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Douglas M Tollefsen - One of the best experts on this subject based on the ideXlab platform.
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proteolytic activation transforms Heparin Cofactor II into a host defense molecule
Journal of Immunology, 2013Co-Authors: Martina Kalle, Gopinath Kasetty, Martin Malmsten, Praveen Papareddy, Douglas M Tollefsen, Matthias Morgelin, Artur SchmidtchenAbstract:The abundant serine proteinase inhibitor Heparin Cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.
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glycosaminoglycan binding properties and kinetic characterization of human Heparin Cofactor II expressed in escherichia coli
Analytical Biochemistry, 2010Co-Authors: Suryakala Sarilla, Douglas M Tollefsen, Sally Y Habib, David B Friedman, Diana R Arnett, Ingrid M VerhammeAbstract:Abstract Irreversible inactivation of α-thrombin (T) by the serpin, Heparin Cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) Heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine–Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for Heparin and DS were compared. Recombinant and deglycosylated HCII bound Heparin with dissociation constants (KD) of 6 ± 1 and 7 ± 1 μM, respectively, ∼6-fold tighter than plasma HCII, with KD 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with KD 4 ± 1 μM, was ∼4-fold tighter than for plasma HCII, with KD 15 ± 4 μM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn169 N-glycan with the HCII Heparin-binding site.
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vascular dermatan sulfate and Heparin Cofactor II
Progress in Molecular Biology and Translational Science, 2010Co-Authors: Douglas M TollefsenAbstract:Abstract Heparin Cofactor II (HCII) is a plasma protease inhibitor of the serpin family that inactivates thrombin by forming a covalent 1:1 complex. The rate of complex formation increases more than 1000-fold in the presence of dermatan sulfate (DS). Endothelial injury allows circulating HCII to enter the vessel wall, where it binds to DS and presumably becomes activated. Mice that lack HCII develop carotid artery thrombosis more rapidly than wild-type mice after oxidative damage to the endothelium. These mice also have increased arterial neointima formation following mechanical injury and develop more extensive atherosclerotic lesions when made hypercholesterolemic. Similarly, low plasma HCII levels appear to be a risk factor for atherosclerosis and in-stent restenosis in human subjects. These observations suggest that a major function of the HCII–DS system is to regulate the physiologic response to arterial injury.
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vascular dermatan sulfate regulates the antithrombotic activity of Heparin Cofactor II
Blood, 2008Co-Authors: Li He, Tusar Giri, Cristina P Vicente, Douglas M TollefsenAbstract: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.
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accelerated atherogenesis and neointima formation in Heparin Cofactor II deficient mice
Blood, 2007Co-Authors: Cristina P Vicente, Li He, Douglas M TollefsenAbstract: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.
Frank C Church - One of the best experts on this subject based on the ideXlab platform.
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Heparin Cofactor II discovery properties and role in controlling vascular homeostasis
Seminars in Thrombosis and Hemostasis, 2011Co-Authors: Jennifer W Mitchell, Frank C Church, Yolanda M FortenberryAbstract:Abstract Heparin Cofactor II (HCII) is a serine protease inhibitor (serpin) found in high concentrations in human plasma. Despite its discovery >30 years ago, its physiological function is still poorly understood. It is known to inhibit thrombin, the predominant coagulation protease, and HCII-thrombin complexes have been found in plasma, yet it is thought to contribute little to normal hemostasis. However, thrombin has several other physiological functions, and therefore many biological roles for HCII need consideration. The unique structure and mechanism of action of HCII have helped guide our understanding of HCII. In particular, HCII binds many glycosaminoglycans (GAGs) such as Heparin and Heparin sulfate as well as several different polyanions to enhance its inhibition of thrombin. Distinctly, HCII is able to use the GAG dermatan sulfate for accelerated thrombin inhibition. Dermatan sulfate is found in high concentrations in the walls of blood vessels as well as in placental tissue. This knowledge has led to research indicating that HCII may play a protective role in atherosclerosis and placental thrombosis. Additionally, pharmaceuticals are being developed that use the dermatan sulfate activation of HCII for anticoagulation. Although much research is still needed to fully understand HCII, this humble protein may have significant impact in our medical future. This article reviews the laboratory history, protein characteristics, structure-activity relationships, protease inhibition, physiological function, and medical relevance of HCII in hopes of regenerating interest in this sometimes forgotten serpin.
