The anticoagulant effect of heparan sulfate and dermatan sulfate

The potency of the various glycosaminoglycans (GAGs) as anticoagulants in the activated partial thromboplastin time (APTT) test system has been investigated.

Heparin, heparan sulfate (HS) and dermatan sulfate (DS) exhibit anticoagulant effect. About 70 times higher concentrations of HS and DS were required to obtain the effect of heparin. The other GAGs, including chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate and hyaluronic acid (HA) did not prolong the APTT at the concentrations used.

The influence of the GAGs on thrombin and activated factor X (Xa), and on the inactivation of these clotting enzymes by antithrombin III (At-III) has been studied, using chromogenic substrates for the assay of enzyme activity. All GAGs, except keratan sulfate, had inhibitory effect on the amidolytic activity of thrombin. However, no GAG affected the amidolytic activity of Xa.

Heparin and HS accelerate the inactivation of thrombin and Xa by At-III. About 75 times higher concentration of HS was required to obtain the effect of heparin. DS and HA had a slight accelerating effect on the inactivation of thrombin by At-III, but only at a high concentration (500 μg/ml). At concentrations where DS exhibited anticoagulant activity in the APTT test system, no accelerating effect on At-III activity could be detected. Thus, the anticoagulant effect of DS is probably not exerted via At-III, and may in this respect differ from the anticoagulant activity of heparin and HS.     

Standard heparin enhances the antithrombotic activity of dermatan sulfate in the rabbit but CY 216 does not

Standard heparin (SH) and dermatan sulfate (DS) two glycosaminoglycans with different pharmacological targets are effective antithrombotic agents in the rabbit. We have investigated the antithrombotic activity of the association DS plus SH. It was found that doses as low as 25 micrograms/kg for DS and 10 micrograms/kg for SH were ineffective when injected separately but generated a high and significant antithrombotic activity when injected together. These results were confirmed when higher doses of each compound were delivered in association. Further experiments were performed to determine if the enhancement of the antithrombotic activity of DS by HS resulted from its anti-factor IIa or anti-factor Xa activity or from its moiety without affinity to AT III. A low molecular weight heparin (CY 216) with an anti-factor Xa/anti-factor IIa ratio of 5, the synthetic pentasaccharide bearing the minimum binding sequence to antithrombin III, and a low affinity fraction of SH to AT III did not increase the antithrombotic activity of DS; in contrast a high affinity fraction of SH to AT III had the same effect than SH. We conclude that the enhancement of the antithrombotic activity of DS by SH mainly results from its anti-factor IIa activity.     

Neutralization of the antithrombotic effects of heparin and Fraxiparin by protamine sulfate.

In general, the in vitro anti Xa activity of low molecular weight heparins is neutralized to a lesser degree than the anti Xa activity of unfractionated heparin. To determine whether these differences occur in vivo, a rabbit stasis thrombosis model and a rat laser-induced thrombosis model were utilized. In the laser model, a similar degree of neutralization of the antithrombotic activity of heparin and Fraxiparin was obtained. However, in the stasis thrombosis model, significant antithrombotic activity of Fraxiparin remained after equigravimetric protamine administration. Ex vivo APTT, thrombin time, Heptest, amidolytic anti Xa and anti IIa assays were performed. A coefficient (r = .806) was obtained for the correlation of Heptest activity to antithrombotic effect in the stasis thrombosis model, while the coefficients obtained for the other tests ranged from .152-.570. However, after neutralization by protamine, the thrombin time exhibited the highest correlation coefficient (r = .685) between ex vivo activity and residual antithrombotic effect. Since Fraxiparin retains antithrombotic activity after protamine administration, clinical benefit may be observed for this low molecular weight heparin as compared to unfractionated heparin after neutralization.     

The relationship between the structure of dermatan sulfate derivatives and their antithrombotic activities

To study the relationship between the structure of dermatan sulfate (DS) derivatives and their anti-thrombotic activities, DS-derived oligosaccharides (with different structures and relative molecular weight (M(r))) were prepared, and the effects of the DS-derived oligosaccharides on the activities of heparin cofactor II (HCII), activated protein C (APC), blood platelet, and vascular endothelial cells were studied. The major disaccharides of DS and polysulfated dermatan sulfate (PSDS) were IdoA-1-->3-GalNAc-4-OSO(3) and IdoA-2OSO(3)-1-->3-GalNAc4, 6-diOSO(3), respectively. The results showed that the consequence of the thrombotic inhibitory effects of DS and its derivatives were as follows: PSDS>low molecular weight polysulfated dermatan sulfate (LPSDS)>DS. Both DS and PSDS inhibited platelet aggregation in the concentration-dependent manner, and the IC(50) value of DS and PSDS is 12.7+/-1.3 and 28.6+/-0.9 mg/mL, respectively. DS oligosaccharides (DSOSs) and PSDS oligosaccharides (PSDSOSs) both significantly inhibited P-selectin expression on platelet surface (P<0.01), while DSOSs have no different effect compared with PSDSOSs. DSOSs and PSDSOSs significantly enhanced the activity of HCII in inhibiting thrombin in the plasma. The most active PSDSOS was PSDSOS(1) with M(r) of 4959, which enhanced the HCII activity by 91% (P<0.01). The experiments on APC activity showed that DS and its derivatives enhanced APC activity. The most active PSDSOS was PSDSOS(3) with M(r) of 2749, which enhanced the APC activity to 331+/-27% (P<0.01). DSOSs and PSDSOSs enhanced tissue plasminogen activator (t-PA) activity and reduced the plasminogen activator inhibitor (PAI) activity from cultured human umbilical vein endothelial cells (HUVEC), resulting in the ratio of t-PA/PAI going up. PSDSOSs which have the same M(r) as DSOSs produced more active effects in above assays, except for platelet aggregation.     

Potentiation of the antithrombotic action of dermatan sulphate by small amounts of heparin

We have examined the effect in impairing thrombus formation of a preparation of dermatan sulphate (DS) alone and DS plus small amounts of unfractionated heparin (UFH). In rabbits given a dose of 150 micrograms/kg of DS alone, there was minimal reduction in serum-induced stasis thrombosis (Wessler model), with an overall score of 92.5% (100% = no impairment of thrombus formation). When the same dose of DS containing UFH was given (two different subgroups given DS containing 0.25 and 2.5% heparin by dry weight, respectively), the overall thrombus score was reduced to 60% (P less than 0.003), with no significant difference between the two subgroups. To achieve a comparable result with DS alone, a dose of 1 mg/kg was required. We conclude that very small amounts of UFH significantly enhance the antithrombotic action of DS, by a mechanism that has yet to be determined.     

Experimental studies on the relative efficacy of dermatan sulphate and heparin as antithrombotic agents

In this study, the anticoagulant and antithrombotic properties of unfractionated heparin (UFH) and dermatan sulphate (DS) were compared. The ability of UFH and DS to impair thrombin generation in vitro and in ex vivo plasma samples was also studied. DS has minimal anticoagulant activity by conventional assays but impairs thrombin generation both in vitro and in ex vivo plasma samples. However, thrombin generation could not be suppressed below about 35% of control values at all doses of DS studied. While this was sufficient to impair experimental venous thrombosis during 10 minutes' stasis, DS was ineffective in preventing thrombosis following 20 minutes' stasis in doses up to 1.25 mg/kg. In contrast, 1 microgram/ml of UFH completely suppressed thrombin generation in vitro, and 150 micrograms/kg prevented thrombogenesis over a period of 20 minutes' stasis. Neither drug prolonged the bleeding time (BT) at effective antithrombotic doses, but 2.5 mg/kg UFH significantly increased the BT, whereas DS did not. While DS has antithrombotic activity, it is less effective than UFH in inhibiting thrombin generation, and as an antithrombotic agent.     

Comparison of the in vivo hemorrhagic and antithrombotic effects of a low antithrombin-iii affinity heparin fraction

Antithrombin III (ATIII) affinity chromatography of commercial grade heparin yields fractions of high and low affinity for ATIII. In vitro, the high affinity fraction accounts for most of the anticoagulant effect while there is evidence that the low affinity material interferes with platelet function. We have studied the relative antithrombotic and hemorrhagic effects of low affinity heparin. The low affinity heparin fraction, specific activity 43 USP units/mg, was compared with standard heparin (150 USP units/mg) in rabbit experimental models. A residual 12.5% by weight of this low affinity heparin showed high affinity for ATIII. Inhibition of thrombosis in a stasis-hypercoagulability model was directly related to the weight (mg) of high affinity material in each of the heparins. In the bleeding model, when similar weights (mg) of high affinity material were infused, significantly more bleeding was demonstrated with the low affinity fraction which contained a 5-fold excess by weight of low affinity material. We have demonstrated that a low affinity heparin depleted of in vitro anticoagulant and in vivo antithrombotic activity significantly contributes to hemorrhage.     

Inhibition of agrin-induced acetylcholine-receptor aggregation by heparin, heparan sulfate, and other polyanions.

Heparin and heparan sulfate have been shown to block nerve-induced acetylcholine-receptor (AChR) aggregation at developing neuromuscular junctions. We found that heparin, heparan sulfate, and a wide variety of other polyanions also inhibited agrin-induced AChR aggregation. The more highly charged the polyanion, the more potent it was as an inhibitor. Inhibition of agrin-induced AChR aggregation was due, at least in part, to the formation of a complex between the polyanion and agrin that was inactive. These findings are consistent with the hypothesis that nerve-induced aggregation of AChRs is mediated by the release of agrin, or a closely related protein, from axon terminals and suggest that a polyanion, such as a sulfated proteoglycan, may be involved in the interaction of agrin with its receptor on the myotube surface.     

Consumption of heparin cofactor II (dermatan sulfate cofactor) and antithrombin during coagulation

In vitro coagulation produces a consumption of heparin co factor II (HC II) of 8-10% both in plasma from normal individuals and patients whereas antithrombin (AT) consumption ranges 40-50%. In blood from heparin treated patients consumption is similar or greater. In blood from warfarin treated patients, consumption is decreased. Addition of heparin prior to clotting has little effect on HC II consumption, but high heparin concentration reduces AT consumption. Addition of dermatan sulfate has no effect on AT consumption, but increases HC II consumption dramatically. In consumption coagulopathy, the HC II levels are as low as AT, possibly reflecting intravascular consumption accelerated by vascular glycosaminoglycans.     

Anticoagulant and antithrombotic effects of heparin and low molecular weight heparin fragments in rabbits

Heparin and heparin fragments of different molecular weight and with different anti-factor X_a/APTT activity ratios were studied with respect to their ability to inhibit thrombus formation in an animal model. It is concluded that: a) Neither anti-factor X_a nor the APTT activity alone is a good reflector of the antithrombotic activity. b) Anti-factor X_a active fragments must have a minimum molecular weight in order to elicit good antithrombotic activity. c) High affinity for antithrombin III is important for good antithrombotic activity. d) A heparin fragment of molecular weight 4 000 has the same antithrombotic activity as heparin but less effect on the clotting time.     

Comparison of antithrombotic and hemorrhagic effects of edoxaban,a novel factor Xa inhibitor,with unfractionated heparin,dalteparin, lepirudin and warfarin in rats

Background

Edoxaban is a novel, potent and orally active direct Factor Xa (FXa) inhibitor under development for prophylaxis and treatment of thromboembolic diseases. Properties of dose response and margin of safety of anticoagulants are the key factors for a positive risk/benefit of novel oral anticoagulants.

Objectives

To compare the dose response of antithrombotic effect and margin of safety between antithrombotic and hemorrhagic effects of edoxaban with conventional anticoagulants, unfractionated heparin (UFH), dalteparin (low molecular weight heparin), lepirudin, and warfarin in rat models of thrombosis and hemorrhage.

Methods

Rats were treated with edoxaban, UFH, dalteparin, and lepirudin by continuous intravenous (iv) infusion, or with oral warfarin for 4 days before inducing thrombosis or bleeding. Thrombosis was induced by inserting a platinum wire into the inferior vena cava for 60 minutes. Tail template bleeding time was measured after making an incision on the tail.

Results

In rats, iv infusion of edoxaban inhibited venous thrombosis in a dose-dependent manner. The other anticoagulants also exerted dose-dependent antithrombotic effects. The slopes of the dose–response curves of edoxaban were significantly shallower than the slopes of UFH, dalteparin, and warfarin. At supratherapeutic doses, edoxaban prolonged bleeding time in a rat tail bleeding model. To determine bleeding risk, the margins between antithrombotic and bleeding-time prolongation were compared. The margins of safety of edoxaban were wider than those of UFH, dalteparin, lepirudin, and warfarin.

Conclusions

These results suggest that edoxaban may be more easily controlled and has the potential for a more positive risk/benefit ratio compared to conventional anticoagulants.     

Assay of dermatan sulfate cofactor (heparin cofactor II) activity in human plasma

An assay measuring the thrombin inactivating effect of human plasma in the presence of dermatan sulfate (DS) is described. Test plasma, diluted $\text{1}\text{50}$, is incubated with human thrombin in the presence of DS. Remaining thrombin is determined with chromogenic substrate 2AcOH. H-D-CHG-Ala-Arg-pNA. Three dilutions of reference plasma suffice and the standard curve is linear. Antithrombin III (AT) exerts a small (3–8%) effect in the assay.When test plasma contains heparin above 0.05 U/ml, this unspesific effect of AT increases, but it may be abolished by antibodies against AT. In a normal material (n=50), the SD of DS cofactor activity was greater (15%) than that of AT (8.7%). DS cofactor was normal in hereditary AT deficiency and in 15 patients with deep venous thrombosis. In liver cirrhosis and in DIC, both inhibitors were markedly depressed, to similar degrees (r=0.84).     

Use of purified dermatan sulfate for heparin cofactor II (HC II) assay.

HC II functional assays are generally preferred with respect to immunochemical assays. Nevertheless functional assays can be biased by the antithrombin III (AT III)-heparin complex activity; in fact trace amounts of heparin generally contaminate dermatan sulfate (DS) commercial preparations used as HC II reaction activators. We have employed a purified DS preparation showing these features: DS concentration 94.4%, chondroitin sulfate 5.6%, M.W. 21.4 kDa. The absence of any interference due to AT III-heparin complexes was verified in a kinetic HC II assay of some human plasma pools. The immunological inhibition of AT III by anti-AT III caused a minimal decrease (6-8%) of the reaction slope, attributable to AT III activity. Progressive increase of heparin concentration in the assay was effective only starting from 30 U/ml (the assay was carried out in the presence of polybrene to prevent any AT III activation). The reference interval (mean +/- SD) obtained from 157 normal subjects was 100.8 +/- 20.2%; there was a good correlation with immunoreactive HC II. The purified DS we have used seems suitable for routinary assays of HC II where a minimal interference due to AT III-heparin is required.     

Effects of heparin, dermatan sulfate and of their association on the inhibition of venous thrombosis growth in the rabbit.

This study compares the ability of unfractionated heparin, of dermatan sulfate, and of their simultaneous administration delivered as continuous intravenous infusion or as a single bolus injection to inhibit the growth of a standardized venous thrombosis in the rabbit. When delivered as continuous intravenous infusion for 4 h, heparin and dermatan sulfate inhibited thrombus growth in a dose dependent manner. The maximum antithrombotic effect of heparin was achieved at the dose of 0.15 mg kg-1 h-1 (25 U kg-1 h-1) which generated a mean plasma concentration of 1.8 micrograms ml-1 (0.31 U ml-1) and a 1.8 fold prolongation of the activated partial thromboplastin time (APTT) in comparison to the pretreatment value. A comparable antithrombotic effect was obtained with dermatan sulfate at the dose of 2 mg kg-1 h-1. This dose generated a mean plasma concentration of 30 micrograms ml-1 and a 1.3 fold APTT prolongation. Increasing these doses up to 10 fold did not improve the antithrombotic effect which did not overpass 60-70% of the controls. When the compounds were delivered simultaneously, the maximum antithrombotic effect (64%) was obtained with the following association: 0.06 mg kg-1 h-1 (10 U kg-1 h-1) for heparin and 1 mg kg-1 h-1 for dermatan sulfate. Increasing these doses up to 4 to 5 fold did not improve the antithrombotic effect. Heparin, dermatan sulfate and the association of both were also delivered as single bolus injections and the resultant antithrombotic effect was determined 4 h after saline infusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Additive thrombin inhibition by fast moving heparin and dermatan sulfate explains the anticoagulant effect of sulodexide,a natural mixture of glycosaminoglycans

Introduction: The aim of the study was to evaluate the mechanism of the anticoagulant action of sulodexide, a mixture of glycosaminoglycans (GAGs) composed of dermatan sulfate (DS) and fast moving heparin (FMH), in vitro. Materials and methods: Thrombin clotting time (TCT) was measured in human platelet poor plasma (PPP). A chromogenic substrate assay was used to determine the pseudo-first order constant kinetic of thrombin inhibition (k′=k_obs/min) either in defibrinated PPP or antithrombin (AT) or heparin cofactor II (HCII) depleted defibrinated PPP in the absence and presence of sulodexide or its components, alone and in combination. The interaction between DS and FMH was analysed by both the algebraic fractional and isobole graphical methods. Results: Sulodexide, DS and FMH produced a dose-dependent prolongation of TCT with unclottable TCT at sulodexide above 4 μg/ml and at DS or FMH above 5 μg/ml. Sulodexide and its components alone and in combination produced a dose-dependent linear increase in the rate of thrombin inhibition in defibrinated PPP. The algebraic fractional and the isobole graphical methods indicated an additive effect between DS and FMH. In AT depleted PPP, the dose-dependent increase in k′ produced by sulodexide was significantly lower than in PPP, while the dose-dependent increase in k′ produced by DS was similar to the increase produced in PPP. In HCII depleted PPP, the dose-dependent increase in k′ produced by sulodexide was significantly lower than in PPP, while the dose-dependent increase in k′ produced by FMH was similar to the increase produced in PPP. Conclusions: Thrombin inhibition produced by sulodexide is due to the additive effect of its components, namely, HCII catalysis by DS and AT catalysis by FMH.     

