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Anup Kumar Misra - One of the best experts on this subject based on the ideXlab platform.
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Expedient synthesis of the Pentasaccharide repeating unit of the O-antigen of Escherichia coli O86 and its conformational analysis
Glycoconjugate Journal, 2016Co-Authors: Ishani Bhaumik, Anirban Bhunia, Anup Kumar MisraAbstract:Synthesis of the Pentasaccharide with a 2-aminoethyl linker attached to the reducing end corresponding to the cell wall O -antigen of Escherichia coli O86 strain is reported. The synthetic strategy involves sequential glycosylation of suitably protected monosaccharide intermediates under similar glycosylation reaction conditions. Thioglycosides have been used as glycosyl donor throughout the synthetic strategy. Conformational analysis of the synthesized Pentasaccharide has been carried out using 2D ROESY NMR spectral analysis and all atom explicit molecular dynamics (MD) simulation technique. Graphical abstract Facile synthesis of the Pentasaccharide with a 2-aminoethyl linker attached to the reducing end corresponding to the cell wall O -antigen of Escherichia coli O86 strain is reported. Conformational analysis of the synthesized Pentasaccharide has been carried out using 2D ROESY NMR spectral analysis and all atom explicit molecular dynamics (MD) simulation technique.
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Concise synthesis of a Pentasaccharide repeating unit corresponding to the O-antigen of Escherichia coli O102
Tetrahedron-asymmetry, 2013Co-Authors: Abhijit Sau, Debashis Dhara, Anup Kumar MisraAbstract:An efficient synthetic strategy has been developed for the synthesis of a Pentasaccharide repeating unit of the O-antigen of Escherichia coli O102 strain. The target Pentasaccharide 1 has been synthesized using a [2+3] block glycosylation strategy. All glycosylation steps are highly stereoselective and high yielding. Concept of armed-disarmed and orthogonal glycosylation strategies has been applied during the synthesis. The target compound has been synthesized using the minimum number of steps.
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Convergent synthesis of a common Pentasaccharide corresponding to the O-antigen of Escherichia coli O168 and Shigella dysenteriae type 4
Glycoconjugate Journal, 2011Co-Authors: Goutam Guchhait, Anup Kumar MisraAbstract:A convenient synthetic strategy of the common acidic Pentasaccharide repeating unit corresponding to the O -antigen of enterotoxigenic E. coli O168 and Shigella dysenteriae type 4 has been successfully developed. A stereoselective [2 + 3] block glycosylation method has been exploited to get the target Pentasaccharide derivative. Most of the synthetic intermediates were solid and prepared in high yields from commercially available reducing sugars following a series of protection-deprotection reactions. A α-D-mannose moiety has been used as the source of α-D-glucosamine moiety. A late-stage TEMPO mediated selective oxidation reaction finally resulted in the Pentasaccharide containing a glucuronic acid unit. A convenient synthetic strategy of the common acidic Pentasaccharide repeating unit corresponding to the O -antigen of enterotoxigenic E. coli O168 and Shigella dysenteriae type 4 has been successfully developed using stereoselective [2+3] block glycosylation technique.
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Synthesis of tri- and Pentasaccharide fragments corresponding to the O-antigen of Shigella boydii type 6
Tetrahedron-asymmetry, 2010Co-Authors: Abhishek Santra, Anup Kumar MisraAbstract:Abstract A convenient synthetic strategy for the synthesis of the acidic Pentasaccharide repeating unit and its trisaccharide fragment corresponding to the O-antigen of Shigella boydii type 6 has been successfully developed. A stereoselective sequential glycosylation method has been exploited to obtain the target tri- and Pentasaccharide derivatives. Most of the synthetic intermediates were solid and prepared in high yields from commercially available reducing sugars following a series of protection–deprotection reactions. A late-stage TEMPO mediated selective oxidation reaction finally resulted in the Pentasaccharide containing a glucuronic acid unit. A 2-(4-methoxyphenoxy) ethyl group has been chosen as the anomeric protecting group to provide trisaccharide and Pentasaccharide derivatives linked to an ethylene glycol linker.
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convergent synthesis of a common Pentasaccharide repeating unit corresponding to the o specific polysaccharide of escherichia coli o4 k3 o4 k6 and o4 k12
Tetrahedron-asymmetry, 2010Co-Authors: Rajib Panchadhayee, Anup Kumar MisraAbstract:Abstract A convergent chemical synthesis of a Pentasaccharide found in the O-specific polysaccharide of Escherichia coli O4:K3, O4:K6, and O4:K12 has been achieved in excellent yield. A [3+2] block synthetic strategy has been adopted to couple a disaccharide donor 11 with a trisaccharide acceptor 10 for the construction of the Pentasaccharide derivative 12 which on deprotection furnished target Pentasaccharide 1 as its 4-methoxyphenyl glycoside. Disaccharide thioglycoside donor 11 and trisaccharide acceptor 10 were prepared from suitably protected monosaccharide intermediates. Yields were excellent in all steps.
Laurence Mulard - One of the best experts on this subject based on the ideXlab platform.
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synthesis of two tetra and four Pentasaccharide fragments of shigella flexneri serotypes 3a and x o antigens from a common tetrasaccharide intermediate
European Journal of Organic Chemistry, 2008Co-Authors: Julien Boutet, Laurence MulardAbstract:Relying on trichloroacetimidate chemistry, six tetra- and Pentasaccharide fragments of the {2)-[α-D-Glcp-(1→3)]-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-[Ac→2]-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→}n ((E)ABAcCD)n polymer were synthesized as their propyl glycosides by use of a common fully protected (E)ABAcC intermediate (9). Tetrasaccharide 9 derived from the condensation of an EA donor and a BAcC acceptor. Partial and full deprotection gave free tetrasaccharides (E)ABAcC and (E)ABC, respectively. Alternatively, 9 was converted into a trichloroacetimidate donor, which provided linear Pentasaccharides (E)ABAcCD and (E)ABCD, following a reaction with a D acceptor and subsequent partial or total deprotection, respectively. Additionally, the selective removal of the 2A-levulinoyl protecting group in 9 allowed for chain elongation at this position. The glycosylation of the resulting acceptor with a D donor, and subsequent partial or total deprotection, gave branched Pentasaccharides D(E)ABAcC and D(E)ABC. All targets are parts of the O-antigen of Shigellaflexneri 3a, a prevalent serotype. Non-O-acetylated oligosaccharides are shared by the S.flexneri serotype X O-antigen. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)
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Conformational studies of the O-specific polysaccharide of Shigella flexneri 5a and of four related synthetic Pentasaccharide fragments using NMR and molecular modeling.
Journal of Biological Chemistry, 2003Co-Authors: Marie-jeanne Clément, Armelle Phalipon, Laurence Mulard, Anne Imberty, Serge Pérez, Catherine Simenel, Muriel DelepierreAbstract:As part of a program for the development of synthetic vaccines against the pathogen Shigella flexneri, we used a combination of NMR and molecular modeling methods to study the conformations of the O-specific polysaccharide (O-SP) of S. flexneri 5a and of four related synthetic Pentasaccharide fragments. The NMR study, based on the analysis of 1H and 13C chemical shifts, the evaluation of inter-residue distances, and the measurement of one- and three-bond heteronuclear coupling constants, showed that the conformation of one of the four Pentasaccharides is similar to that of the native O-SP in solution. Interestingly, inhibition enzyme-linked immunosorbent assay demonstrated that a protective monoclonal antibody specific for S. flexneri 5a has a greater affinity for this Pentasaccharide than for the others. We carried out a complete conformational search on the Pentasaccharides using the CICADA algorithm interfaced with MM3 force field. We calculated Boltzmann-averaged inter-residue distances and 3JC,H coupling constants for the different conformational families and compared the results with NMR data for all Pentasaccharides. Our experimental data are consistent with only one conformational family. We also used molecular modeling data to build models of the O-SP with the molecular builder program POLYS. The models that are in agreement with NMR data adopt right-handed 3-fold helical structures in which the branched glucosyl residue points outwards.
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Synthesis of the methyl glycoside of a branched octasaccharide fragment specific for the Shigella flexneri serotype 2a O-antigen
Tetrahedron Letters, 2002Co-Authors: Frédéric Bélot, Corina Costachel, Karen Wright, Armelle Phalipon, Laurence MulardAbstract:An efficient synthesis of the methyl glycoside of a branched octasaccharide representative of Shigella flexneri serotype 2a O-specific polysaccharide is described. The synthesis is based on the use of the trichloroacetimidate methodology, and involves the condensation of a Pentasaccharide acceptor and a trisaccharide donor as the key step. The target octasaccharide was synthesized efficiently according to a strategy based on the condensation of a Pentasaccharide acceptor and a trisaccharide donor as the key step.
