Antithrombotic properties of a dermatan sulfate hexadecasaccharide fractionated by affinity for heparin cofactor II

The relationship between the antithrombotic activity of dermatan sulfate (DS) in vivo and its catalytic effect on the inhibition of thrombin by heparin cofactor II (HC II) in vitro was investigated. DS was depolymerized by Smith degradation and the fragments obtained were separated by gel filtration. The fragment of minimal size with full catalytic activity was a hexadecasaccharide, which was further fractionated by affinity for immobilized HC II. Only a small proportion by weight (6.7%) was recovered in the high-affinity fraction, which had about 10 times more catalytic activity than the unfractionated oligosaccharide; the change in activity was primarily caused by the removal of inert materials, recovered in the low-affinity fraction. 1H- NMR spectra indicated strengthening of the signal given by Ido A (2S04) in the high-affinity fraction compared with that of the low-affinity fraction. The anticoagulant activity of the high-affinity fraction was exclusively HC II-dependent. The antithrombotic potency was evaluated in rabbits using the Wessler-thromboplastin model. Half-maximal prevention of thrombosis was obtained after injection of 250 micrograms/kg DS, of 500 micrograms/kg hexadecasaccharide, or of 60 micrograms/kg of its high-affinity fraction. The low-affinity fraction was ineffective at the highest dose tested (1,200 micrograms/kg) and did not potentiate the effect of the high-affinity fraction. These results show that the antithrombotic effect of DS is essentially dependent on HC II binding and activation and that HC II is therefore a suitable target for antithrombotic drugs.     

Fibrinogen inhibits the heparin cofactor II-mediated antithrombin activity of dermatan sulfate

Dermatan sulfate is a naturally occurring antithrombotic glycosaminoglycan. The antithrombin activity of several dermatan sulfate preparations has been measured in whole human plasma and found to be -55% of that in purified systems. Kinetic studies under pseudo- first-order conditions indicated that the reduction in antithrombin activity of dermatan sulfate in plasma compared with that in buffer was due to noncompetitive inhibition with respect to dermatan sulfate. Analysis of the protein profile bound to immobilized dermatan sulphate showed that on a molar basis, histidine-rich glycoprotein and apolipoprotein E were the most abundant proteins specifically bound, together with significant amounts of fibrinogen and vitronectin. Addition of these proteins to the purified system showed that only fibrinogen inhibited the antithrombin activity of dermatan sulfate and that it did so in a concentration-dependent manner over the physiologic range of plasma fibrinogen levels. These results indicate that the anticoagulant activity of dermatan sulfate may be modulated in human plasma by fibrinogen.     

Vascular dermatan sulfate regulates the antithrombotic activity of heparin cofactor II

Heparin cofactor II (HCII)-deficient mice form occlusive thrombi more rapidly than do wild-type mice following injury to the carotid arterial endothelium. Dermatan sulfate (DS) and heparan sulfate (HS) increase the rate of inhibition of thrombin by HCII in vitro, but it is unknown whether vascular glycosaminoglycans play a role in the antithrombotic effect of HCII in vivo. In this study, we found that intravenous injection of either wild-type recombinant HCII or a variant with low affinity for HS (K173H) corrected the abnormally short thrombosis time of HCII-deficient mice, while a variant with low affinity for DS (R189H) had no effect. When HCII was incubated with frozen sections of the mouse carotid artery, it bound specifically to DS in the adventitia. HCII was undetectable in the wall of the uninjured carotid artery, but it became concentrated in the adventitia following endothelial injury. These results support the hypothesis that HCII interacts with DS in the vessel wall after disruption of the endothelium and that this interaction regulates thrombus formation in vivo.     

Placental dermatan sulfate: isolation, anticoagulant activity, and association with heparin cofactor II

Pregnancy is associated with hemostatic challenges that may lead to thrombosis. Heparin cofactor II (HCII) is a glycosaminoglycan-dependent thrombin inhibitor present in both maternal and fetal plasma. HCII activity increases during pregnancy, and HCII levels are significantly decreased in women with severe pre-eclampsia. Dermatan sulfate (DS) specifically activates HCII and is abundant in the placenta, but the locations of DS and HCII in the placenta have not been determined. We present evidence that DS is the major anticoagulant glycosaminoglycan in the human placenta at term. DS isolated from human placenta contains disaccharides implicated in activation of HCII and has anticoagulant activity similar to that of mucosal DS. Immunohistochemical studies revealed that DS is associated with fetal blood vessels and stromal regions of placental villi but is notably absent from the syncytiotrophoblast cells in contact with the maternal circulation. HCII colocalizes with DS in the walls of fetal blood vessels and is also present in syncytiotrophoblast cells. Our data suggest that DS is in a position to activate HCII in the fetal blood vessels or in the stroma of placental villi after injury to the syncytiotrophoblast layer and thereby inhibit fibrin generation in the placenta.     

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

Summary The 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.     

Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4- sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.     

Accelerated atherogenesis and neointima formation in heparin cofactor II deficient mice

Heparin cofactor II (HCII) is a plasma protein that inhibits thrombin when bound to dermatan sulfate or heparin. HCII-deficient mice are viable and fertile but rapidly develop thrombosis of the carotid artery after endothelial injury. We now report the effects of HCII deficiency on atherogenesis and neointima formation. HCII-null or wild-type mice, both on an apolipoprotein E-null background, were fed an atherogenic diet for 12 weeks. HCII-null mice developed plaque areas in the aortic arch approximately 64% larger than wild-type mice despite having similar plasma lipid and glucose levels. Neointima formation was induced by mechanical dilation of the common carotid artery. Thrombin activity, determined by hirudin binding or chromogenic substrate hydrolysis within 1 hour after injury, was higher in the arterial walls of HCII-null mice than in wild-type mice. After 3 weeks, the median neointimal area was 2- to 3-fold greater in HCII-null than in wild-type mice. Dermatan sulfate administered intravenously within 48 hours after injury inhibited neointima formation in wild-type mice but had no effect in HCII-null mice. Heparin did not inhibit neointima formation. We conclude that HCII deficiency promotes atherogenesis and neointima formation and that treatment with dermatan sulfate reduces neointima formation in an HCII-dependent manner.     

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.     

Antithrombotic activity, bleeding effect and pharmacodynamics of a succinyl derivative of dermatan sulphate in rabbits.

This paper compares the pharmacological properties of a new succinyl dermatan sulphate derivative (Suc-DS) to those of the natural dermatan sulphate (DS). Suc-DS was on average 2-3 times more potent than DS in catalysing the inhibition of thrombin by heparin cofactor II and in prolonging the activated partial thromboplastin time and the thrombin clotting time. After bolus injection, Suc-DS was also 2-3 times more potent than DS to prevent experimental venous thrombosis in a Wessler model. Thromboplastin or human serum were used as the thrombogenic stimulus. In contrast, the bleeding effect assessed by rat tail transection technique was comparable. After bolus intravenous injection, the pharmacodynamics of Suc-DS indicated a lower volume of distribution, which was close to the plasma volume, and a slightly lower clearance of elimination. Therefore this chemical alteration of natural DS yields a new compound with an improved antithrombotic benefit/haemorrhagic risk ratio.     

Antithrombotic properties of dermatan sulphate in a rat venous thrombosis model

It has been suggested that glycosaminoglycans (GAG) such as heparan sulphate (HS), dermatan sulphate (DS), chondroitin-4-sulphate and chondroitin-6-sulphate contribute to the nonthrombogenic properties of the vascular wall. We have investigated the potential role of DS and HS as antithrombotic agents in an experimental model of stasis-induced venous thrombosis in rats. We utilized a range of doses of both DS and HS (0.25-4 mg/kg BW) to test both their antithrombotic activity and potential bleeding effects. The results were evaluated with reference to an unfractionated heparin (0.5-2 mg/kg BW). We report that the antithrombotic activity of DS is not related to its anticoagulant activity as measured by the activated partial thromboplastin time (APTT), thrombin time (TT) and anti-Xa tests. The dose of DS which was able to inhibit thrombus formation by 70% did not prolong the bleeding time measured using two techniques (template and tail transection); in contrast, with HS a prolongation of both times could clearly be seen. On the other hand, standard unfractionated heparin, at a dose which is equipotent to that of DS in preventing thrombus formation, significantly prolonged the bleeding time. These results suggest that DS may be a useful antithrombotic agent with a lower haemorrhagic effect than heparin, unlike HS which expresses a haemorrhagic risk similar to heparin.     

Low-molecular-weight heparin and dermatan sulfate end group-labeled with tyramine and fluorescein. Biochemical and biological characterization of the fluorescent-labeled heparin derivative

To improve the understanding of the biological functions and pharmacology of heparin and dermatan sulfate, low-molecular-weight heparin (LMWH) and low-molecular-weight dermatan sulfate (LMWDS) were labeled with tyramine (T) by covalently linking T to the terminal residue of 2,5-anhydromannose (or 2,5-anhydrotalose for dermatan sulfate). The covalent labeling was demonstrated by nuclear magnetic resonance spectroscopy. The tyramine-labeled LMWH (LMWH-T) was also labeled with fluorescein (F) by further reacting it with fluorescein isothiocyanate. The fluoresceinated LMWH-T (LMWH-T,F ) was used to analyze biological functions on blood coagulation and binding to leukocytes. The biological activities on factor Xa and thrombin inhibition remained unchanged compared with the parent compound. Flow cytometric analysis of leukocytes demonstrated binding of the modified heparin to granulocytes, monocytes, and lymphocytes, the half-live being twice as long as the antifactor Xa activity. F-labeled heparin was displaced by unlabeled heparin from all three populations of leukocytes. Binding of heparin to leukocytes may play an important role in inflammation and atherosclerosis.     

Pharmacodynamics and pharmacokinetics of dermatan sulfate in humans

Dermatan sulfate (DS), a catalyst of the thrombin-heparin cofactor II interaction, has antithrombotic activity and is devoid of significant hemorrhagic risk in several animal models. We investigated the pharmacodynamic and pharmacokinetic properties of DS in humans. DS was injected in single bolus intravenous injections of four increasing doses (0.5, 1, 1.5, 2 mg/kg) to six healthy volunteers. The resulting anticoagulant activities were assessed by the activated partial thromboplastin time (APTT) and the thrombin clotting time (TCT). There were dose-dependent prolongations of the APTT and TCT, and the anticoagulant activities disappeared in less than three hours. The pharmacokinetic parameters were calculated from the plasma concentrations of DS measured with a new chromogenic assay. The volume of distribution was approximately 1.8 times greater than the theoretical plasma volume and was independent of dose. In contrast, the clearance decreased with dose and the terminal half-life ranged from 0.45 +/- 0.08 hours at 0.5 mg/kg to 0.72 +/- 0.11 hours (mean +/- SD) at 2 mg/kg. The bioavailabilities of subcutaneous (SC) and intramuscular (IM) administration relative to those of intravenous administration were determined in 12 other volunteers. The respective bioavailabilities were 24.7% +/- 12.9% and 12.4% +/- 9.2% for SC and IM administration. There was no detectable change in the APTT and the TCT when the volunteers were injected with 1.5 mg/kg SC or IM. In addition, the pharmacokinetic parameters derived from plasma concentrations of DS showed considerable interindividual variations by the two later routes of administration. Peak concentrations were noted 2.7 +/- 1.3 hours after SC injection and 4.3 +/- 4.9 hours after IM injection. The average peak concentrations were 0.7 +/- 0.3 and 0.4 +/- 0.2 mg/L after SC and IM injections, respectively. The half-lives of DS were 7.9 +/- 6.5 hours (SC) and 6.3 +/- 7.4 hours (IM). No adverse reaction to DS was recorded during this study.     

Antithrombotic effects of heparin and related agents.

Native heparin was very effective in models of arterial and venous thrombosis as well as in a model demonstrating the effect on endothelial stability in rats. The effect on venous thrombosis was particularly prominent. The activities of LMW-heparin were about the same in all three models, the absolute effective dose against arterial thrombosis being much lower than with native heparin. A heparan sulfate-related preparation (suleparoide) was much less effective in both thrombosis models, especially in the venous one, while the activity was most specifically directed to the maintenance of endothelial stability.     

Methodology and clinical significance of heparin cofactor II. Probable heparin cofactor II deficiency in a patient with cerebrovascular thrombosis

HC II was functionally determined by thrombin inhibition in the presence of heparin in AT III-free plasma prepared by immunoadsorption on anti-AT III-Sepharose 4B column. HC II antigen concentration was assayed using specific antibodies to HC II. Simultaneously, AT III was measured. Plasma levels of HC II and AT III were determined in 110 patients with thrombotic tendency and two patients with obstetric complications and DIC. Highly significant correlations between activity and antigen prove the suitability of the methods. Reduced levels of HC II to about 50% with normal AT III values were repeatedly found in one patient with thrombotic tendency. The course of AT III and HC II during the process of DIC suggests that HC II may function as a thrombin inhibitor reserve when AT III becomes subnormally low.     

Plasma heparin cofactor II in alcoholic liver disease

The coagulation inhibitor heparin cofactor II (HC II) was measured in various liver diseases and compared with antithrombin (AT), Normotest (NT), albumin and bilirubin. The lowest mean HC II level was found in alcoholic cirrhosis and the reduction reflected the degree of liver failure. A statistically significant association was found between HC II and AT (r = 0.79), NT (r = 0.71) and albumin (r = 0.66) (P less than 0.001), and there was a negative association between HC II and bilirubin (r = -0.55, P less than 0.001). HC II values below 42% in alcoholic cirrhosis seem to indicate a poor prognosis, since nine out of 18 patients with such recordings died. In contrast, no association between mortality and AT, NT and albumin levels was observed. In conclusion, HC II seems to be a reliable liver function test. It may serve as a prognostic indicator and in that respect it may be superior to NT and AT. The site of production is most probably the hepatocyte.     

Antithrombotic activity of orally administered low molecular weight heparin (Logiparin) in a rat model

Previous studies in rats demonstrated that orally administered, unfractionated bovine lung heparin is absorbed and has a dose-dependent antithrombotic effect. The objective of this study was to determine if an oral low molecular weight heparin had a similar antithrombotic effect in the same model. Thrombosis was induced in rats by application of 10% formalin in 65% methanol to the exposed jugular vein. Immediately following, saline, unfractionated heparin (3.3-60 mg/kg) or the low molecular weight heparin, Logiparin (0.025-15 mg/kg; 20-30 rats per group) was placed in the stomach and 4 h later the jugular vein was inspected for a thrombus. Compared to saline, oral Logiparin reduced the incidence of thrombosis at all doses with a dose-dependent effect suggested. A significant increase was observed in the activated partial thromboplastin time and in plasma heparin concentrations, determined by Accuclot Heptest and anti-factor Xa chromogenic assay for rats given oral Logiparin versus saline. A dose-dependent increase in plasma heparin concentration was observed when estimated by the anti-Xa chromogenic assay. Heparin was recovered in 9% of aortic endothelial samples when > or = 0.8 mg/kg Logiparin was administered. A 50% reduction in thrombosis was observed at 0.1 mg/kg for oral Logiparin versus 7.5 mg/kg for unfractionated bovine lung heparin indicating that oral Logiparin is an effective antithrombotic agent at doses lower than unfractionated heparin. Orally administered low molecular weight heparin may be useful for the prevention and treatment of thrombosis.     

Alterations of fibrin network structure mediated by dermatan sulfate

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4–20 μg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.