G20210A prothrombin gene polymorphism and prothrombin activity in subjects with or without angiographically documented coronary artery disease.

BACKGROUND: G20210A prothrombin mutation has been associated with high prothrombin levels and an increased risk of venous thrombosis. The role of this common polymorphism, as well as that of prothrombin levels, in determining the risk of arterial disease is still somewhat controversial. METHODS AND RESULTS: We determined the prevalence of the G20210A mutation and prothrombin activity in 660 individuals, of whom 436 had angiographically documented severe coronary artery disease (CAD patients) and 224 had normal coronary angiography (CAD-free control subjects). Heterozygosity for the 20210A allele was found in 5.3% of the CAD patients versus 3.1% of the CAD-free subjects (P=0.21). Similarly, no statistically significant difference was found between CAD patients with or without previous myocardial infarction (4.5% versus 5.3%, respectively; P=0.73). The genotype-phenotype correlation study showed a significant influence of the 20210A allele on prothrombin activity, with higher levels in carriers compared with noncarriers (153.2% versus 122.2%, respectively; P<0.001). Prothrombin activity was significantly higher in CAD patients than in CAD-free subjects (132.8% versus 123.3%, respectively; P<0.005). By multiple logistic regression, prothrombin activity in the upper tertile of the control distribution was significantly associated with CAD compared with prothrombin activity in the lower tertile (adjusted odds ratio 1.86, 95% CI 1.01 to 3.4). CONCLUSIONS: In a population with a clear-cut definition of the phenotype, the G20210A prothrombin mutation was not significantly associated, per se, with either angiographically documented CAD or myocardial infarction, whereas it significantly influenced prothrombin activity. In our population, high prothrombin activity itself was independently associated with CAD but not with the presence or absence of previous myocardial infarction.

Levels of plasma factor VII and factor VII activated forms as a function of plasma triglyceride levels.

It has been shown that triglyceride levels are one of the determinants of factor VII levels. In this study we have simultaneously evaluated, in a group of 102 healthy individuals, the different forms of factor VII, namely factor VII mass, factor VII coagulant activity, activated factor VII double-chain form and factor VII-phospholipid complex, in relation to triglyceridaemia. The data showed a highly significant correlation of factor VII mass, factor VII coagulant activity and factor VII-phospholipid complex with triglycerides. No correlation was observed between the activated factor VII double-chain form and triglycerides. These data, together with analysis of the linear and orthogonal regression slopes, suggest that increase of plasma factor VII coagulant activity as a function of plasma triglyceride levels is attributable to an increase in both mass and activity of factor VII and that the increase in activity is dependent on an increase of factor VII-phospholipid complex rather than activated factor VII double-chain form. The ratio between the slopes of the regression straight line of factor VII mass and factor VII-phospholipid complex in relation to triglycerides was 2.23 (95% confidence limits 1.74-2.50), thus indicating that the contribution of factor VII mass is prevalent over that of the factor VII-phospholipid complex.

Expression of plasminogen activator inhibitor-1 in human adipose tissue: a role for TNF-alpha?

Elevated plasminogen activator inhibitor-1 (PAI-1) plasma levels, responsible for reduced fibrinolysis, are associated with animal and human obesity and with increased cardiovascular disease. The expression of PAI-1 has been found recently in animal and human adipose tissue. Factors and mechanisms regulating such an expression remain to be elucidated. In omental and/or subcutaneous biopsies from obese non-diabetic patients, incubated in Medium 199, we have confirmed that human adipose tissue expresses PAI-1 protein and mRNA; furthermore we have demonstrated that such an expression is clearly evident also in collagenase isolated human adipocytes and that it is stimulated by incubation itself and enhanced by exogenous human tumor necrosis factor-alpha (h-TNF-alpha). Since human adipose tissue produces TNF-alpha, to further characterize the relationship of PAI-1 to TNF-alpha, human fat biopsies were also incubated with Pentoxifylline (PTX) or Genistein, both known to inhibit endogenous TNF-alpha through different mechanisms. PTX caused a dose-dependent decrease of basal PAI-1 protein release, reaching 80% maximal inhibitory effect at 10(-3)M, the same inhibitory effect caused by Genistein at 100 microg/ml. This was associated to a marked inhibition of PAI-1 mRNA and of endogenous TNF-alpha production. Furthermore, when human fat biopsies were incubated in the presence of polyclonal rabbit neutralizing anti-human TNF-alpha antibody (at a concentration able to inhibit 100 UI/ml human TNF-alpha activity), a modest but significant decrease of the incubation induced expression of PAI-1 mRNA was observed (19.8+/-19.0% decrease, P = 0.04, n = 7). In conclusion, the results of this study demonstrate that PAI-I expression is present in human isolated adipocytes and that it is enhanced in human adipose tissue in vitro by exogenous TNF-alpha. Furthermore our data support the possibility of a main role of endogenous TNF-alpha on human adipose tissue PAI-1 expression. This cytokine, produced by human adipose tissue and causing insulin resistance, may be a link in the clinical relationship between insulin-resistance syndrome and increased PAI-1 plasma levels.

Study of platelet adhesion in patients with uncomplicated hypertension.

OBJECTIVE: To evaluate platelet function in patients with essential hypertension by sensitive methods investigating platelet adhesion and expression of some platelet glycoproteins (GP), namely GPIIb/IIIa (CD41/alpha 2 beta 3) and GMP-140 (CD62/P-selectin/PADGEM). Other markers of platelet (beta-thromboglobulin) and endothelium activation (von Willebrand factor) were also measured. METHODS: We studied 21 uncomplicated essential hypertensive patients and 20 healthy normotensive control subjects, non-smokers, matched for age and sex. Resting and stimulated platelet adhesion was performed with a colorimetric method using the activity of platelet acid phosphatase for the determination of the number of platelets adhering to human plasma- or fibrinogen-coated microwells. Platelet activation was characterized by flow cytometric measurement of GPIIb/IIIa and GMP-140 in whole blood and washed platelets suspensions, with antihuman fluorescent monoclonal antibodies. RESULTS: Thrombin-stimulated platelet adhesion to human plasma-coated microwells was significantly higher in hypertensive patients than in control subjects (0.05 U/ml thrombin: 13.4 +/- 1.0 versus 7.7 +/- 0.6% adhesion; 0.1 U/ml thrombin: 19.4 +/- 2.3 versus 12.6 +/- 1.8%; means +/- SEM), whereas platelet adhesion to fibrinogen-coated wells did not differ in the two groups. Flow-cytometry analysis of whole blood demonstrated a significantly increased expression of GMP-140 in hypertensive patients compared with normal subjects (percentage of CD62+ platelets: 7.3 +/- 1.2 versus 3.7 +/- 1; means +/- SEM), whereas the expression of GPIIb/IIIa did not differ in the two groups (percentage of CD41a+ platelets: 72.5 +/- 4.5 versus 70.4 +/- 3.9). Moreover, flow cytometry showed an increased size of platelets in hypertensive patients compared with that in control subjects (forwards scattering: 46.5 +/- 1.5 versus 38.9 +/- 1.1; means +/- SEM). Flow-cytometric evaluation of washed platelet suspensions showed no statistically significant differences between the expression of GMP-140 and GPIIb/IIIa in the two groups. beta-Thrombo-globulin plasma levels were higher in hypertensive patients than they were in normal subjects (36.3 +/- 2.0 versus 28.2 +/- 1.3 ng/ml; means +/- SEM). Von Willebrand factor plasma levels were not significantly different in the two groups (101.2 +/- 10.3 versus 86.3 +/- 5.6 U/dl). CONCLUSIONS: These findings provide further evidence that there is a significant, albeit weak, platelet activation in hypertensive patients compared with normal subjects.

Interrelation between angiographic severity of coronary artery disease and plasma levels of insulin, C-peptide and plasminogen activator inhibitor-1.