Antithrombotic activity of avian crown dermatan sulfate

The antithrombotic activity of dermatan sulfate from avian crown with the mean molecular weight of 38000 was compared to those from bovine intestine with the mean molecular weight of 16000 in vivo and in vitro. In an in vitro test, bovine intestine dermatan sulfate exhibited stronger effects on stimulation of heparin cofactor II and activation of Glu-plasminogen by tissue plasminogen activator. In vivo, avian crown dermatan sulfate more effectively prevented the development of thrombus in a rat deep vein thrombosis model. The measurement of plasma levels of these two kinds of dermatan sulfate revealed that avian crown dermatan sulfate circulated in higher concentration and longer duration than bovine intestine dermatan sulfate after intravenous administration to rats.     

Expression of a heparan sulfate remodeling enzyme, heparan sulfate 6-O-endosulfatase sulfatase FP2, in the rat nervous system

A new class of sulfatases, SulfFP1/sulf-1 and SulfFP2/sulf-2, has been implicated in the modulation of extracellular signaling by remodeling heparan sulfate through 6-O-endosulfatase activity. Here, we isolated rat SulfFP2 cDNA and examined its expression by in situ hybridization. SulfFP2 expression is abundant and dynamically regulated in the nervous system during development, whereas it is confined to the cerebral cortex, hippocampal CA3 region, and medial habenular nucleus in the adult brain.     

Arterial antithrombotic effects of aspirin, heparin, enoxaparin and clopidogrel alone, or in combination, in the rat

INTRODUCTION: Many antithrombotic drugs may have a deleterious effect on normal haemostasis leading to bleeding complications. The aim of this study was to determine if sub-therapeutic (low) doses of antithrombotic agents, when administered in combination, have enhanced efficacy without augmentation of bleeding time. MATERIALS AND METHODS: The antithrombotic effects of i.v. aspirin (4-30 mg/kg), heparin (100-500 U/kg), enoxaparin (4-30 mg/kg) and clopidogrel (10-20 mg/kg) were studied in a rat Folts-like preparation of carotid arterial thrombosis. The frequency of cyclic flow reductions (CFRs; indicating occlusive thrombus formation) and bleeding time were measured. Drug doses that were singly ineffective at preventing occlusive thrombus formation were tested in the following combinations: aspirin (10 mg/kg) with heparin (250 U/kg); aspirin (4 mg/kg) with enoxaparin (4 mg/kg); and aspirin (10 mg/kg) with clopidogrel (10 mg/kg). RESULTS: Control period (pretreatment) CFRs were not significantly different between groups; average 7.0+/-0.3 CFRs/30 min (n=64). Tail bleeding time before drug(s) was 3.1+/-0.1 min (n=86). When administered alone, aspirin (4-30 mg/kg), heparin (250 U/kg) or enoxaparin (4 mg/kg) had no effect on CFRs or bleeding time. Heparin (500 U/kg), enoxaparin (10 and 30 mg/kg) and clopidogrel (20 mg/kg) significantly decreased CFRs. Single administration of heparin (500 U/kg) or enoxaparin (30 mg/kg) increased bleeding time by 4- or 11-fold. When co-administered, aspirin 10 mg/kg and heparin 250 U/kg decreased CFRs, but also increased bleeding time by 11-fold. However, combination of aspirin and enoxaparin (4 mg/kg each), or aspirin and clopidogrel (10 mg/kg each), decreased CFRs with no effect on bleeding. CONCLUSIONS: In a preparation of arterial thrombosis in the rat, combinations of sub-efficacious (low) doses of aspirin with enoxaparin or clopidogrel inhibited thrombus formation without augmenting bleeding time. However, low-dose aspirin combined with heparin, whilst inhibiting thrombus formation, exacerbated bleeding time. If these findings translate into the clinic, the use of effective low-dose combinations may have therapeutic advantages.