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Per Olsson - One of the best experts on this subject based on the ideXlab platform.
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influence of high and low wall shear rates on the inhibition of factor xa and thrombin at surfaces coated with Immobilized Heparin
Artificial Organs, 2008Co-Authors: Claes Arnander, Anders Rasmuson, Boris Pasche, Kenji Kodama, Per OlssonAbstract:: The thromboresistant function of a surface with end-point attached Heparin is based upon interaction among the Immobilized Heparin, antithrombin, and at least factor Xa or thrombin. Heparinized arteriovenous shunts were implanted in dogs. By compressing a segment of the shunt, high and low wall shear rate regions were obtained in each shunt. After removal, the tubings were tested for their factor Xa and thrombin inhibitory capacity. It was found that on a molar basis, the factor Xa and thrombin inhibitory capacity were similar in low wall shear rate segments. In high wall shear rate segments, the thrombin inhibitory capacity was decreased, thus indicating that the AT-mediated inhibition of the serine protease is dependent on the wall shear rate.
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studies of adsorption activation and inhibition of factor xii on Immobilized Heparin
Thrombosis Research, 1998Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:Abstract The aim of the present investigation was to clarify whether Immobilized Heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of Heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound α-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound α-FXIIa was slightly slower than with purified components. Nevertheless, neither β-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from Heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound α-FXIIa within 1 second. In the liquid phase, β-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of Heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the β-FXIIa inhibition rate and prevent FXI activation in plasma, although β-FXIIa and FXIa-AT complex formation occurred. It is concluded that surface-Immobilized Heparin, unlike Heparin in solution, effectively inhibits the initial contact activation enzymes by an antithrombin-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway.
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inhibition of the plasma contact activation system of Immobilized Heparin relation to surface density of functional antithrombin binding sites
Journal of Biomedical Materials Research, 1997Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:End-point immobilization of Heparin to artificial materials gives rise to a surface that prevents triggering of the plasma contact activation system and, presumably as a result thereof, generally has thrombo-resistant properties. The present investigation was undertaken to determine what density of Immobilized Heparin molecules expressing functionally intact antithrombin binding sites is required to achieve these blood compatible properties. Six different Heparin surfaces were prepared on polyethylene tubing and studied in contact with human plasma. The content of bound Heparin was the same on all surfaces while the densities of antithrombin binding sites ranged from 1 to 28 pmol/cm2. The surfaces expressing 4 pmol/cm2 or more of specific antithrombin binding sites generated no measurable enzymatic activity in contact with plasma, either on the exposed surfaces or in the plasma phases. Below this level, the degree of activation gradually increased with decreasing densities, and in parallel the thrombo-resistant properties deteriorated. Addition of Heparin to the plasma phase reduced the capacity of the Heparin surfaces to bind antithrombin, leading to a diminished ability of the surfaces to prevent contact activation. This finding supports the hypothesis that antithrombin is the critical coagulation inhibitor for the suppression of contact activation on end-point Immobilized Heparin.
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control of contact activation on end point Immobilized Heparin the role of antithrombin and the specific antithrombin binding sequence
Journal of Biomedical Materials Research, 1995Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:The uptake and activation of FXII from blood plasma was studied in small-diameter polyethylene tubing, surface-modified by end-point immobilization of Heparin. Two preparations of Heparin were used to modify the contact-activating properties of the plastic tubing: unfractionated, functionally active Heparin and low-affinity Heparin, lacking the specific antithrombin-binding sequence and virtually devoid of anticoagulant activity. The uptakes of FXII on the two Heparin surfaces were similar. No activated FXII could be demonstrated on the unfractionated Heparin surface, whereas on the low-affinity Heparin surface nearly all FXII underwent spontaneous activation. The suppression of FXII activation on the unfractionated Heparin surface was investigated by using plasma depleted of antithrombin, complement C1 esterase inhibitor, or both. The removal of antithrombin resulted in extensive activation of FXII, whereas the depletion of C1 esterase inhibitor had only a minor effect. Experiments with recalcified plasma showed rapid clot formation during exposure to the low-affinity Heparin surface. After depletion of antithrombin, but not complement C1 esterase inhibitor, the recalcified plasma clotted in contact with the unfractionated Heparin surface as well. We conclude that antithrombin and the antithrombin-binding sequence in the surface-Immobilized Heparin are essential for the prevention of surface activation of FXII and triggering of the intrinsic coagulation system. © 1995 John Wiley & Sons, Inc.
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On the mechanism of coagulation inhibition on surfaces with end point Immobilized Heparin.
Thrombosis and Haemostasis, 1993Co-Authors: Graciela Elgue, Margareta Blomback, Per Olsson, J RiesenfeldAbstract:: A well established technique to improve blood compatibility of artificial materials for use in the circulation is to coat the surface with Heparin. The present report describes the antithrombin mediated inhibition of thrombin and factor Xa by surfaces modified with end point Immobilized Heparin. The reaction was followed by conventional chromogenic substrate based enzyme assays as well as by immunological measurement of the enzyme inhibitor (thrombin-antithrombin) complex formation. Both enzymes were rapidly inactivated by Heparin surfaces after selective presaturation with antithrombin on the Immobilized high affinity Heparin molecules. The thrombin inhibitory capacity was enhanced when both high and low affinity Heparin were preadsorbed with inhibitor. The main part of the thrombin-antithrombin complex formed remained bound to the surface, however, without functionally blocking the activity of the high affinity sequence of the Immobilized Heparin. Aliquots of recalcified plasma were slowly rotated in loops of Heparinized tubing to investigate whether the main thromboresistant function of the surface was exerted at the level of thrombin or by inactivation of preceding enzymes. After 1 h no visible clotting occurred and only trace amounts of thrombin (0.07 IU/ml), measured as thrombin-antithrombin complexes, had been formed. In non-Heparinized loops and in the test tube plasma clotted after 20 min. The thrombin generation when clotting occurred was in the order of 10 IU/ml. It is concluded that the Immobilized Heparin mediates inhibition of the coagulation cascade prior to prothrombin activation.
Robert J Linhardt - One of the best experts on this subject based on the ideXlab platform.
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glycosaminoglycan binding motif at s1 s2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus sars cov 2 host cell entry
bioRxiv, 2020Co-Authors: Amika Sood, David W Montgomery, Oliver C Grant, Mark M Fuster, Li Fu, Jonathan S Dordick, Robert J Woods, Fuming Zhang, Robert J LinhardtAbstract: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 might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind 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. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.
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glycosaminoglycan binding motif at s1 s2 proteolytic cleavage site on spike glycoprotein may facilitate novel coronavirus sars cov 2 host cell entry
bioRxiv, 2020Co-Authors: So Young Kim, Amika Sood, David W Montgomery, Oliver C Grant, Mark M Fuster, Jonathan S Dordick, Robert J Woods, Fuming Zhang, Weihua Jin, Robert J LinhardtAbstract: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 might be involved in host cell entry of SARS-CoV-2. Using a surface plasmon resonance direct binding assay, we found that both monomeric and trimeric SARS-CoV-2 spike more tightly bind 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. Our study augments our knowledge in SARS-CoV-2 pathogenesis and advances carbohydrate-based COVID-19 therapeutic development.
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binding between the integrin αxβ2 cd11c cd18 and Heparin
Journal of Biological Chemistry, 2007Co-Authors: Thomas Vorupjensen, Robert J Linhardt, Lianli Chi, Louise C Gjelstrup, Uffe Birk Jensen, Craig A Jewett, Can Xie, Motomu Shimaoka, Timothy A SpringerAbstract:Abstract The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor αXβ2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within Heparin that constitute high affinity binding sites for the I domain of integrin αXβ2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the αX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic Heparinoid fondaparinux were not able to block the binding. Semipurified Heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 μm affinity for the αX I domain. In studies of direct binding by the αX I domain to Immobilized Heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated Heparin fragment induced conformational change in the ectodomain of the αXβ2 receptor, also demonstrating allosteric linkage between Heparin binding and integrin conformation.
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probing the interaction of dengue virus envelope protein with Heparin assessment of glycosaminoglycan derived inhibitors
Journal of Medicinal Chemistry, 2001Co-Authors: Rory M Marks, Hong Lu, Renuka Sundaresan, Toshihiko Toida, Atsushi Suzuki, Toshio Imanari, Maria J Hernaiz, Robert J LinhardtAbstract:A structure−activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a Heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to Immobilized Heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope prote...
Javier Sanchez - One of the best experts on this subject based on the ideXlab platform.
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anchoring of vascular endothelial growth factor to surface Immobilized Heparin on pancreatic islets implications for stimulating islet angiogenesis
Tissue Engineering Part A, 2010Co-Authors: Sanja Cabric, Javier Sanchez, Ulrika Johansson, Rolf Larsson, Bo Nilsson, Olle Korsgren, Peetra U MagnussonAbstract:In pancreatic islet transplantation, early revascularization is necessary for long-term graft function. We have shown in in vitro and in vivo models that modification with surface-attached Heparin protects the islets from acute attack by the innate immune system of the blood following intraportal islet transplantation. In this study, we have investigated the ability of an Immobilized conjugate composed of Heparin to bind the angiogenic growth factor vascular endothelial growth factor-A (VEGF-A) as a means of attracting endothelial cells (ECs) to induce angiogenesis and revascularization. We analyzed the capacity of VEGF-A to bind to Immobilized Heparin and how this affected the proliferation and adherence of ECs to both artificial glass surfaces and islets. Quartz crystal microbalance with dissipation monitoring and slot-blot demonstrated the binding of VEGF-A to Heparin-coated surfaces upon which ECs showed protein-dependent proliferation. Also, ECs cultured on Heparin-coated glass surfaces exhibited effects upon focal contacts. Heparinized islets combined with VEGF-A demonstrated unaffected insulin release. Further, covering islets with Heparin also increased the adhesion of ECs to the islet surface. Immobilized Heparin on the islet surface may be a useful anchor molecule for achieving complete coverage of islets with angiogenic growth factors, ultimately improving islet revascularization and engraftment in pancreatic islet transplantation.
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functionality and stability of Heparin Immobilized onto poly dimethylsiloxane
Colloids and Surfaces B: Biointerfaces, 2005Co-Authors: Sara Thorslund, Javier Sanchez, Rolf Larsson, Fredrik Nikolajeff, Jonas BergquistAbstract:Poly(dimethylsiloxane) (PDMS) has become an attractive material when working in the field of microfluidics, mainly because of the rapid prototyping process it involves. The increased surface volume ratio in microchannels makes the interaction between sample and material surface highly important, evident when handling complex biological samples such as plasma or blood. This study demonstrates a new grade of non-covalent Heparin surface that adds efficient anticoagulant property to the PDMS material. The surface modification is a simple and fast one-step process performed at neutral pH, optimal when working with closed microsystems. The Heparin formed a uniform and functional coating on hydrophobic PDMS with comparatively high level of antithrombin-binding capacity. In addition, long-term studies revelaed that the Immobilized Heparin was more or less stable in the microchannels over a time of three weeks. Recalcified plasma in contact with native PDMS showed complete coagulation after 1 h, while no fibrin formation was detected in plasma incubated on Heparin-coated PDMS within the same time. In conclusion, we see the Heparin coating developed and evaluated in this study as a tool that greatly facilitates the use of PDMS in microfluidics dealing with plasma or blood samples.
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studies of adsorption activation and inhibition of factor xii on Immobilized Heparin
Thrombosis Research, 1998Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:Abstract The aim of the present investigation was to clarify whether Immobilized Heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of Heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound α-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound α-FXIIa was slightly slower than with purified components. Nevertheless, neither β-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from Heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound α-FXIIa within 1 second. In the liquid phase, β-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of Heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the β-FXIIa inhibition rate and prevent FXI activation in plasma, although β-FXIIa and FXIa-AT complex formation occurred. It is concluded that surface-Immobilized Heparin, unlike Heparin in solution, effectively inhibits the initial contact activation enzymes by an antithrombin-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway.
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inhibition of the plasma contact activation system of Immobilized Heparin relation to surface density of functional antithrombin binding sites
Journal of Biomedical Materials Research, 1997Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:End-point immobilization of Heparin to artificial materials gives rise to a surface that prevents triggering of the plasma contact activation system and, presumably as a result thereof, generally has thrombo-resistant properties. The present investigation was undertaken to determine what density of Immobilized Heparin molecules expressing functionally intact antithrombin binding sites is required to achieve these blood compatible properties. Six different Heparin surfaces were prepared on polyethylene tubing and studied in contact with human plasma. The content of bound Heparin was the same on all surfaces while the densities of antithrombin binding sites ranged from 1 to 28 pmol/cm2. The surfaces expressing 4 pmol/cm2 or more of specific antithrombin binding sites generated no measurable enzymatic activity in contact with plasma, either on the exposed surfaces or in the plasma phases. Below this level, the degree of activation gradually increased with decreasing densities, and in parallel the thrombo-resistant properties deteriorated. Addition of Heparin to the plasma phase reduced the capacity of the Heparin surfaces to bind antithrombin, leading to a diminished ability of the surfaces to prevent contact activation. This finding supports the hypothesis that antithrombin is the critical coagulation inhibitor for the suppression of contact activation on end-point Immobilized Heparin.
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control of contact activation on end point Immobilized Heparin the role of antithrombin and the specific antithrombin binding sequence
Journal of Biomedical Materials Research, 1995Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:The uptake and activation of FXII from blood plasma was studied in small-diameter polyethylene tubing, surface-modified by end-point immobilization of Heparin. Two preparations of Heparin were used to modify the contact-activating properties of the plastic tubing: unfractionated, functionally active Heparin and low-affinity Heparin, lacking the specific antithrombin-binding sequence and virtually devoid of anticoagulant activity. The uptakes of FXII on the two Heparin surfaces were similar. No activated FXII could be demonstrated on the unfractionated Heparin surface, whereas on the low-affinity Heparin surface nearly all FXII underwent spontaneous activation. The suppression of FXII activation on the unfractionated Heparin surface was investigated by using plasma depleted of antithrombin, complement C1 esterase inhibitor, or both. The removal of antithrombin resulted in extensive activation of FXII, whereas the depletion of C1 esterase inhibitor had only a minor effect. Experiments with recalcified plasma showed rapid clot formation during exposure to the low-affinity Heparin surface. After depletion of antithrombin, but not complement C1 esterase inhibitor, the recalcified plasma clotted in contact with the unfractionated Heparin surface as well. We conclude that antithrombin and the antithrombin-binding sequence in the surface-Immobilized Heparin are essential for the prevention of surface activation of FXII and triggering of the intrinsic coagulation system. © 1995 John Wiley & Sons, Inc.
Innkyu Kang - One of the best experts on this subject based on the ideXlab platform.
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in vitro blood compatibility of Heparin Immobilized polyurethane containing ester groups in the side chain
Journal of Materials Science: Materials in Medicine, 2004Co-Authors: Meng Wan, Innkyu Kang, Dong Ki Baek, Jinho Cho, K H KimAbstract:In a previous study, we reported on the synthesis of Heparin-Immobilized polyetherurethanes containing ester groups in the side chain. In this study, the blood compatibility of Heparin-Immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by polyethylene oxide (PEO) grafting and further prolonged by Heparin immobilization. The APTT was prolonged on the PU-C-H and PU-P-H, suggesting the binding of Immobilized Heparin to the antithrombin III. The percentage of platelet adhesion on the PU was almost the same as that on carboxylic acid-introduced PU (PU-C), but was slightly decreased by PEO grafting and further decreased by Heparin immobilization. The release of serotonin from the adhering platelets was slightly suppressed on the PEO-grafted PU yet significantly suppressed on the Heparin-Immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were significantly suppressed on the Heparin-Immobilized PUs, and the amount of interleukin-6 (IL-6) released from the PBMCs stimulated with the surface-modified PUs decreased with a decrease in the PBMC adhesion.
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interaction of blood components with Heparin Immobilized polyurethanes prepared by plasma glow discharge
Journal of Biomaterials Science-polymer Edition, 2001Co-Authors: Innkyu Kang, E J Seo, M W Huh, K H KimAbstract:The blood compatibility of poly(ethylene oxide) (PEO)-grafted and Heparin (Hep) Immobilized polyurethanes was investigated using in vitro plasma recalcification time (PRT), activated partial thromboplastin time (APTT), platelet adhesion and activation, and peripheral blood mononuclear cell (PBMC) adhesion and activation. In the experiment with plasma proteins, the PRT of the polyurethane (PU) surface was prolonged by PEO grafting and further prolonged by Heparin immobilization. The APTT was prolonged on PU-Hep, suggesting the binding of Immobilized Heparin to antithrombin III. The percentage of platelet adhesion on PU was not much different from that on acrylic acid- and PEO-grafted PUs (PU-C, PU-6, PU-33), yet was substantially decreased by Heparin immobilization (PU-6-Hep, PU-33-Hep). The release of serotonin from adhering platelets was slightly suppressed on PEO-grafted PUs yet significantly suppressed on Heparin-Immobilized PUs. In the PBMC experiments, the adhesion and activation of the cells were si...
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surface characterization and in vitro blood compatibility of poly ethylene terephthalate Immobilized with insulin and or Heparin using plasma glow discharge
Biomaterials, 2000Co-Authors: Innkyu Kang, Sung Chul YoonAbstract:Poly(ethylene terephthalate)(PET) "lm was exposed to oxygen plasma glow discharge to produce peroxides on its surfaces. These peroxides were then used as catalysts for the polymerization of acrylic acid (AA) in order to prepare a carboxylic acid groupintroduced PET (PET-AA). Insulin and Heparin co-Immobilized PET (PET-I-H) was prepared by the grafting of poly(ethylene oxide) (PEO) on to PET-AA, followed by reaction "rst with insulin and then Heparin. These surface-modi"ed PETs were characterized by attenuated total re#ection Fourier transform infrared (ATR-FTIR) spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of the Heparin (1.23 lg/cm2) bound to the PEO-grafted PET (PET-PEO) was higher than that (0.77 lg/cm2) on the insulin-Immobilized PET (PET-In). The blood compatibilities of the surface-modi"ed PETs were examined using in vitro thrombus formation, plasma recalci"cation time (PRT), activated partial thromboplastin time (APTT), and platelet adhesion and activation. In the experiment with plasma proteins, the PRT and APTT were signi"cantly prolonged for both the Heparin-Immobilized PET (PET-He) and the PET-I-H, suggesting the binding of Immobilized Heparin to antithrombin III. The percentage of platelet adhesion slightly increased with the introduction of AA on the PET surfaces, decreased with the
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surface characterization and in vitro blood compatibility of poly ethylene terephthalate Immobilized with insulin and or Heparin using plasma glow discharge
Biomaterials, 2000Co-Authors: Youngjin Kim, Innkyu Kang, M W Huh, Sung Chul YoonAbstract:Abstract Poly(ethylene terephthalate)(PET) film was exposed to oxygen plasma glow discharge to produce peroxides on its surfaces. These peroxides were then used as catalysts for the polymerization of acrylic acid (AA) in order to prepare a carboxylic acid group-introduced PET (PET-AA). Insulin and Heparin co-Immobilized PET (PET-I-H) was prepared by the grafting of poly(ethylene oxide) (PEO) on to PET-AA, followed by reaction first with insulin and then Heparin. These surface-modified PETs were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of the Heparin (1.23 μg/cm2) bound to the PEO-grafted PET (PET-PEO) was higher than that (0.77 μg/cm2) on the insulin-Immobilized PET (PET-In). The blood compatibilities of the surface-modified PETs were examined using in vitro thrombus formation, plasma recalcification time (PRT), activated partial thromboplastin time (APTT), and platelet adhesion and activation. In the experiment with plasma proteins, the PRT and APTT were significantly prolonged for both the Heparin-Immobilized PET (PET-He) and the PET-I-H, suggesting the binding of Immobilized Heparin to antithrombin III. The percentage of platelet adhesion slightly increased with the introduction of AA on the PET surfaces, decreased with the introduction of PEO and insulin, and decreased further with the immobilization of Heparin. The release of serotonin was highly suppressed on PET-He and PET-I-H, and on surface-modified PETs the percentage of its release increased with an increase in platelet adhesion.
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synthesis and surface characterization of Heparin Immobilized polyetherurethanes
Journal of Polymer Science Part A, 1998Co-Authors: Innkyu Kang, Young Moo Lee, Donc Ki Baek, Yong Kiel SungAbstract:Novel poly(ether urethanes) containing diester groups in the side chains (PU) were synthesized from 4,4'-diphenylmethyl diisocyanate, polytetramethylene glycol, and diethyl bis(hydroxymethyl)malonate as a chain extender. The surface modification of the PU film was carried out by a hydrolysis reaction, poly(ethylene oxide) (PEO) grafting, and Heparin immobilization, and the surface-modified PUs were then characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, electron spectroscopy for chemical analysis (ESCA), and a contact angle goniometer. The concentration of carboxylic acid groups introduced on the PU surfaces as determined by the rhodamine interaction method was 61 nmol/cm 2 when treated with 4N NaOH/methanol (1: 2 v/v) for 30 min and subsequently with a citric acid-methanolic aqueous solution. The amounts of Heparin coupled to the carboxyl groups on the PU surfaces and to the terminus amino groups on the PU-PEO were 0.92 and 0.84 μ g/cm 2 , respectively. There was almost no Heparin released from the Immobilized surface of a physiological solution for 100 h, thereby indicating the strong stability of Immobilized Heparin.
Graciela Elgue - One of the best experts on this subject based on the ideXlab platform.
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studies of adsorption activation and inhibition of factor xii on Immobilized Heparin
Thrombosis Research, 1998Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:Abstract The aim of the present investigation was to clarify whether Immobilized Heparin does, as previously suggested, prevent triggering of the plasma contact activation system. Purified FXII in the absence or presence of antithrombin and/or C1 esterase inhibitor as well as plasma was exposed for 1 to 600 seconds to a surface modified by end-point immobilization of Heparin. With purified reagents, a process including surface adsorption and activation of FXII occurred within 1 second. In the presence of antithrombin, the resulting surface-bound α-FXIIa was inhibited within that time. Likewise, the adsorption of native FXII from plasma was a rapid process. However, the inhibition of surface-bound α-FXIIa was slightly slower than with purified components. Nevertheless, neither β-FXIIa nor FXIa were found in the plasma phase. Exposure of a surface prepared from Heparin molecules, lacking antithrombin binding properties, to plasma resulted in surface-bound α-FXIIa within 1 second. In the liquid phase, β-FXIIa was detected after 2.5 seconds and, 12 seconds later, FXIIa and FXIa in complex with the C1 esterase inhibitor appeared. Addition of Heparin to plasma prior to surface exposure did not prevent activation of surface-bound FXII, nor did it increase the β-FXIIa inhibition rate and prevent FXI activation in plasma, although β-FXIIa and FXIa-AT complex formation occurred. It is concluded that surface-Immobilized Heparin, unlike Heparin in solution, effectively inhibits the initial contact activation enzymes by an antithrombin-mediated mechanism, thereby suppressing the triggering of the intrinsic plasma coagulation pathway.
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inhibition of the plasma contact activation system of Immobilized Heparin relation to surface density of functional antithrombin binding sites
Journal of Biomedical Materials Research, 1997Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:End-point immobilization of Heparin to artificial materials gives rise to a surface that prevents triggering of the plasma contact activation system and, presumably as a result thereof, generally has thrombo-resistant properties. The present investigation was undertaken to determine what density of Immobilized Heparin molecules expressing functionally intact antithrombin binding sites is required to achieve these blood compatible properties. Six different Heparin surfaces were prepared on polyethylene tubing and studied in contact with human plasma. The content of bound Heparin was the same on all surfaces while the densities of antithrombin binding sites ranged from 1 to 28 pmol/cm2. The surfaces expressing 4 pmol/cm2 or more of specific antithrombin binding sites generated no measurable enzymatic activity in contact with plasma, either on the exposed surfaces or in the plasma phases. Below this level, the degree of activation gradually increased with decreasing densities, and in parallel the thrombo-resistant properties deteriorated. Addition of Heparin to the plasma phase reduced the capacity of the Heparin surfaces to bind antithrombin, leading to a diminished ability of the surfaces to prevent contact activation. This finding supports the hypothesis that antithrombin is the critical coagulation inhibitor for the suppression of contact activation on end-point Immobilized Heparin.
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control of contact activation on end point Immobilized Heparin the role of antithrombin and the specific antithrombin binding sequence
Journal of Biomedical Materials Research, 1995Co-Authors: Javier Sanchez, Graciela Elgue, J Riesenfeld, Per OlssonAbstract:The uptake and activation of FXII from blood plasma was studied in small-diameter polyethylene tubing, surface-modified by end-point immobilization of Heparin. Two preparations of Heparin were used to modify the contact-activating properties of the plastic tubing: unfractionated, functionally active Heparin and low-affinity Heparin, lacking the specific antithrombin-binding sequence and virtually devoid of anticoagulant activity. The uptakes of FXII on the two Heparin surfaces were similar. No activated FXII could be demonstrated on the unfractionated Heparin surface, whereas on the low-affinity Heparin surface nearly all FXII underwent spontaneous activation. The suppression of FXII activation on the unfractionated Heparin surface was investigated by using plasma depleted of antithrombin, complement C1 esterase inhibitor, or both. The removal of antithrombin resulted in extensive activation of FXII, whereas the depletion of C1 esterase inhibitor had only a minor effect. Experiments with recalcified plasma showed rapid clot formation during exposure to the low-affinity Heparin surface. After depletion of antithrombin, but not complement C1 esterase inhibitor, the recalcified plasma clotted in contact with the unfractionated Heparin surface as well. We conclude that antithrombin and the antithrombin-binding sequence in the surface-Immobilized Heparin are essential for the prevention of surface activation of FXII and triggering of the intrinsic coagulation system. © 1995 John Wiley & Sons, Inc.
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On the mechanism of coagulation inhibition on surfaces with end point Immobilized Heparin.
Thrombosis and Haemostasis, 1993Co-Authors: Graciela Elgue, Margareta Blomback, Per Olsson, J RiesenfeldAbstract:: A well established technique to improve blood compatibility of artificial materials for use in the circulation is to coat the surface with Heparin. The present report describes the antithrombin mediated inhibition of thrombin and factor Xa by surfaces modified with end point Immobilized Heparin. The reaction was followed by conventional chromogenic substrate based enzyme assays as well as by immunological measurement of the enzyme inhibitor (thrombin-antithrombin) complex formation. Both enzymes were rapidly inactivated by Heparin surfaces after selective presaturation with antithrombin on the Immobilized high affinity Heparin molecules. The thrombin inhibitory capacity was enhanced when both high and low affinity Heparin were preadsorbed with inhibitor. The main part of the thrombin-antithrombin complex formed remained bound to the surface, however, without functionally blocking the activity of the high affinity sequence of the Immobilized Heparin. Aliquots of recalcified plasma were slowly rotated in loops of Heparinized tubing to investigate whether the main thromboresistant function of the surface was exerted at the level of thrombin or by inactivation of preceding enzymes. After 1 h no visible clotting occurred and only trace amounts of thrombin (0.07 IU/ml), measured as thrombin-antithrombin complexes, had been formed. In non-Heparinized loops and in the test tube plasma clotted after 20 min. The thrombin generation when clotting occurred was in the order of 10 IU/ml. It is concluded that the Immobilized Heparin mediates inhibition of the coagulation cascade prior to prothrombin activation.
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binding of antithrombin to Immobilized Heparin under varying flow conditions
Artificial Organs, 1991Co-Authors: Boris Pasche, Graciela Elgue, J Riesenfeld, Per Olsson, Anders RasmusonAbstract:Rheologic factors are likely to influence the balance between thrombotic and antithrombotic forces at the level of the vascular wall. In this study, the effects of flow-velocity/wall shear stress on the interaction of antithrombin (AT) with surface-Immobilized Heparin were investigated. The binding of AT to low-affinity and high-affinity Heparin could be discriminated by measurements at physiological of elevated ionic strength. Under low shear stress conditions, substantial binding of AT to both high- and low-affinity Heparin was observed, in relative quantities largely reflecting the proportion of these polysaccharide populations on the surface. With increasing shear stress, the binding to high-affinity sites was relatively constant, while total low-affinity binding decreased. Furthermore, under the highest shear stress (greater than 1,000 N/m2), the binding of AT to low-affinity Heparin completely disappeared while binding to the high-affinity fraction persisted. These results were related to values obtained in a mathematical model, describing the theoretical maximum transport of AT from the liquid phase to the surface under the conditions used in the experimental system.
