Roberta Giacomello

Hyperglycemia-Induced Thrombin Formation in Diabetes: The Possible Role of Oxidative Stress

Diabetes is characterized by the existence of a thrombosis-prone condition, possibly related to hyperglycemia. However, the mechanism linking hyperglycemia to the activation of the coagulation cascade is still unclear. It has been recently suggested that diabetes is accompanied by increased oxidative stress. In this work, the possibility that oxidative stress may be involved in the hyperglycemia-induced coagulation activation has been evaluated. Prothrombin fragment 1 + 2 (F1+2), which represents a reliable marker of the amount of thrombin released in the circulation, has been chosen for studying thrombin formation in vivo. In nine type II diabetic patients and in seven healthy control subjects, matched for age and body mass index, three different experiments were performed: oral glucose tolerance test (OGTT), intravenous antioxidant glutathione (GSH) administration for 2 h, and OGTT plus intravenous GSH administration. Samples were drawn at −15 min and every 30 min from 0 to 180 min. During the OGTT, F1+2 significantly increased in both diabetic and healthy subjects. GSH administration during OGTT normalized this phenomenon. GSH administered alone significantly decreased F1+2 in diabetic patients, while no effect was observed in the normal subjects. These data suggest that hyperglycemia may induce thrombin activation, possibly inducing an oxidative stress, and that antioxidant GSH may counterbalance this effect.

Post-meal coagulation activation in diabetes mellitus: the effect of acarbose

SummaryIt has been previously demonstrated that hyperglycaemia activates haemostasis; diabetes mellitus is considered a thrombosis-prone state. Acarbose, by inhibiting dietary carbohydrate absorption, reduces post-meal hyperglycaemia. In this study we evaluated the effect of post-meal hyperglycaemia on two markers of coagulation activation: prothrombin fragments 1 + 2 and D-dimer. Seventeen non-insulin-dependent diabetic patients maintained on diet therapy alone were randomly assigned to receive — with a cross-over study design — acarbose (100 mg orally) or placebo before a standard meal. Blood samples for measurement of plasma glucose, insulin, prothrombin fragments 1 + 2 and D-dimer were drawn at 0, 60, 120 and 240 min. After both placebo and acarbose, hyperglycaemia and hyperinsulinaemia which followed a standard meal were accompanied by a significant increase of plasma concentration of prothrombin fragments 1 + 2 and D-dimer in comparison to their baseline values. Acarbose administration significantly reduced the rise of glucose, insulin, prothrombin fragments 1 + 2 and D-dimer from 0 to 240 min in comparison to placebo. We conclude that post-meal hyperglycaemia, at the level reached by many diabetic patients on diet therapy alone, induces a coagulation activation. Acarbose, by decreasing post-meal hyperglycaemia, may be useful in reducing meal-induced activation of haemostasis in diabetic patients.

Fibrinogen Plasma Levels as a Marker of Thrombin Activation in Diabetes

This study attempted to verify the existence of a correlation between fibrinogen, a major cardiovascular risk factor in diabetes, and indexes of thrombin generation and action, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer (D-D), in a group of diabetic subjects compared with a matched control group. Forty insulin-dependent diabetes mellitus patients and 30 matched healthy control subjects participated in this study. The subjects were tested for the following parameters: fibrinogen, prothrombin F1 + 2, D-D, fasting glycemia, and HbA1c. In addition, 5 diabetic subjects who maintained stable fibrinogen plasma levels > 300 mg/dl for at least 6 months before the study were treated with 12,500 U/day subcutaneous heparin for 7 days. Diabetic subjects showed increased levels of fibrinogen, prothrombin F1 + 2, and D-D plasma levels. Simple linear regression analysis detected a positive correlation between fibrinogen and prothrombin F1 + 2, D-D, and glycosylated HbA1c. In the five diabetic subjects treated with heparin fibrinogen, prothrombin F1 + 2 and D-D levels decreased at the end of the treatment. All these parameters returned to baseline after 7 days of washout. These data indicate that fibrinogen plasma levels are correlated to parameters of thrombin activation in plasma in diabetic patients and suggest that high fibrinogen plasma levels might be a risk marker for cardiovascular disease in diabetes because it is an expression of an existing thrombophilia.

Total Plasma Antioxidant Capacity Predicts Thrombosis-Prone Status in NIDDM Patients

OBJECTIVE To explore the hypothesis that a relationship exists between free radical activity and abnormalities in hemostasis in NIDDM. RESEARCH DESIGN AND METHODS The use of the total radical-trapping antioxidant parameter (TRAP) has very recently been proposed to explore the antioxidant property of a plasma and their mutual cooperation. In the present study, TRAP, vitamin E, vitamin C, vitamin A, uric acid, protein-bound SH (thiol) groups, fibrinogen, prothrombin fragments F1 + 2, and D-dimer have been evaluated in 46 NIDDM patients and 47 healthy matched control subjects. RESULTS In NIDDM patients, TRAP, vitamin A, SH groups, and uric acid were significantly reduced, whereas the level of vitamin E was significantly increased. Vitamin C was similar in the two groups. Fibrinogen, prothrombin fragment 1 + 2, and D-dimer were increased in diabetic patients. TRAP, but no single other antioxidant, had a strong inverse association with fibrinogen, prothrombin fragment 1 + 2, and D-dimer. CONCLUSIONS These findings are consistent with the hypothesis that oxidative stress may condition coagulation activation in diabetics. However, the data suggest that it is the total antioxidant capacity rather than any single plasma antioxidant that is the most relevant parameter.

Frequency of Factor V, Prothrombin and Methylenetetrahydrofolate Reductase Gene Variants in Preeclampsia

Background: The association between thrombophilic variants (Leiden mutation of the factor V gene, G20210A mutation of the prothrombin gene and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene) with preeclampsia was investigated in a north-eastern Italian population. Methods: Fifty-eight preeclamptic (PE) women and 74 normal pregnancies were evaluated. Genotypes were determined by polymerase chain reaction. Results: The frequency of heterozygous carriers of the factor V Leiden was similar between PE women (5.2%) compared to the control subjects (4.1%; p 0.76). Also the frequencies of G20210A and C677T mutations were similar between PE and control subjects. Conclusions: In this population, we found no difference in the prevalence of genetic risk factors for thrombosis in women with preeclampsia compared with control subjects.

Increased prothrombin fragment 1 + 2 in type I diabetic patients.

Dr. Antonio Ceriello, Cattedra di Medicina Interna, Facoltà di Medicina, Università di Udine, P.le S. Maria della Misericordia, I-33100 Udine (Italy) The pathogenesis of vascular lesions in diabetic patients has been considered to be at least partly dependent on the alterations in the hemostatic system [1]. However, the existence and the relevance of a hypercoagulable state in diabetes mellitus have been the subject of much debate [1,2]. It has been demonstrated that the conversion of the coagulation zymogen prothrombin to thrombin is associated with the prominent production of a cleavage product namely prothrombin fragment 1+2 (Fl+2) [3]. It has been proposed recently that prothrombin Fl+2 plasma levels may be considered a very sensitive marker for hypercoagulable states in humans [4]. We evaluated prothrombin Fl+2‚(ELISA) levels in 19 insulin-dependent diabetic patients without signs of vascular complications (12 males and 7 females: age 23.4 ± 1.9 years, mean ± SE; body mass index 22.8 ± 1.4; duration of diabetes 6.4 ± 1.3 years; insulin regimen 25-55 U/day, mean 32.8 ± 2.5 U/ day; all subjects had 24-hour urinary albumin excretion rates less than 30 μg/min, and no microaneurysms were detected on full fundal photographs or íluorescein fundal angiogra-phy; they had systolic blood pressures < 120 mm Hg and diastolic blood pressures < 90 mm Hg; ischemic heart disease and peripheral vascular occlusion were excluded according to local criteria) compared to 10 matched healthy normal subjects (6 males and 4 females: age 24.2 ± 1.8 years, body mass index 23.2 ± 1.3). Prothrombin Fl+2 levels were significantly elevated in diabetic patients (0.69 ± 0.11 vs. 0.27 ± 0.03 nmol/l;p < 0.01), but no correlation was found between prothrombin Fl+2 plasma levels and both fasting glycemia (r = 0.17) and glycosylated Hb A1 c (r = 0.16). Increased FPA levels have been reported in diabetes [5]. These findings were considered more suggestive of a thrombin hyperac-tivity than of an increase of thrombin production [6]. This hypothesis was sustained by the evidence of normal [7] or depressed [6] levels of thrombinantithrombin complex in the presence of increased FPA [6] and fibrin monomers [7] in diabetes.

Effect of intensive glycaemic control on fibrinogen plasma concentrations in patients with Type II diabetes mellitus. Relation with β-fibrinogen genotype

Summary Recent studies show that in diabetic subjects an increase of plasma fibrinogen concentration is associated with a high risk of cardiovascular complications. Environmental and genetic factors contribute to the plasma fibrinogen concentration. Several studies indicate a relation between the polymorphism in the 5 ′ region of the β-fibrinogen gene and plasma protein concentrations and in diabetes the possible influence of hyperglycaemia on fibrinogen is still debated. In this study we investigated these relations. Hind III polymorphism was evaluated by a polymerase chain reaction-technique. On the basis of the observed allelic combination of fibrinogen β-gene polymorphism and the existence of poor metabolic control (glycated haemoglobin ≥ 7.5 %), 50 Type II diabetic patients were selected. They were divided into three groups according to their β-gene polymorphism (α1α1: n = 20, α1α2: n = 15, α2α2: n = 15) and then intensive insulin therapy was started. After 3 months of intensive treatment, the improvement in glycaemic control was equivalent, in terms of glycated haemoglobin, in all the three groups. A fibrinogen reduction was observed in α1α2 and α2α2 but not in α1α1 subjects. These results underline a possible relation between fibrinogen genotypes and glycaemic control in determining plasma fibrinogen concentrations in diabetic patients. [Diabetologia (1998) 41: 1270–1273]

Lupus anticoagulant: a multicenter study for a standardized and harmonized reporting.

Laboratory assessment of Lupus anticoagulant (LAC) is very challenging because of inter and intralaboratory variability, which makes it difficult to standardize and harmonize results expression. Five hospital laboratories in North-eastern Italy shared their efforts and their experience in a cross-laboratory study, conducting the diagnostic process as homogeneously as possible and providing a better interpretation for LAC positivity. Hundred normal samples from healthy subjects (20 from each center) were processed to confirm negative upper limits and calculate positivity cutoffs of LAC integrated assays, that is dilute Russells viper venom time (dRVVT) and silica clotting time (SCT). Moreover, 311 samples previously diagnosed by the laboratories as positive for LAC were analyzed to characterize different positivity levels for each assay. As far as the analysis of healthy subjects is concerned, negative upper limits are set at 1.17 and 1.19 for dRVVT and SCT screen ratio, respectively. Positivity cutoffs are set at 1.20 for dRVVT and 1.23 for SCT, expressed as Test Ratio calculated on screen and confirm integrated tests. Positive results for each integrated assay are subsequently divided into three subgroups: weak, moderate and strong; the results obtained are presented as a score proposal that can provide LAC interpretation. The combined use of both dRVVT and SCT assays and the definition of different positivity levels may lead to clearer, more objective LAC reporting. An interpretative table for LAC-proposed score provides LAC-positive results and it is now adopted by all centers involved in the study.