seems that this early atherosclerotic changes may be reversed by normalization of body weight.

Seasonal variation of some metabolic and haemostatic risk factors in subjects with and without coronary artery disease.

Acute myocardial infarction (AMI) is more frequent in winter months than in summer months. The aetiologic mechanisms underlying this seasonal pattern are poorly understood. We investigate whether seasonal variation of metabolic and haemostatic coronary risk factors exists, and whether this variation is more pronounced in subjects with coronary artery disease (CAD). In 82 subjects (47 free of clinical signs of CAD and in 35 survivors of AMI), measurements of body mass index (BMI), lipoproteins, glucose, insulin, plasminogen activator inhibitor-1, tissue-type plasminogen activator (t-PA), euglobulin clot lysis time, fibrinogen, and platelet count were performed twice in the cold months (December and March) and twice in the warm months (June and September). Significantly higher BMI (26.8 versus 26.2 kg/m2, P < 0.01), glucose (5.5 versus 5.1 mmol/l, P < 0.01), total cholesterol (5.61 versus 5.32 mmol/l, P < 0.05), low-density lipoprotein cholesterol (3.63 versus 3.34 mmol/l, P < 0.05), triglycerides (1.79 versus 1.61 mmol/l, P < 0.01), Lp(a) (270.7 versus 237.5 mg/l, P < 0.01), fibrinogen level (3.50 versus 2.95 g/l, P < 0.00001), platelet count (212 x 10(9) versus 173 x 10(9)/l, P < 0.01) and significantly lower high-density lipoprotein cholesterol level (1.22 versus 1.28 mmol/l, P < 0.05) were observed in the cold months compared with the warm months. Significant seasonal variation of t-PA activity (1.19 versus 0.87 IU/ml, P = 0.015) and t-PA antigen (8.5 versus 7.3 ng/ml, P = 0.05) was demonstrated only in subjects with CAD. Clustering of peak values of several metabolic and haemostatic coronary risk factors was observed in winter months. This variation might be of aetiopathogenetic importance for the seasonal pattern of acute myocardial infarction.

Do baseline serum leptin levels predict weight regain after dieting in obese women?

AIM: Weight loss achieved during weight reduction programme is difficult to maintain. We investigated the possible role of circulating leptin in failure or success in maintaining weight loss. METHODS: Serum leptin levels were measured in 30 healthy premenopausal obese women before and after 12 weeks of dietary intervention and after 5 months of follow-up. RESULTS: After intervention body mass index (BMI) decreased from 30.6 to 25.4 kg/m2 (p < 0.01) and leptin levels decreased from 16.7 to 7.7 ng/ml (p < 0.01). After 5 months follow-up 12 women regained reduced weight and 18 women maintained weight loss. In the regainers leptin levels increased again, but remained low in the maintainers. Baseline leptin concentrations were lower in the regainers than in the maintainers (12.1 vs. 21.2 ng/ml, p = 0.04). During intervention leptin levels decreased three times more in the maintainers than in the regainers, although weight loss was similar in both groups. CONCLUSIONS: This study shows that obese women who regain weight after dieting have significantly lower baseline leptin levels than women who maintain weight loss. Our results suggest that differences in leptin resistance might exist in similarly obese women which could influence the success of dieting.

Screening for selective thrombin inhibitors in mushrooms.

Thrombin is the key serine proteinase of the coagulation cascade and therefore a suitable target for inhibition of blood coagulation. A number of pharmacologically active secondary metabolites from mushrooms have already been isolated, thus providing the rationale for screening for new thrombin inhibitors in mushrooms. In this study, inhibitory activities of mushroom extracts on thrombin and trypsin were measured using the chromogenic substrates H-D-phenylalanine-L-pipecolyl-L-arginine-paranitroaniline dihydrochloride (S-2238) for thrombin and N-benzoyl-D,L-Arg-p-nitroanilide (BAPNA) for trypsin. The inhibitory activities of extracts from 95 Basidiomycete species have been determined. The majority of samples inhibited trypsin and thrombin with various potencies; however, some extracts showed no activity against one or both of the enzymes. An aqueous extract of Gleophyllum odoratum exhibited high inhibitory activity on both thrombin and trypsin (72 and 60%, respectively), while extracts of Clitocybe gibba, Amanita virosa, Cantharellus lutescens, Suillus tridentinus, Hypoloma fasciculare and Lactarius badiosanguineus considerably inhibited thrombin (49, 48, 36, 34, 32 and 31%, respectively) and showed no inhibitory activity on trypsin. The results at this point are promising for further research with the objective of finding an effective and safe thrombin inhibitor.

Mild hyperhomocysteinemia and fibrinolytic factors in patients with history of venous thromboembolism.

Mild hyperhomocysteinemia is recognized as a risk factor for venous thromboembolism (VTE), though its role in the thrombogenic processes is not understood. Its possible association with impaired fibrinolysis was investigated in 157 patients (61 women, 96 men) below the age of 60 years (43+/-11, mean+/-SD) with a history of objectively confirmed VTE. Patients had significantly higher fasting total plasma homocysteine (tHcy) levels than 138 apparently healthy subjects (8.0, 6.6-9.9 micromol/L vs. 7.2, 5.9-8.6 micromol/L, P=0. 001; median, range between first and third quartile). In 17 of 157 patients (12%) hyperhomocysteinemia (tHcy>11.4 micromol/L for women and tHcy>12.6 micromol/L for men) was established. The adjusted odds ratio as an estimate of relative risk for VTE was 2.3 (0.8-7.0; 95% confidence interval). When patients with hyperhomocysteinemia were compared to patients without hyperhomocysteinemia, no significant differences in t-PA (antigen 9.2+/-5.5 microg/L and 9.7+/-4.7 microg/L, respectively; activity 1.3+/-0.5 IU/mL and 1.3+/-0.7 IU/mL, respectively) and PAI-1 (antigen 19.3+/-17.5 microg/L and 22.6+/-20. 4 microg/L, respectively; activity 15.0+/-12.6 and 15.8+/-13.3 IU/mL, respectively) were observed. In conclusion, this study showed an association between mild hyperhomocysteinemia and VTE, but provided no evidence for an independent association between hyperhomocysteinemia and alterations in fibrinolytic proteins.

Alteration of the fibrinolytic system in patients with peripheral arterial occlusive disease.

BACKGROUND: The fibrinolytic system may play an important role in the development and progression of peripheral arterial occlusive disease. PATIENTS AND METHODS: The fibrinolytic system of the whole blood and a diseased leg was investigated in twenty men with chronic peripheral atherosclerotic occlusive arterial disease (PAOD, clinical stage II according to Fontaine), aged from 46 to 66 years (x = 55.3) [symbol: see text]. The diagnosis of PAOD was established by clinical examination and segmental systolic blood pressure measurements using a Doppler ultrasound detector. Twenty age-matched (x = 53.4) male volunteers with normal arterial circulation of the lower limbs and without risk factors of atherosclerosis, served as controls. In both groups fibrinolytic system was investigated in basal conditions and during provocation. Release of tissue-type plasminogen activator (t-PA) was provoked by 20 min venous occlusion of the arm and the leg and by infusion of DDAVP (1-desamino-8-D-arginine-vasopressin, 0.4 ug/kg of body weight). Blood samples were obtained from the arm and the leg before and after each stimulus. The fibrinolytic parameters: euglobulin clot lysis time, t-PA activity (amidolytic assay) and antigen (ELISA) and t-PA inhibitor (PAI) activity (amidolytic assay) were determined. RESULTS: With the exception of a boderline increase in PAI activity in patients, no other differences between the two groups were observed in basal conditions. The most prominent deterioration of the fibrinolytic system detected in male PAOD patients was a significantly higher residual PAI activity registered during venous occlusion of the arm and two minutes after combined stimulation. Two minutes after combined stimulation (DDAVP and venous occlusion of the arm) significantly lower t-PA activity was observed in patients. In patients t-PA antigen response to venous occlusion and DDAVP was not significantly different from the response observed in healthy volunteers. The fibrinolytic response of the leg to venous occlusion was poor and after DDAVP application it was comparable to the arm. The fibrinolytic response of the diseased leg in men was not significantly different from the healthy leg. CONCLUSION: The results of our study indicate that alteration of the fibrinolytic system in atherosclerotic disease is predominantly a generalised phenomenon and is not directly related to a local atherosclerotic process.

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.

The 4G/5G sequence polymorphism in the promoter of plasminogen activator inhibitor-1 (PAI-1) gene: relationship to plasma PAI-1 level in venous thromboembolism.

Impaired fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) is observed in up to 40% of patients with venous thromboembolism and might be causally related to the disease. There is evidence that genetic variations in the promoter of the PAI-1 gene and metabolic factors contribute to increased plasma PAI-1 levels. A single nucleotide insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene and metabolic factors were studied in 158 unrelated patients below the age of 61 years (43 +/- 11 years, mean +/- standard deviation) with history of objectively confirmed venous thromboembolism and in 145 apparently healthy controls. Patients had on average two times higher PAI activity (11.9 vs. 6.1 IU/ml) and by 40% higher PAI-1 antigen (14.8 vs. 10.7 ng/ml) than healthy controls, and also higher body mass index, lipid levels, fasting glucose and insulin. Patients differed significantly from healthy controls neither in the frequency of the 4G and 5G alleles (0.57/0.43 in patients and 0.52/0.48 in controls) nor in the distribution of the 4G/5G genotypes. Possession of the 4G/4G or the 4G/5G genotype did not increase relative risk for venous thromboembolic disease and the distribution of the 4G/5G genotypes was neither associated with recurrent nor with spontaneous disease. In patients association between the 4G/5G genotypes and PAI activity (adjusted for body mass index, triglyceride and glucose level) was observed, with the highest PAI activity values in the 4G/4G genotype (14.6 IU/ml), intermediate in the 4G/5G genotype (13.3 IU/ml) and the lowest in the 5G/5G genotype (5.2 IU/ml, all values means). Association between PAI activity and triglyceride level was the strongest in the 4G/4G genotype (correlation coefficient r = 0.47, p < 0.01) and the weakest in the 5G/5G genotype (r = -0.04, not significant). In conclusion, the present case-control study shows an association between the 4G/5G polymorphism in the promoter of the PAI-1 gene and plasma PAI-1 levels in patients with venous thromboembolism. Similar distribution of the 4G/5G genotypes in patients and healthy controls suggests that this genetic variation by itself is not a major risk factor for venous thromboembolism.

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.

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.

Reproducibility of fibrinolytic response to venous occlusion in healthy subjects.

Fibrinolytic response to venous occlusion is used to assess the efficiency of the fibrinolytic system. Reproducibility of fibrinolytic variables after 20 min upper arm venous occlusion was investigated in 40 apparently healthy subjects tested twice in a period of 11-23 (mean 15) days. Resting and post-venous occlusion euglobulin clot lysis time, tissue-type plasminogen activator (t-PA) activity, t-PA antigen, plasminogen activator inhibitor (PAI) activity and plasminogen activator inhibitor type 1 (PAI-1) antigen determined on the two occasions did not differ significantly. Positive correlation coefficients for variables before (r = 0.43-0.74, all p < 0.01) and after venous occlusion (r = 0.07-0.66) indicated low to moderate associations between repeated measurements. Differences between repeated measurements relative to the initial measurement were greater after venous occlusion than before venous occlusion and were for euglobulin activity 41 (0-826)%, t-PA activity 27 (2-398)%, and for t-PA antigen 27 (0-179)% (medians and ranges). Poor responses were defined by the lowest (euglobulin activity, t-PA activity and t-PA antigen) or the highest (PAI activity) fifth percentile of the distribution. Almost no agreement between poor responses was observed: poor responses at the first occasion were not determined in the same subjects when re-examined after two weeks. It was concluded that due to relatively low reproducibility of the variables measured after venous occlusion the test needs improvement in order to be potentially clinically useful.