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).     

Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray (Raja radula) skin dermatan sulfate

Introduction

The kinetics of the thrombin inhibition by heparin cofactor II (HCII) and antithrombin (AT) have been studied as a function of the concentration of a dermatan sulfate (DS) from the skin of the ray Raja radula.

Materials and methods

The initial concentrations of inhibitor (I), HCII or AT, and thrombin (E) were set at equimolecular levels (3.10^{- 9} M). Analysis of the experimental data obtained for DS concentrations ranging from 10^{- 8} to 10^{- 4} M was performed according to a previously described model in which DS binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin.

Results

The apparent rate constant of the thrombin inhibition, k_app, by either HCII or AT, increased in a concentration-dependent manner for DS concentrations up to 10^{- 5} M or 10^{- 6} M, respectively. At higher DS concentrations, k_app remained unchanged for thrombin inhibition by HCII whereas a decrease in k_app was observed for the thrombin-AT reaction. The dissociation constant of the polysaccharide-inhibitor complex, K_DSI, and the rate constant of the thrombin inhibition by this complex, k, were (7.81 ± 0.75).10^{- 7} M and (2.84 ± 0.42).10⁹ M^{- 1}.min^{- 1}, whereas they were (4.93 ± 0.31).10^{- 7} M and (2.47 ± 0.28).10⁸ M^{- 1}.min^{- 1}, when the inhibitor was either HCII or AT, respectively.

Conclusion

DS from ray skin catalyzes the thrombin inhibition by HCII or AT primarily by forming a DS-inhibitor complex more reactive than the free inhibitor towards the protease. The affinity of DS for HCII was approximately 2-fold higher whereas the catalyzed reaction rate constant was approximately 20-fold higher when compared to AT.     

Purification and biological property of heparin cofactor II: Activation of heparin cofactor II and antithrombin III by dextran sulfate and various glycosaminoglycans

Heparin cofactor II (HC II) has been purified from human plasma by a mocification of the method described by Tollefsen $\text{et al}$.(J. Biol. Chem., $\text{257}$, 2162, 1982) and abilities of dextran sulfate and various glycosaminoglycans to activate the antithrombin activities of HC II and antithrombin III (AT III) were studied. By the purification method described here, highly purified HC II with the same specific activity as reported by Tollefsen $\text{et al}$. was obtained with a higher yield and in a shorter purification time. Heparin, dextran sulfate and chondroitin polysulfates 1 and 5 activated both HC II and AT III, while dermatan sulfate activated only HC II. Dextran sulfate was almost as active as heparin in the activation of HC II and AT III, indicating that in the interactions of heparin with HC II and AT III, sulfate groups of heparin are more important than carboxyl groups. When mixed with thrombin in the presence of dermatan sulfate, normal human plasma showed antithrombin activity which was not due to AT III but to HC II only. HC II did not inhibit factor Xa or plasmin in the presence of any glycosaminoglycans or dextran sulfate, suggesting that HC II would be a specific inhibitor of thrombin.     

Anticoagulant activities and effects on platelets of a heparin fragment with high affinity for antithrombin

A fragment of heparin containing 10–16 sugar units and retaining ability to bind to antithrombin III has been prepared by degrading standard heparin with nitrous acid. This fragment greatly potentiated the inhibition of factor X_a by antithrombin III but had virtually no effect on the inhibition of thrombin. Studies on heparin neutralization showed that the fragment was affected to a much lesser extent than standard heparin by heparin neutralizing components in plasma. The heparin-potentiated aggregation of platelets by low concentrations of ADP was measured for a number of heparin fractions and the fragment. The molecular weight of the heparin was found to be the most important factor determining the platelet aggregation activity, low molecular weight fractions including the fragment being much less active than high molecular weight ones.     

Identification of heparin cofactor II as the principal plasma cofactor for the antithrombin activity of pentosan polysulphate (SP54)

In antithrombin III depleted plasma only a slight reduction was observed in thrombin inhibitory activity due to potentiation by dermatan sulphate or by pentosan polysulphate (SP54). On gel filtration of normal plasma the peak of pentosan polysulphate dependent activity eluted with a higher apparent molecular weight than that observed with low concentrations of heparin. During purification of heparin cofactor II (1) good coincidence was observed between yields of its activity and the pentosan polysulphate dependent inhibitory activity and cochromatography of the two activities was observed also. Maximal second order rate constant of 2.5 X 10(8) M-1 min-1 (with apparent Kd of 1.8 microM) was found for the potentiation of the thrombin/heparin cofactor II interaction by pentosan polysulphate. A polysaccharide of high charge density may therefore potentiate heparin cofactor II activity and this may be of importance in vivo.     

