Characterization Of Heparin Research Articles

Characterization of Heparin's Conformational Ensemble by Molecular Dynamics Simulations and Nuclear Magnetic Resonance Spectroscopy.

Heparin is a highly charged, polysulfated polysaccharide and serves as an anticoagulant. Heparin binds to multiple proteins throughout the body, suggesting a large range of potential therapeutic applications. Although its function has been characterized in multiple physiological contexts, heparin's solution conformational dynamics and structure-function relationships are not fully understood. Molecular dynamics (MD) simulations facilitate the analysis of a molecule's underlying conformational ensemble, which then provides important information necessary for understanding structure-function relationships. However, for MD simulations to afford meaningful results, they must both provide adequate sampling and accurately represent the energy properties of a molecule. The aim of this study is to compare heparin's conformational ensemble using two well-developed force fields for carbohydrates, known as GLYCAM06 and CHARMM36, using replica exchange molecular dynamics (REMD) simulations, and to validate these results with NMR experiments. The anticoagulant sequence, an ultra-low-molecular-weight heparin, known as Arixtra (fondaparinux, sodium), was simulated with both parameter sets. The results suggest that GLYCAM06 matches experimental nuclear magnetic resonance three-bond J-coupling values measured for Arixtra better than CHARMM36. In addition, NOESY and ROESY experiments suggest that Arixtra is very flexible in the sub-millisecond time scale and does not adopt a unique structure at 25 C. Moreover, GLYCAM06 affords a much more dynamic conformational ensemble for Arixtra than CHARMM36.

Сложность патогенеза коагулопатии при COVID-19

Введение. Коронавирус SARS-CoV-2 является новым вирусом, обладающим способностью осуществлять трансмиссию воздушно-капельным путем, вызывая тяжелое течение атипичной пневмонии, нередко сочетающейся с коагулопатиями. Роль структурных белков коронавируса в их патогенезе неизвестна. Цель исследования: с помощью биоинформационного анализа выявить в структурных белках коронавируса SARS-CoV-2 последовательности, гомологичные белкам системы гемостаза, и рассмотреть возможные сценарии их участия в патогенезе коагулопатий при COVID-19, а также объяснить существование вирусостатического эффекта гепарина. Материалы и методы. Для компьютерного анализа были использованы доступные в Интернете базы данных первичных структур белков коронавирусов и их рецепторов, а также поверхностных белков других вирусов, белков системы гемостаза и иммунной системы. Сравнивали аминокислотный состав белков и распределение оснόвных аминокислот (аргинина и лизина) в их первичных последовательностях. С целью выявления пептидного (иммуноэпитопного) родства структурных белков коронавирусов с белками системы гемостаза человека был выполнен поиск гомологичных последовательностей в их белках. Результаты. В структурных белках коронавируса SARS-CoV-2 выявлено множество последовательностей, гомологичных белкам системы гемостаза и иммунной системы. В отличие от коронавирусов SARS-CoV и MERS-CoV, S1-субъединица S-белка коронавируса SARS-CoV-2 имеет положительную полярность. Заключение. Множество последовательностей в структурных белках коронавируса SARS-CoV-2, гомологичных белкам системы гемостаза, потенциально способны вы- зывать различные сценарии патогенеза коагулопатий. Положительная полярность S1-субъединицы S-белка коронавируса SARS-CoV-2 позволяет объяснить неспецифическое взаимодействие ее с гепарином и его вирусостатический (неантикоагулянтный) эффект. Background. The coronavirus SARS-CoV-2 is a new virus capable of human-human transmission and inducing a severe atypical pneumonia often associated with coagulopathy. A role of SARS-CoV-2 structural proteins in coagulopathy pathogenesis is unknown. Objectives: to use a bioinformation analysis to identify SARS-CoV-2 sequences in the structural proteins that are homologous to hemostasis system proteins, regard their possible participation in coagulopathy pathogenesis and explain the antiviral effect of heparin. Materials / Methods. For computer analysis, Internet databases were used of the primary structures of coronavirus proteins and their receptors, as well as surface proteins of other viruses, proteins of hemostasis and immune systems. The amino acid composition of proteins and the distribution of basic amino acids (arginine and lysine) in their primary sequences were compared. For detection of peptide (immunoepitopic) relationship of coronaviruses structural proteins with human hemostasis proteins, a search for homologous sequences in their proteins was performed. Results. Many sequences have been identified in structural proteins of SARS-CoV-2 coronavirus that are homologous to the proteins of hemostasis and immune systems. In contrast with SARS-CoV and MERS-CoV coronaviruses, the S1-subunit of SARS-CoV-2 coronavirus S-protein has a positive polarity. Conclusions. Many sequences in SARS-CoV-2 structural proteins that homologous to hemostasis system proteins are potentially responsible for coagulopathy pathogenesis. The positive polarity of the S1-subunit of SARS-CoV-2 S-protein explains its nonspecific interaction with heparin and its virostatic (non-anticoagulant) effect.

Characterization of heparin and severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) spike glycoprotein binding interactions

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread around the globe at an unprecedented rate. To date, no effective therapeutic is available to fight its associated disease, COVID-19. Our discovery of a novel insertion of glycosaminoglycan (GAG)-binding motif at S1/S2 proteolytic cleavage site (681–686 (PRRARS)) and two other GAG-binding-like motifs within SARS-CoV-2 spike glycoprotein (SGP) led us to hypothesize that host cell surface GAGs may interact SARS-CoV-2 SGPs to facilitate host cell entry. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 SGP bind more tightly to immobilized heparin (KD = 40 pM and 73 pM, respectively) than the SARS-CoV and MERS-CoV SGPs (500 nM and 1 nM, respectively). In competitive binding studies, the IC50 of heparin, tri-sulfated non-anticoagulant heparan sulfate, and non-anticoagulant low molecular weight heparin against SARS-CoV-2 SGP binding to immobilized heparin were 0.056 μM, 0.12 μM, and 26.4 μM, respectively. Finally, unbiased computational ligand docking indicates that heparan sulfate interacts with the GAG-binding motif at the S1/S2 site on each monomer interface in the trimeric SARS-CoV-2 SGP, and at another site (453–459 (YRLFRKS)) when the receptor-binding domain is in an open conformation. The current study serves a foundation to further investigate biological roles of GAGs in SARS-CoV-2 pathogenesis. Furthermore, our findings may provide additional basis for further heparin-based interventions for COVID-19 patients exhibiting thrombotic complications.

An integrated approach using orthogonal analytical techniques to characterize heparan sulfate structure

Heparan sulfate (HS), a glycosaminoglycan present on the surface of cells, has been postulated to have important roles in driving both normal and pathological physiologies. The chemical structure and sulfation pattern (domain structure) of HS is believed to determine its biological function, to vary across tissue types, and to be modified in the context of disease. Characterization of HS requires isolation and purification of cell surface HS as a complex mixture. This process may introduce additional chemical modification of the native residues. In this study, we describe an approach towards thorough characterization of bovine kidney heparan sulfate (BKHS) that utilizes a variety of orthogonal analytical techniques (e.g. NMR, IP-RPHPLC, LC-MS). These techniques are applied to characterize this mixture at various levels including composition, fragment level, and overall chain properties. The combination of these techniques in many instances provides orthogonal views into the fine structure of HS, and in other instances provides overlapping / confirmatory information from different perspectives. Specifically, this approach enables quantitative determination of natural and modified saccharide residues in the HS chains, and identifies unusual structures. Analysis of partially digested HS chains allows for a better understanding of the domain structures within this mixture, and yields specific insights into the non-reducing end and reducing end structures of the chains. This approach outlines a useful framework that can be applied to elucidate HS structure and thereby provides means to advance understanding of its biological role and potential involvement in disease progression. In addition, the techniques described here can be applied to characterization of heparin from different sources.

Heparin Characterization: Challenges and Solutions

Although heparin is an important and widely prescribed pharmaceutical anticoagulant, its high degree of sequence microheterogeneity and size polydispersity make molecular-level characterization challenging. Unlike nucleic acids and proteins that are biosynthesized through template-driven assembly processes, heparin and the related glycosaminoglycan heparan sulfate are actively remodeled during biosynthesis through a series of enzymatic reactions that lead to variable levels of O- and N-sulfonation and uronic acid epimers. As summarized in this review, heparin sequence information is determined through a bottom-up approach that relies on depolymerization reactions, size- and charge-based separations, and sensitive mass spectrometric and nuclear magnetic resonance experiments to determine the structural identity of component oligosaccharides. The structure-elucidation process, along with its challenges and opportunities for future analytical improvements, is reviewed and illustrated for a heparin-derived hexasaccharide.

Isolation and Characterization of Heparin From Tuna Skins

This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation. Anticoagulant activity was determined using the activated partial thromboplastin time and Heptest assays. Potency was determined using amidolytic antifactor IIa and antifactor Xa assays. The presence of heparin in the extracted tuna skin glycosaminoglycans was confirmed using (13)C-nuclear magnetic resonance. The activated partial thromboplastin time and Heptest clotting times were doubled at concentrations of about 4 and 1 microg/mL, respectively. The clotting time prolongation and antiprotease activity induced by tuna heparin was readily neutralized by 25 microg/mL protamine sulfate. These results demonstrate that biologically active heparin with properties similar to clinical grade heparin can be derived from tuna skin, a raw material with otherwise relatively little economic value.

Isolation and characterization of heparin and gelatin binding buffalo seminal plasma proteins and their effect on cauda epididymal spermatozoa

Seventy semen ejaculates were obtained from 14 Murrah buffalo bulls and were subjected to plasma separation immediately after collection by centrifugation at 2000 rpm for 20 min and stored in liquid nitrogen until analysis. In the seminal plasma the total protein concentration were estimated and the heparin and gelatin binding (HB and GB) proteins were isolated using heparin and gelatin affinity column chromatography. The molecular weight of individual isolated HB and GB protein was determined by SDS–PAGE analysis. Buffalo bull spermatozoa was collected from cauda epididymis under aseptic conditions and was used for the in vitro fertility tests (i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic swelling test (HOST)). The heparin and gelatin binding buffalo seminal plasma proteins were used in six concentrations i.e. 10, 20, 30, 40, 50 and 60 μg/ml to test their effect on in vitro fertility assessment of cauda epididymal spermatozoa. The overall mean values of total protein, HB and GB proteins were recorded as 29 ± 2.7, 2.61 and 0.2 mg/ml, respectively. Eighteen total protein bands were observed in the range of 12–127 kDa. Eight major HB proteins were isolated in the range of 13–71 kDa. Seven major GB proteins were isolated in the range of 13–61 kDa in the buffalo seminal plasma. The mean penetration distance (mm) travelled by the buffalo cauda spermatozoa was maximum in HB proteins (26.9 ± 0.6) followed by GB proteins (25.4 ± 0.6) and control (21.2 ± 1.4). The difference in BCMPT values between protein treated and control group was significant ( P < 0.05). Almost similar trend in the effect of protein on values of HOST percentage in both HB and GB proteins treated semen samples were recorded (66.4 ± 0.65 and 66.1 ± 0.6, respectively). The difference in HOST values between proteins treated and control group (50.4 ± 2.0) was significant ( P < 0.05). The present results indicate that among the isolated proteins, 4 proteins were commonly seen in both the heparin and gelatin–sepharose affinity column chromatography, and the addition of buffalo seminal plasma proteins improved the in vitro sperm functions (40 μg/ml gave best results) of buffalo cauda spermatozoa.

Purification and Characterization of Heparin from the Italian ClamCallistachione

An unusual heparin (approximately 1.9 mg/g of dry tissue) was isolated from the marine italian bivalve mollusk Callista chione. Agarose gel electrophoresis showed a high content of the fast-moving heparin component (85 +/- 7.6%) and 15 +/- 1.3% of the slow-moving species. An average molecular mass of 10 950 was calculated by PAGE analysis. The anticoagulant properties were measured as APTT (97 +/- 12.1 IU/mg) and anti-Xa activity (52 +/- 7.4 IU/mg). Structural analysis of clam heparin, performed by depolymerizing heparin samples with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by SAX-HPLC, revealed the presence of low amounts of the trisulfated disaccharide [DeltaUA2S(1-->4)-alpha-d-GlcN2S6S] and a significant increase of the disaccharides bearing nonsulfated iduronic and glucuronic acids, [-->4)-alpha-l-IdoA(1-->4)-alpha-d-GlcNAc6S(1-->] and [-->4)-alpha-l-IdoA(1-->4)-alpha-d-GlcN2S6S(1-->], and [-->4)-beta-d-GlcA(1-->4)-alpha-d-GlcN2S6S(1-->]. As a consequence, Callista chione heparin is a low-sulfated polysaccharide showing a specific decrease of the sulfatation in position 2 of the uronic acid units.

Mast cells are present in epithelial layers of different tissues of the mollusc Anomalocardia brasiliana. In situ characterization of heparin and a correlation of heparin and histamine concentration.

Heparin, other sulphated glycosaminoglycans and histamine were extracted from various dissected organs of Anomalocardia brasiliana, a mollusc from the South Atlantic, and quantified. A good correlation between heparin and histamine content was found in the labial palp, intestine, ctenidium, mantle and foot tissues. The tissue location of metachromatic cells, putatively containing heparin, was identified histologically with Alcian Blue, Toluidine Blue, Masson trichrome, Haematoxylin-Eosin and PAS. Except for the foot, cells containing metachromatic granules were found in the epithelium surfaces of all the organs analysed. An in situ identification of heparin using nitrous acid and heparinase degradation has established unequivocally the presence of this compound in the metachromatic cells. The location of 'mast-like' cells at the epithelium surface of mollusc tissues exposed to the environment are very similar to the distribution of mammalian and other vertebrate mast cells and gives support to the suggestion for a role of mast cells in defense mechanisms.

Characterization of Heparin and Heparan Sulfate Domains Binding to the Long Splice Variant of Platelet-derived Growth Factor A Chain

Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.

Characterization of heparins with different relative molecular masses (from 11 600 to 1600) by various analytical techniques

Heparin was extracted and purified from beef intestinal mucosa, and its structure and physico-chemical properties, e.g. disaccharide pattern (by specific enzymatic cleavage), relative molecular mass and sulfate-to-carboxyl ratio, were evaluated by different techniques. Heparin fractions with different relative molecular mass (from M r = 7560 to M r = 1600) were prepared by chemical degradation and gel-permeation chromatography. The fractions were characterized with respect to relative molecular mass, disaccharide pattern, sulfate-to-carboxyl ratio and percentage of slow moving and fast moving components by agarose-gel electrophoresis. The percentage of the two heparin species was calculated by densitometric analysis and specific calibration curves. The amount of the slow moving component decreases with relative molecular mass. The disaccharide pattern is different for the two heparin fractions. The percentage of trisulfated disaccharide decreases and the amount of mono- and disulfated disaccharides increases with a decrease of the relative molecular mass. The charge density, evaluated as the sulfate-to-carboxyl ratio, also decreases with the molecular mass of the fractions. This study confirms the heterogeneity of the structure, as evaluated by the constituent disaccharides, of the physico-chemical properties, such as relative molecular mass and charge density, and of the relative amount of the two heparin components also for low molecular mass heparins particularly produced by chemical depolymerization in the presence of free radicals.

Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides

A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505–1510) ; this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH ∼ 1.5 ( J. E. Shively, and H. E. Conrad, 1976, Biochemistry 15, 3932–3942 ) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH ∼ 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH ∼ 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5–8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.

Characterization of heparins by high-performance size exclusion liquid chromatography

A method for the determination of the molecular weight distribution of heparins using heparin oligosaccharides as calibration standards and high-performance size exclusion liquid chromatography has been developed. An organic microparticulate stationary phase (TSK 3000), an aqueous mobile phase and UV detection at 206 nm were adopted. Commercial heparins, low-molecular-weight heparins and polysulphated mucopolysaccharides with heparin-like activities were characterized by this procedure. Average molecular weights [weight average ( M w ), number average ( M n ), z average ( M z )] and molecular weight distribution (cumulative weight fraction and differential weight fraction) were calculated from the chromatographic data.

Isolation and Characterization of Heparin from Human Mastocytoma Tissue

Polysaccharide was isolated from human spleen mastocytoma by proteolytic digestion, precipitation with cetylpyridinium chloride, digestion with chondroitinase ABC, and ion-exchange chromatography on DEAE-cellulose. The final product (0.7 mg per g of starting material, MW 8000) behaved like standard heparin on ion-exchange chromatography and on electrophoresis, and contained D-glucuronic acid, L-iduronic acid, D-glucosamine and sulfate in the proportions expected for heparin. Affinity chromatography on antithrombin-Sepharose separated a distinct high-affinity fraction (4-5% of the total material). Structural analysis of this fraction showed that about 10% of the D-glucosamine residues were N-acetylated, the remainder N-sulfated. The anticoagulant activity of the isolated heparin was 71 B.P. units per mg (whole-blood system), or 30 units per mg (anti-thrombin and chromogenic substrate). 205 and 10-15 units per mg (chromogenic assay) were found for high and low affinity fractions, respectively. These results demonstrate conclusively the occurrence of heparin in a human tissue.

Isolation and characterization of heparin from human lung.

Heparin as measured by azure A metachromasia and anticoagulant activity has been extracted with 1 M NaCl from (35)S-labeled human lung fragments or dispersed human lung cells enriched for mast cells. The (35)S-labeled metachromatic material in the 3 M NaCl eluate from Dowex-1 chromatography of the extract from lung fragments exhibited an average mol wt of 20,000 by Sepharose 4B gel filtration. The (35)S-labeled metachromatic material with the charge characteristics of commercial porcine heparin on DEAE cellulose chromatography was entirely heparin by the criteria of resistance to degradation by chondroitin ABC lyase and complete degradation by purified heparinase. Antithrombin affinity chromatography of purified heparin with an anticoagulant activity of 137 U/mg, revealed that the one-third that was bound and eluted had a 273 U/mg sp act, whereas the unbound activity was 31 U/mg. Thus, the previously observed heterogeneity of commercial porcine heparin for binding to human antithrombin was also observed with human heparin. The mast cell-enriched human lung cell preparations yielded [(35)S]mucopolysaccharides with an average mol wt of 60,000 by Sepharose 4B gel filtration. Approximately 30% of this fraction was degraded by chondroitin ABC lyase, and the residual 70% was degraded by purified heparinase. When the chondroitin ABC lyase-resistant fraction was subjected to alkali degradation the average mol wt was reduced to 20,000. The calculated human lung mast cell heparin content of 2.4-7.8 mug/10(6) cells gave a ratio to histamine on a weight basis similar to that of intact lung fragments, thereby implying that heparin in the lung fragments was largely restricted to the mast cells.