Marcelo A Dankert - One of the best experts on this subject based on the ideXlab platform.
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sequential assembly and polymerization of the polyprenol linked Pentasaccharide repeating unit of the xanthan polysaccharide in xanthomonas campestris
Journal of Bacteriology, 1993Co-Authors: Luis Ielpi, Roberto O. Couso, Marcelo A DankertAbstract:Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating Pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear Pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of Pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially Pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new Pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked Pentasaccharide, generating a branched polymer with a trisaccharide side chain.
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sequential assembly and polymerization of the polyprenol linked Pentasaccharide repeating unit of the xanthan polysaccharide in xanthomonas campestris
Journal of Bacteriology, 1993Co-Authors: Luis Ielpi, Roberto O. Couso, Marcelo A DankertAbstract:Lipid-linked intermediates are involved in the synthesis of the exopolysaccharide xanthan produced by the bacterium Xanthomonas campestris (L. Ielpi, R. O. Couso, and M. A. Dankert, FEBS Lett. 130:253-256, 1981). In this study, the stepwise assembly of the repeating Pentasaccharide unit of xanthan is described. EDTA-treated X. campestris cells were used as both enzyme preparation and lipid-P acceptor, and UDP-Glc, GDP-Man, and UDP-glucuronic acid were used as sugar donors. A linear Pentasaccharide unit is assembled on a polyprenol-P lipid carrier by the sequential addition of glucose-1-P, glucose, mannose, glucuronic acid, and mannose. The in vitro synthesis of Pentasaccharide-P-P-polyprenol was also accompanied by the incorporation of radioactivity into a polymeric product, which was characterized as xanthan, on the basis of gel filtration and permethylation studies. Results from two-stage reactions showed that essentially Pentasaccharide-P-P-polyprenol is polymerized. In addition, the direction of chain elongation has been studied by in vivo experiments. The polymerization of lipid-linked repeat units occurs by the successive transfer of the growing chain to a new Pentasaccharide-P-P-polyprenol. The reaction involves C-1 of glucose at the reducing end of the polyprenol-linked growing chain and C-4 of glucose at the nonreducing position of the newly formed polyprenol-linked Pentasaccharide, generating a branched polymer with a trisaccharide side chain.
Maurice Petitou - One of the best experts on this subject based on the ideXlab platform.
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from heparin to ep217609 the long way to a new Pentasaccharide based neutralisable anticoagulant with an unprecedented pharmacological profile
Thrombosis and Haemostasis, 2009Co-Authors: Maurice Petitou, G M T Vogel, C. A. A. Van Boeckel, V Nancyportebois, G Dubreucq, V Motte, Meuleman Dirk Gerrit, De M Kort, Jaj WisseAbstract:The elucidation of the structure of the antithrombin binding sequence in heparin has given a large impulse to the rational design of heparin related drugs. De novo chemical synthesis of the corresponding Pentasaccharide as well as simplified analogues has provided very specific, antithrombin-mediated inhibitors of factor Xa with various pharmacokinetic profiles. Fondaparinux and idraparinux are examples of such compounds that have found clinical application as antithrombotics. Because of the very specific binding to antithrombin the pharmacokinetics of Pentasaccharides can be predicted and transferred to other molecules covalently bound to them. The new chemical entities thus obtained display a wide array of antithrombotic activities, giving improved heparin molecules as well as new anticoagulants, devoid of the undesired side effects of heparin and with unprecedented pharmacological profiles. In this context, a direct thrombin inhibitor was covalently coupled to a Pentasaccharide by an inert spacer. This compound, EP42675 exerts antithrombin mediated anti-factor Xa activity together with direct thrombin inhibiting capacity. It displays favourable pharmacokinetics as imposed by the Pentasaccharide. EP42675 was further modified by the introduction of a biotin moiety in its structure. The new entity obtained, EP217609 exerts the same pharmacological profile as EP42675 and it can be instantaneously neutralised by injection of avidin. Due to this unprecedented mechanism of anticoagulant activity and its ability to be neutralised, EP217609 deserves to be investigated in clinical settings where direct thrombin inhibition is required.
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The Effect of a Reducing-end Extension on Pentasaccharide Binding by Antithrombin
Journal of Biological Chemistry, 2000Co-Authors: Klara J. Belzar, Robin W Carrell, Timothy R. Dafforn, Maurice Petitou, Jennifer A. HuntingtonAbstract:Abstract Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific Pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate Pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural Pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG′H′). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths ≥0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the Pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.
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The Effect of a Reducing-end Extension on Pentasaccharide Binding by Antithrombin
Journal of Biological Chemistry, 2000Co-Authors: Klara J. Belzar, Robin W Carrell, Timothy R. Dafforn, Maurice Petitou, Jennifer A. HuntingtonAbstract:Abstract Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific Pentasaccharide domain in a two-step mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate Pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural Pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHG′H′). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths ≥0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the Pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.
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A synthetic heparan sulfate Pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers.
Bioorganic & medicinal chemistry, 1999Co-Authors: José Kovensky, Maurice Petitou, Philippe Duchaussoy, Françoise Bono, Markku Salmivirta, Philippe Sizun, Jean-marc Herbert, Pierre SinaÿAbstract:It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated Pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible Pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The Pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other Pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.
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mechanism of heparin activation of antithrombin role of individual residues of the Pentasaccharide activating sequence in the recognition of native and activated states of antithrombin
Journal of Biological Chemistry, 1998Co-Authors: Umesh R. Desai, Maurice Petitou, Ingemar Björk, Steven T. OlsonAbstract:Abstract To determine the role of individual saccharide residues of a specific heparin Pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting Pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in Pentasaccharide binding energy of ∼15–75% but did not affect the oligosaccharide’s ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the Pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin Pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.
Jawed Fareed - One of the best experts on this subject based on the ideXlab platform.
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Comparative Studies on the HIT Antibody Mediated Platelet Aggregation / Serotonin Release by Synthetic Pentasaccharide and Two Chemoenzymatically Synthesized Heptasaccharides
Blood, 2011Co-Authors: Jeanine M. Walenga, Chris Aranda, Mary Lewis, Debra Hoppensteadt, Robert J. Linhardt, Jawed FareedAbstract:Abstract 2231 Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT Pentasaccharide-binding sites within their structures which is comparable to the Pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to Pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to Pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the Pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The Pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the Pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the Pentasaccharide in heparin compromised patients. Disclosures: No relevant conflicts of interest to declare.
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comparative studies on the hit antibody mediated platelet aggregation serotonin release by synthetic Pentasaccharide and two chemoenzymatically synthesized heptasaccharides
Blood, 2011Co-Authors: Jeanine M. Walenga, Chris Aranda, Mary Lewis, Debra Hoppensteadt, Robert J. Linhardt, Jian Liu, Jawed FareedAbstract:Abstract 2231 Synthetic oligosaccharides such as the Pentasaccharide (Arixtra) and its derivatives are antithrombotic agents which are clinically used in the management of thrombotic indications. These agents are claimed to be devoid of triggering the generation of HIT antibodies and therefore do not produce HIT syndrome. Several additional synthetic oligosaccharides are also developed for the management of thrombotic indications. More recently, two novel ultra low molecular weight heparins (ULMWHs) were synthesized chemoenzymatically. These ULMWHs are both heptasaccharides with AT Pentasaccharide-binding sites within their structures which is comparable to the Pentasaccharide. The IC50 of the anti-Xa effects of the agents are comparable to Pentasaccharide, ranging from 0.7 to 1.0 ug/ml in comparison to Pentasaccharide which is 0.8 ug/ml. All agents produced comparable anticoagulant effects in the Heptest clotting time. The purpose of this study is to compare the effects of the Pentasaccharide and the two heptasaccharides namely ULMWH1 and ULMW2 in the HIT mediated platelet aggregation and serotonin release assays. In addition platelet factor 4 release in whole blood was also studied. The HIT mediated platelet aggregation studies were carried out utilizing a HIT antibody positive pool plasma preparation. PRP collected from 10 individual donors (250ul) was mixed with 200ul of HIT pool plasma and equilibrated at 37° C for 3 minutes. 50ul of 1, 10, and 100 ug/ml of each of these agents was added to trigger the platelet aggregation responses. Enoxaparin was used as a positive control in the same concentration ranges. The serotonin release assay was carried out using the standard method in the same concentration range monitoring the release of 14C serotonin with each of these agents. The PF4 release was also measured using an ELISA method for serotonin measurement in whole blood samples incubated with each of these agents at concentrations of 0, 10 and 100ug/ml. The Pentasaccharide and the two heptasaccharides did not produce any aggregation of platelets in the HIT aggregation assay at all concentrations whereas Enoxaparin at concentrations of > 1ug/ml produces positive aggregation responses. In the 14C assay none of the agents produced any release of serotonin however Enoxaparin produced 14C release at all concentration studied. Similarly, the pentasachhardide and heptasaccharides did not produce any platelet factor 4 from the whole blood incubation studies, however Enoxaparin produced a measurable release of platelet factor 4. Interestingly, unlike Enoxaparin, the anti-Xa and heptest effects of these agents were not neutralized by platelet factor 4 or protamine sulfate. These results demonstrate that the Pentasaccharide and chemoenzymatically synthesized ULMWH1 and ULMWH2 do not meditate HIT antibody induced aggregation and serotonin release. Therefore, these heptasaccharides may exhibit comparable safety profile to the Pentasaccharide in heparin compromised patients. Disclosures: No relevant conflicts of interest to declare.
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Synthetic Heparin Pentasaccharide Depolymerization by Heparinase 1: Molecular and Biological Implications
Clinical and applied thrombosis hemostasis : official journal of the International Academy of Clinical and Applied Thrombosis Hemostasis, 2001Co-Authors: Asif N. Daud, Jeanine M. Walenga, Ahmad Ahsan, Omer Iqbal, Paul J. Silver, Sarfraz Ahmad, Jawed FareedAbstract:A synthetic Pentasaccharide (SR90107/ ORG31540) representing the antithrombin III (ATIII) binding sequence in heparin is under clinical development for the prophylaxis and management of venous thromboembolism. This Pentasaccharide exhibits potent anti-factor Xa (AXa) effects (>750 lU/mg> and does not exhibit any anti-factor IIa (AIIa) activity. Previous reports have suggested that synthetic heparin Pentasaccharides are resistant to the digestive effects of heparinase 1. To investigate the effect of heparinase I on the AXa activity of Pentasaccharide SR90107/ORG31540. graded concentrations (1.25-100 μg/ml) were incubated with a fixed amount of heparinase I (0.1 U/ml). Heparinase I produced a strong neutralizing effect on this Pentasaccharide, as measured by AXa activity. This observation led to further studies where high performance liquid chromatography (HPLC) analysis was eniployed to determine the potential breakdown products of the Pentasaccharide. The experiment with the Pentasaccharide included incub...
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Heparinase I acts on a synthetic heparin Pentasaccharide corresponding to the antithrombin III binding site.
Thrombosis research, 2000Co-Authors: Laurie A. Lebrun, Jeanine M. Walenga, Debra Hoppensteadt, Jawed Fareed, Nur Sibel Gunay, Robert J. LinhardtAbstract:A synthetic Pentasaccharide, containing an intact antithrombin III (ATIII) binding site that is in clinical studies a specific antifactor Xa agent, serves as a substrate for a heparin lyase (heparinase I, EC 4.2.2.7) from Flavobacterium heparinum. Heparinase I, currently being assessed as a heparin reversal agent, also reverses the antifactor Xa activity of this synthetic Pentasaccharide by breaking it down to inactive disaccharide and trisaccharide products.
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Laboratory monitoring of Pentasaccharide in a dog model of hemodialysis.
Thrombosis research, 1999Co-Authors: Debra Hoppensteadt, Jeanine M. Walenga, Jean-marc Herbert, Walter Jeske, Liu Hui Yang, Todd S. Ing, Jawed FareedAbstract:Varying dosages of Pentasaccharide (400-800 nmol/kg) were compared to a 250-U/kg single bolus dosage of unfractionated heparin (UFH) in a dog model of hemodialysis. Several laboratory assays were used to monitor the effects of Pentasaccharide and UFH. The Pentasaccharide did not produce any anticoagulant effects as measured by the activated partial thromboplastin time. However, in the anti-Xa chromogenic assay and the Heptest assays, there was a dose-dependent prolongation after Pentasaccharide administration. In the group of dogs administered 800 nmol/kg of Pentasaccharide, there was a 50% decrease in the thrombin antithrombin (TAT) complex level after 60 minutes on dialysis. In the UFH-treated dogs, wide variations in assays were observed. There was a marked elevation in the activated partial thromboplastin time and Heptest assays up to 6 hours after UFH administration. Both anti-Xa and anti-IIa activity was measured up to 4 hours. In the TAT assay, UFH was found to have a stronger effect in suppressing the formation of TAT in comparison to the Pentasaccharide. These results suggest that Pentasaccharide can be used as a replacement for UFH in a dog model of hemodialysis to keep the dialysis circuit patent. In addition, the anti-Xa-based assays such as the Heptest and the chromogenic anti-Xa assays can be used to monitor the effects of Pentasaccharide in this model.