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.     

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.     

Effects of antithrombin and heparin cofactor II levels on anticoagulation with Intimatan

Heparin is the current mainstay drug for anticoagulation during cardiac surgery, but it requires normal levels of antithrombin (AT) for optimal anticoagulation. Heparin anticoagulation may be less effective in cardiac surgical patients because of decreased AT levels due to the prolonged heparin therapy. Therefore, other anticoagulants that would work well in AT deficient patients may be more desirable. One such agent currently being evaluated is Intimatan, which catalyzes heparin cofactor II (HCII) dependent inhibition of thrombin. In the current in vitro study we examined the effects of Intimatan (20 microg/ml), heparin (0.25 U/ml), or both drugs in combination on thrombin generation in plasma with decreasing levels of AT, HCII or both cofactors, using a novel method based on the continuous measurement of thrombin generation. For the study, we collected blood samples from healthy volunteers, isolated platelet poor plasma by centrifugation and mixed it with AT, HCII, or AT-HCII deficient plasma samples to achieve different levels of AT, HCII and AT-HCII. Thrombin generation was inhibited equally well with heparin or Intimatan when the level of their respective cofactors was within the normal range. With decreasing levels of AT or HCII, heparin and Intimatan became less effective in thrombin inhibition, respectively. With the absence of both cofactors, neither agent alone or in combination had any effect on thrombin generation. We conclude that Intimatan may be an effective adjunct to heparin therapy under low AT conditions.     

Adenovirus-mediated expression of heparin cofactor II inhibits thrombin-induced cellular responses in fibroblasts and vascular smooth muscle cells

Heparin cofactor II functions as a physiological inhibitor of thrombin activity. The rate of inactivation of thrombin by heparin cofactor II is increased in the presence of dermatan sulfate, which is produced by fibroblasts or smooth muscle cells. To elucidate the role of heparin cofactor II in the extravascular cells, we induced expression of heparin cofactor II in cultured human fibroblasts or vascular smooth muscle cells using adenovirus-mediated gene transfer. After infection of adenovirus vector, these cells secreted heparin cofactor II protein into culture medium. The expressed heparin cofactor II formed the complex with exogenous thrombin and inhibited the proteolytic activity of thrombin. Expression of heparin cofactor II by infection of adenovirus vector inhibited thrombin-induced tissue-type plasminogen activator and interleukin-6 releases from fibroblasts and thrombin-induced interleukin-6 release from vascular smooth muscle cells. These findings show that fibroblasts and vascular smooth muscle cells expressing heparin cofactor II are resistant to thrombin-induced cellular responses.     

Mechanism of potentiation of antithrombin III and heparin cofactor II inhibition by sulfated xylans

Kinetic analyses of antithrombin III (AT-III)-thrombin or heparin cofactor II (HC-II)-thrombin or AT-III-factor Xa interactions were carried out in the absence or in the presence of one of the sulfated xylans or unfractionated heparin or low molecular weight (LMW) heparin utilizing chromogenic substrates. These studies demonstrated that under pseudo first order conditions the inhibitions were proportional to the AT-III or HC-II concentrations used and the apparent second order rate constants determined from the slopes of the pseudo first order plots of log of thrombin or Xa remaining as a function of time were significantly elevated in presence of the sulfated compounds. On a molar basis oat spelts xylan sulfate was the most effective compound in accelerating the rate of thrombin-AT-III interaction followed by commercial heparin while the latter was most effective in accelerating the rate of thrombin-HC-II interaction. Heparin and LMW heparin were more effective in that order in accelerating the rate of Xa-AT-III interaction while oat spelts xylan sulfate, corn cob xylan sulfate, SP-54 were less effective than the heparins in that order. Studies were also conducted on the concentrations of the sulfated compounds required to inhibit by 50% the thrombin activity by AT-III or HC-II or that required to inhibit by 50% the factor Xa activity by AT-III. The results showed an inverse relationship between the increase in the rate of acceleration by the sulfated compound with the decrease in the amount required for 50% inhibition. SDS-polyacrylamide gel study of the reaction mixture containing thrombin, AT-III or HC-II along with heparin or oat spelts xylan sulfate showed that like heparin, oat spelts xylan sulfate potentiated the formation of thrombin-AT-III or thrombin-HC-II complexes which were stable in presence of denaturing or reducing agents. Chemical modification of arginine or lysine of AT-III significantly lowered its potentiation of thrombin or Xa inhibition by oat spelts xylan sulfate.     

Comparative study on heparin and a synthetic thrombin inhibitor no.805 (MD-805) in experimental antithrombin III-deficient animals

Antithrombotic effect of heparin and a synthetic thrombin inhibitor, MD-805, was studied in experimental antithrombin III (AT III)-deficient animals which were prepared by intravenous injection of anti-AT III δ-globulin.The reduction of plasma AT III levels accelerated venous thrombosis in rats which was induced by insertion of a stainless steel wire coil into the inferior vena cava, but it gave no effect on the development of thrombin-induced acute pulmonary thromboembolism in mice.In these animals with lowered AT III levels (about 20 % of control mice and about 60 % of control rats), heparin was no longer effective in preventing thrombus formation, in contrast to MD-805 which was still effective regardless of plasma AT III levels. MD-805 was also effective in preventing the increase in complexed forms of AT III and the decrease in free (active) form of AT III in plasma of these thrombosed animals, while heparin gave no effect on these changes in plasma AT III profile.These findings strongly suggest the clinical usefulness of MD-805 in treatment of thrombotic disorders which were often accompanied by low plasma AT III levels.     

Comparative turn-over of heparin cofactor II and antithrombin III in baboons. Influence of heparin and pentosan polysulfate administration

Heparin and pentosan polysulfate (PPS) interact in plasma with antithrombin III (AT III) and Heparin cofactor II (HC II) respectively. To assess the influence of heparin or PPS treatment on the metabolism of their respective cofactors, we performed a double tracer study in baboons receiving heparin or PPS. Purified AT III and HC II from human plasma were labelled with 131I and 125I respectively by the lactoperoxidase-glucose oxidase technique. The tracers had unchanged biological activities, were homogeneous in SDS-PAGE, migrated as native proteins by crossed immunoelectrophoresis in the presence of heparin or PPS and virtually coeluted with endogenous baboon proteins from heparin-agarose. Nine animals were randomly allocated to receive, during the metabolic study, heparin (500 IU/kg/d, n = 3), PPS (5 mg/kg,d, n = 3) or a placebo (n = 3) given in 2 daily subcutaneous injections. Heparin levels and anticoagulant effects were similar in extent and duration to those usually achieved in man. The plasma concentrations of AT III and HC II did not vary under treatment. The half-life of the elimination phase in the placebo group ranged from 1.95 to 2.33 d for AT III and from 1.96 to 2.21 d for HC II. There was no significant difference in the half-lives of the 2 inhibitors between the placebo group and the animals receiving heparin or PPS. This suggest that clinical conditions associated with heparin treatment may be important for the effect of heparin on AT III metabolism previously reported in patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective degradation of heparin and heparan sulphate, and reactivity of products in the competitive binding assay

Heparin and heparan sulphate were degraded by various chemical methods, including sodium meta-periodate oxidation, alkaline treatment, Smith degradation, and treatment with acids such as nitrous and hydrochloric acid. The products were assessed by gel filtration and in the competitive binding assay (CBA) developed recently by Dawes and Pepper (3). With the exception of nitrous acid treatment, all the chemical methods were nonselective in their effect on heparin and heparan sulphate. However, nitrous acid treatment exhibited a degree of selectivity which may be useful in enhancing the specificity of CBA. The results also highlighted the significance of sulphated groups in the binding of heparin and heparan sulphate in CBA.     

Enhancement by heparin of thrombin-induced antithrombin III proteolysis: Its relation to the molecular weight and anticoagulant activity of heparin

Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. We now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of α-thrombin with a molar excess of ¹²⁵I AT III. Proteolysis of the inhibitor was assessed by SDS polyacreylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% of AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistant with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.