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Heparin Cofactor II in atherosclerotic lesions from the pathobiological determinants of atherosclerosis in youth pday study
Experimental and Molecular Pathology, 2009Co-Authors: Carolyn Deans, Maureane Hoffman, David B Thomas, Gray T Malcom, Arthur W Zieske, Jack P Strong, Gary G Koch, Frank C ChurchAbstract:Abstract Heparin Cofactor II (HCII) is a serine protease inhibitor (serpin) that has been shown to be a predictor of decreased atherosclerosis in the elderly and protective against atherosclerosis in mice. HCII inhibits thrombin in vitro and HCII–thrombin complexes have been detected in human plasma. Moreover, the mechanism of protection against atherosclerosis in mice was determined to be the inhibition of thrombin. Despite this evidence, the presence of HCII in human atherosclerotic tissue has not been reported. In this study, using samples of coronary arteries obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, we explore the local relationship between HCII and (pro)thrombin in atherosclerosis. We found that HCII and (pro)thrombin are co-localized in the lipid-rich necrotic core of atheromas. A significant positive correlation between each protein and the severity of the atherosclerotic lesion was present. These results suggest that HCII is in a position to inhibit thrombin in atherosclerotic lesions where thrombin can exert a proatherogenic inflammatory response. However, these results should be tempered by the additional findings from this, and other studies, that indicate the presence of other plasma proteins (antithrombin, albumin, and α 1 -protease inhibitor) in the same localized region of the atheroma.
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designed polyanionic coiled coil proteins acceleration of Heparin Cofactor II inhibition of thrombin
International Journal of Peptide and Protein Research, 2009Co-Authors: Laura G Melton, Frank C Church, Bruce W EricksonAbstract:Novel polyanionic proteins were designed to increase the rate of Heparin Cofactor II (HC) inhibition of α-thrombin, an essential protease in the coagulation cascade. Two α-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue dimer containing 14 Glu residues (E14C), plus two 31-residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two-chain α-helical coiled coils at pH 7 and 25 °C. The control peptides were only partially α-helical. E14C remained folded at 90 °C but E8C was half unfolded at 49 °C. Coiled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fold, respectively, the rate of HC inhibition of α-thrombin. None of these compounds accelerated antithrombin inhibition of α-thrombin, and neither control peptide accelerated HC inhibition of α-thrombin. Acceleration of the HC inhibition of α-thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary complex. The results suggest that antithrombotic polyanionic α-helical coiled-coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biologcal activity. © Munksgaard 1995.
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molecular mapping of the thrombin Heparin Cofactor II complex
Journal of Biological Chemistry, 2004Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Holly R Gentry, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:Abstract We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) Heparin Cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 × 108 m-1 min-1 for HCII-Heparin when compared with 2.36 × 108 m-1 min-1 with wild-type thrombin and 0.03-0.53 × 108 m-1 min-1 for HCII-dermatan sulfate when compared with 5.23 × 108 m-1 min-1 with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a “double bridge” mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the “hirudin-like” domain of HCII with thrombin exosite-1.
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crystal structures of native and thrombin complexed Heparin Cofactor II reveal a multistep allosteric mechanism
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Trevor Baglin, Frank C Church, Robin W Carrell, Charles T Esmon, James A HuntingtonAbstract:The serine proteases sequentially activated to form a fibrin clot are inhibited primarily by members of the serpin family, which use a unique β-sheet expansion mechanism to trap and destroy their targets. Since the discovery that serpins were a family of serine protease inhibitors there has been controversy as to the role of conformational change in their mechanism. It now is clear that protease inhibition depends entirely on rapid serpin β-sheet expansion after proteolytic attack. The regulatory advantage afforded by the conformational mobility of serpins is demonstrated here by the structures of native and S195A thrombin-complexed Heparin Cofactor II (HCII). HCII inhibits thrombin, the final protease of the coagulation cascade, in a glycosaminoglycan-dependent manner that involves the release of a sequestered hirudin-like N-terminal tail for interaction with thrombin. The native structure of HCII resembles that of native antithrombin and suggests an alternative mechanism of allosteric activation, whereas the structure of the S195A thrombin–HCII complex defines the molecular basis of allostery. Together, these structures reveal a multistep allosteric mechanism that relies on sequential contraction and expansion of the central β-sheet of HCII.
Mauro S G Pavao - One of the best experts on this subject based on the ideXlab platform.
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dermatan sulfate in tunicate phylogeny order specific sulfation pattern and the effect of 4idoa 2 sulfate β 1 3galnac 4 sulfate β 1 motifs in dermatan sulfate on Heparin Cofactor II activity
BMC Biochemistry, 2011Co-Authors: Eliene O. Kozlowski, P C Lima, Tito Monteiro Da Cruz Lotufo, Cristina P Vicente, Kazuyuki Sugahara, Mauro S G PavaoAbstract:Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [→4IdoA(2S)β-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for Heparin Cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low Heparin Cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how Heparin Cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfate)β-1→3GalNAcβ-1→] modulate Heparin Cofactor II activity of dermatan sulfate polymers. Thus, high and low Heparin Cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units. Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [→4IdoA(2S)β-1→3GalNAcβ-1→] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [→4IdoA(2S)β-1→3GalNAc(4S)β-1→] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased Heparin Cofactor II stimulating activity.
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selective cleavage and anticoagulant activity of a sulfated fucan stereospecific removal of a 2 sulfate ester from the polysaccharide by mild acid hydrolysis preparation of oligosaccharides and Heparin Cofactor II dependent anticoagulant activity
Glycobiology, 2005Co-Authors: Vitor H. Pomin, Mariana S Pereira, Ana Paula Valente, Douglas M Tollefsen, Mauro S G PavaoAbstract:A linear sulfated fucan with a regular repeating sequence of [3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2,4SO₄)-(1[rightwards arrow]3)-[alpha]-L-Fucp-(2SO₄)-(1[rightwards arrow]][subscript n] is an anticoagulant polysaccharide mainly due to thrombin inhibition mediated by Heparin Cofactor II. No specific enzymatic or chemical method is available for the preparation of tailored oligosaccharides from sulfated fucans. We employ an apparently nonspecific approach to cleave this polysaccharide based on mild hydrolysis with acid. Surprisingly, the linear sulfated fucan was cleaved by mild acid hydrolysis on an ordered sequence. Initially a 2-sulfate ester of the first fucose unit is selectively removed. Thereafter the glycosidic linkage between the nonsulfated fucose residue and the subsequent 4-sulfated residue is preferentially cleaved by acid hydrolysis, forming oligosaccharides with well-defined size. The low-molecular-weight derivatives obtained from the sulfated fucan were employed to determine the requirement for interaction of this polysaccharide with Heparin Cofactor II and to achieve complete thrombin inhibition. The linear sulfated fucan requires significantly longer chains than mammalian glycosaminoglycans to achieve anticoagulant activity. A slight decrease in the molecular size of the sulfated fucan dramatically reduces its effect on thrombin inactivation mediated by Heparin Cofactor II. Sulfated fucan with [approximately] 45 tetrasaccharide repeating units binds to Heparin Cofactor II but is unable to link efficiently the plasma inhibitor and thrombin. This last effect requires chains with [approximately] 100 or more tetrasaccharide repeating units. We speculate that the template mechanism may predominate over the allosteric effect in the case of the linear sulfated fucan inactivation of thrombin in the presence of Heparin Cofactor II.
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antithrombotic activity of dermatan sulfate in Heparin Cofactor II deficient mice
Blood, 2004Co-Authors: Cristina P Vicente, Mauro S G Pavao, Li He, Douglas M TollefsenAbstract:Heparin Cofactor II (HCII) is a plasma protein that inhibits thrombin rapidly in the presence of dermatan sulfate or Heparin. We previously reported that the time to thrombotic occlusion of the carotid artery after photochemical injury was shorter in HCII-deficient mice than in wild-type control animals. In this paper, we describe the antithrombotic activity of dermatan sulfate in wild-type and HCII-deficient mice. Intravenous administration of porcine skin dermatan sulfate induced a dose-dependent prolongation of the carotid artery occlusion time in HCII+/+ mice that was not observed in HCII-/- animals. Pharmacokinetic studies suggested that porcine skin dermatan sulfate expresses antithrombotic activity after being transferred from the plasma to sites in the vessel wall. Using invertebrate dermatan sulfate preparations, we showed that N-acetylgalactosamine-4-O-sulfate residues are required for the HCII-dependent antithrombotic effect. Furthermore, the invertebrate dermatan sulfates, which have higher charge densities than mammalian dermatan sulfate, slightly prolonged the thrombotic occlusion time of HCII-/- mice. These results indicate that HCII mediates the antithrombotic effect of porcine skin dermatan sulfate after injury to the carotid arterial endothelium in mice, whereas more highly charged dermatan sulfates possess weak antithrombotic activity independent of HCII. (Blood. 2004;104:3965-3970)
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the medicinal plant porana volubilis contains polysaccharides with anticoagulant activity mediated by Heparin Cofactor II
Thrombosis Research, 2002Co-Authors: Seonjoo Yoon, Mariana S Pereira, Yu Ryang Pyun, Jaekwan Hwang, Mauro S G Pavao, Paulo A S MouraoAbstract:Abstract We searched for polysaccharides with anticoagulant activity and inhibitory action on platelet aggregation induced by collagen in 59 species of medicinal plants. We then concentrated our studies on the polysaccharide from the species Porana volubilis , which showed the highest anticoagulant activity among the plants tested. The polysaccharide from this species has an average molecular mass of ∼10 kDa, contains mainly galactose, galacturonic acid, and mannose but no sulfate esters. Its anticoagulant activity is mediated by the enhancement of thrombin inhibition that in turn is mediated by Heparin Cofactor II but not by antithrombin. The galacturonic acid residues are essential for activity since after reduction of its carboxyl groups the anticoagulant activity disappears. Our report is the first description of a natural nonsulfated polysaccharide from higher plants with anticoagulant activity, which may constitute a new source of compounds with action on coagulation and, perhaps, on thrombosis.
