Egidio Marchi

Evidence for a hyperglycaemia-dependent decrease of antithrombin III-thrombin complex formation in humans.

SummaryIn the presence of increased levels of fibrinopeptide A, decreased antithrombin III biological activity, and thrombin-antithrombin III complex levels are seen in diabetic patients. Induced-hyperglycaemia in diabetic and normal subjects decreased antithrombin III activity and thrombin-antithrombin III levels, and increased fibrinopeptide A plasma levels, while antithrombin III concentration did not change; heparin was shown to reduced these phenomena. In diabetic patients, euglycaemia induced by insulin infusion restored antithrombin III activity, thrombin-antithrombin III complex and fibrinopeptide A concentrations; heparin administration had the same effects. These data stress the role of a hyperglycaemia-dependent decrease of antithrombin III activity in precipitating thrombin hyperactivity in diabetes mellitus.

Hyperglycemia may determine fibrinopeptide A plasma level increase in humans

The effects of hyperglycemia on plasma fibrinopeptide A (FPA) levels in normal subjects are reported. An increase of FPA concentration parallel to sustained hyperglycemia was observed; when the glycemia returned to basal values, FPA showed values in normal range. Heparin infusion was able to significantly decrease the hyperglycemia-induced augment of FPA levels. Isovolumic-isotonic NaCl solution infusion produced a slight (NS) increase in FPA levels; however, mild hyperglycemia, achieved by glucagon, was also able to produce a significant increase in FPA concentration. These data demonstrate the direct role of hyperglycemia in conditioning FPA level, and suggest that hyperglycemia, by itself, is a sufficient stimulus to produce thrombin activation in humans.

Alkali-Induced Optical Rotation Changes in Heparins and Heparan Sulfates, and Their Relation to Iduronic Acid-Containing Sequences

Abstract Under specific basic conditions, the glucosaminoglycans heparin and heparan sulfate, containing α-L-iduronic acid 2-O-sulfate, undergo selective epoxidation between C-2 and C-3 of this residue, with formation of a residue of 2,3-anhydro-α-L-guluronic acid. The epoxidation reaction was studied by means of 13C NMR and optical rotation measurements. The optical rotation values correlate well with the composition of the reaction products as determined by 13C NMR, thus indicating that the heparin- or heparan-like character of both natural and semisynthetic polysaccharides can be easily determined also through optical rotation measurements.

Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

SummaryThe effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfatedependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

Antithrombotic activity of Desmin 370. Comparison with a high molecular weight dermatan sulfate

Dermatans are endogenous glycosaminoglycans which catalyze thrombin-heparin cofactor II interaction (1) and may promote fibrinolysis by induction of PA release (2). In different animal models dermatans have been shown to prevent thrombus formation (3,4,5,6). Since they are less haemorrhagic than standard heparin (4,7), dermatans may represent a class of antithrombotic agents with reduced bleeding risk. Low molecular weight heparins have longer biological halflives than standard heparins (8). Recently it has been found that potentiation of thrombin-heparin cofactor II interaction by a low molecular weight dermatan sulfate lasted longer than that induced by a native dermatan sulfate, both in rats and in monkeys (9), suggesting that the two molecular weight forms have different pharmacokinetic profiles. The aim of the present study was to investigate whether the antithrombotic activity of Desmin 370, a new low molecular weight dermatan sulfate (IO, 11,12) and DS435, the native dermatan, was influenced by route of administration, In addition, the concentration-time curves of the two dermatans, given s.c., were evaluated ex vivo by a coagulation assay.

Antithrombotic and thrombolytic activity of sulodexide in rats

SummaryWe evaluated the ability of sulodexide, an extracted glycosaminoglycan, to prevent thrombus formation and to reduce a stabilized thrombus in a rat venous thrombosis model (vena cava ligature). Injection of sulodexide 10 min before induction of venous stasis, prevented thrombus formation in a dose-dependent manner (median effective dose 0.55 mg/kg). When given to rats with 6-h-old thrombi, sulodexide caused a marked reduction in thrombus size which reached 70% after 2 h with the highest dose tested (2 mg/kg). The effect of sulodexide on established thrombi appears to be due, at least in part, to a fibrinolysis-mediated mechanism, since it was significantly inhibited by ε-aminocaproic acid, a well-known antifibrinolytic drug. Treatment with sulodexide did not noticeably affect plasma levels of plasminogen activator and its specific inhibitor. We also showed that fluorescein-labelled sulodexide, when given to animals with 6-h-old thrombi, was present within the thrombi harvested 2 h later, but was then absent from blood. The fluorescence was mainly located in areas filled with amorphous material, that was identified as fibrin by staining with phosphotungstic acid-hematoxylin. No fluoresceinlabelled material could be detected in rats treated with fluorescein alone. These findings indicate that, besides preventing venous thrombus formation, sulodexide is able to promote thrombus dissolution by a mechanism that is partly related to local fibrinolysis stimulation.

Effects of glycosaminoglycans on platelet and leucocyte function: Role of N-sulfation

The effect of glycosaminoglycans (GAGs) such as sulodexide, low molecular mass dermatan sulfate, heparin and some derivatives with different degrees and types of sulfation was studied on cathepsin G- or thrombin-stimulated platelets and n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leucocytes (PMNs). All GAGs (0.01-20 micrograms/mL) inhibited both platelet aggregation induced by cathepsin G and its catalytic activity. Thrombin-induced platelet aggregation in contrast was only prevented by heparin, sulodexide and dermatan (2-100 micrograms/mL). All GAGs, except 2-O,N-desulfated heparin, inhibited beta-glucuronidase and lysozyme release, as well as beta-glucuronidase activity and PMN superoxide production by the peptide fMLP. The efficacy of GAGs was clearly dependent on the degree and type of sulfation since dermatan and N-desulfated heparins were comparatively less effective. The observation that heparin and other GAGs inhibit platelet activation induced by the PMN protease cathepsin G may help determine whether mechanisms of action other than anticoagulation are critical in the antithrombotic activity of heparin and related compounds.

The role of hyperglycaemia-induced alterations of antithrombin III and factor X activation in the thrombin hyperactivity of diabetes mellitus

Factor X concentration and factor X activation, antithrombin III anti‐Xa activity and plasma concentration, and fibrinopeptide A were measured in 20 diabetic patients and 20 normal subjects. Although factor X activation (81.3 ± 2.2 vs 97.3 ± 2.1 %, p < 0.01; mean ± SE) and antithrombin III activity (76.5 ± 2.2 vs 96.3 ± 1.8 %, p < 0.01) were reduced in the diabetic patients, fibrinopeptide A concentration was increased (3.7 ± 0.4 vs 1.7 ± 0.2 ng ml−1, p < 0.01). The ratio of factor X activation to antithrombin III anti‐factor Xa activity was increased in the diabetic patients (1.10 ± 0.01 vs 1.01 ± 0.02, p < 0.01). Induced hyperglycaemia was able to mimic all these abnormalities, without changing factor X or antithrombin III concentration. The results suggest that in vivo hyperglycaemia produces a decrease of factor X activation, but at the same time increases fibrinopeptide A formation due to a greater decrease of antithrombin III anti‐Xa activity.

Possible Role for Increased C4b-Binding–Protein Level in Acquired Protein S Deficiency in Type I Diabetes

In this study, total protein S (PS) immunological levels, free-PS and C4b-binding–protein (C4bBP) concentrations, and PS functional activity were investigated in insulin-dependent (type I) diabetic patients and compared with nondiabetic subjects. Mean total PS antigen concentration was not different between diabetic patients and nondiabetic subjects, whereas free-PS levels and PS functional activity were significantly reduced in diabetic patients. C4bBP was increased in diabetic patients and correlated with HbA1 levels. This study shows that type I diabetic patients have depressed free PS and PS activity despite the presence of normal total PS concentration and suggests that this phenomenon is probably linked to the increase of circulating C4bBP.

Evidence for a reduced heparin cofactor II biological activity in diabetes

A reduction of heparin cofactor II (HCII) biological activity, despite its normal plasma concentration, is reported in insulin-dependent diabetic patients. A good linear correlation between HCII activity and concentration is present in normal controls but not in diabetics. In these subjects HCII activity correlates inversely with fasting blood glucose and glycated proteins but not with Hb A1. These data demonstrate the presence of a depressed HCII activity in the presence of its normal plasma concentration in insulin-dependent diabetics and suggest a role for short-term metabolic control in conditioning this phenomenon.