Plasma insulin, C-peptide and plasminogen activator inhibitor-1 (PAI-1) levels were measured in 64 men with coronary artery disease (CAD) documented by angiography. Coronary arteriograms were analyzed, and the severity and diffusion of coronary lesions were quantified by score systems. C-peptide and PAI-1 levels in patients with CAD were significantly higher than in 30 control subjects. Insulin, C-peptide and PAI-1 showed a highly significant correlation with the severity scores for coronary lesions (C-peptide more than insulin), but only a weak correlation with diffusion scores. Highly significant correlations were found between insulin and PAI-1, and even greater ones between C-peptide and PAI-1. It has been proposed that hyperinsulinemia may be involved in the etiology of atherosclerotic cardiovascular disease by dysregulating lipoprotein metabolism and blood pressure. These findings support that hypothesis and suggest that insulin secretion may be an index of the severity of CAD. Because a direct effect of insulin on the cells that synthesize PAI-1 has been shown, the present data further indicate that the effect of insulin on fibrinolysis may be another way by which hyperinsulinemia accelerates atherogenesis.

Procedure for effective separation of high-density lipoproteins in normal serum and hypertriglyceridemic samples.

A chemical procedure for high-density lipoprotein (HDL) separation in normo- and hyperlipidemic sera has been developed. The procedure is effective up to 25 mmol/L of serum triglycerides and the constituents of the precipitation mixture (dextran sulfate, PEG-6000 and MgCl2) do not interfere with enzymatic assay of the HDL cholesterol.

Severe preeclampsia in antithrombin III deficiency with no history of venous thromboembolism.

Complications of pregnancy, such as preeclampsia, placental abruption, fetal growth retardation, still-birth and fetal death are associated with an increased frequency of pro-thrombotic abnormalities. We describe a case of severe preeclampsia and multiple placental infarctions in a 28-year-old woman at 31 weeks' gestation. Despite a negative personal history for venous thromboembolism, coagulation screening for thrombophilia detected an isolated antithrombin III deficiency. In view of the high prevalence of pro-thrombotic complications, laboratory screening for thrombophilia would be advantageous in women with complicated pregnancies, to ensure adequate management in high-risk situations, as suggested by larger-scale clinical investigations.

Prothrombotic effects and clinical implications of third-generation oral contraceptives use.

Although the use of oral contraceptives has been frequently associated with an increased risk of thromboembolic events, definitive prothrombotic mechanisms have not so far been fully elucidated. The aim of our investigation was the evaluation of the activities of antithrombin, protein C, protein S and the resistance to activated protein C in 137 healthy users of third-generation oral contraceptives and in 170 healthy women who were not consuming oral contraceptives. Women on oral contraceptives showed a marked prothrombotic pattern, characterized by reduced activities of antithrombin and protein S, and increased resistance to activated protein C. Nearby 50% of oral contraceptive users displayed activities of protein S below the lower value of the reference range (controls, 10%; P < 0.001). No significant differences were observed between two progestagens (desogestrel or gestodene) on the coagulation parameters tested. We believe that, due to the adverse effect on haemostasis, the administration of third-generation oral contraceptives should be carefully considered in women carrying prothrombotic abnormalities.

Resistance to activated protein C, associated with oral contraceptives use; effect of formulations, duration of assumption, and doses of oestro-progestins.

Resistance to activated protein C (APC-R) is at present considered the most frequent laboratory abnormality in patients with deep vein thrombosis. An increased risk for venous thrombosis is associated with the use of oral contraceptives (OCs). We recently described a statistically significant association between APC-R status and oral contraceptives use in a healthy group of women. We re-evaluated 50 healthy women taking low-dose combination OCs in order to consider a possible correlation between the APC sensitivity ratio (APC-SR) and different oral contraceptive formulations. Seven women showed an APC ratio < or = 2 (APC-resistant). Only one of the seven women was found to be heterozygous for Leiden factor V mutation. We observed no significant differences between normally sensitive and APC-resistant women in terms of duration of OC use, amount of estrogenic or progestogenic dose, or type of formulation. We conclude that APC-resistance associated with oral contraceptives use seems to occur only in predisposed subjects (in our results, about 12% of the healthy population).

Variation of plasma D-dimer following surgery: implications for prediction of postoperative venous thromboembolism.

The prognosis of venous thromboembolism is considerably influenced by an accurate and fast diagnosis. Although the role of D-dimer testing in the diagnosis of suspected venous thromboembolism is well established for outpatients, there is controversial evidence on the clinical usefulness of its measurement in surgical patients. In order to recognize patterns of variation of D-dimer following surgery and identify potential pitfalls in prediction of venous thromboembolic complications, plasma D-dimer was assayed in 30 patients undergoing major elective hip surgery and 20 patients undergoing laparoscopic cholecystectomy for acute cholecystitis. The postoperative variation of plasma D-dimer differed widely between the two subgroups. Patients undergoing laparoscopic cholecystectomy showed D-dimer concentrations persistently increased from the baseline to the 15th postoperative day, whereas patients undergoing hip surgery were characterized by a double peak, on the 1st and 7th postoperative days. Mean inter-individual daily coefficient of variations of plasma D-dimer throughout the postoperative period were 49% (range 39%-61%) for laparoscopic cholecystectomy and 101% (range 72%-156%) for orthopedic surgery. The markedly heterogeneous fluctuation of plasma D-dimer suggests that the postoperative activation of the hemostatic system depends on the type and time since surgery, thus limiting the clinical usefulness of D-dimer testing in the diagnostic approach to postoperative venous thromboembolism.

A new chemical procedure for selective sequential separation of serum lipoproteins.

A new chemical procedure for selective sequential separation of serum lipoproteins is described. Low density lipoproteins (LDL) were precipitated by polyvinylsulfate and the precipitates were redissolved in NaCl 0.45 mol/l. Very low density lipoproteins (VLDL) were precipitated from LDL-free supernatants by PEG-6000 at 100 g/l final concentration, pH 10, and the precipitates were redissolved in NaCl 0.15 mol/l. For the precipitation of high density lipoproteins (HDL), apolipoprotein B-free supernatants were treated by PEG-6000 at 266 g/l final concentration and the precipitates were redissolved in NaCl 0.15 mol/l. The procedure was effective up to a serum triglyceride concentration of 4.0 mmol/l as well as in serum samples without chylomicrons or remnants. The determination of cholesterol and triglyceride contents in separated lipoproteins allows the study of their chemical composition in normo- and moderately hypertriglyceridemic serum samples.

Evaluation of the activated partial thromboplastin time (APTT) sensitivity to heparin using five commercial reagents: implications for therapeutic monitoring.

Heparin is an effective drug for prevention and treatment of thromboembolic conditions. Although several biological assays have been proposed for monitoring unfractionated heparin therapy, the measurement of the activated partial thromboplastin time (APTT) is the most widely employed test, and the overall risk of thromboembolic episodes was markedly reduced by maintaining APTT ratios above 1.5. However, the adjustment of the heparin therapy on the basis of APTT presents several questions which are still unresolved. Major discrepancies were found in APTTs performed using different reagents in both ex vivo and in vitro heparinized samples and occasionally with different lots of the same reagents; poor correlation was observed between APTT values and plasma heparin concentrations. In order to gain further insights into this phenomenon, we analysed the sensitivity to heparin of five commercial reagents for APTT measurement in 19 ex vivo heparinized samples. Differences were observed; correlation coefficients ranged from 0.820 to 0.985 and slopes of linear regressions from 0.26 to 1.14. Moreover, unsatisfactory correlations were obtained when APTT ratios were compared with heparin plasma concentrations in the same patients' samples. In the heparin therapeutic range of 0.35 - 0.70 U/ml, reagent-dependent differences were observed in the corresponding APTT values. These results point out a critical role of the assay methodology in monitoring heparin therapy by APTT. We suggest that reference materials and methods should be urgently identified, a universally agreed scale for reporting results should be established and reference ranges for the unfractionated heparin therapy should be reconsidered taking on account the reagent employed.

Lipoprotein(a) concentration is not associated with venous thromboembolism in a case control study.