Seonjoo Yoon - One of the best experts on this subject based on the ideXlab platform.
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a sulfated fucan from the brown alga laminaria cichorioides has mainly Heparin Cofactor II dependent anticoagulant activity
Carbohydrate Research, 2007Co-Authors: Seonjoo Yoon, Yu Ryang Pyun, Jaekwan HwangAbstract:Abstract The major acidic polysaccharide from the brown alga Laminaria cichorioides is a complex and heterogeneous sulfated fucan. Its preponderant structure is a 2,3-disulfated, 4-linked α-fucose unit. The purified polysaccharide has a potent anticoagulant activity, as estimated by APTT assay (∼40 IU/mg), which is mainly mediated by thrombin inhibition by Heparin Cofactor II. It also accelerates thrombin and factor Xa inhibition by antithrombin but at a lower potency. Sulfated fucan from L. cichorioides is a promising anticoagulant polysaccharide and a possible alternative for an antithrombotic compound due to its preferential Heparin Cofactor II-dependent activity.
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a sulfated fucan from the brown alga laminaria cichorioides has mainly Heparin Cofactor II dependent anticoagulant activity
Carbohydrate Research, 2007Co-Authors: Seonjoo Yoon, Yu Ryang Pyun, Jaekwan Hwang, Paulo A S MouraoAbstract:The major acidic polysaccharide from the brown alga Laminaria cichorioides is a complex and heterogeneous sulfated fucan. Its preponderant structure is a 2,3-disulfated, 4-linked alpha-fucose unit. The purified polysaccharide has a potent anticoagulant activity, as estimated by APTT assay ( approximately 40 IU/mg), which is mainly mediated by thrombin inhibition by Heparin Cofactor II. It also accelerates thrombin and factor Xa inhibition by antithrombin but at a lower potency. Sulfated fucan from L. cichorioides is a promising anticoagulant polysaccharide and a possible alternative for an antithrombotic compound due to its preferential Heparin Cofactor II-dependent activity.
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the medicinal plant porana volubilis contains polysaccharides with anticoagulant activity mediated by Heparin Cofactor II
Thrombosis Research, 2002Co-Authors: Seonjoo Yoon, Mariana S Pereira, Yu Ryang Pyun, Jaekwan Hwang, Mauro S G Pavao, Paulo A S MouraoAbstract:Abstract We searched for polysaccharides with anticoagulant activity and inhibitory action on platelet aggregation induced by collagen in 59 species of medicinal plants. We then concentrated our studies on the polysaccharide from the species Porana volubilis , which showed the highest anticoagulant activity among the plants tested. The polysaccharide from this species has an average molecular mass of ∼10 kDa, contains mainly galactose, galacturonic acid, and mannose but no sulfate esters. Its anticoagulant activity is mediated by the enhancement of thrombin inhibition that in turn is mediated by Heparin Cofactor II but not by antithrombin. The galacturonic acid residues are essential for activity since after reduction of its carboxyl groups the anticoagulant activity disappears. Our report is the first description of a natural nonsulfated polysaccharide from higher plants with anticoagulant activity, which may constitute a new source of compounds with action on coagulation and, perhaps, on thrombosis.
Hermann Ragg - One of the best experts on this subject based on the ideXlab platform.
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Zinc ions promote the interaction between Heparin and Heparin Cofactor II
FEBS Letters, 2003Co-Authors: Ralf Eckert, Hermann RaggAbstract:The effects of bivalent cations on Heparin binding, structure, and thrombin inhibition rates of Heparin Cofactor II were examined. Zn2+ – and to a lesser extent Cu2+ and Ni2+ – enhanced the interaction between Heparin Cofactor II and Heparin as demonstrated by Heparin affinity chromatography and surface plasmon resonance experiments. Metal chelate chromatography and increased intrinsic protein fluorescence in the presence of Zn2+ indicated that Heparin Cofactor II has metal ion-binding properties. The results are compatible with the hypothesis that Zn2+ induces a conformational change in Heparin Cofactor II that favors its interaction with Heparin.
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tyrosine sulfation and n glycosylation of human Heparin Cofactor II from plasma and recombinant chinese hamster ovary cells and their effects on Heparin binding
FEBS Journal, 2002Co-Authors: Christoph Bohme, Manfred Nimtz, Eckart Grabenhorst, Harald S Conradt, Annemarie Strathmann, Hermann RaggAbstract:The structure of post-translational modifications of human Heparin Cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on Heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of Heparin Cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with α26 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLex motif. Proximal α16 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in > 90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively α23-linked N-acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of Heparin Cofactor II molecules that eluted with higher ionic strength from Heparin–Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans.