S PROTEIN/VITRONECTIN NEUTRALIZES THE ANTICOAGULANT ACTIVITY OF GLYCOSAMINOGLYCANS IN THE INHIBITION OF THROMBIN BY HEPARIN COFACTOR II

The complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin against fast inactivation by antithrombin III. The interference of S protein with glycos-aminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in a purified system. In the presence of 0.3 μg/ml heparin, or 0.5 μg/ml pentosan polyphosphate (SP 54), or 2 μg/ml dermatan sulfate, S protein induced a concentration-dependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo-first order rate constants by about 17-fold (heparin), or about 7-fold (SP 54), but only by about 2-fold for dermatan sulfate at a physiological ratio of S protein to heparin cofactor II. Likewise, S protein significantly counteracted the anticoagulant activity of heparin and SP 54 bot not of dermatan sulfate when tested in an inhibition assay using various concentrations of glycosaminoglycans. For heparin, the activity of S protein at the point of 50% inhibition of thrombin was expressed in the range 0.06-0.6 μg/ml (0.01-0.1 U/ml) and for SP 54 in the range 0.3-2 pg/ml. Exposure of the glycos-aminoglycan-binding region of S protein by reduction and carb-oxymethylation of the protein even increased the neutralizing activity of S protein towards heparin and SP 54. S protein not only was found together with thrombin in a binary complex. S protein also became incorporated into a ternary complex with thrombin and heparin cofactor II as judged by crossed immunoelectrophoresis, regardless whether complex formation was initiated by heparin or dermatan sulfate. These findings underline the role of S protein as potent glycosaminoglycan-neutral-izing protein in plasma and as scavenger protein which may bind to enzyme-inhibitor complexes of the coagulation system.

Modulation of Heparin Cofactor II Function by S Protein (Vitronectin) and Formation of a Ternary S Protein-Thrombin-Heparin Cofactor II Complex

10.1055/s-0038-1646979 ◽

1988 ◽

Vol 60 (03) ◽

pp. 399-406 ◽

Cited By ~ 15

Author(s):

Klaus T Preissner ◽

Pierre Sié

Keyword(s):

Dermatan Sulfate ◽

Enzyme Inhibitor ◽

Anticoagulant Activity ◽

Factor Xa ◽

Binding Domain ◽

Coagulation System ◽

Pentosan Polysulfate ◽

Heparin Cofactor Ii ◽

S Protein ◽

SummaryThe complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 μg/ml heparin, 0.5 μg/ml pentosan polysulfate, or 2 μg/ml dermatan sulfate, S protein induced a concentrationdependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity. Independent on the polysaccharide used, S protein was found incorporated within a ternary complex with thrombin and heparin cofactor II during the inhibition reaction as judged by crossed immunoelectrophoresis, ultracentrifugation as well as ELISA analysis, emphazising the function of S protein as scavenger protein for enzyme-inhibitor complexes of the coagulation system. These findings demonstrate the role of S protein as effective neutralising plasma protein of the anticoagulant activity of various glycosaminoglycans also with respect to heparin cofactor II. Although the glycosaminoglycan-binding domain of S protein readily neutralized different dermatan sulfate compounds, physiological modulation of heparin cofactor-II-dependent inhibition of thrombin by native S protein appears to be restricted to the vascular compartments, where other glycosaminoglycans than dermatan sulfate appear to be operative.

Inhibition of Thrombin by Antithrombin III and Heparin Cofactor II In Vivo

10.1055/s-0038-1653789 ◽

1995 ◽

Vol 73 (03) ◽

pp. 405-412 ◽

Cited By ~ 27

Author(s):

Longbin Liu ◽

Lori Dewar ◽

Yingqi Song ◽

Myron Kulczycky ◽

Blajchman A Morris ◽

...

Keyword(s):

Pregnant Women ◽

Antithrombin Iii ◽

Dermatan Sulfate ◽

Critical Role ◽

Experimental Studies ◽

Thrombin Inhibitor ◽

Heparin Cofactor Ii ◽

SummaryThe critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate.

Serpin-independent anticoagulant activity of a fucosylated chondroitin sulfate

10.1160/th08-04-0210 ◽

2008 ◽

Vol 100 (09) ◽

pp. 420-428 ◽

Cited By ~ 47

Author(s):

Bianca F. Glauser ◽

Mariana S. Pereira ◽

Robson Q. Monteiro ◽

Paulo A. S. Mourão

Keyword(s):

Chondroitin Sulfate ◽

Anticoagulant Activity ◽

Factor Xa ◽

Anticoagulant Effect ◽

Coagulation System ◽

Heparin Cofactor Ii ◽

Thrombin Inhibition ◽

Coagulation Proteins ◽

Dependent Inhibition ◽

Structure Versus

SummaryFucosylated chondroitin sulfate is a glycosaminoglycan from sea cucumber composed of a chondroitin sulfate-like core with branches of sulfated fucose. This glycosaminoglycan has high anticoagulant and antithrombotic activities. Its serpin-dependent anticoagulant activity is mostly due to activating thrombin inhibition by heparin cofactor II. Here, we evaluated the anticoagulant activity of fucosylated chondroitin sulfate using antithrom-bin- and heparin cofactor II-free plasmas. In contrast to mammalian heparin, the invertebrate glycosaminoglycan is still able to prolong coagulation time and delay thrombin and factor Xa generation in serpin-free plasmas. These observations suggest that fucosylated chondroitin sulfate has a serpin-independent anticoagulant effect. We further investigated this effect using purified blood coagulation proteins. Clearly, fucosylated chondroitin sulfate inhibits the intrinsic tenase and prothrombinase complexes, which are critical for thrombin generation. It is possiblethat the invertebrate chondroitin sulfate inhibits interactions between cofactor Va and factor Xa. We also employed chemically modified polysaccharides in order to trace a structure versus activity relationship. Removal of the sulfated fucose branches, but not reduction of the glucuronic acid residues to glucose, abolished its activity. In conclusion, fucosylated chondroitin sulfate has broader effects on the coagulation system than mammalian glycosaminoglycans. In addition to its serpin-dependent inhibition of coagulation protease, it also inhibits the generation of factor Xa and thrombin by the tenase and prothrombinase complexes, respectively. In plasma systems, the serpin-independent anticoagulant effect of fucosylated chondroitin sulfate predominates over its serpin-dependent action. This glycosaminoglycan opens new avenues for the development of antithrombotic agents.

The Additive Effect of Low Molecular Weight Heparins on Thrombin Inhibition by Dermatan Sulfate

10.1055/s-0038-1649602 ◽

1993 ◽

Vol 70 (03) ◽

pp. 443-447 ◽

Cited By ~ 6

Author(s):

Benilde Cosmi ◽

Giancarlo Agnelli ◽

Edward Young ◽

Jack Hirsh ◽

Jeffrey Weitz

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Additive Effect ◽

Low Molecular Weight ◽

Low Molecular Weight Heparins ◽

Heparin Cofactor Ii ◽

Thrombin Inhibition ◽

Sds Page

SummaryThe aim of this study was to investigate the mechanism by which the anticoagulant activity of dermatan sulfate (DS) is increased by low molecular weight heparin (LMWH). In platelet poor plasma, LMWH enhances the effect of DS on thrombin (IIa) inhibition as determined by thrombin clotting times and with a chromogenic substrate assay. Analysis of the results of the chromogenic assays using either the algebraic fractional or the graphic isobole method suggests that LMWH has an additive effect on the anti-IIa activity of DS. This additive effect was lost when the experiments were repeated in plasma immunodepleted of antithrombin III (ATIII), indicating that the anti-IIa activity of LMWH is ATIII-dependent. To further explore the mechanism of the interaction between LMWH and DS, 125I-labeled IIa was added to plasma in the presence or absence of DS and/or LMWH and the formation of IIa-inhibitor complexes was assessed using SDS-PAGE followed by autoradiography. DS addition selectively increases the formation of heparin cofactor II (HCII)-IIa complexes, whereas LMWH enhances ATIII-IIa complex generation. Compared to plasma containing DS alone, the formation of ATIII-IIa complexes also is increased when the combination of DS and LMWH is added. These findings suggest that the additive effect of LMWH on the anti-IIa activity of DS reflects their different modes of IIa inhibition; DS potentiates IIa inhibition by HCII, while LMWH catalyses ATIII-dependent IIa inactivation. The potential clinical significance of these findings requires further investigation.

A Low Molecular Weight Heparin Alters the Fetal Coagulation System in the Pregnant Sheep

10.1055/s-0038-1661560 ◽

1986 ◽

Vol 55 (03) ◽

pp. 342-346 ◽

Cited By ~ 14

Author(s):

M Andrew ◽

F Ofosu ◽

F Fernandez ◽

A Jefferies ◽

J Hirsh ◽

...

Keyword(s):

Anticoagulant Activity ◽

Factor Xa ◽

Coagulation System ◽

Dermatan Sulphate ◽

Protamine Sulphate ◽

Pregnant Sheep ◽

Pregnant Ewe ◽

SummaryStandard heparin and a LMWH, CY222 do not cross the placenta nor alter fetal coagulation when injected into the pregnant ewe. We found that another LMWH, Pharmuka-10169 (PK-10169) alters fetal coagulation without crossing the placenta in the pregnant sheep. To characterize this anticoagulant we measured the in vitro and in vivo effects of 125I-PK-10169 in maternal and fetal plasmas following administration of PK-10169 to the mother or fetus. The fetal anticoagulant activity was not neutralizable by protamine sulphate and was attributable to the inhibition of thrombin but not factor Xa. In vitro, the fetal anticoagulant activity had properties similar to dermatan sulphate : both catalyzed the inhibition of thrombin but not factor Xa by sheep plasma; and neither was neutralizable by protamine sulphate. These effects were due to the enhanced neutralization of thrombin by heparin cofactor II. We conclude that PK-10169 does not cross the placenta, but does induce the release of an endogenous dermatan sulphate-like substance which alters fetal coagulation.

Plasma dermatan sulfate proteoglycan in a patient on chronic hemodialysis

Blood ◽

10.1182/blood.v82.11.3380.3380 ◽

1993 ◽

Vol 82 (11) ◽

pp. 3380-3385 ◽

Cited By ~ 8

Author(s):

MA Delorme ◽

N Saeed ◽

A Sevcik ◽

L Mitchell ◽

L Berry ◽

...

Keyword(s):

Molecular Weight ◽

Antithrombin Iii ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Psoas Muscle ◽

Chronic Hemodialysis ◽

Gel Chromatography ◽

Thrombin Time ◽

Heparin Cofactor Ii ◽

Dermatan Sulfate Proteoglycan

Abstract A 68-year-old man on chronic hemodialysis for 6 years, presented with a spontaneous psoas muscle hemorrhage. Investigations showed intermittently elevated activated partial-thromboplastin time and thrombin time. Preliminary investigations suggested a heparin-like inhibitor in the patient's plasma, but no anti-Xa activity could be detected. Investigation of the ability of patient plasma to inhibit exogenous thrombin showed that most thrombin was inhibited by heparin cofactor II, in contrast to normal plasma in which most thrombin was inhibited by antithrombin III. Treatment of plasma with glycosaminoglycan-degrading enzymes suggested the presence of dermatan sulfate (DS) in patient plasma. This was confirmed in a heparin cofactor II-dependent antithrombin assay for DS that showed anticoagulant equivalent to 2.2 +/- 0.3 micrograms/mL (mean +/- SD) of porcine mucosal DS. Of this activity, approximately 90% was sensitive to enzymes that degrade DS. The glycosaminoglycan containing fraction of plasma was isolated and subjected to gel chromatography. Anticoagulant activity eluted from Sephadex G-100 (Pharmacia, Montreal, Quebec, Canada) as two peaks with Kav of 0.10 and 0.45. After treatment with base, the Kav of the higher molecular weight species was increased to 0.55. This activity was completely sensitive to enzymes that degrade DS. Thus, the active DS was present as a proteoglycan. The lower molecular weight material was not sensitive to enzymes that degrade DS or heparan sulfate and it was active in the heparin cofactor II- dependent antithrombin assay but not in an antithrombin III-dependent antithrombin assay. This activity was not degraded by heating. Subsequently, measurement of DS activity was performed in plasmas obtained from eight other patients on hemodialysis before administration of heparin that showed that all patients had DS activity present that varied from 0.05 to 0.4 microgram/mL. No enzyme-resistant activity could be shown in these patients. In summary, a circulating anticoagulant with properties of DS is present in patients requiring hemodialysis.

EFFECT OF RABBIT THROMBOMODULIN ON THE INHIBITION OF THROMBIN BY ANTITHROMBIN IN PRESENCE OF EXOGENOUS HEPARIN: ROLE OF THE ACIDIC DOMAIN OF THROMBOMODULIN

10.1055/s-0038-1643964 ◽

1987 ◽

Author(s):

M C Bourin ◽

I Bjôrk ◽

M C Boffa ◽

U Lindahl

Keyword(s):

Protein C ◽

Anticoagulant Activity ◽

Proteolytic Degradation ◽

High Affinity ◽

Acidic Domain ◽

Acidic Form ◽

Cell Membrane Protein ◽

A Site ◽

Thrombomodulin (TM) is an endothelial cell membrane protein chat acts as a cofactor for Protein C (PC) activation by thrombin (T). Rabbit TM also prevents fibrinogen clotting by T (direct anticoagulant activity) and accelerates T inhibition by antithrombin (AT-dependent anticoagulant activity). Rabbit TM was previously found to contain an acidic domain (presumably a heparin-like polysaccharide), separated from the PC cofactor site but required for the direct and AT-dependent anticoagulant activities. Although binding of TM to T modifies the specificity of T, it does not involve a modification of the catalytic site or a global conformational change of the enzyme.A non-acidic form of rabbit TM was obtained by limited proteolytic degradation of the acidic form. The non-acidic form retained only the PC cofactor activity. The acidic and non-acidic forms of rabbit TM were compared with regard to their effects on the inhibition of T by AT in presence of exogenous heparin (hep). The AT-heparin complex was preformed by incubation for 5 min at 37°C of AT (0.1 μM) with high-affinity heparin (HA-hep, 250 ng/ml) and then added to a solution of T (90 nM) or T preincubated with either the non-acidic form (250 nM) or the acidic form (160 nM) of TM. Residual T activity was determined after various times for up to 5 min. Controls were performed by substituting buffer for TM. It was found that T bound to the non-acidic form of TM was inhibited at the same rate as free T (90% T inhibited within 30 sec), whereas T bound to the acidic form of TM was inhibited at a much slower rate (40 to 50% T inhibited after 5 min). Similar results were obtained even when HA-hep was used in excess (up to 750 ng/ml). The data suggest that the acidic domain of rabbit TM is primarily responsible for the retardation of T-AT complex formation in presence of exogenous hep. It is proposed that the polyanionic (endogenous heparin-like) component of rabbit TM blocks a site on T required for binding of T to hep in the ternary T-Hep-AT complex.

Role of Thrombin Exosites in Protection from Inhibition by Antithrombin in the Presence of Heparin and Fibrin.

Blood ◽

10.1182/blood.v104.11.1721.1721 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1721-1721

Author(s):

James C. Fredenburgh ◽

Alan R. Stafford ◽

Jeffrey I. Weitz

Keyword(s):

Ternary Complex ◽

Dermatan Sulfate ◽

Order Rate Constant ◽

Thrombin Inhibitors ◽

Direct Thrombin Inhibitors ◽

Heparin Cofactor Ii ◽

Fold Reduction ◽

The Individual ◽

Two Populations

Abstract The γ-chain of fibrinogen exists in two forms, γA and γ′, such that circulating fibrinogen consists of two populations, γA/γA (90%) and γA/γ′ (10%). The 16 amino acid extension at the COOH-terminus of the γ′ chain contains numerous negatively-charged residues. This alteration endows γA/γ′-fibrin (Fn) with a greater capacity to bind thrombin (IIa), a feature that may render thrombi prothrombotic. The purpose of this study was to explore how the various mechanisms by which IIa binds to Fn impact on IIa protection from inactivation by antithrombin/heparin. IIa binds weakly to γA/γA-Fn utilizing exosite 1. IIa binds with higher affinity to γA/γ′-Fn due to the additional exosite 2/γ′ interaction. In the presence of heparin, IIa can bind to γA/γA-Fn with high affinity by forming a ternary complex wherein heparin bridges IIa to Fn via exosite 2, thereby heightening the interaction of IIa with Fn via exosite 1. Consequently, the amount of IIa bound to γA/γA-Fn clots increases in the presence of heparin. Formation of the γA/γA-Fn/heparin/IIa ternary complex reduces the second order rate constant of IIa inhibition by antithrombin (AT) 11-fold compared with the heparin-catalyzed rate of inhibition of free IIa (1.1x108 M−1 min−1). This reduction reflects the inability of AT-bound heparin to access exosite 2. When γA/γ′-Fn is used in place of γA/γA-Fn, the heparin-catalyzed rate is reduced 55-fold. The enhanced protection with γA/γ′-Fn is due to the exosite 2-mediated interaction of IIa with the γ′-chain because addition of an antibody against the γ′ sequence that blocks this interaction reduces the protection from AT to the level seen with γA/γA-Fn. Thus, both γA/γ′-Fn/IIa and γA/γA-Fn/heparin/IIa complexes restrict the access of heparin to exosite 2, thereby impairing inhibition by heparin/AT. Heparin cofactor II (HCII) also utilizes heparin to bridge to IIa, but, unlike AT, HCII must also directly engage exosite 1 to effect IIa inhibition. Consequently, the heparin-catalyzed rate of IIa inhibition by HCII is reduced 27-fold in the presence of γA/γA-Fn compared with the 11-fold reduction with AT. To examine the contribution of the heparin-Fn interaction to this phenomenon, dermatan sulfate (DS) was used to catalyze HCII because, unlike heparin, DS does not bind Fn. γA/γA-Fn produced only a 5-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This suggests that in the absence of heparin, occupation of exosite 1 by γA/γA-Fn only modestly impairs inhibition by HCII. In contrast, γA/γ′-Fn produced a 28-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This protection is abolished by addition of the antibody that blocks binding of IIa to the γ′ chain. Therefore, although exosite 1 mediates IIa binding to both γA/γA- and γA/γ′-Fn, interaction of exosite 2 with the γ′-chain in γA/γ′-Fn heightens exosite 1-mediated binding. These findings provide independent confirmation that ligation of both exosites on IIa accentuates the affinity of the individual exosite 1 and 2 interactions. Thus, reactants that require access to either exosite will be restricted when IIa binding is mediated by both exosites as occurs with γA/γA-Fn/heparin or with γA/γ′-Fn. These results confirm that Fn can serve as a reservoir of active IIa and that bound IIa is protected from inhibition by circulating inhibitors. Our data also highlight the limitations of physiological inhibitors of IIa and validate the need for development of direct thrombin inhibitors.

Heparin Cofactor II (HCII) Levels and the Risk of Ischemic Stroke. No Deleterious Role of HCII Deficiency but Potential Protective Role of High Plasma Level

Blood ◽

10.1182/blood.v112.11.5344.5344 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5344-5344

Author(s):

Pierre A. Toulon ◽

Mathieu Zuber ◽

Raphael Adda ◽

Jean-Louis Mas

Keyword(s):

Ischemic Stroke ◽

Dermatan Sulfate ◽

Ethnic Origin ◽

High Plasma ◽

Protective Role ◽

Thrombin Inhibitor ◽

Normal Range ◽

Heparin Cofactor Ii ◽

Chromogenic Assay

Abstract Background: Heparin cofactor II (HCII) is a thrombin inhibitor which activity is dramatically enhanced by heparin and more specifically by dermatan sulfate, another glycosaminoglycan. Its pathophysiological role remains unknown even if cases of HCII deficiency were reported in patients with a history of venous or arterial thrombosis. Recently, it was suggested that high plasma HCII level could play a protective role against atherosclerosis in elderly individuals and protect from restenosis after femoro-popliteal or coronary artery stenting. The aim of this case-control study was to investigate the significance of plasma HCII level in the development of ischemic stroke. Methods: 81 cases (44 M and 37 F, mean age of 43 years, range: 22–63) and 80 age-and sex-matched healthy controls of the same ethnic origin. HCII activity was evaluated as antithrombin dermatan sulfate cofactor activity using a chromogenic assay. Test results were expressed in arbitrary unit per mL: 1 U/mL is the activity found in 1 mL of pooled normal human plasma. Results: Plasma HCII activity was not significantly different in cases and in controls (1.04±0.17 vs. 1.00±0.23). The same applied after stratification in males and in females. The normal range for HCII, previously defined in a large cohort of healthy individuals, was between 0.60 and 1.40 U/mL. The frequency of HCII deficiency was not significantly different in cases and in controls [n=3/81 (3.7%) vs. n=2/80, (2.5%); p=1.00] and the same applied to the frequency of HCII level above 1.40 U/mL [n=0/81 (0%) vs. n=2/80 (2.5%); p=0.25]. Interestingly, HCII activity was found above 1.30 U/mL in 7 controls and in only 1 case (p<0.05), none presenting with any of pathophysiological state usually associated with increased HCII levels such as pregnancy, oral contraceptive, inflammation, infection, or acute phase of illness. Conclusions: These results suggest that HCII deficiency is not in itself a risk factor for the development of ischemic stroke. Even if the frequency of HCII level above the normal range was not statistically different in cases and in controls, the significance of a higher proportion of controls than cases presenting with a HCII level in the high end of the normal range deserves to be further evaluated in connection with a potential protective role against ischemic stroke.

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

Blood ◽

10.1182/blood.v81.7.1771.1771 ◽

1993 ◽

Vol 81 (7) ◽

pp. 1771-1777 ◽

Cited By ~ 13

Author(s):

P Sie ◽

D Dupouy ◽

C Caranobe ◽

M Petitou ◽

B Boneu

Keyword(s):

Catalytic Activity ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Gel Filtration ◽

Heparin Cofactor Ii ◽

High Affinity ◽

Suitable Target ◽

The Relationship

Abstract 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.