BACKGROUND AND OBJECTIVE: Lipoprotein(a) is an LDL-like particle displaying strong athero-thrombotic properties. Although Lp(a) plays a pivotal role in the genesis and progression of thrombosis in the arterial district, its role in promoting thrombosis in the venous district is still unclear. DESIGN AND METHODS: To give further insight into the thrombotic potential of Lp(a), 100 potentially eligible consecutive outpatients who had suffered from previous episodes of venous thrombosis (deep vein thrombosis with or without pulmonary embolism) were enrolled into the study. Thirty-six of these patients who did not fulfil the entry criteria were then excluded from the study. The concentration of Lp(a) was thus measured in 64 patients, and compared to that of 64 control subjects, matched for sex (p=0.46), age (p=0.25) and pharmacological treatment; no subject belonging to the control group had a familial or personal history of venous thromboembolism. Exclusion criteria for both groups included: diabetes mellitus, liver or kidney diseases and malignancy, as established by both laboratory analysis and physical examination. To rule out false elevations of Lp(a) due to the presence of a concurrent acute phase response, C reactive protein (CRP) was measured in both groups using a commercial immunonephelometric assay. RESULTS: No statistically significant differences were observed in the median Lp(a) concentration between patients and controls (median: 69 vs 83 mg/L, respectively; p=0.34). Neither were any significant differences found between patients who had suffered from deep venous thrombosis with (n=18) or without (n=46) pulmonary embolism (median: 73 vs 69 mg/L, respectively; p=0. 83). The concentration of CRP did not differ significantly between cases and controls (median: 1.8 vs 2.3 g/L, respectively; p=0.37). INTERPRETATION AND CONCLUSIONS: Although there are several plausible biological mechanisms to explain the strong thrombogenicity of Lp(a) in vitro, we failed to demonstrate a convincing association between Lp(a) and thrombosis in the venous district. Besides the proven prothrombotic role of Lp(a) in some selected clinical settings, it is thus conceivable that the contribution of Lp(a) to genesis and progression of the venous thrombosis might be marginal or efficiently counterbalanced in vivo. The clinical usefulness of including the measurement of Lp(a) among the screening tests for thrombophilic patients, therefore, remains questionable.

A colorimetric method for the measurement of platelet adhesion in microtiter plates.

A procedure for the determination of the adhesion of human platelets to protein-coated culture microwells was developed. The number of platelets was quantitated by measuring the activity of acid phosphatase, a platelet enzyme whose activity is stable independently of platelet stimulation and is not released. Isolated and washed platelets were incubated in 96-well microtiter plates with flat-bottom wells that had been precoated with various compounds, including collagen, fibrinogen, human plasma, and human albumin. At the end of incubation (optimal time: 40-60 min), nonadherent platelets were washed out, adherent platelets were solubilized with Triton X-100, and the acid phosphatase activity was measured by using the substrate p-nitrophenyl phosphate. The p-nitrophenol produced was measured with a microplate reader at 405 nm and the percentage of adhesion was calculated with reference to known platelet standards. ADP and thrombin stimulated platelet adhesion in a dose-dependent manner to fibrinogen and human plasma, but not to human albumin. Platelets adhered to collagen even in the absence of stimulants. Simultaneous evaluation of adhesion and aggregation demonstrated that with ADP as stimulus, but not with thrombin, the two platelet responses were dissociated. Microscopic examination of culture wells showed that most of platelets adhered as single cells and not as aggregates. The sensitivity of this method allowed the assay of platelet adhesion by using only 2.5 x 10(5) platelets/well.

Determination of high density lipoprotein cholesterol in venous and capillary whole blood.

A procedure is presented and evaluated for separation of plasma high density lipoprotein from either capillary or venous whole blood. The lipoprotein is separated by adding 50 microliter of sample to 250 microliter of 0.15 M NaCl solution containing 99.9 g/l polyethyleneglycol 6000, 0.0374 g/l dextran sulfate (Mr 15,000) and 2.6 mM Mg2+. After gentle mixing for a few minutes and standing 10 min at room temperature, mixtures are centrifuged (1,500 g) for 10 min and cholesterol is measured on 200 microliter of supernatant by an enzymatic-colorimetric method. Comparison studies demonstrate a good correlation between high density lipoprotein cholesterol in plasma and capillary or venous whole blood. The procedure is simple, has the advantage of using either K3-EDTA-anticoagulated whole blood, without the need of centrifugation, or capillary whole blood which can also be collected away from the laboratory.