Antithrombin stabilisation by sulfated carbohydrates correlates with anticoagulant activity

Thermal stabilisation of native antithrombin-III (AT), determined using differential scanning fluorimetry, correlated with the anticoagulant activity of heparin and heparin-related saccharides. Similar conformational changes were induced in native AT by a variety of active and inactive heparin-related sulfated carbohydrates, measured in solution using synchrotron radiation circular dichroism, and their anticoagulant activities. Measurement of native AT stabilisation provides a convenient assay for prospective anticoagulants and represents an additional parameter by which to compare biosimilar heparins.National Science Foundation's National High Magnetic Field Laboratory User Program in the Advanced Magnetic Resonance Imaging and Spectroscopy (AMRIS) Facility (McKnight Brain Institute, University of Florida, USA)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo UNIFESP, Dept Biochem, BR-04044020 São Paulo, BrazilDiamond Light Source Ltd, Beam Line Circular Dichroism 23, Didcot OX11 ODE, Oxon, EnglandUniv Liverpool, Dept Struct & Chem Biol, Liverpool L69 7ZB, Merseyside, EnglandIst Ric Chim & Biochim G Ronzoni, I-20133 Milan, ItalyUniv Fed Rio Grande do Norte, Dept Biochem, BR-59072970 Natal, RN, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc

A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

The year 2007 was marked by widespread adverse clinical responses to heparin use, leading to a global recall of potentially affected heparin batches in 2008. Several analytical methods have since been developed to detect impurities in heparin preparations; however, many are costly and dependent on instrumentation with only limited accessibility. A method based on a simple UV-scanning assay, combined with principal component analysis (PCA), was developed to detect impurities, such as glycosaminoglycans, other complex polysaccharides and aromatic compounds, in heparin preparations. Results were confirmed by NMR spectroscopy. This approach provides an additional, sensitive tool to determine heparin purity and safety, even when NMR spectroscopy failed, requiring only standard laboratory equipment and computing facilities

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k2 = 21±1 s−1) was much higher than the HNE deacylation step (k3 = 0.57±0.05 s−1). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k1 2.4-fold and reducing k−1 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k2 value, whereas the k3 value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs

Cysteine proteinase activity regulation - A possible role of heparin and heparin-like glycosaminoglycans

Papain is considered to be the archetype of cysteine proteinases. the interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. the conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. the presence of heparin significantly increases the a-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. the incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. in order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced a-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce a-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.Univ Mogi Cruzes, Ctr Interdisciplinar Invest Bioquim, Ctr Ciencias Biomed, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Inst Nacl Farmacol, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Inst Nacl Farmacol, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Inst Nacl Farmacol, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Inst Nacl Farmacol, Disciplina Biol Mol, BR-04044020 São Paulo, BrazilWeb of Scienc

Chemical reduction of carboxyl groups in heparin abolishes its vasodilatory activity

Previous studies have shown that heparin induces vascular relaxation via integrin-dependent nitric oxide (NO)-mediated activation of the muscarinic receptor. The aim of this study was to identify the structural features of heparin that are necessary for the induction of vasodilatation. To address this issue, we tested heparin from various sources for their vasodilatation activities in the rat aorta ring. Structural and chemical characteristics of heparin, such as its molecular weight and substitution pattern, did not show a direct correlation with the vasodilation activity. Principal component analysis (PCA) of circular dichroism (CD), 1H-nuclear magnetic resonance (NMR) and vasodilation activity measurements confirmed that there is no direct relationship between the physico-chemical nature and vasodilation activity of the tested heparin samples. To further understand these observations, unfractionated heparin (UFH) from bovine intestinal mucosa, which showed the highest relaxation effect, was chemically modified. Interestingly, non-specific O- and N-desulfation of heparin reduced its anticoagulant, antithrombotic, and antihemostatic activities, but had no effect on its ability to induce vasodilation. On the other hand, chemical reduction of the carboxyl groups abolished heparin-induced vasodilation and reduced the affinity of heparin toward the extracellular matrix (ECM). In addition, dextran and dextran sulfate (linear non-sulfated and highly sulfated polysaccharides, respectively) did not induce significant relaxation, showing that the vasodilation activity of polysaccharides is neither charge-dependent nor backbone unspecific. Our results suggest that desulfated heparin molecules may be used as vasoactive agents due to their low side effects. J. Cell. Biochem. 113: 13591367, 2012. (c) 2011 Wiley Periodicals, Inc.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2006/61006-2]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Financiadora de Estudos e Projetos (FINEP)Financiadora de Estudos e Projetos (FINEP)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES