Hyperhomocysteinemia, obesity and cryptogenic stroke.

BACKGROUND: The pathogenic role of hyperhomocysteinemia in cryptogenic stroke is not well established. We aimed to determine homocysteine levels in patients with cryptogenic stroke considering the possible variables that may act as confounders and analyze the influence of obesity on this association. PATIENTS AND METHODS: We conducted a case-control study in 123 patients with cryptogenic stroke aged 42 ± 12 years and in 153 control subjects aged 42 ± 13 years. Serum homocysteine was determined by fluorescence polarization immunoassay. RESULTS: Patients showed statistically higher levels of homocysteine, creatinine and higher BMI than controls (p = 0.045, p = 0.014, p = 0.013), respectively. After multivariate adjustment the differences in homocysteine levels disappeared (p = 0.774). When subjects were classified according to the presence or absence of obesity, the differences in the prevalence of hyperhomocysteinemia (homocysteine >15 µM) were highly significant, being higher in patients than in controls (p = 0.009). Likewise, mean values of homocysteine in obese were higher in cases than in controls (16.9 ± 9.5 µM vs. 10.12 ± 2.5 µM; p = 0.020), remaining significant after adjusting for the above mentioned confounders. CONCLUSION: Although in general, hyperhomocysteinemia does not seem to constitute an independent risk factor for cryptogenic stroke, it significantly increases the risk in obese subjects; therefore it is convenient to decrease its levels in this sub-group to minimize the risk.

Erythrocyte membrane composition in patients with primary hypercholesterolemia.

There are conflicting results regarding the erythrocyte membrane cholesterol and phospholipid content in patients with primary hypercholesterolemia (PHC), due to methodological problems in obtaining haemoglobin-free ghosts. At the same time, the different units used and the fact that the cholesterol and phospholipids are not expressed in relation with integral protein membrane content, produces contradictory results. We have analysed in 33 patients with PHC (12 male, 31 female) aged 43+/-12 years and in 33 healthy normolipaemic volunteers (9 male, 24 female) aged 43+/-13 years plasma lipids, along with, erythrocyte membrane cholesterol, phospholipids and integral proteins. PHC patients showed increased erythrocyte membrane cholesterol: 0.36+/-0.15 mg/mg when compared with controls: 0.29+/-0.75 mg/mg; p=0.018. Phospholipid membrane content, although higher in the cases, did not reach statistical significance (PHC patients: 0.38+/-0.15 mg/mg vs. 0.33+/-0.72 mg/mg; p=0.098). The cholesterol/phospholipids ratio (Chol/Ph) was 0.99+/-0.22 in PHC patients versus 0.92+/-0.28 in controls; p=0.127. Our results suggest that there is a slight increase in erythrocyte membrane cholesterol in patients with PHC. Given the increasing importance of erythrocyte membrane cholesterol in the stability of the atheroma plaque due its possible contribution to the clinical signs of ischaemic heart disease, it seems relevant to determine this parameter in risk populations. Therefore, a simple and reproducible method needs to be standardised which would enable comparisons between laboratories and facilitate further studies aimed to it as a marker of acute coronary syndromes.

Fibrinogen, plasma viscosity and blood viscosity in obesity. Relationship with insulin resistance.

Plasma viscosity (PV) and blood viscosity (BV) have been scarcely evaluated in morbid obese patients with no other concomitant cardiovascular risk factors. Contradictory results have been published regarding the influence of insulin resistance on these rheological parameters in obesity. In 67 severe or morbid obese patients without other cardiovascular risk factors (51 women and 11 men, aged 34+/-11 years), fibrinogen, PV and BV at native (nBV) and corrected 45% hematocrit (cBV) have been determined, and insulin resistance has been calculated with homeostasis model assessment (HOMA) index, in basal conditions and after a three month diet period. The same determinations were performed in 67 healthy volunteers (45 women, 22 men, aged 32+/-10 years) at baseline and three months later. When cases and controls were compared, obese patients showed higher fibrinogen levels (P<0.001), PV (P=0.050) and cBV (P=0.035), and showed a higher insulin resistance than the control group (P<0.001). Differences in PV were maintained after adjusting for BMI (P=0.001), but disappeared after adjusting for HOMA (P=0.391) fibrinogen (P=0.367) and LDL-chol (P=0.097). Differences between obese patients and the control group for cBV disappeared after adjusting for BMI (P=0.739), HOMA (P=0.744), fibrinogen (P=0.907), LDL-chol (P=0.283) and PV (P=0.112). The achieved weight loss (8.7+/-3.53%) was not accompanied by any changes in these rheological parameters (P>0.050). Obese patients show increased fibrinogen levels, PV and cBV. These rheological disturbances seem to be associated with insulin resistance and the metabolic syndrome, and do not seem to improve with moderate weight loss.

Erythrocyte deformability in obesity measured by ektacytometric techniques.

Erythrocyte deformability (ED) has been scarcely evaluated in obese patients without other concomitant cardiovascular risk factors and contradictory results have been published regarding the influence of plasma lipids on the erythrocyte membrane lipid composition and insulin resistance on this rheological parameter. In 67 severe or morbid obese patients without other cardiovascular risk factors (51 women and 11 men, aged 34+/-11 years) and in 67 controls (45 women and 22 men, aged 32+/-10 years), ED has been determined by ektacytometric techniques in a Rheodyn SSD, the elongation index (EI) being measured at 12, 30 and 60 Pa, along with plasma lipids, red blood cell membrane lipids (cholesterol and phospholipids) and insulin resistance indexes in basal conditions and after a three month diet period. No significant differences were obtained in the EI between obese patients and the control group at any of the shear stresses tested (P>0.05). The cholesterol and phospholipid content of the red blood cell membrane did not significantly differ between cases and controls (P>0.05). Obese patients with metabolic syndrome showed lower EI at 30 and 60 Pa than those without metabolic syndrome (P=0.014 and P=0.031 respectively). Weight loss was not accompanied by any changes in these rheological parameters. Obesity itself does not seem to modify ED. However, metabolic syndrome seems to decrease ED, possibly through insulin resistance.

Platelet activation and red blood cell phosphatidylserine exposure evaluated by flow cytometry in patients with Behçet's disease: are they related to thrombotic events?

Behçet's disease (BD) is associated with an increased risk of venous and arterial thromboses that are associated with morbidity and mortality increase, although the mechanisms are not well established. In the present study, we used whole blood cytometry to determine the exposure of CD62 on the surface of platelets and the expression of phosphatidylserine (PS) on the surface of circulating red blood cells. Microparticle and microaggregate formation from platelets were also determined in a well-classified group of 72 patients (39 males, 33 females, aged 46.5 +/- 12.5 years) with BD, in comparison with a well-matched control group of 72 healthy volunteers. Results showed no differences in the above-mentioned parameters when BD patients and controls were compared. However, when we compared BD patients with/without thrombosis using these parameters, there were significant differences between both groups. BD patients with previous thrombosis had a higher percentage of circulating CD62-positive platelets and a higher number of circulating microaggregates than those without thrombosis, suggesting that platelet activation may be involved in the development of thrombotic events in these patients.

Erythrocyte aggregability and disaggregability in thalassemia trait carriers analyzed by a laser backscattering technique.

Erythrocyte aggregability was determined by a laser backscattering light technique in 23 beta minor thalassemia carriers and in 36 age and sex matched controls. The aggregation time (Ta) was statistically higher in cases than in controls (2.8 +/- 1.0 vs 2.3 +/- 0.4, p < 0.05) and the aggregation index at 10 sec (AI10) was statistically lower (25.1 +/- 5.7 vs 28.2 +/- 3.8, p < 0.05), suggesting both parameters a statistically lower erythrocyte aggregability tendency. However, the total disaggregation threshold (gammaD) was statistically higher in cases than in controls (134.4 +/- 34.1 vs 105.1 +/- 33.1, p < 0.05), indicating that once aggregates are formed a higher shear rate is needed to break them up. No differences were observed in plasmatic factors, i.e., fibrinogen, total cholesterol and triglycerides, that could have influenced erythrocyte aggregation. A negative statistically significant correlation was found between erythrocyte indexes and the total disaggregation threshold. The lower erythrocyte aggregation found in minor thalassemia carriers could be attributed in part to the morphological alterations, although others mechanisms such as modifications in the membrane structure of the RBC can not be ruled out.

Plasma lipids and blood fluidity in patients with polygenic hypercholesterolaemia treated with fluvastatin.

The clinical benefit brought about by HMG-CoA reductase inhibitors (statins) may not entirely be due to their lipid-lowering effect. Further investigation is necessary in order to determine the significance of ancillary effects to the clinical benefit of statin treatment. We studied 27 polygenic hypercholesterolaemia (PHC) patients before and 3 and 6 months after fluvastatin treatment. A control group of 38 normal, sex and age matched, subjects were also studied. The following parameters were measured: haematimetry, serum lipids and general biochemistry, apo-A/B and lipoproteins, fibrinogen, blood filterability, red blood cell aggregation, blood and plasma viscosity. PHC patients showed lower blood filterability (16.00+/-0.99 vs 19.90+/-2.90 microl/s), higher plasma fibrinogen (274.8+/-41.5 vs 241.6+/-43.2 mg/dl), increased erythrocyte aggregation at low shear stress (8.10+/-1.15 vs 7.19+/-1.29) and increased plasma viscosity (1.26+/-0.06 vs 1.23+/-0.05 mPa.s). Notable lipid changes after 6 months fluvastatin treatment were not accompanied by measurable changes in the haemorheological alterations of the PHC patients.

Factor V Leiden and prothrombin G20210A in relation to arterial and/or vein rethrombosis: two cases.

The factor V Leiden (FV Leiden) and prothrombin G20210A mutations, are the most common established genetic risk factors for deep vein thrombosis (DVT). However, the relationship between these mutations and arterial thrombotic syndromes (coronary heart disease, myocardial infarction, stroke) has not been established. Some studies have suggested a relationship between them, but other authors have considered it unlikely that these anomalies are a major risk factor for arterial thrombosis. From the clinical point of view, a question arises concerning the risk of repeated thrombosis in patients carrying one of these two mutations. The question is whether the recurrence is attributable to the mutations or to the presence of additional circumstantial risk factors. As the risk of repeated thrombosis varies considerably from one patient to another, decisions about long-term treatment require weighing the persistence of risk factors for vascular disease (venous and arterial), especially in selected cases such as young patients or patients with thrombosis of unusual localization.

The effect of acenocoumarol on hemorheological parameters.

The effect of acenocoumarol on hemorheological variables was measured in 35 non-valvular chronic atrial fibrillation patients before starting oral anticoagulant therapy (basal) and one and two months after beginning treatment (INR-2,3). Fibrinogen increased significantly from the basal situation: 332+/-99 mg/dl to 386+/-96 mg/dl in the second month (p<0.05). However, this small increase in fibrinogen is not large enough to mediate other rheological changes, and whole blood filterability, blood viscosity, plasma viscosity and erythrocyte deformability and aggregability remained unchanged after treatment. These results suggest that acenocoumarol does not affect rheological parameters and can therefore be used as a "neutral drug" for rheological studies in cardiovascular patients under oral anticoagulant therapy.

Spontaneous cervical epidural hematoma associated with oral anticoagulant therapy.

A 54-year-old woman who was on anticoagulant treatment with acenocoumarol for a mitral prothesis developed a cervical spinal epidural hematoma, probably triggered by coughing fits together with supratherapeutic anticoagulation. Because of the subacute evolution of the hematoma, it was not diagnosed until the patient was admitted to the hospital with profuse hemorrhages. Given the subacute nature of the hematoma, along with the favorable evolution, conservative treatment with dexamethasone was decided upon, and it was resolved with almost no sequelae. This unusual clinical entity definitely should be suspected in patients on anticoagulants who complain of severe localized neck pain, most often with radicular irradiation.

Upper extremity deep vein thrombosis in a young patient double heterozygous for factor V Leiden and prothrombin G20210A mutation.

We report on a 19-year-old girl with upper extremity deep vein thrombosis after catheter indwelling whose thrombophilic study disclosed the coexistence of factor V Leiden and the prothrombin G20210A mutation. The family study identified five other members who were also heterozygous for both mutations. This is the first case of upper extremity deep vein thrombosis with the co-inheritance of both genetic defects. It provides further evidence that thrombophilic defects mostly require additional triggering factors to induce a thrombotic event and suggests that in young patients with this venous thrombotic location, a thrombophilic search should be performed even when there are other acquired thrombotic risk factors.

Low level of circulating activated protein C is a risk factor for venous thromboembolism.

The levels of circulating activated protein C (APC) reflect in vivo protein C activation. The aim of this study was to determine whether a low APC level is an independent risk factor for venous thromboembolism (VTE). We measured APC in 160 patients with a history of VTE and without recognized thrombophilic defects, and in 199 healthy individuals. The mean (+/- SD) APC level was lower in patients (0.99 +/- 0.44 ng/ml) than in controls (1.19 +/- 0.41 ng/ml) (p < 0.0001), and showed a different distribution in the two groups. Thirty-eight patients (23.7%) had APC levels below the 5th percentile of the control group (<0.69 ng/ml) and 57 patients (35.6%) had APC levels below the 10th percentile (<0.77 ng/ml). APC levels <0.69 ng/ml increased the risk of a single or recurrent episode of VTE 4.2-fold (95% confidence interval, 2.0-9.0) or 6.9-fold (2.6-17.9). respectively, and APC levels <0.77 ng/ml increased these risks 3.4-fold (1.9-6.2) or 5.1-fold (2.3-11.2), respectively, compared with controls. Familial studies revealed that in some cases the low APC phenotype seems to be hereditary. We conclude that a low level of circulating APC in individuals without any of the most recognized thrombophilic defects is a prevalent, independent risk factor for VTE, and that it predisposes to recurrent VTE.

Relationship between fibrinogen protein and fibrinogen function in postmyocardial infarction patients.

The present study investigates the association between increases in the concentration and function of plasma fibrinogen in two groups of patients with chronic ischemic heart disease (11 with recurrent ischemic events and 19 free of these episodes) and in 34 healthy controls. The fibrinogen function index (fibrinogen function per unit of fibrinogen protein) (FgFI) was used as a measure of the fibrinogen clotting potential. The prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin (TAT) were used as procoagulant markers. Plasma sialic acid (SA) was also evaluated as an inflammatory marker. No differences were found between FgFI (1.06+/-0.13 vs. 1.02+/-0.13), F1+2 (1.2+/-0.5 vs. 1.1+/-0.4 nmol/l) and TAT (2.5+/-1.3 vs. 2.5+/-0.7 microg/ml) in postinfarction patients without recurrent coronary ischemic events and the control group. However, postinfarction patients who suffered recurrent coronary ischemic events had significantly higher FgFI than patients without these symptoms (1.19+/-0.09 vs. 1.06+/-0.13), P<.01) and than the control group (1.19+/-0.09 vs. 1.02+/-0.13, P<.001). Moreover, the F1+2 (1.4+/-0.5 vs. 1.1+/-0.4 nmol/l, P<.05) and TAT (3.6+/-3.3 vs. 2.5+/-0.7 microg/ml, P<.05) were significantly higher in patients who suffered recurrent coronary ischemic events than in the control group. However, F1+2 and TAT were not different between patients with and without these symptoms. The fibrinogen protein (Fg-protein) concentration and high molecular weight fibrinogen (HMW-Fg) levels were significantly higher in both postinfarction patient groups than in the control group and in postinfarction patients with recurrent coronary ischemic events than in postinfarction patients without these symptoms. The plasma SA levels were significantly increased in postinfarction patients with and without recurrent coronary ischemia as compared with the control group. A positive correlation was found between fibrinogen and SA levels (r=.5, P<.01). In conclusion, our study indicates that the procoagulant factors, among which we include fibrinogen, F1+2 and TAT play a very active role in recurrent ischemic events in postmyocardial infarction patients. High plasma concentrations of both fibrinogen and SA suggests that fibrinogen becomes elevated as a consequence of inflammatory processes. The FgFI as an indicator of clotting potential of fibrinogen appears to be associated with ischemic events in chronic coronary artery disease.

Risk of venous thrombosis in carriers of the prothrombin G20210A variant and factor V Leiden and their interaction with oral contraceptives.

BACKGROUND AND OBJECTIVES: The prothrombin G20210A mutation and factor V Leiden have been found to be associated with an increased risk of venous thrombosis, but the reported prevalences of the prothrombin gene variant both in the normal population and in patients with deep venous thrombosis (DVT) vary greatly in the literature. Moreover, the influence of oral contraceptives (OC) on thrombotic events in patients with the prothrombin G20210A variant has not been well established. In this study we evaluate both circumstances. DESIGN AND METHODS: A case-control study was run on 229 patients with DVT and 246 healthy controls. The patients' history of thrombosis and acquired thrombotic risk factors, especially OC, were recorded. Prothrombin G20210A mutation, factor V Leiden, antithrombin, heparin II cofactor, plasminogen and proteins C and S were evaluated. RESULTS: Seven and a half percent of the patients and 2.9% of the controls were carriers of the prothrombin mutation, while 12.2% of the patients and 1.6% of the controls had factor V Leiden. Among the 229 DVT patients there were 130 patients with clinically suspected thrombophilia (first thrombotic event occurring before the age of 45 years or positive family history of thrombosis or recurrent venous thrombosis). Ten percent of these 130 patients were carriers of the prothrombin G20210A mutation and 18.5% had the factor V Leiden mutation. The odds ratios (OR) for DVT risk were: 2.4 (95% CI, 1.0-6.3) for the total DVT patients and 5.2 (95% CI, 1.4-19.5) for the patients with clinically suspected thrombophilia with the prothrombin mutation. The risk of thrombosis was 6.9 (95% CI, 2.3-20.6) for the DVT patients and 14.3 (95% CI, 3.3-64.6) for the patients with clinically suspected thrombophilia with factor V Leiden. Fifty-five percent of the patients with combined congenital defects (prothrombin mutation G20210A plus another congenital defect) had recurrent thrombosis. In women receiving OC the risk of DVT was 3.5 (95% CI, 1.5-8.2) that of the patients not receiving OC. When women with combined defects were also taking OC, the risk of thrombosis increased significantly. INTERPRETATION AND CONCLUSIONS: The prevalence of the prothrombin G20210A mutation in the healthy population in our study is similar to that observed in other southern European countries. The prothrombin G20210A mutation does not by itself seem to be a high thrombotic risk factor. However, when it is present together with other thrombotic risk factors, the predicted risk of thrombotic events increases. The use of OC by women with the prothrombin G20210A variant or FV Leiden, either alone or combined with other thrombotic risk factors, was associated with a significant increase in the risk of venous thrombosis.

PAI-1 promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects with genetic thrombophilic defects.

Impaired fibrinolysis as a result of increased plasminogen activator inhibitor-1 (PAI-1) levels in plasma is a common finding in patients with deep vein thrombosis (DVT). A 4G/5G polymorphism in the promoter region of the PAI-1 gene has been reported to influence the levels of PAI-1. The 4G allele was found to be associated with higher plasma PAI-1 activity (act), but contradictory results on the incidence of the 4G allele in DVT patients have been reported. The aim of this study was to analyse whether the PAI-1 promoter 4G/5G genotype increases the risk of venous thrombosis in subjects with thrombophilic defects, and to determine the distribution of the PAI-1 4G/5G genotype and its relation to plasma PAI-1 levels in 190 unrelated patients with DVT in comparison with a control group of 152 healthy subjects. No differences between the 4G/5G allele distribution in the DVT group (0.43/0.57) and in the control group (0.42/0.58) were observed. However, the presence of the 4G allele significantly increased the risk of thrombosis in patients with other thrombophilic defects. Significantly higher PAI-1 levels were observed in DVT patients than in the controls. Our results also showed significant differences in the plasma levels of PAI-1 antigen (ag) and PAI-1 act among the 4G/5G genotypes in DVT patients. A multivariate analysis revealed that, in the DVT group, PAI-1 ag levels were influenced by the 4G allele dosage, triglyceride levels and body mass index (BMI). The influence of the 4G allele dosage on PAI-1 levels was independent of the triglyceride levels and BMI. In the control group, no significant correlation between PAI-1 levels and 4G allele dosage was observed. In conclusion, the PAI-1 promoter polymorphism was found to have an influence on PAI-1 levels in DVT patients and on the risk of venous thrombosis in subjects with other genetic thrombophilic defects.