Heparin Facilitates Dissociation of Complexes between Thrombin and a Reactive Site Mutant (L444R) of Heparin Cofactor II

Han, Jin-Hua; Deerlin, Vivianna M.D. Van; Tollefsen, Douglas M.

doi:10.1074/jbc.272.13.8243

Cited by 17 publications

(27 citation statements)

References 24 publications

(12 reference statements)

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“…Whereas unfractionated DS, desmin, and low molecular weight fractions of desmin increase the rate of thrombin inactivation by pHCII Ͼ 1000-fold, Mut D inactivates thrombin at a rate only 130-fold greater than the basal rate of thrombin inactivation by pHCII. These observations raise the possibility that the allosteric changes induced by the binding of unfractionated and low molecular weight DS to HCII are more extensive than those produced by heparin or by the mutations introduced into the GAG-binding domains of Mut D. This concept is supported by the observation that a reactive site HCII variant with a Leu 444 3 Arg mutation is stimulated by DS to a greater extent than by heparin (15,39). Further support comes from our findings that the heparin-and DS-binding sites on HCII are not identical.…”

Section: Discussionsupporting

confidence: 67%

“…This is corroborated by reports that heparin produces little stimulation in the rate of inactivation of ␥-thrombin (32, 33) or chymotrypsin (41) by HCII. Furthermore, because conversion of Leu 444 to Arg makes HCII 100-fold more efficient at inactivating thrombin (15,39), it is possible that the reactive site loop of HCII is in a more accessible conformation than that of AT. These observations suggest that displacement of the amino-terminal domain is the predominant mechanism by which HCII is allosterically activated by GAGs.…”

Section: Discussionmentioning

confidence: 99%

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Comparison of Heparin- and Dermatan Sulfate-mediated Catalysis of Thrombin Inactivation by Heparin Cofactor II

Liaw¹,

Austin²,

Fredenburgh³

et al. 1999

Journal of Biological Chemistry

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Heparin and dermatan sulfate activate heparin cofactor II (HCII) comparably, presumably by liberating the amino terminus of HCII to bind to exosite I of thrombin. To explore this model of activation, we systematically substituted basic residues in the glycosaminoglycanbinding domain of HCII with neutral amino acids and measured the rates of thrombin inactivation by the mutants. Mutant , demonstrated a ϳ130-fold increased rate of thrombin inactivation that was unaffected by the presence of glycosaminoglycans. The increased rate reflects displacement of the amino terminus of mutant D because (a) mutant D inactivates ␥-thrombin at a 65-fold slower rate than ␣-thrombin, (b) hirudin-(54 -65) decreases the rate of thrombin inactivation, and (c) deletion of the amino terminus of mutant D reduces the rate of thrombin inactivation ϳ100-fold. We also examined the contribution of glycosaminoglycan-mediated bridging of thrombin to HCII to the inhibitory process. Whereas activation of HCII by heparin was chain-length dependent, stimulation by dermatan sulfate was not, suggesting that dermatan sulfate does not utilize a template mechanism to accelerate the inhibitory process. Fluorescence spectroscopy revealed that dermatan sulfate evokes greater conformational changes in HCII than heparin, suggesting that dermatan sulfate stimulates HCII by producing more effective displacement of the amino terminus.Heparin cofactor II (HCII), 1 a serpin found in human plasma at a concentration of 1.2 M, selectively inactivates thrombin in a reaction that is accelerated Ͼ1000-fold by glycosaminoglycans (GAGs) such as heparin, dermatan sulfate, and heparan sulfate (1). A second serpin, antithrombin (AT), also inactivates thrombin but differs from HCII in four important ways. First, whereas HCII only inactivates thrombin, AT inactivates other coagulation enzymes including factors Xa and IXa (2). Second, the high affinity interaction of heparin with AT is mediated by a unique pentasaccharide sequence found only in a subpopulation of heparin molecules (3-5). In contrast, heparin does not possess a high affinity sequence for HCII (6). Furthermore, dermatan sulfate (DS), a GAG found in the extracellular matrix of connective tissue (7, 8), activates HCII, but has no effect on AT (1). Third, the uncatalyzed rate of thrombin inactivation by AT is about 10-fold faster than that for HCII, probably reflecting differences in the amino acid residue at their P-1 position, with AT containing an Arg residue and HCII a Leu (9). Fourth, HCII possesses a unique 75-amino acid domain at its amino terminus that binds to thrombin exosite I, an interaction analogous to the binding of the carboxyl terminus of hirudin to exosite I (1).Although the uncatalyzed rate of thrombin inactivation by HCII is slower than that for AT, in the presence of heparin or DS, HCII inactivates thrombin at a rate similar to that at which AT inactivates thrombin when heparin is present (1, 10). The current model to explain GAG-mediated catalysis of HCII inactivation of thrombin suggests ...

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Comparison of Heparin- and Dermatan Sulfate-mediated Catalysis of Thrombin Inactivation by Heparin Cofactor II

Liaw¹,

Austin²,

Fredenburgh³

et al. 1999

Journal of Biological Chemistry

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“…Consistent with this result is the observation that the related serpin, heparin cofactor II (L444R variant), inhibits thrombin with a high stoichiometry in the presence of heparin, but not dermatan sulphate. 40 This can best be interpreted as an allosteric effect of heparin binding to thrombin since thrombin does not bind to dermatan sulfate with appreciable affinity.…”

Section: Discussionmentioning

confidence: 99%

Heparin-induced substrate behavior of antithrombin Cambridge II

Mushunje

Zhou

Carrell

et al. 2003

Blood

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Cambridge II (A384S) is a highly prevalent antithrombin variant in the British population (1.14 per 1000) and predisposes carriers to a mild but significant increased risk of thrombosis. To determine if the association of Cambridge II with thrombophilia is due to a perturbation of the antithrombin inhibitory mechanism, we expressed and characterized the variant. Antithrombin Cambridge II was found to be normal in its affinity for heparin, its ability to form sodium dodecyl sulfate-stable complexes with factor Xa and thrombin, and its uncatalyzed stoichiometries and rates of inhibition. However, in the presence of full-length heparin there was a 3-and 7-fold increase in stoichiometry of inhibition of factor Xa and thrombin. The stoichiometries were not affected by pen

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“…Human heparin cofactor II and thrombin were purified as described previously (24). The full-length cDNA for human heparin cofactor II was cloned in the expression vector pET-3d (Novagen, Madison, WI) and mutated to produce the Lys 173 3 Glc, Lys 185 3 Asn, and Arg 189 3 His variants as described previously (25,26). The mutations and ligation sites were verified by dideoxynucleotide sequencing.…”

Section: Methodsmentioning

confidence: 99%

Highly Sulfated Dermatan Sulfates from Ascidians

Pavão¹,

Aiello²,

Werneck³

et al. 1998

Journal of Biological Chemistry

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170

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Dermatan sulfate from A. nigra has no discernible anticoagulant activity, which indicates that 4-O-sulfation of the N-acetyl-␤-D-galactosamine is essential for the anticoagulant activity of this glycosaminoglycan. In contrast dermatan sulfates from S. plicata and H. pyriformis are potent anticoagulants due to potentiation of thrombin inhibition by heparin cofactor II. These ascidian dermatan sulfates have ϳ10-fold and ϳ6-fold higher activity with heparin cofactor II than native and an oversulfated mammalian dermatan sulfate, respectively. They have no effect on thrombin or factor Xa inhibition by antithrombin. These naturally oversulfated ascidian dermatan sulfates are sulfated at selected sites required for interaction with heparin cofactor II and thus have specific and potent anticoagulant activity.Sulfated polysaccharides constitute a complex group of macromolecules known to possess a wide range of important biological properties. These anionic polymers are widespread in nature, occurring in a great variety of organisms. Marine invertebrates are a rich source of sulfated polysaccharides with novel structures (1-15).The ascidians (Chordata-Tunicata) are marine invertebrates covered by an external supportive tissue called the tunic, surrounding a body. The tunic contains large amounts of a unique high molecular mass sulfated ␣-L-galactan (1, 2, 5-7). Recently, we reported the occurrence of dermatan sulfate-like glycosaminoglycan in the body of this invertebrate (11, 13).Mammalian dermatan sulfate is an anticoagulant due to selective inhibition of thrombin by potentiating heparin cofactor II activity (16,17). Although of lower in vitro anticoagulant potency than heparin, it has efficacy in vivo with less hemorrhagic risk (18). Thus several authors have suggested using mammalian dermatan sulfate as an alternative antithrombotic polysaccharide (18 -21).In view of the increasing interest in the anticoagulant and antithrombotic actions of dermatan sulfate, we have characterized the fine chemical structure of dermatan sulfates extracted from the ascidian body and tested this compound in coagulation assays, including activation of heparin cofactor II. In addition, there is now more interest in therapeutics prepared from nonmammalian sources, thus reducing the risk of contamination with pathogenic agents.In the present work, we report that dermatan sulfates isolated from different ascidian species have distinguishable patterns and proportions of sulfate substitution. These ascidian dermatan sulfates (together with native and oversulfated mammalian dermatan sulfate), where the extent and position of sulfate substitution have been fully characterized, are a valuable tool to trace the relationship between structure versus anticoagulant activity of this glycosaminoglycan. Dermatan sulfates with potent anticoagulant potency and high heparin cofactor II activity have been found. EXPERIMENTAL PROCEDURESMaterials-Heparan sulfate from human aorta was extracted and purified as described previously (22). Chondroitin 4-sulfate fro...

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Heparin Facilitates Dissociation of Complexes between Thrombin and a Reactive Site Mutant (L444R) of Heparin Cofactor II

Cited by 17 publications

References 24 publications

Comparison of Heparin- and Dermatan Sulfate-mediated Catalysis of Thrombin Inactivation by Heparin Cofactor II

Comparison of Heparin- and Dermatan Sulfate-mediated Catalysis of Thrombin Inactivation by Heparin Cofactor II

Heparin-induced substrate behavior of antithrombin Cambridge II

Highly Sulfated Dermatan Sulfates from Ascidians

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