The US regulatory and pharmacopeia response to the global heparin contamination crisis.

PubMed

Szajek, Anita Y; Chess, Edward; Johansen, Kristian; Gratzl, Gyöngyi; Gray, Elaine; Keire, David; Linhardt, Robert J; Liu, Jian; Morris, Tina; Mulloy, Barbara; Nasr, Moheb; Shriver, Zachary; Torralba, Pearle; Viskov, Christian; Williams, Roger; Woodcock, Janet; Workman, Wesley; Al-Hakim, Ali

2016-06-09

The contamination of the widely used lifesaving anticoagulant drug heparin in 2007 has drawn renewed attention to the challenges that are associated with the characterization, quality control and standardization of complex biological medicines from natural sources. Heparin is a linear, highly sulfated polysaccharide consisting of alternating glucosamine and uronic acid monosaccharide residues. Heparin has been used successfully as an injectable antithrombotic medicine since the 1930s, and its isolation from animal sources (primarily porcine intestine) as well as its manufacturing processes have not changed substantially since its introduction. The 2007 heparin contamination crisis resulted in several deaths in the United States and hundreds of adverse reactions worldwide, revealing the vulnerability of a complex global supply chain to sophisticated adulteration. This Perspective discusses how the US Food and Drug Administration (FDA), the United States Pharmacopeial Convention (USP) and international stakeholders collaborated to redefine quality expectations for heparin, thus making an important natural product better controlled and less susceptible to economically motivated adulteration.

Heparin-induced thrombocytopenia

PubMed Central

2017-01-01

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Pathogenic antibodies to PF4/heparin bind and activate cellular FcγRIIA on platelets and monocytes to propagate a hypercoagulable state culminating in life-threatening thrombosis. It is now recognized that anti-PF4/heparin antibodies develop commonly after heparin exposure, but only a subset of sensitized patients progress to life-threatening complications of thrombocytopenia and thrombosis. Recent scientific developments have clarified mechanisms underlying PF4/heparin immunogenicity, disease susceptibility, and clinical manifestations of disease. Insights from clinical and laboratory findings have also been recently harnessed for disease prevention. This review will summarize our current understanding of HIT by reviewing pathogenesis, essential clinical and laboratory features, and management. PMID:28416511

High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface*

PubMed Central

Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W.; Linhardt, Robert J.; Sharp, Joshua S.

2015-01-01

Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes. PMID:25752613

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

PubMed

Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

2015-06-01

The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamic properties of biologically active synthetic heparin-like hexasaccharides.

PubMed

Angulo, Jesús; Hricovíni, Milos; Gairi, Margarida; Guerrini, Marco; de Paz, José Luis; Ojeda, Rafael; Martín-Lomas, Manuel; Nieto, Pedro M

2005-10-01

A complete study of the dynamics of two synthetic heparin-like hexasaccharides, D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (1) and -->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHAc-6-SO4-alpha-(1-->4)-L-IdoA-alpha-(1-->4)-D-GlcNHSO3-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (2), has been performed using 13C-nuclear magnetic resonance (NMR) relaxation parameters, T1, T2, and heteronuclear nuclear Overhauser effect (NOEs). Compound 1 is constituted from sequences corresponding to the major polysaccharide heparin region, while compound 2 contains a sequence never found in natural heparin. They differ from each other only in sulphation patterns, and are capable of stimulating fibroblast growth factors (FGFs)-1 induced mitogenesis. Both oligosaccharides exhibit a remarkable anisotropic overall motion in solution as revealed by their anisotropic ratios (tau /tau||), 4.0 and 3.0 respectively. This is a characteristic behaviour of natural glycosaminoglycans (GAG) which has also been observed for the antithrombin (AT) binding pentasaccharide D-GlcNHSO3-6-SO4-alpha-(1-->4)-D-GlcA-beta-(1-->4)-D-GlcNHSO3-(3,6-SO4)-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-1-->Me (3) (Hricovíni, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11-23). The motional properties observed for 1 and 2 provide additional support to the suitability of these compounds as heparin models in agreement with previous structural (de Paz, J.L., Angulo, J., Lassaletta, J.M., Nieto, P.M., Redondo-Horcajo, M., Lozano, R.M., Jiménez-Gallego, G., and Martín-Lomas, M. (2001) The activation of fibroblast growth factors by heparin: synthesis, structure and biological activity of heparin-like oligosaccharides. Chembiochem, 2, 673-685; Ojeda, R., Angulo, J., Nieto, P.M., and Martin-Lomas. M. (2002) The activation of fibroblast growth factors by heparin: synthesis and structural study of rationally modified heparin-like oligosaccharides. Can. J. Chem,. 80, 917-936; Lucas, R., Angulo, J., Nieto, P.M., and Martin-Lomas, M. (2003) Synthesis and structural studies of two new heparin-like hexasaccharides. Org. Biomol. Chem., 1, 2253-2266) and biological data (Angulo, J., Ojeda, R., de Paz, J.L., Lucas, R., Nieto, P.M., Lozano, R.M., Redondo-Horcajo, M., Giménez-Gallego, G., and Martín-Lomas, M. (2004) The activation of fibroblast growth factors (FGFs) by glycosaminoglycans: influence of the sulphation pattern on the biological activity of FGF-1. Chembiochem, 5, 55-61). Fast internal motions observed for the less sulphated compound 2, as compared with 1, may be related to their different behavior in stimulating FGF1-induced mitogenic activity.

Effect of SanOrg123781A, a synthetic hexadecasaccharide, on clot-bound thrombin and factor Xa in vitro and in vivo.

PubMed

Hérault, J-P; Cappelle, M; Bernat, A; Millet, L; Bono, F; Schaeffer, P; Herbert, J-M

2003-09-01

Factor (F)Xa and thrombin bound to the clot during its formation contribute to the propensity of thrombi to activate the coagulation system. The aim of this work was to study the inhibition of clot-bound FXa and clot-bound thrombin by SanOrg123781A, a synthetic hexadecasaccharide that enhances the inhibition of thrombin and FXa by antithrombin (AT). SanOrg123781A, designed to exhibit low non-specific binding to proteins other than AT, was compared with heparin. In buffer, heparin and SanOrg123781A inhibited FXa and thrombin at similar concentrations [concentration inhibiting 50% (IC50) of Xa and IIa activity were, respectively: heparin 120 +/- 7 and 3 +/- 1 ng mL-1; SanOrg123781A 77 +/- 5 and 4 +/- 1 ng mL-1]. In human plasma, the activity of both compounds was reduced, although the activity of heparin was much more affected than that of SanOrg123781A (IC50 values for inhibition of FXa and FIIa activity were, respectively: heparin 100 +/- 5 and 800 +/- 40 ng mL-1; SanOrg123781A 10 +/- 5 and 30 +/- 3 ng mL-1). We demonstrated, in agreement with our previous results, that the procoagulant activity of the clot is essentially due to clot-bound FXa and to some extent to clot-bound thrombin. We showed that heparin and SanOrg123781A were able to inhibit fragment F1+2 generation induced by clot-bound FXa with IC50 values of 2 +/- 0.5 micro g mL-1 and 0.6 +/- 0.2 micro g mL-1, respectively. Both compounds also inhibited clot-bound thrombin activity, the IC50 values of heparin and SanOrg123781A being 1 +/- 0.01 micro g mL-1 and 0.1 +/- 0.1 micro g mL-1, respectively. Moreover, both heparin and SanOrg123781A significantly inhibited fibrinopeptide A generated by the action of clot-bound thrombin on fibrinogen but also by free thrombin generated from prothrombin by clot-bound FXa with IC50 values of 4 +/- 0.6 and 1 +/- 0.1 micro g mL-1, respectively. As with clot-bound enzymatic activities, SanOrg123781A was three times more active than heparin in vivo on fibrinogen accretion onto a pre-existing thrombus and as activators of recombinant tissue-type plasminogen activator-induced thrombolysis. In conclusion, due to the specific activities of SanOrg123781A, this compound is much more active than heparin in the presence of plasma proteins, on clot-bound enzymes and in in vivo models of thrombosis/thrombolysis.

Advanced nanocarriers based on heparin and its derivatives for cancer management.

PubMed

Yang, Xiaoye; Du, Hongliang; Liu, Jiyong; Zhai, Guangxi

2015-02-09

To obtain a satisfying anticancer effect, rationally designed nanocarriers are intensively studied. In this field, heparin and its derivatives have been widely attempted recently as potential component of nanocarriers due to their unique biological and physiochemical features, especially the anticancer activity. This review focuses on state-of-the-art nanocarriers with heparin/heparin derivatives as backbone or coating material. At the beginning, the unique advantages of heparin used in cancer nanotechnology are discussed. After that, different strategies of heparin chemical modification are reviewed, laying the foundation of developing various nanocarriers. Then a systematic summary of diverse nanoparticles with heparin as component is exhibited, involving heparin-drug conjugate, polymeric nanoparticles, nanogels, polyelectrolyte complex nanoparticles, and heparin-coated organic and inorganic nanoparticles. The application of these nanoparticles in various novel cancer therapy (containing targeted therapy, magnetic therapy, photodynamic therapy, and gene therapy) will be highlighted. Finally, future challenges and opportunities of heparin-based biomaterials in cancer nanotechnology are discussed.

Investigation of a Potential Protective Mechanism Against Heparin-Induced Thrombocytopenia in Patients on Chronic Intermittent Hemodialysis

PubMed Central

Tanhehco, Yvette C.; Cuker, Adam; Rudnick, Michael; Sachais, Bruce S.

2015-01-01

BACKGROUND Heparin-induced thrombocytopenia (HIT) develops as a result of platelet (PLT) activation by anti-platelet factor 4 (PF4)/heparin complex antibodies. Despite repeated exposure to heparin, patients undergoing chronic intermittent hemodialysis (HD) rarely develop HIT. We investigated the possibility that HD decreases/removes PF4 from PLT surfaces and/or plasma, thereby disfavoring immune complex formation as a mechanism of protection against HIT. MATERIALS AND METHODS We enrolled 20 patients undergoing chronic HD at the Penn Presbyterian Medical Center. Blood samples were drawn before, during and after treatment in the presence and absence of heparin. PF4, PF4/heparin antibody, heparin, and P-selectin levels were measured. RESULTS No patients demonstrated clinical symptoms of HIT. PLT surface PF4 levels decreased and plasma PF4 levels increased concurrently with increase in plasma heparin concentration. In the absence of heparin, PLT surface and plasma PF4 levels were unchanged. Anti-PF4/heparin antibodies, which were non-functional by the serotonin release assay, were detectable in 8 patients. PLT surface P-selectin levels did not change during treatment. CONCLUSIONS Removal of PLT surface and/or plasma PF4 as a mechanism of protection against HIT in patients undergoing HD is not supported by the results of our study, although the transient decrease in PLT surface PF4 in the presence of large amounts of heparin remains a candidate mechanism. The small sample size, single type of dialyzer membrane, and early sampling time points may have led to the inability to detect changes in PF4 levels. Future studies should explore other potential protective mechanisms. PMID:23305841

Development of hydrophilic interaction chromatography with quadruple time-of-flight mass spectrometry for heparin and low molecular weight heparin disaccharide analysis.

PubMed

Ouyang, Yilan; Wu, Chengling; Sun, Xue; Liu, Jianfen; Linhardt, Robert J; Zhang, Zhenqing

2016-01-30

Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts. Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products. HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes. Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products. Copyright © 2015 John Wiley & Sons, Ltd.

Atomic features of an autoantigen in heparin-induced thrombocytopenia (HIT).

PubMed

Cai, Zheng; Zhu, Zhiqiang; Greene, Mark I; Cines, Douglas B

2016-07-01

Autoantigen development is poorly understood at the atomic level. Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). In solution, PF4 exists as an equilibrium among monomers, dimers and tetramers. Structural studies of these interacting components helped delineate a multi-step process involved in the pathogenesis of HIT. First, heparin binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation; exposing the 'open' end. Second, PF4 arrays along heparin/GAG chains, which approximate tetramers, form large antigenic complexes that enhance antibody avidity. Third, pathogenic HIT antibodies bind to the 'open' end of stabilized PF4 tetramers to form an IgG/PF4/heparin ternary immune complex and also to propagate the formation of 'ultralarge immune complexes' (ULCs) that contain multiple IgG antibodies. Fourth, ULCs signal through FcγRIIA receptors, activating platelets and monocytes directly and generating thrombin, which transactivates hematopoietic and endothelial cells. A non-pathogenic anti-PF4 antibody prevents tetramer formation, binding of pathogenic antibody, platelet activation and thrombosis, providing a new approach to manage HIT. An improved understanding of the pathogenesis of HIT may lead to novel diagnostics and therapeutics for this autoimmune disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis.

PubMed

Padmanabhan, Anand; Jones, Curtis G; Bougie, Daniel W; Curtis, Brian R; McFarland, Janice G; Wang, Demin; Aster, Richard H

2015-01-01

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are the hallmark of heparin-induced thrombocytopenia and thrombosis (HIT), but many antibody-positive patients have normal platelet counts. The basis for this is not fully understood, but it is believed that antibodies testing positive in the serotonin release assay (SRA) are the most likely to cause disease. We addressed this issue by characterizing PF4-dependent binding of HIT antibodies to intact platelets and found that most antibodies testing positive in the SRA, but none of those testing negative, bind to and activate platelets when PF4 is present without any requirement for heparin (P < .0001). Binding of SRA-positive antibodies to platelets was inhibited by chondroitinase ABC digestion (P < .05) and by the addition of chondroitin-4-sulfate (CS) or heparin in excess quantities. The findings suggest that although all HIT antibodies recognize PF4 in a complex with heparin, only a subset of these antibodies recognize more subtle epitopes induced in PF4 when it binds to CS, the major platelet glycosaminoglycan. Antibodies having this property could explain "delayed HIT" seen in some individuals after discontinuation of heparin and the high risk for thrombosis that persists for weeks in patients recovered from HIT. © 2015 by The American Society of Hematology.

Heparin-independent, PF4-dependent binding of HIT antibodies to platelets: implications for HIT pathogenesis

PubMed Central

Jones, Curtis G.; Bougie, Daniel W.; Curtis, Brian R.; McFarland, Janice G.; Wang, Demin; Aster, Richard H.

2015-01-01

Antibodies specific for platelet factor 4 (PF4)/heparin complexes are the hallmark of heparin-induced thrombocytopenia and thrombosis (HIT), but many antibody-positive patients have normal platelet counts. The basis for this is not fully understood, but it is believed that antibodies testing positive in the serotonin release assay (SRA) are the most likely to cause disease. We addressed this issue by characterizing PF4-dependent binding of HIT antibodies to intact platelets and found that most antibodies testing positive in the SRA, but none of those testing negative, bind to and activate platelets when PF4 is present without any requirement for heparin (P < .0001). Binding of SRA-positive antibodies to platelets was inhibited by chondroitinase ABC digestion (P < .05) and by the addition of chondroitin-4-sulfate (CS) or heparin in excess quantities. The findings suggest that although all HIT antibodies recognize PF4 in a complex with heparin, only a subset of these antibodies recognize more subtle epitopes induced in PF4 when it binds to CS, the major platelet glycosaminoglycan. Antibodies having this property could explain “delayed HIT” seen in some individuals after discontinuation of heparin and the high risk for thrombosis that persists for weeks in patients recovered from HIT. PMID:25342714

Cell-mediated Delivery and Targeted Erosion of Noncovalently Crosslinked Hydrogels

NASA Technical Reports Server (NTRS)

Kiick, Kristi L. (Inventor); Yamaguchi, Nori (Inventor)

2013-01-01

A method for targeted delivery of therapeutic compounds from hydrogels is presented. The method involves administering to a cell a hydrogel in which a therapeutic compound is noncovalently bound to heparin.

Heparin-induced thrombocytopenia

PubMed Central

Shaikh, Nissar

2011-01-01

In the last 7 decades heparin has remained the most commonly used anticoagulant. Its use is increasing, mainly due to the increase in the number of vascular interventions and aging population. The most feared complication of heparin use is heparin-induced thrombocytopenia (HIT). HIT is a clinicopathologic hypercoagulable, procoagulant prothrombotic condition in patients on heparin therapy, and decrease in platelet count by 50% or to less than 100,000, from 5 to 14 days of therapy. This prothrombotic hypercoagulable state in HIT patient is due to the combined effect of various factors, such as platelet activation, mainly the formation of PF4/heparin/IgG complex, stimulation of the intrinsic factor, and loss of anticoagulant effect of heparin. Diagnosis of HIT is done by clinical condition, heparin use, and timing of thrombocytopenia, and it is confirmed by either serotonin release assay or ELISA assay. Complications of HIT are venous/arterial thrombosis, skin gangrene, and acute platelet activation syndrome. Stopping heparin is the basic initial treatment, and Direct Thrombin Inhibitors (DTI) are medication of choice in these patients. A few routine but essential procedures performed by using heparin are hemodialysis, Percutaneous Coronary Intervention, and Cardiopulmonary Bypass; but it cannot be used if a patient develops HIT. HIT patients with unstable angina, thromboembolism, or indwelling devices, such as valve replacement or intraaortic balloon pump, will require alternative anticoagulation therapy. HIT can be prevented significantly by keeping heparin therapy shorter, avoiding bovine heparin, using low-molecular weight heparin, and stopping heparin use for flush and heparin lock. PMID:21633576

Adeno-associated virus-2 and its primary cellular receptor-Cryo-EM structure of a heparin complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

2009-03-15

Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R{sub 448/585}, R{sub 451/588} and R{sub 350/487} from another subunit cluster at the center of the heparin footprint. The footprint is much more extensivemore » than apparent through mutagenesis, including R{sub 347/484}, K{sub 395/532} and K{sub 390/527} that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.« less

In vitro and in vivo characterization of a reversible synthetic heparin analog.

PubMed

Whelihan, Matthew F; Cooley, Brian; Xu, Yongmei; Pawlinski, Rafal; Liu, Jian; Key, Nigel S

2016-02-01

The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

Anti-Tumor Activity of a Novel HS-Mimetic-Vascular Endothelial Growth Factor Binding Small Molecule

PubMed Central

Sugahara, Kazuyuki; Thimmaiah, Kuntebommanahalli N.; Bid, Hemant K.; Houghton, Peter J.; Rangappa, Kanchugarakoppal S.

2012-01-01

The angiogenic process is controlled by variety of factors of which the vascular endothelial growth factor (VEGF) pathway plays a major role. A series of heparan sulfate mimetic small molecules targeting VEGF/VEGFR pathway has been synthesized. Among them, compound 8 (2-butyl-5-chloro-3-(4-nitro-benzyl)-3H-imidazole-4-carbaldehyde) was identified as a significant binding molecule for the heparin-binding domain of VEGF, determined by high-throughput-surface plasmon resonance assay. The data predicted strong binding of compound 8 with VEGF which may prevent the binding of VEGF to its receptor. We compared the structure of compound 8 with heparan sulfate (HS), which have in common the functional ionic groups such as sulfate, nitro and carbaldehyde that can be located in similar positions of the disaccharide structure of HS. Molecular docking studies predicted that compound 8 binds at the heparin binding domain of VEGF through strong hydrogen bonding with Lys-30 and Gln-20 amino acid residues, and consistent with the prediction, compound 8 inhibited binding of VEGF to immobilized heparin. In vitro studies showed that compound 8 inhibits the VEGF-induced proliferation migration and tube formation of mouse vascular endothelial cells, and finally the invasion of a murine osteosarcoma cell line (LM8G7) which secrets high levels of VEGF. In vivo, these effects produce significant decrease of tumor burden in an experimental model of liver metastasis. Collectively, these data indicate that compound 8 may prevent tumor growth through a direct effect on tumor cell proliferation and by inhibition of endothelial cell migration and angiogenesis mediated by VEGF. In conclusion, compound 8 may normalize the tumor vasculature and microenvironment in tumors probably by inhibiting the binding of VEGF to its receptor. PMID:22916091

In vitro induction of protein complexes between bevacizumab, VEGF-A¹⁶⁵ and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

PubMed

Julien, Sylvie; Biesemeier, Antje; Schraermeyer, Ulrich

2013-04-01

By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

Atomic description of the immune complex involved in heparin-induced thrombocytopenia

DOE PAGES

Cai, Zheng; Yarovoi, Serge V.; Zhu, Zhiqiang; ...

2015-09-22

Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the ‘closed’ end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the ‘open’ end of the tetramer. Fondaparinux and KKO thereby collaborate to ‘stabilize’ the ternary pathogenic immune complex. Bindingmore » of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. Lastly, the atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.« less

Prevention of equine herpesvirus myeloencephalopathy - Is heparin a novel option? A case report.

PubMed

Walter, Jasmin; Seeh, Christoph; Fey, Kerstin; Bleul, Ulrich; Osterrieder, Nikolaus

2016-10-12

Equine herpesvirus myeloencephalopathy (EHM) is a severe manifestation of equine herpesvirus 1 (EHV-1) infection. Prevention and treatment of EHM during EHV-1 outbreaks is critical, but no reliable and tested specific medication is available. Due to the thromboischemic nature of EHM and due to the fact that EHV-1 entry in cells is blocked by heparin, it was hypothesized that this compound may be useful in reduction of EHM incidence and severity. Therefore, during an acute EHV-1 outbreak with the neuropathogenic G 2254 /D 752 Pol variant, metaphylactic treatment with heparin to prevent EHM was initiated. Clinical signs were present in 61 horses (fever n = 55; EHM n = 8; abortion n = 6). Heparin (25000 IU subcutaneously twice daily for 3 days) was given to 31 febrile horses from day 10 of the outbreak, while the first 30 horses exhibiting fever remained untreated. Treatment outcome was analyzed retrospectively. Heparin-treated horses showed a lower EHM incidence (1/31; 3.2%) than untreated horses (7/30; 23.3%; p = 0.03). Results indicate that heparin may be useful for prevention of EHM during an EHV-1 outbreak. These promising data highlight the need for randomized and possibly blinded studies for the use of heparin in EHV-1 outbreaks.

Biotechnological engineering of heparin/heparan sulphate: a novel area of multi-target drug discovery.

PubMed

Rusnati, Marco; Oreste, Pasqua; Zoppetti, Giorgio; Presta, Marco

2005-01-01

Heparin is a sulphated glycosaminoglycan currently used as an anticoagulant and antithrombotic drug. It consists largely of 2-O-sulphated IdoA not l&r arrow N, 6-O-disulphated GlcN disaccharide units. Other disaccharides containing unsulphated IdoA or GlcA and N-sulphated or N-acetylated GlcN are also present as minor components. This heterogeneity is more pronounced in heparan sulphate (HS), where the low-sulphated disaccharides are the most abundant. Heparin/HS bind to a variety of biologically active polypeptides, including enzymes, growth factors and cytokines, and viral proteins. This capacity can be exploited to design multi-target heparin/HS-derived drugs for pharmacological interventions in a variety of pathologic conditions besides coagulation and thrombosis, including neoplasia and viral infection. The capsular K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor N-acetyl heparosan. The possibility of producing K5 polysaccharide derivatives by chemical and enzymatic modifications, thus generating heparin/HS-like compounds, has been demonstrated. These K5 polysaccharide derivatives are endowed with different biological properties, including anticoagulant/antithrombotic, antineoplastic, and anti-AIDS activities. Here, the literature data are discussed and the possible therapeutic implications for this novel class of multi-target "biotechnological heparin/HS" molecules are outlined.

Molecular dynamics-based model of VEGF-A and its heparin interactions.

PubMed

Uciechowska-Kaczmarzyk, Urszula; Babik, Sándor; Zsila, Ferenc; Bojarski, Krzysztof Kamil; Beke-Somfai, Tamás; Samsonov, Sergey A

2018-06-01

We present a computational model of the Vascular Endothelial Growth Factor (VEGF), an important regulator of blood vessels formation, which function is affected by its heparin interactions. Although structures of a receptor binding (RBD) and a heparin binding domain (HBD) of VEGF are known, there are structural data neither on the 12 amino acids interdomain linker nor on its complexes with heparin. We apply molecular docking and molecular dynamics techniques combined with circular dichroism spectroscopy to model the full structure of the dimeric VEGF and to propose putative molecular mechanisms underlying the function of VEGF/VEGF receptors/heparin system. We show that both the conformational flexibility of the linker and the formation of HBD-heparin-HBD sandwich-like structures regulate the mutual disposition of HBDs and so affect the VEGF-mediated signalling. Copyright © 2018 Elsevier Inc. All rights reserved.

Hydrogels with covalent and noncovalent crosslinks

NASA Technical Reports Server (NTRS)

Kilck, Kristi L. (Inventor); Yamaguchi, Nori (Inventor)

2013-01-01

A method for targeted delivery of therapeutic compounds from hydrogels is presented. The method involves administering to a cell a hydrogel in which a therapeutic compound is noncovalently bound to heparin. The hydrogel may contain covalent and non-covalent crosslinks.

Hydrazinolysis of heparin and other glycosaminoglycans.

PubMed Central

Shaklee, P N; Conrad, H E

1984-01-01

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed. PMID:6421280

New Insights in Thrombin Inhibition Structure-Activity Relationships by Characterization of Octadecasaccharides from Low Molecular Weight Heparin.

PubMed

Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Sizun, Philippe; Viskov, Christian

2017-03-08

Low Molecular Weight Heparins (LMWH) are complex anticoagulant drugs that mainly inhibit the blood coagulation cascade through indirect interaction with antithrombin. While inhibition of the factor Xa is well described, little is known about the polysaccharide structure inhibiting thrombin. In fact, a minimal chain length of 18 saccharides units, including an antithrombin (AT) binding pentasaccharide, is mandatory to form the active ternary complex for LMWH obtained by alkaline β-elimination (e.g., enoxaparin). However, the relationship between structure of octadecasaccharides and their thrombin inhibition has not been yet assessed on natural compounds due to technical hurdles to isolate sufficiently pure material. We report the preparation of five octadecasaccharides by using orthogonal separation methods including size exclusion, AT affinity, ion pairing and strong anion exchange chromatography. Each of these octadecasaccharides possesses two AT binding pentasaccharide sequences located at various positions. After structural elucidation using enzymatic sequencing and NMR, in vitro aFXa and aFIIa were determined. The biological activities reveal the critical role of each pentasaccharide sequence position within the octadecasaccharides and structural requirements to inhibit thrombin. Significant differences in potency, such as the twenty-fold magnitude difference observed between two regioisomers, further highlights the importance of depolymerisation process conditions on LMWH biological activity.

CXCL4-platelet factor 4, heparin-induced thrombocytopenia and cancer.

PubMed

Sandset, Per Morten

2012-04-01

Platelet factor 4 (CXCL4-PF4) is a chemokine that binds to and neutralizes heparin and other negatively charged proteoglycans, but is also involved in angiogenesis and cancer development. In some patients exposed to heparin, antibodies are generated against the CXCL-PF4/heparin complex that may activate platelets and coagulation and lead to thrombocytopenia and arterial or venous thrombosis, a condition commonly named heparin induced thrombocytopenia (HIT). HIT has been investigated in numerous clinical settings, but there is limited data on the epidemiology and phenotype of HIT in cancer patients. The present review describes the role of CXCL4-PF4 in cancer, the immunobiology, clinical presentation and diagnosis of HIT, and the specific problems faced in cancer patients. Copyright © 2012 Elsevier Ltd. All rights reserved.

Induction of anti-PF4/heparin antibodies after arthroplasty for rheumatic diseases.

PubMed

Migita, Kiyoshi; Asano, Tomoyuki; Sato, Shuzo; Motokawa, Satoru

2018-04-17

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. These pathogenic antibodies against PF4/heparin bind and activate cellular FcγRIIa on platelets to induce a hypercoagulable state culminating in thrombosis. Recent studies indicate several conditions, including joint surgery, induce spontaneous HIT, which can occur without exposure to heparin. To determine the real-world evidences concerning the incidences of venous thromboembolism (VTE) after total joint arthroplasty for rheumatic disease, we conducted a multicenter cohort study (J-PSVT) designed to document the VTE and seroconversion rates of anti-PF4/heparin antibody in 34 Japanese National hospital organization (NHO) hospitals. J-PSVT indicated that prophylaxis with fondaparinux, not enoxaparin, reduces the risk of deep vein thrombosis in patients undergoing arthroplasty. Multivariate analysis revealed that dynamic mechanical thromboprophylaxis (intermittent plantar device) was an independent risk factor for seroconversion of anti-PF4/heparin antibodies, which was also confirmed by propensity-score matching. Seroconversion rates of anti-PF4/heparin antibodies were significantly reduced in rheumatoid arthritis (RA) patients compared with osteoarthritis (OA) patients, which may link with the findings that IgG fractions isolated from RA patients not OA patients contained PF4. Our study indicated that a unique profile of anti-PF4/heparin antibodies is induced by arthroplasty for rheumatic diseases.

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

PubMed

Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

2018-01-24

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Novel HIT antibody detection method using Sonoclot® coagulation analyzer.

PubMed

Wanaka, Keiko; Asada, Reiko; Miyashita, Kumiko; Kaneko, Makoto; Endo, Hirokazu; Yatomi, Yutaka

2015-01-01

Since heparin-induced thrombocytopenia (HIT), caused by the generation of antibodies against platelet factor 4 (PF4)/heparin complexes (HIT antibodies), may induce serious complications due to thrombosis, a prompt diagnosis is desirable. Functional tests with platelet activation to detect HIT antibodies are useful for diagnosis of HIT, in particular (14)C-selotonin release assay (SRA). However, they are complicated and so can be performed only in limited laboratories. We tested if a blood coagulation test using Sonoclot® analyzer can serve for the detection of HIT antibodies. A murine monoclonal antibody (HIT-MoAb) against PF4/heparin complexes was used as an alternative to human HIT antibodies. To the mixture of HIT-MoAb and heparin (0.5 U/mL, final), whole blood obtained from a healthy volunteer was added, and then the activated clotting time (ACT), clot rate (CR), and area under the curve (AUC) were measured with Sonoclot® analyzer for 30minutes. The HIT-MoAb (30 to 100μg/mL, final) concentration dependently suppressed the anticoagulation activity (prolongation of ACT and decrease of CR and AUC) of heparin. The suppression of anticoagulation effect of heparin by HIT-MoAb was demonstrated by measurements using Sonoclot® analyzer. This method may provide a new tool for screening of HIT antibodies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recent advances in the diagnosis and treatment of heparin-induced thrombocytopenia

PubMed Central

Bakchoul, Tamam

2012-01-01

Heparin-induced thrombocytopenia (HIT) is a drug-mediated, prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After their binding to PF4/heparin complexes on the platelet surface, HIT antibodies are capable of intravascular platelet activation by cross-linking Fcγ receptor IIA leading to a platelet count decrease and/or thrombosis. Diagnosis of HIT is often difficult. This, and the low specificity of the commercially available immunoassays, leads currently to substantial overdiagnosis of HIT. Timing of onset, the moderate nature of thrombocytopenia, and the common concurrence of thrombosis are very important factors, which help to differentiate HIT from other potential causes of thrombocytopenia. A combination of a clinical pretest scoring system and laboratory investigation is usually necessary to diagnose HIT. Although HIT is considered to be a rare complication of heparin treatment, the very high number of hospital inpatients, and increasingly also hospital outpatients receiving heparin, still result in a considerable number of patients developing HIT. If HIT occurs, potentially devastating complications such as life-threatening thrombosis make it one of the most serious adverse drug reactions. If HIT is strongly suspected, all heparin must be stopped and an alternative nonheparin anticoagulant started at a therapeutic dose to prevent thromboembolic complications. However, the nonheparin alternative anticoagulants bear a considerable bleeding risk, especially if given to patients with thrombocytopenia due to other reasons than HIT. While established drugs for HIT are disappearing from the market (lepirudin, danaparoid), bivalirudin, fondaparinux and potentially the new anticoagulants such as dabigatran, rivaroxaban and apixaban provide new treatment options. PMID:23606934

Corrosion resistance and biocompatibility of magnesium alloy modified by alkali heating treatment followed by the immobilization of poly (ethylene glycol), fibronectin and heparin.

PubMed

Pan, Changjiang; Hu, Youdong; Hou, Yu; Liu, Tao; Lin, Yuebin; Ye, Wei; Hou, Yanhua; Gong, Tao

2017-01-01

In recent years, magnesium alloys are attracting more and more attention as a kind of biodegradable metallic biomaterials, however, their uncontrollable biodegradation speed in vivo and the limited surface biocompatibility hinder their clinical applications. In the present study, with the aim of improving the corrosion resistance and biocompatibility, the magnesium alloy (AZ31B) surface was modified by alkali heating treatment followed by the self-assembly of 3-aminopropyltrimethoxysilane (APTMS). Subsequently, poly (ethylene glycol) (PEG) and fibronectin or fibronectin/heparin complex were sequentially immobilized on the modified surface. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) confirmed that the above molecules were successfully immobilized on the magnesium alloy surface. An excellent hydrophilic surface was obtained after the alkali heating treatment while the hydrophilicity decreased to some degree after the self-assembly of APTMS, the surface hydrophilicity was gradually improved again after the immobilization of PEG, fibronectin or fibronectin/heparin complex. The corrosion resistance of the control magnesium alloy was significantly improved by the alkali heating treatment. The self-assembly of APTMS and the following immobilization of PEG further enhanced the corrosion resistance of the substrates, however, the grafting of fibronectin or fibronectin/heparin complex slightly lowered the corrosion resistance. As compared to the pristine magnesium alloy, the samples modified by the immobilization of PEG and fibronectin/heparin complex presented better blood compatibility according to the results of hemolysis assay and platelet adhesion as well as the activated partial thromboplastin time (APTT). In addition, the modified substrates had better cytocompatibility to endothelial cells due to the improved anticorrosion and the introduction of fibronectin. The substrates modified by fibronectin or fibronectin/heparin complex can significantly promote endothelial cell adhesion and proliferation. Taking all these results into consideration, the method of the present study can be used for the surface modification of the magnesium alloy to simultaneously impart it better corrosion resistance, favorable blood compatibility and good cytocompatibility to endothelial cells. Copyright © 2016 Elsevier B.V. All rights reserved.

A Turn-on Fluorescence Sensor for Heparin Detection Based on a Release of Taiwan Cobra Cardiotoxin from a DNA Aptamer or Adenosine-Based Molecular Beacon.

PubMed

Shi, Yi-Jun; Wang, Liang-Jun; Lee, Yuan-Chin; Huang, Chia-Hui; Hu, Wan-Ping; Chang, Long-Sen

2018-02-19

This study presents two sensitive fluorescent assays for sensing heparin on the basis of the electrostatic interaction between heparin and Naja naja atra cardiotoxin 3 (CTX3). Owing to CTX3-induced folded structure of an adenosine-based molecular beacon (MB) or a DNA aptamer against CTX3, a reduction in the fluorescent signal of the aptamer or MB 5'-end labeled with carboxyfluorescein (FAM) and 3'-end labeled with 4-([4-(dimethylamino)phenyl]azo)-benzoic acid (DABCYL) was observed upon the addition of CTX3. The presence of heparin and formation of the CTX3-heparin complex caused CTX3 detachment from the MB or aptamer, and restoration of FAM fluorescence of the 5'-FAM-and-3'-DABCYL-labeled MB and aptamer was subsequently noted. Moreover, the detection of heparin with these CTX3-aptamer and CTX3-MB sensors showed high sensitivity and selectivity toward heparin over chondroitin sulfate and hyaluronic acid regardless of the presence of plasma. The limit of detection for heparin in plasma was determined to be 16 ng/mL and 15 ng/mL, respectively, at a signal-to-noise ratio of 3. This study validates the practical utility of the CTX3-aptamer and CTX3-MB systems for determining the concentration of heparin in a biological matrix.

The heparin-Ca(2+) interaction: the influence of the O-sulfation pattern on binding.

PubMed

Chevalier, Franck; Lucas, Ricardo; Angulo, Jesús; Martin-Lomas, Manuel; Nieto, Pedro M

2004-04-02

The specific binding of Ca(2+) to synthetic hexasaccharide models of modified heparin has been investigated by NMR and molecular modeling and compared with previous results on a model of regular heparin. These two models represent the regular region of heparin lacking one type of O-sulfate group, either at C-6 of glucosamine or at C-2 of iduronate. The NMR experiments show different responses to the presence of Ca(2+). In the case of the compound lacking O-sulfate groups at C-2, the results are indicative of specific binding similar to that observed for the regular heparin, while the model lacking sulfate groups in position 6 interacts more weakly with Ca(2+). In order to understand the basis of this difference, a molecular modeling study based on a rigid body docking approach of the interaction of these carbohydrates with Ca(2+) and Na(+) was performed. We have found that the results are strongly dependent on the starting orientation of the lateral side chains of the charged groups of the carbohydrate, and that the best agreement with the experimental results is obtained when the starting conformations are taken from previous simulations in the presence of Ca(2+).

Binding of heparin to plasma proteins and endothelial surfaces is inhibited by covalent linkage to antithrombin.

PubMed

Chan, Anthony K C; Paredes, Nethnapha; Thong, Bruce; Chindemi, Paul; Paes, Bosco; Berry, Leslie R; Monagle, Paul

2004-05-01

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are used for prophylaxis and treatment of thrombosis. However, UFH has a short plasma half-life and variable anticoagulant response in vivo due to plasma or vessel wall protein binding and LMWH has a decreased ability to inactivate thrombin, the pivotal enzyme in the coagulation cascade. Covalent linkage of antithrombin to heparin gave a complex (ATH) with superior anticoagulant activity compared to UFH and LMWH, and longer intravenous half-life compared to UFH. We found that plasma proteins bound more to UFH than ATH, and least to LMWH. Also, UFH bound significantly more to endothelial cells than ATH, with 100% of UFH and 94% of ATH binding being on the cell surface and the remainder was endocytosed. Competition studies with UFH confirmed that ATH binding was likely through its heparin moiety. These findings suggest that differences in plasma protein and endothelial cell binding may be due to available heparin chain length. Although ATH is polydisperse, the covalently-linked antithrombin may shield a portion of the heparin chain from association with plasma or endothelial cell surface proteins. This model is consistent with ATH's better bioavailability and more predictable dose response.

Positive modulator of bone morphogenic protein-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zamora, Paul O.; Pena, Louis A.; Lin, Xinhua

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

Positive modulator of bone morphogenic protein-2

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY; Takahashi, Kazuyuki [Germantown, MD

2009-01-27

Compounds of the present invention of formula I and formula II are disclosed in the specification and wherein the compounds are modulators of Bone Morphogenic Protein activity. Compounds are synthetic peptides having a non-growth factor heparin binding region, a linker, and sequences that bind specifically to a receptor for Bone Morphogenic Protein. Uses of compounds of the present invention in the treatment of bone lesions, degenerative joint disease and to enhance bone formation are disclosed.

Heparin-induced thrombocytopenia in solid organ transplant recipients: The current scientific knowledge

PubMed Central

Assfalg, Volker; Hüser, Norbert

2016-01-01

Exposure to heparin is associated with a high incidence of immunization against platelet factor 4 (PF4)/heparin complexes. A subgroup of immunized patients is at risk of developing heparin-induced thrombocytopenia (HIT), an immune mediated prothrombotic adverse drug effect. Transplant recipients are frequently exposed to heparin either due to the underlying end-stage disease, which leads to listing and transplantation or during the transplant procedure and the perioperative period. To review the current scientific knowledge on anti-heparin/PF4 antibodies and HIT in transplant recipients a systematic PubMed literature search on articles in English language was performed. The definition of HIT is inconsistent amongst the publications. Overall, six studies and 15 case reports have been published on HIT before or after heart, liver, kidney, and lung transplantation, respectively. The frequency of seroconversion for anti-PF4/heparin antibodies ranged between 1.9% and 57.9%. However, different methods to detect anti-PF4/heparin antibodies were applied. In none of the studies HIT-associated thromboembolic events or fatalities were observed. More importantly, in patients with a history of HIT, reexposure to heparin during transplantation was not associated with thrombotic complications. Taken together, the overall incidence of HIT after solid organ transplantation seems to be very low. However, according to the current knowledge, cardiac transplant recipients may have the highest risk to develop HIT. Different alternative suggestions for heparin-free anticoagulation have been reported for recipients with suspected HIT albeit no official recommendations on management have been published for this special collective so far. PMID:27011914

Effect of heparin and heparan sulphate on open promoter complex formation for a simple tandem gene model using ex situ atomic force microscopy.

PubMed

Chammas, Oliver; Bonass, William A; Thomson, Neil H

2017-05-01

The influence of heparin and heparan sulphate (HepS) on the appearance and analysis of open promoter complex (RP o ) formation by E. coli RNA polymerase (RNAP) holoenzyme (σ 70 RNAP) on linear DNA using ex situ imaging by atomic force microscopy (AFM) has been investigated. Introducing heparin or HepS into the reaction mix significantly reduces non-specific interactions of the σ 70 RNAP and RNAP after RP o formation allowing for better interpretation of complexes shown within AFM images, particularly on DNA templates containing more than one promoter. Previous expectation was that negatively charged polysaccharides, often used as competitive inhibitors of σRNAP binding and RP o formation, would also inhibit binding of the DNA template to the mica support surface and thereby lower the imaging yield of active RNAP-DNA complexes. We found that the reverse of this was true, and that the yield of RP o formation detected by AFM, for a simple tandem gene model containing two λ PR promoters, increased. Moreover and unexpectedly, HepS was more efficient than heparin, with both of them having a dispersive effect on the sample, minimising unwanted RNAP-RNAP interactions as well as non-specific interactions between the RNAP and DNA template. The success of this method relied on the observation that E. coli RNAP has the highest affinity for the mica surface of all the molecular components. For our system, the affinity of the three constituent biopolymers to muscovite mica was RNAP>Heparin or HepS>DNA. While we observed that heparin and HepS can inhibit DNA binding to the mica, the presence of E. coli RNAP overcomes this effect allowing a greater yield of RP o s for AFM analysis. This method can be extended to other DNA binding proteins and enzymes, which have an affinity to mica higher than DNA, to improve sample preparation for AFM studies. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mapping of low molecular weight heparins using reversed phase ion pair liquid chromatography-mass spectrometry.

PubMed

Li, Daoyuan; Chi, Lequan; Jin, Lan; Xu, Xiaohui; Du, Xuzhao; Ji, Shengli; Chi, Lianli

2014-01-01

Low molecular weight heparins (LMWHs) are structurally complex, highly sulfated and negatively charged, linear carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. They are widely used as anticoagulant drugs possessing better bioavailability, longer half-life, and lower side effects than heparin. Comprehensive structure characterization of LMWHs is important for drug quality assurance, generic drug application, and new drug research and development. However, fully characterization of all oligosaccharide chains in LMWHs is not feasible for current available analytical technologies due to their structure complexity and heterogeneity. Fingerprinting profiling is an efficient way for LMWHs' characterization and comparison. In this work, we present a simple, sensitive, and powerful analytical approach for structural characterization of LMWHs. Two different LMWHs, enoxaparin and nadroparin, were analyzed using reversed phase ion pair electrospray ionization mass spectrometry (RPIP-ESI-MS). More than 200 components were identified, including major structures, minor structures, and process related impurities. This approach is robust for high resolution and complementary fingerprinting analysis of LMWHs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Current insights into the laboratory diagnosis of HIT.

PubMed

Bakchoul, T; Zöllner, H; Greinacher, A

2014-06-01

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction and prothrombotic disorder caused by immunization against platelet factor 4 (PF4) after complex formation with heparin or other polyanions. After antibody binding to PF4/heparin complexes, HIT antibodies are capable of intravascular platelet activation by cross-linking Fc gamma receptor IIa (FcγRIIa) on the platelet surface leading to a platelet count decrease and/or thrombosis. In contrast to most other immune-mediated disorders, the currently available laboratory tests for anti-PF4/heparin antibodies show a high sensitivity also for clinically irrelevant antibodies. This makes the diagnosis of HIT challenging and bears the risk to substantially overdiagnose HIT. The strength of the antigen assays for HIT is in ruling out HIT when the test is negative. Functional assays have a higher specificity for clinically relevant antibodies, but they are restricted to specialized laboratories. Currently, a Bayesian approach combining the clinical likelihood estimation for HIT with laboratory tests is the most appropriate approach to diagnose HIT. In this review, we give an overview on currently available diagnostic procedures and discuss their limitations. © 2014 John Wiley & Sons Ltd.

Laboratory tests for identification or exclusion of heparin induced thrombocytopenia: HIT or miss?

PubMed

Favaloro, Emmanuel J

2018-02-01

Heparin induced thrombocytopenia (HIT) is a potentially fatal condition that arises subsequent to formation of antibodies against complexes containing heparin, usually platelet-factor 4-heparin ("anti-PF4-heparin"). Assessment for HIT involves both clinical evaluation and, if indicated, laboratory testing for confirmation or exclusion, typically using an initial immunological assay ("screening"), and only if positive, a secondary functional assay for confirmation. Many different immunological and functional assays have been developed. The most common contemporary immunological assays comprise enzyme-linked immunosorbent assay [ELISA], chemiluminescence, lateral flow, and particle gel techniques. The most common functional assays measure platelet aggregation or platelet activation events (e.g., serotonin release assay; heparin-induced platelet activation (HIPA); flow cytometry). All assays have some sensitivity and specificity to HIT antibodies, but differ in terms of relative sensitivity and specificity for pathological HIT, as well as false negative and false positive error rate. This brief article overviews the different available laboratory methods, as well as providing a suggested approach to diagnosis or exclusion of HIT. © 2017 Wiley Periodicals, Inc.

Binding of anti-prion agents to glycosaminoglycans: Evidence from electronic absorption and circular dichroism spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zsila, Ferenc; Gedeon, Gabor

2006-08-11

The polyanionic glycosaminoglycans (GAGs) are intimately involved in the pathogenesis of protein conformational disorders such as amyloidosis and prion diseases. Several cationic agents are known to exhibit anti-prion activity but their mechanism of action is poorly understood. In this study, UV absorption and circular dichroism (CD) spectroscopic techniques were used to investigate the interaction between heparin and chondroitin-6-sulfate and anti-prion drugs including acridine, quinoline, and phenothiazine derivatives. UV band hypochromism of ({+-})-quinacrine, ({+-})-primaquine, tacrine, quinidine, chlorpromazine, and induced CD spectra of ({+-})-quinacrine upon addition of GAGs provided evidence for the GAG binding of these compounds. The association constants ({approx}10{sup 6}-10{supmore » 7} M{sup -1}) estimated from the UV titration curves show high-affinity drug-heparin interactions. Ionic strength-dependence of the absorption spectra suggested that the interaction between GAGs and the cationic drugs is principally electrostatic in nature. Drug binding differences of heparin and chondroitin-6-sulfate were attributed to their different negative charge density. These results call the attention to the alteration of GAG-prion/GAG-amyloid interactions by which these compounds might exert their anti-prion/anti-amyloidogenic activities.« less

Heparin-associated thrombocytopenia: antibody binding specificity to platelet antigens.

PubMed

Lynch, D M; Howe, S E

1985-11-01

Sera from four patients with heparin-associated thrombocytopenia (HAT) were evaluated by a quantitative enzyme-linked immunosorbent assay (ELISA) to detect heparin-dependent serum platelet-bindable immunoglobulin (S-PBIg) and by Western blotting and immunoprecipitation to investigate the specificity of the antibody binding. All HAT sera showed mildly increased S-PBIg (mean, 7.8 fg per platelet; normal, less than 6.0 fg per platelet) to intact target platelets in the ELISA, which was markedly increased in the presence of heparin (mean, 20.9 fg per platelet). This increase was 20-fold greater than normal control sera, which showed a mean differential increase of only 0.5 fg per platelet. Immunoglobulin binding specificity to platelet antigens was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of platelet lysate with transfer of the platelet fractions onto nitrocellulose strips (Western blotting) and subsequent immunoassay using HAT and normal sera. In the presence of heparin, the four HAT patients demonstrated increased binding of immunoglobulin to platelet antigens of apparent molecular weights of 180, 124, and 82 kd. Radiolabeled heparin when incubated with HAT sera, normal sera, or albumin blanks bound to platelet proteins of the same apparent molecular weights. These observations are consistent with current hypotheses suggesting that HAT antibody is directed to heparin-platelet complexes or, alternatively, that heparin induces conformational change of antigenic sites on the platelet membrane.

Current Trends in Heparin Use During Arterial Vascular Interventional Radiology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Durran, Alexandra C., E-mail: durranjobs@hotmail.com; Watts, Christopher, E-mail: Christopher.watts@salisbury.nhs.uk

2012-12-15

Purpose: This study was designed to assess the current use of heparinized saline and bolus doses of heparin in non-neurological interventional radiology and to determine whether consensus could be reached to produce guidance for heparin use during arterial vascular intervention. Methods: An interactive electronic questionnaire was distributed to members of the British Society of Interventional Radiology regarding their current practice in the use, dosage, and timing of heparin boluses and heparinized flushing solutions.ResultsA total of 108 completed questionnaires were received. More than 80% of respondents used heparinized saline with varying concentrations; the most prevalent was 1,000 IU/l (international units ofmore » heparin per liter) and 5,000 IU/l. Fifty-one percent of interventionalists use 3,000 IU as their standard bolus dose; however, the respondents were split regarding the timing of bolus dose with {approx}60% administering it after arterial access is obtained and 40% after crossing the lesion. There was no consensus on altering dose according to body weight, and only 4% monitored clotting parameters. Conclusions: There seems to be some coherence among practicing interventionalists regarding heparin administration. We hypothesize that heparinized saline should be used at a recognized standard concentration of 1,000 IU/l as a flushing concentration in all arterial vascular interventions and that 3,000 IU bolus is considered the standard dose for straightforward therapeutic procedures and 5000 IU for complex, crural, and endovascular aneurysm repair work. The bolus should be given after arterial access is obtained to allow time for optimal anticoagulation to be achieved by the time of active intervention and stenting. Further research into clotting abnormalities following such interventional procedures would be an interesting quantifiable follow-up to this initial survey of opinions and practice.« less

Coagulation and complement activation.

PubMed

Christensen, K; Larsson, R; Emanuelsson, H; Elgue, G; Larsson, A

2001-02-01

The purpose of this investigation was to assess the effect of heparin coating of a new stent construction (Stent Graft, Jomed Implantate GmbH, Germany) on platelet and coagulation activity. Stent grafts with an ePTFE membrane interfoliated between two stents were deployed in tubings to form Chandler loops. Fresh human blood with a low concentration of heparin was rotated for 1 h, then collected and used for measurements of platelet number, thrombin-antithrombin complex (TAT), CD11b, C3a and C5b-9. There were five study groups: Group 1, conventional unmodified stents (n = 8); Group 2, untreated stent grafts (n = 8); Group 3, heparin-coated stents and untreated membrane (n = 7); Group 4, heparin-coated stents and membrane (n = 8); Group 5, heparin-coated PVC tubings with no stents (n = 8). There was a significant drop in platelet count, increase in TAT-values and CD11b expression in Groups 1-3 but not in Group 4 compared to Group 5. Examination by scanning electron microscopy revealed extensive activation on non-modified stents but almost no deposition of thrombotic material on heparin-modified stent grafts. With unmodified stents and membrane there were signs of significant activation of platelets and coagulation. In contrast, the heparin-coated stent graft induced much less alterations, indicating improved blood compatibility.

Reversed-phase ion-pair ultra-high-performance-liquid chromatography-mass spectrometry for fingerprinting low-molecular-weight heparins.

PubMed

Langeslay, Derek J; Urso, Elena; Gardini, Cristina; Naggi, Annamaria; Torri, Giangiacomo; Larive, Cynthia K

2013-05-31

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC-MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC-MS fingerprints are presented and compared for tinzaparinand enoxaparin. Copyright © 2013 Elsevier B.V. All rights reserved.

New strategies for the treatment of acute venous thromboembolism.

PubMed

Prandoni, Paolo; Lensing, Anthonie W A; Pesavento, Raffaele

2006-11-01

In recent years, new opportunities have emerged that have the potential to change rapidly the therapeutic scenario of patients with acute venous thromboembolism (VTE). Selected patients with deep vein thrombosis (DVT) can be treated effectively and safely at home with fixed doses of low molecular weight heparins. The prompt administration of compression elastic stockings in addition to anticoagulant drugs in patients with acute DVT has the potential to halve the rate of late postthrombotic sequelae. The long-term use of low molecular weight heparins is likely to be more effective than oral anticoagulants for the secondary prevention of VTE in patients with advanced malignancy. Patients with pulmonary embolism and right ventricular dysfunction might benefit from the early administration of thrombolytic drugs in combination with heparin to a greater extent than from heparin alone. Despite an impressive amount of clinical information on the proper duration of oral anticoagulants in patients with unprovoked VTE, the optimal long-term treatment of these patients remains undefined. Finally, new categories of drugs are emerging that have the potential to replace conventional anticoagulants in the near future. They include compounds that inhibit factor Xa or thrombin.

High-throughput differentiation of heparin from other glycosaminoglycans by pyrolysis mass spectrometry.

PubMed

Nemes, Peter; Hoover, William J; Keire, David A

2013-08-06

Sensors with high chemical specificity and enhanced sample throughput are vital to screening food products and medical devices for chemical or biochemical contaminants that may pose a threat to public health. For example, the rapid detection of oversulfated chondroitin sulfate (OSCS) in heparin could prevent reoccurrence of heparin adulteration that caused hundreds of severe adverse events including deaths worldwide in 2007-2008. Here, rapid pyrolysis is integrated with direct analysis in real time (DART) mass spectrometry to rapidly screen major glycosaminoglycans, including heparin, chondroitin sulfate A, dermatan sulfate, and OSCS. The results demonstrate that, compared to traditional liquid chromatography-based analyses, pyrolysis mass spectrometry achieved at least 250-fold higher sample throughput and was compatible with samples volume-limited to about 300 nL. Pyrolysis yielded an abundance of fragment ions (e.g., 150 different m/z species), many of which were specific to the parent compound. Using multivariate and statistical data analysis models, these data enabled facile differentiation of the glycosaminoglycans with high throughput. After method development was completed, authentically contaminated samples obtained during the heparin crisis by the FDA were analyzed in a blinded manner for OSCS contamination. The lower limit of differentiation and detection were 0.1% (w/w) OSCS in heparin and 100 ng/μL (20 ng) OSCS in water, respectively. For quantitative purposes the linear dynamic range spanned approximately 3 orders of magnitude. Moreover, this chemical readout was successfully employed to find clues in the manufacturing history of the heparin samples that can be used for surveillance purposes. The presented technology and data analysis protocols are anticipated to be readily adaptable to other chemical and biochemical agents and volume-limited samples.

A Nitric Oxide-Releasing Heparin Conjugate for Delivery of a Combined Antiplatelet/Anticoagulant Agent

PubMed Central

2015-01-01

Heparin is a widely used anticoagulant due to its ability to inhibit key components in the coagulation cascade such as Factor Xa and thrombin (Factor IIa). Its potential to preferentially bind to antithrombin (ATIII) results in a conformational change and activation that leads to the prevention of fibrin formation from fibrinogen and ultimately obstructs a hemostatic plug from forming. Nitric oxide (NO) exhibits potent antiplatelet activity attributed to its capacity to increase the amount of cyclic guanosine monophosphate (cGMP) within platelets, which decreases the Ca2+ concentration required for platelet activation. Currently there is no single agent that combines the functions of both antiplatelet and anticoagulant (anti-Xa and anti-IIa) activities to effectively block both the extrinsic and the intrinsic coagulation pathways. The research reported herein demonstrates the ability to combine the physiological capabilities of both heparin and NO into one functional compound via use of a spermine derivative of heparin, thus enabling formation of a novel diazeniumdiolate (NONOate). The heparin–spermine NONOate has a half-life of 85 min at 25 °C (pH 7.4). The heparin backbone of the conjugate maintains its anticoagulant activity as demonstrated via an anti-Xa assay, providing an anticoagulant conversion of 3.6 μg/mL of the heparin–spermine–NONO conjugate being equivalent to 2.5 μg/mL (0.50 IU/mL) of underivatized heparin in terms of anti-Xa activity. Using standard platelet aggregometry, it is shown that the functionality of the NO release portion of the heparin conjugate prevents (nearly 100%) platelet aggregation in the presence of adenosine diphosphate (ADP, platelet agonist). PMID:24423090

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects variousmore » (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.« less

Comparison of biological activities of human antithrombins with high-mannose or complex-type nonfucosylated N-linked oligosaccharides

PubMed Central

Yamada, Tsuyoshi; Kanda, Yutaka; Takayama, Makoto; Hashimoto, Akitoshi; Sugihara, Tsutomu; Satoh-Kubota, Ai; Suzuki-Takanami, Eri; Yano, Keiichi; Iida, Shigeru; Satoh, Mitsuo

2016-01-01

The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the β-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the β-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The β-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the β-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The β-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood. PMID:26747427

Comparison of biological activities of human antithrombins with high-mannose or complex-type nonfucosylated N-linked oligosaccharides.

PubMed

Yamada, Tsuyoshi; Kanda, Yutaka; Takayama, Makoto; Hashimoto, Akitoshi; Sugihara, Tsutomu; Satoh-Kubota, Ai; Suzuki-Takanami, Eri; Yano, Keiichi; Iida, Shigeru; Satoh, Mitsuo

2016-05-01

The structure of the N-linked oligosaccharides attached to antithrombin (AT) has been shown to affect its anticoagulant activity and pharmacokinetics. Human AT has biantennary complex-type oligosaccharides with the unique feature of lacking a core fucose, which affects its biological activities by changing its heparin-binding affinity. In human plasma, AT circulates as a mixture of the α-form bearing four oligosaccharides and the β-form lacking an oligosaccharide at Asn135. However, it remains unclear how the immature high-mannose-type oligosaccharides produced by mammalian cells affect biological activities of AT. Here, we succeeded in directly comparing the activities between the high-mannose and complex types. Interestingly, although there were no substantial differences in thrombin inhibitory activity, the high-mannose type showed higher heparin-binding affinity. The anticoagulant activities were increased by heparin and correlated with the heparin-binding affinity, resulting in the strongest anticoagulant activity being displayed in the β-form with the high-mannose type. In pharmacokinetic profiling, the high-mannose type showed a much shorter plasma half-life than the complex type. The β-form was found to have a prolonged plasma half-life compared with the α-form for the high-mannose type; conversely, the α-form showed a longer half-life than the β-form for the complex-type. The present study highlights that AT physiological activities are strictly controlled not only by a core fucose at the reducing end but also by the high-mannose-type structures at the nonreducing end. The β-form with the immature high-mannose type appears to function as a more potent anticoagulant than the AT typically found in human plasma, once it emerges in the blood. © The Author 2016. Published by Oxford University Press.

Effect of a plaster containing DHEP and heparin in acute ankle sprains with oedema: a randomized, double-blind, placebo-controlled, clinical study.

PubMed

Coudreuse, J-M; de Vathaire, F

2010-09-01

Ankle sprains are the most frequent injuries in sport and daily life, and are usually treated with anti-inflammatory drugs or compounds that have an effect on microcirculation. The efficacy and tolerability of a novel plaster containing both diclofenac epolamine (DHEP) and heparin in the treatment of acute painful ankle sprains with oedema was investigated in a randomized, double-blind, placebo-controlled study. This study, carried out in 32 French medical centres, enrolled 233 patients (148 male and 86 female, aged 18-65 years) with an ankle sprain that had occurred within the previous 48 hours. Patients were treated once daily with DHEP heparin or placebo plaster for 7 days. Reduction in ankle joint swelling measured by submalleolar circumference was the primary efficacy endpoint; secondary endpoints were pain (at rest, in active mobilization, by passive stretch and by pressure), functional disability and global judgement of efficacy and tolerability. DHEP heparin-treated patients experienced a significantly greater reduction in joint swelling compared with placebo (p = 0.005). The reduction in pain was also in favour of DHEP heparin patients, with significantly lower pain in DHEP heparin-treated than placebo-treated patients within 3 hours of the first application (p < 0.05). Only two patients in the DHEP heparin plaster group and six in the placebo group experienced minor adverse events, all of which resolved spontaneously. By design, the study was limited to a placebo-controlled comparison, and there was no test for possible selection bias (subsequently ruled out by choice of efficacy parameters and measures) that may have resulted in a baseline imbalance between patient groups. Results confirm the efficacy of DHEP heparin plaster compared with placebo for the treatment of painful ankle sprain with oedema. Prompt control of pain and oedema may shorten the time to initiation of a rehabilitation programme, thus reducing the risk of ankle disability recurrence and the development of chronic injury.

Low-molecular-weight heparin biosimilars: potential implications for clinical practice. Australian Low-Molecular-Weight Heparin Biosimilar Working Group (ALBW).

PubMed

Nandurkar, H; Chong, B; Salem, H; Gallus, A; Ferro, V; McKinnon, R

2014-05-01

A working group of clinicians and scientists was formed to review the clinical considerations for use of low-molecular-weight heparin (LMWH) biosimilars. LMWH are biological molecules of significant complexity; the full complexity of chemical structure is still to be elucidated. LMWH biosimilars are products that are biologically similar to their reference product and rely on clinical data from a reference product to establish safety and efficacy. The complex nature of LMWH molecules means that it is uncertain whether a LMWH biosimilar is chemically identical to its reference product; this introduces the possibility of differences in activity and immunogenicity. The challenge for regulators and clinicians is to evaluate the level of evidence required to demonstrate that a LMWH is sufficiently similar to the reference product. The consensus opinion of the working group is that prior to clinical use a LMWH biosimilar should have proven efficacy and safety, similar to the reference product with prospective studies, which should be confirmed with a proactive post-marketing pharmacovigilance programme. © 2014 The Authors; Internal Medicine Journal © 2014 Royal Australasian College of Physicians.

Evidence for a hyperglycaemia-dependent decrease of antithrombin III-thrombin complex formation in humans.

PubMed

Ceriello, A; Giugliano, D; Quatraro, A; Marchi, E; Barbanti, M; Lefèbvre, P

1990-03-01

In the presence of increased levels of fibrinopeptide A, decreased antithrombin III biological activity, and thrombin-antithrombin III complex levels are seen in diabetic patients. Induced-hyperglycaemia in diabetic and normal subjects decreased antithrombin III activity and thrombin-antithrombin III levels, and increased fibrinopeptide A plasma levels, while antithrombin III concentration did not change; heparin was shown to reduced these phenomena. In diabetic patients, euglycaemia induced by insulin infusion restored antithrombin III activity, thrombin-antithrombin III complex and fibrinopeptide A concentrations; heparin administration had the same effects. These data stress the role of a hyperglycaemia-dependent decrease of antithrombin III activity in precipitating thrombin hyperactivity in diabetes mellitus.

Emissive H-Aggregates of an Ultrafast Molecular Rotor: A Promising Platform for Sensing Heparin.

PubMed

Mudliar, Niyati H; Singh, Prabhat K

2016-11-23

Constructing "turn on" fluorescent probes for heparin, a most widely used anticoagulant in clinics, from commercially available materials is of great importance, but remains challenging. Here, we report the formation of a rarely observed emissive H-aggregate of an ultrafast molecular rotor dye, Thioflavin-T, in the presence of heparin, which provides an excellent platform for simple, economic and rapid fluorescence turn-on sensing of heparin. Generally, H-aggregates are considered as serious problem in the field of biomolecular sensing, owing to their poorly emissive nature resulting from excitonic interaction. To the best of our knowledge, this is the first report, where contrastingly, the turn-on emission from the H-aggregates has been utilized in the biomolecule sensing scheme, and enables a very efficient and selective detection of a vital biomolecule and a drug with its extensive medical applications, i.e., heparin. Our sensor system offers several advantages including, emission in the biologically advantageous red-region, dual sensing, i.e., both by fluorimetry and colorimetry, and most importantly constructed from in-expensive commercially available dye molecule, which is expected to impart a large impact on the sensing field of heparin. Our system displays good performance in complex biological media of serum samples. The novel Thioflavin-T aggregate emission could be also used to probe the interaction of heparin with its only clinically approved antidote, Protamine.

Differentiation of parenteral anticoagulants in the prevention and treatment of venous thromboembolism.

PubMed

Fareed, Jawed; Adiguzel, Cafer; Thethi, Indermohan

2011-03-28

The prevention of venous thromboembolism has been identified as a leading priority in hospital safety. Recommended parenteral anticoagulant agents with different indications for the prevention and treatment of venous thromboembolism include unfractionated heparin, low-molecular-weight heparins and fondaparinux. Prescribing decisions in venous thromboembolism management may seem complex due to the large range of clinical indications and patient types, and the range of anticoagulants available. MEDLINE and EMBASE databases were searched to identify relevant original articles. Low-molecular-weight heparins have nearly replaced unfractionated heparin as the gold standard antithrombotic agent. Low-molecular-weight heparins currently available in the US are enoxaparin, dalteparin, and tinzaparin. Each low-molecular-weight heparin is a distinct pharmacological entity with different licensed indications and available clinical evidence. Enoxaparin is the only low-molecular-weight heparin that is licensed for both venous thromboembolism prophylaxis and treatment. Enoxaparin also has the largest body of clinical evidence supporting its use across the spectrum of venous thromboembolism management and has been used as the reference standard comparator anticoagulant in trials of new anticoagulants. As well as novel oral anticoagulant agents, biosimilar and/or generic low-molecular-weight heparins are now commercially available. Despite similar anticoagulant properties, studies report differences between the branded and biosimilar and/or generic agents and further clinical studies are required to support the use of biosimilar low-molecular-weight heparins. The newer parenteral anticoagulant, fondaparinux, is now also licensed for venous thromboembolism prophylaxis in surgical patients and the treatment of acute deep-vein thrombosis; clinical experience with this anticoagulant is expanding. Parenteral anticoagulants should be prescribed in accordance with recommended dose regimens for each clinical indication, based on the available clinical evidence for each agent to assure optimal safety and efficacy.

Prolonged Activated Clotting Time after Protamine Administration Does Not Indicate Residual Heparinization after Cardiopulmonary Bypass in Pediatric Open Heart Surgery.

PubMed

Yamamoto, Tomohiro; Wolf, Hans-Gerd; Sinzobahamvya, Nicodème; Asfour, Boulos; Hraska, Victor; Schindler, Ehrenfried

2015-08-01

In open heart surgery, heparinization is commonly neutralized using an empirical heparin:protamine ratio ranging between 1:1 and 1:1.5. However, these ratios may result in protamine overdose that should be avoided for its negative side effects on the coagulation system. This study aimed to indicate the appropriate treatment for prolonged activated clotting time (ACT) after protamine administration following cardiopulmonary bypass (CPB) in pediatric open heart surgery by investigating the underlying reasons for it. Twenty-seven children (<10 kg) undergoing open heart surgery were included. Heparin was administered only before CPB (400 IU/kg) and in the pump priming volume for CPB (2,000 IU) and was neutralized by 1:1 protamine after CPB. The blood heparin concentration was measured using anti-Xa assay. ACT and blood concentrations of heparin, coagulation factors, thrombin-antithrombin complex, and prothrombin fragment 1 + 2 were assessed. A rotational thromboelastometry (ROTEM; Tem International GmbH, München, Bayern, Germany) was used to confirm the coagulation status and residual heparin after protamine administration. Anti-Xa assay showed that there is no residual heparin in the blood after 1:1 protamine administration. Nevertheless, ACT (128.89 ± 3.09 seconds before heparin administration) remained prolonged (177.14 ± 5.43 seconds at 10 minutes after protamine, 182.00 ± 5.90 seconds at 30 minutes after protamine). The blood concentrations of coagulation factors were significantly lower than those before heparin administration (p < 0.01). The low FIBTEM MCF of ROTEM (4.43 ± 0.32 mm) at 10 minutes after protamine indicated low fibrinogen concentration. Prolonged ACT after heparin neutralization by 1:1 protamine administration does not necessarily indicate residual heparin, but low blood concentrations of coagulation factors should be considered as a reason as well. Accordingly, supply of coagulation factors instead of additional protamine should be considered. Georg Thieme Verlag KG Stuttgart · New York.

Heparin Oligosaccharides as Potential Therapeutic Agents in Senile Dementia

PubMed Central

Ma, Qing; Cornelli, Umberto; Hanin, Israel; Jeske, Walter P.; Linhardt, Robert J.; Walenga, Jeanine M.; Fareed, Jawed; Lee, John M.

2014-01-01

Heparin is a glycosaminoglycan mixture currently used in prophylaxis and treatment of thrombosis. Heparin possesses non-anticoagulant properties, including modulation of various proteases, interactions with fibroblast growth factors, and anti-inflammatory actions. Senile dementia of Alzheimer’s type is accompanied by inflammatory responses contributing to irreversible changes in neuronal viability and brain function. Vascular factors are also involved in the pathogenesis of senile dementia. Inflammation, endogenous proteoglycans, and assembly of senile plagues and neurofibrillary tangles contribute directly and indirectly to further neuronal damage. Neuron salvage can be achieved by anti-inflammation and the competitive inhibition of proteoglycans accumulation. The complexity of the pathology of senile dementia provides numerous potential targets for therapeutic interventions designed to modulate inflammation and proteoglycan assembly. Heparin and related oligosaccharides are known to exhibit anti-inflammatory effects as well as inhibitory effects on proteoglycan assembly and may prove useful as neuroprotective agents. PMID:17504153

Single-molecule dynamic force spectroscopy of the fibronectin-heparin interaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitchell, Gabriel; Lamontagne, Charles-Antoine; Lebel, Rejean

2007-12-21

The integrity of cohesive tissues strongly depends on the presence of the extracellular matrix, which provides support and anchorage for cells. The fibronectin protein and the heparin-like glycosaminoglycans are key components of this dynamic structural network. In this report, atomic force spectroscopy was used to gain insight into the compliance and the resistance of the fibronectin-heparin interaction. We found that this interaction can be described by an energetic barrier width of 3.1 {+-} 0.2 A and an off-rate of 0.2 {+-} 0.1 s{sup -1}. These dissociation parameters are similar to those of other carbohydrate-protein interactions and to off-rate values reportedmore » for more complex interactions between cells and extracellular matrix components. Our results indicate that the function of the fibronectin-heparin interaction is supported by its capacity to sustain significant deformations and considerable external mechanical forces.« less

Towards molecular modeling of the impact of heparin-derived oligosaccharides on hIFN-γ binding

NASA Astrophysics Data System (ADS)

Lilkova, E.; Petkov, P.; Ilieva, N.; Litov, L.

2015-10-01

Human interferon gamma (hIFN-γ) is an important signalling molecule, which plays a key role in the formation and modulation of immune response. The role of the cytokine C-termini in the formation of a complex with the extracellular receptor is still controversial due to the lack of structural information about this domain. Moreover, the C-termini are also responsible for the high affinity interaction of hIFN-γ with the glycosaminoglicans heparan sulfate and heparin. This interaction can drastically change the properties and behaviour of the protein. We performed molecular dynamics simulations in order to model the structure of the hIFN-γ C-terminal part and the interaction of the cytokine with heparin-derived oligosaccharides. For this purpose we reconstructed the missing C-terminal amino acid residues and performed folding simulations to determine their conformation. In order to simulate the interaction with heparin-like fragments, we developed CHARMM 36 compatible force field for the sulfamate anion group that is present in the glucosamine sugar to complete the heparin and heparan sulfate force field. The new topology and parameters reproduce the available experimental structural properties of heparin-like fragments. The simulations show that the oligosaccharides quickly bind the IFN-γ C-termini and reduce their solvent accessible surface area.

The Good the Bad and the Ugly of Glycosaminoglycans in Tissue Engineering Applications

PubMed Central

Ayerst, Bethanie I.; Merry, Catherine L.R.; Day, Anthony J.

2017-01-01

High sulfation, low cost, and the status of heparin as an already FDA- and EMA- approved product, mean that its inclusion in tissue engineering (TE) strategies is becoming increasingly popular. However, the use of heparin may represent a naïve approach. This is because tissue formation is a highly orchestrated process, involving the temporal expression of numerous growth factors and complex signaling networks. While heparin may enhance the retention and activity of certain growth factors under particular conditions, its binding ‘promiscuity’ means that it may also inhibit other factors that, for example, play an important role in tissue maintenance and repair. Within this review we focus on articular cartilage, highlighting the complexities and highly regulated processes that are involved in its formation, and the challenges that exist in trying to effectively engineer this tissue. Here we discuss the opportunities that glycosaminoglycans (GAGs) may provide in advancing this important area of regenerative medicine, placing emphasis on the need to move away from the common use of heparin, and instead focus research towards the utility of specific GAG preparations that are able to modulate the activity of growth factors in a more controlled and defined manner, with less off-target effects. PMID:28608822

Fluorometric "Switch-on" Detection of Heparin Based on a System Composed of Rhodamine-labeled Chitosan Oligosaccharide Lactate, and Graphene Oxide.

PubMed

Yun, Kyusik; Zhong, Linlin

2018-05-16

A novel fluorescence "Switch on" for the detection of heparin based on the RhB-COL/GO system was achieved. A strong fluorescence dye, Rhodamine B, was modified by chitosan oligosaccharide lactate (COL), which plays a major role in the formation of a positively charged RhB-COL complex. RhB-COL was soluble and stable in solution, which was characterized by using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. GO sheets quenched the fluorescence intensity of RhB-COL due to electron transfer from RhB to the GO surface. The decrease in fluorescence intensity of RhB-COL with increasing GO concentration was recorded using a Cary Eclipse fluorescence spectrophotometer. On the other hand, the addition of heparin replaced GO to bind with the RhB-COL surface via an electrostatic and noncovalent bond due to the abundant negative charge, which resulted in recovery of the fluorescence intensity. This RhB-COL/GO system possessed high selectivity and good sensitivity for the detection of heparin compared to other biomolecules, such as glycine, D-glucose, hyaluronic acid, L-glutamic acid, and ascorbic acid. The linear response toward heparin was measured over the range, 0-1.8 U·mL-1, with a low detection limit of 0.04 U·mL-1. The satisfactory sensing performance of RhB-COL/GO for heparin supports new "switch-on" sensor applications in heparin-related biomedical detection. © 2018 IOP Publishing Ltd.

Fluorometric ‘switch-on’ detection of heparin based on a system composed of rhodamine-labeled chitosan oligosaccharide lactate, and graphene oxide

NASA Astrophysics Data System (ADS)

Zhong, Linlin; Yun, Kyusik

2018-07-01

A novel fluorescence ‘Switch on’ for the detection of heparin based on the RhB-COL/GO system was achieved. A strong fluorescence dye, Rhodamine B, was modified by chitosan oligosaccharide lactate (COL), which plays a major role in the formation of a positively charged RhB-COL complex. RhB-COL was soluble and stable in solution, which was characterized by using Fourier transform infrared spectroscopy and x-ray photoelectron spectroscopy. GO sheets quenched the fluorescence intensity of RhB-COL due to electron transfer from RhB to the GO surface. The decrease in fluorescence intensity of RhB-COL with increasing GO concentration was recorded using a Cary Eclipse fluorescence spectrophotometer. On the other hand, the addition of heparin replaced GO to bind with the RhB-COL surface via an electrostatic and noncovalent bond due to the abundant negative charge, which resulted in recovery of the fluorescence intensity. This RhB-COL/GO system possessed high selectivity and good sensitivity for the detection of heparin compared to other biomolecules, such as glycine, D-glucose, hyaluronic acid, L-glutamic acid, and ascorbic acid. The linear response toward heparin was measured over the range, 0–1.8 U · ml‑1, with a low detection limit of 0.04 U · ml‑1. The satisfactory sensing performance of RhB-COL/GO for heparin supports new ‘switch-on’ sensor applications in heparin-related biomedical detection.

Heparin and related polysaccharides: Synthesis using recombinant enzymes and metabolic engineering

PubMed Central

Suflita, Matthew; Fu, Li; He, Wenqin; Koffas, Mattheos; Linhardt, Robert J.

2015-01-01

Glycosaminoglycans are linear anionic polysaccharides that exhibit a number of important biological and pharmacological activities. The two most prominent members of this class of polysaccharides are heparin/heparan sulfate and the chondroitin sulfates (including dermatan sulfate). These polysaccharides, having complex structures and polydispersity, are biosynthesized in the Golgi of most animal cells. The chemical synthesis of these glycosaminoglycans is precluded by their structural complexity. Today, we depend on food animal tissues for their isolation and commercial production. Ton quantities of these glycosaminoglycans are used annually as pharmaceuticals and nutraceuticals. The variability of animal-sourced glycosaminoglycans, their inherent impurities, the limited availability of source tissues, the poor control of these source materials, and their manufacturing processes, suggest a need for new approaches for their production. Over the past decade there have been major efforts in the biotechnological production of these glycosaminoglycans. This mini-review focuses on the use of recombinant enzymes and metabolic engineering for the production of heparin and chondroitin sulfates. PMID:26219501

Major proteins of boar seminal plasma as a tool for biotechnological preservation of spermatozoa.

PubMed

Caballero, I; Vazquez, J M; García, E M; Parrilla, I; Roca, J; Calvete, J J; Sanz, L; Martínez, E A

2008-11-01

Boar seminal plasma is a complex mixture of secretions from the testes, epididymides, and the male accessory reproductive organs which bathe the spermatozoa at ejaculation. The seminal plasma contains factors, mostly proteins, which influence the spermatozoa, the female genital tract, and the ovum. In boars, most of the proteins belong to the spermadhesin family and bind to the sperm surface. Spermadhesins are multifunctional proteins with a wide range of ligand-binding abilities to heparin, phospholipids, protease inhibitors and carbohydrates; the family can be roughly divided into heparin-binding (AQN-1, AQN-3, AWN) and non-heparin-binding spermadhesins (PSP-I/PSP-II heterodimer). These proteins have various effects promoting or inhibiting sperm functions including motility, oviduct binding, zona binding/penetration, and ultimately fertilization. The complexity of the environmental signals that influence these actions have implications for the uses of these proteins in vivo and in vitro, and may lead to uses in improving sperm storage.

A molecular rotor based ratiometric sensor for basic amino acids

NASA Astrophysics Data System (ADS)

Pettiwala, Aafrin M.; Singh, Prabhat K.

2018-01-01

The inevitable importance of basic amino acids, arginine and lysine, in human health and metabolism demands construction of efficient sensor systems for them. However, there are only limited reports on the 'ratiometric' detection of basic amino acids which is further restricted by the use of chemically complex sensor molecules, which impedes their prospect for practical applications. Herein, we report a ratiometric sensor system build on simple mechanism of disassociation of novel emissive Thioflavin-T H-aggregates from heparin surface, when subjected to interaction with basic amino acids. The strong and selective electrostatic and hydrogen bonding interaction of basic amino acids with heparin leads to large alteration in photophysical attributes of heparin bound Thioflavin-T, which forms a highly sensitive sensor platform for detection of basic amino acids in aqueous solution. These selective interactions between basic amino acids and heparin allow our sensor system to discriminate arginine and lysine from other amino acids. This unique mechanism of dissociation of Thioflavin-T aggregates from heparin surface provides ratiometric response on both fluorimetric and colorimetric outputs for detection of arginine and lysine, and thus it holds a significant advantage over other developed sensor systems which are restricted to single wavelength detection. Apart from the sensitivity and selectivity, our system also provides the advantage of simplicity, dual mode of sensing, and more importantly, it employs an inexpensive commercially available probe molecule, which is a significant advantage over other developed sensor systems that uses tedious synthesis protocol for the employed probe in the detection scheme, an impediment for practical applications. Additionally, our sensor system also shows response in complex biological media of serum samples.

Evaluation of flow cytometric HIT assays in relation to an IgG-Specific immunoassay and clinical outcome.

PubMed

Kerényi, Adrienne; Beke Debreceni, Ildikó; Oláh, Zsolt; Ilonczai, Péter; Bereczky, Zsuzsanna; Nagy, Béla; Muszbek, László; Kappelmayer, János

2017-09-01

Heparin-induced thrombocytopenia (HIT) is a severe side effect of heparin treatment caused by platelet activating IgG antibodies generated against the platelet factor 4 (PF4)-heparin complex. Thrombocytopenia and thrombosis are the leading clinical symptoms of HIT. The clinical pretest probability of HIT was evaluated by the 4T score system. Laboratory testing of HIT was performed by immunological detection of antibodies against PF4-heparin complex (EIA) and two functional assays. Heparin-dependent activation of donor platelets by patient plasma was detected by flow cytometry. Increased binding of Annexin-V to platelets and elevated number of platelet-derived microparticles (PMP) were the indicators of platelet activation. EIA for IgG isotype HIT antibodies was performed in 405 suspected HIT patients. Based on negative EIA results, HIT was excluded in 365 (90%) of cases. In 40 patients with positive EIA test result functional tests were performed. Platelet activating antibodies were detected in 17 cases by Annexin V binding. PMP count analysis provided nearly identical results. The probability of a positive flow cytometric assay result was higher in patients with elevated antibody titer. 71% of patients with positive EIA and functional assay had thrombosis. EIA is an important first line laboratory test in the diagnosis of HIT; however, HIT must be confirmed by a functional test. Annexin V binding and PMP assays using flow cytometry are functional HIT tests convenient in a clinical diagnostic laboratory. The positive results of functional assays may predict the onset of thrombosis. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

The effect of heparin rinse on the biocompatibility of continuous veno-venous hemodiafiltration.

PubMed

Opatrný, K; Polanská, K; Krouzecký, A; Vít, L; Novák, I; Kasal, E

2002-06-01

The aims of our cross-over randomized study were (1) to assess hemostasis in patients with acute renal failure (ARF) and (2) to determine whether or not the generally recommended heparin rinse of the extracorporeal circuit (ECC) prior to the procedure affects thrombogenicity, complement activation, and leukocyte count in blood during continuous venovenous hemodiafiltration (CVVHDF). Eleven critically ill ARF patients were treated, in random order, using CVVHDF in postdilution setup following ECC rinse with saline (A) with heparin at a concentration of 2,000 IU/L (10 procedures), (B) with heparin at a concentration of 10,000 IU/L (7 procedures), and (C) without heparin (9 procedures). Except for the rinse, anticoagulation therapy did not differ in individual patients during the procedures. Blood was withdrawn before, and at minutes 15, 60, and 360 invariably at diafilter inlet and outlet. Compared with healthy individuals, patients showed lower blood thrombocyte counts (153 vs 233*10(9)/L, p<0.01, arithmetic means, Student's t test), longer aPTT (44 vs 36 s, p<0.05), higher plasma levels of heparin (0.1 vs 0.0 U/mL, p<0.05), D-dimer (1129 vs 36 ng/mL, p<0.001) and beta-thromboglobulin (BTG) (159 vs 37 U/mL, p<0.001) prior to CVVHDF. The comparison of procedures with different rinsing technique did not reveal any significant difference in their effects on blood thrombocyte and leukocyte counts, aPTT, plasma levels of heparin, BTG, thrombin-antithrombin III complexes, D-dimer, or the C5a complement component. (1) Patients indicated for CVVHDF show impaired hemostasis involving thrombocytes, coagulation, and fibrinolysis, (2) no beneficial effect of heparin rinse on CVVHDF ECC thrombogenicity, complement activation or blood leukocyte counts was demonstrated.

Capillary Electrophoresis-Mass Spectrometry for the Analysis of Heparin Oligosaccharides and Low Molecular Weight Heparin.

PubMed

Sun, Xiaojun; Lin, Lei; Liu, Xinyue; Zhang, Fuming; Chi, Lianli; Xia, Qiangwei; Linhardt, Robert J

2016-02-02

Heparins, highly sulfated, linear polysaccharides also known as glycosaminoglycans, are among the most challenging biopolymers to analyze. Hyphenated techniques in conjunction with mass spectrometry (MS) offer rapid analysis of complex glycosaminoglycan mixtures, providing detailed structural and quantitative data. Previous analytical approaches have often relied on liquid chromatography (LC)-MS, and some have limitations including long separation times, low resolution of oligosaccharide mixtures, incompatibility of eluents, and often require oligosaccharide derivatization. This study examines the analysis of glycosaminoglycan oligosaccharides using a novel electrokinetic pump-based capillary electrophoresis (CE)-MS interface. CE separation and electrospray were optimized using a volatile ammonium bicarbonate electrolyte and a methanol-formic acid sheath fluid. The online analyses of highly sulfated heparin oligosaccharides, ranging from disaccharides to low molecular weight heparins, were performed within a 10 min time frame, offering an opportunity for higher-throughput analysis. Disaccharide compositional analysis as well as top-down analysis of low molecular weight heparin was demonstrated. Using normal polarity CE separation and positive-ion electrospray ionization MS, excellent run-to-run reproducibility (relative standard deviation of 3.6-5.1% for peak area and 0.2-0.4% for peak migration time) and sensitivity (limit of quantification of 2.0-5.9 ng/mL and limit of detection of 0.6-1.8 ng/mL) could be achieved.

Multi-component hybrid hydrogels – understanding the extent of orthogonal assembly and its impact on controlled release† †Electronic supplementary information (ESI) available: Full experimental methods and further data from assays. See DOI: 10.1039/c7sc03301j Click here for additional data file.

PubMed Central

Vieira, Vânia M. P.; Hay, Laura L.

2017-01-01

This paper reports self-assembled multi-component hybrid hydrogels including a range of nanoscale systems and characterizes the extent to which each component maintains its own unique functionality, demonstrating that multi-functionality can be achieved by simply mixing carefully-chosen constituents. Specifically, the individual components are: (i) pH-activated low-molecular-weight gelator (LMWG) 1,3;2,4-dibenzylidenesorbitol-4′,4′′-dicarboxylic acid (DBS–COOH), (ii) thermally-activated polymer gelator (PG) agarose, (iii) anionic biopolymer heparin, and (iv) cationic self-assembled multivalent (SAMul) micelles capable of binding heparin. The LMWG still self-assembles in the presence of PG agarose, is slightly modified on the nanoscale by heparin, but is totally disrupted by the micelles. However, if the SAMul micelles are bound to heparin, DBS–COOH self-assembly is largely unaffected. The LMWG endows hybrid materials with pH-responsive behavior, while the PG provides mechanical robustness. The rate of heparin release can be controlled through network density and composition, with the LMWG and PG behaving differently in this regard, while the presence of the heparin binder completely inhibits heparin release through complexation. This study demonstrates that a multi-component approach can yield exquisite control over self-assembled materials. We reason that controlling orthogonality in such systems will underpin further development of controlled release systems with biomedical applications. PMID:29147525

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

PubMed Central

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-01-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding. PMID:10727405

The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain.

PubMed

Lorentsen, R H; Graversen, J H; Caterer, N R; Thogersen, H C; Etzerodt, M

2000-04-01

Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca(2+)-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca(2+). Exon 2 encodes an alpha-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.

Sulfated, low-molecular-weight lignins are potent inhibitorsof plasmin, in addition to thrombin and factor Xa: Novel opportunity for controlling complex pathologies.

PubMed

Henry, Brian L; Abdel Aziz, May; Zhou, Qibing; Desai, Umesh R

2010-03-01

Recently we prepared sulfated, low-molecular-weight lignins (LMWLs) to mimic the biological activities of heparin and heparan sulfate. Chemo-enzymatically prepared sulfated LMWLs represent a library of diverse non-sugar, aromatic molecules with structures radically different from the heparins, and have been found to potently inhibit thrombin and factor Xa. To assess their effect on the fibrinolytic system, we studied the interaction of LMWLs with human plasmin. Enzyme inhibition studies indicate that the three sulfated LMWLs studied inhibit plasmin with IC50 values in the range of 0.24 and 1.3 mM, which are marginally affected in the presence of antithrombin. Similarly, plasmin degradation of polymeric fibrin is also inhibited by sulfated LMWLs. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of chromogenic substrates decreases nearly 70% in the presence of LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. Competitive binding studies indicate that the sulfated LMWLs compete with full-length heparin. Comparison with thrombin-heparin crystal structure identifies an anionic region on plasmin as a plausible sulfated LMWL binding site. Overall, the chemo-enzymatic origin coupled with coagulation and fibrinolysis inhibition properties of sulfated LMWLs present novel opportunities for designing new pharmaceutical agents that regulate complex pathologies in which both systems are known to play important roles such as disseminated intravascular coagulation.

A molecular rotor based ratiometric sensor for basic amino acids.

PubMed

Pettiwala, Aafrin M; Singh, Prabhat K

2018-01-05

The inevitable importance of basic amino acids, arginine and lysine, in human health and metabolism demands construction of efficient sensor systems for them. However, there are only limited reports on the 'ratiometric' detection of basic amino acids which is further restricted by the use of chemically complex sensor molecules, which impedes their prospect for practical applications. Herein, we report a ratiometric sensor system build on simple mechanism of disassociation of novel emissive Thioflavin-T H-aggregates from heparin surface, when subjected to interaction with basic amino acids. The strong and selective electrostatic and hydrogen bonding interaction of basic amino acids with heparin leads to large alteration in photophysical attributes of heparin bound Thioflavin-T, which forms a highly sensitive sensor platform for detection of basic amino acids in aqueous solution. These selective interactions between basic amino acids and heparin allow our sensor system to discriminate arginine and lysine from other amino acids. This unique mechanism of dissociation of Thioflavin-T aggregates from heparin surface provides ratiometric response on both fluorimetric and colorimetric outputs for detection of arginine and lysine, and thus it holds a significant advantage over other developed sensor systems which are restricted to single wavelength detection. Apart from the sensitivity and selectivity, our system also provides the advantage of simplicity, dual mode of sensing, and more importantly, it employs an inexpensive commercially available probe molecule, which is a significant advantage over other developed sensor systems that uses tedious synthesis protocol for the employed probe in the detection scheme, an impediment for practical applications. Additionally, our sensor system also shows response in complex biological media of serum samples. Copyright © 2017 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cai, Zheng; Yarovoi, Serge V.; Zhu, Zhiqiang

Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by immune complexes containing platelet factor 4 (PF4), antibodies to PF4 and heparin or cellular glycosaminoglycans (GAGs). Here we solve the crystal structures of the: (1) PF4 tetramer/fondaparinux complex, (2) PF4 tetramer/KKO-Fab complex (a murine monoclonal HIT-like antibody) and (3) PF4 monomer/RTO-Fab complex (a non-HIT anti-PF4 monoclonal antibody). Fondaparinux binds to the ‘closed’ end of the PF4 tetramer and stabilizes its conformation. This interaction in turn stabilizes the epitope for KKO on the ‘open’ end of the tetramer. Fondaparinux and KKO thereby collaborate to ‘stabilize’ the ternary pathogenic immune complex. Bindingmore » of RTO to PF4 monomers prevents PF4 tetramerization and inhibits KKO and human HIT IgG-induced platelet activation and platelet aggregation in vitro, and thrombus progression in vivo. Lastly, the atomic structures provide a basis to develop new diagnostics and non-anticoagulant therapeutics for HIT.« less

Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.

PubMed

Li, Lingyun; Zhang, Fuming; Zaia, Joseph; Linhardt, Robert J

2012-10-16

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

Heparin at low concentration acts as antivenom against Bothrops jararacussu venom and bothropstoxin-I neurotoxic and myotoxic actions

PubMed Central

Rostelato-Ferreira, Sandro; Leite, Gildo Bernardo; Cintra, Adélia Cristina Oliveira; da Cruz-Höfling, Maria Alice; Rodrigues-Simioni, Léa; Oshima-Franco, Yoko

2010-01-01

Heparin has been shown to antagonize myotoxic effects of crotaline venoms. Here a very low heparin concentration (LHC) was examined in its ability to antagonize the neurotoxic/myotoxic effects of Bothrops jararacussu venom and its phospholipase A2 myotoxin, bothropstoxin-I (BthTX-I), in an in vitroz nerve-muscle preparation and in mice gastrocnemius. Normalization of results was done by assays with commercial antibothropic antivenom (CBA). LHC (1IU/ml) added to the incubation bath reduced by 4- and 4.5-fold (vs 2.8- and 2.5-fold by CBA) the neuromuscular paralysis, by 5.4 and 4.4-fold (vs 2.5- and 13.3-fold by CBA) the percentage of fibers damaged and by 6- and 1.7-fold (vs 30- and 1.6-fold by CBA) the CK activity induced by B. jararacussu and BthTX-I, respectively. Protamine sulphate added 15min after the incubation of the preparation with LHC+venom, avoided the LHC neutralizing effect against venom neurotoxicity. This strongly attests that given the polycationic nature of protamine, it probably complexed with the polyanionic heparin making it unattainable for binding to basic components of venom, reducing toxicity. Since heparin antagonism is generally stronger against venom effects than is myotoxin we discuss that other venom components than the BthTX-I are likely target for the antagonism promoted by the polyanionic heparin. PMID:21544183

Structural and Functional Basis of CXCL12 (stromal cell-derived factor-1 alpha) Binding to Heparin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy,J.; Cho, Y.; Sachpatzidis, A.

2007-01-01

CXCL12 (SDF-1a) and CXCR4 are critical for embryonic development and cellular migration in adults. These proteins are involved in HIV-1 infection, cancer metastasis, and WHIM disease. Sequestration and presentation of CXCL12 to CXCR4 by glycosaminoglycans (GAGs) is proposed to be important for receptor activation. Mutagenesis has identified CXCL12 residues that bind to heparin. However, the molecular details of this interaction have not yet been determined. Here we demonstrate that soluble heparin and heparan sulfate negatively affect CXCL12-mediated in vitro chemotaxis. We also show that a cluster of basic residues in the dimer interface is required for chemotaxis and is amore » target for inhibition by heparin. We present structural evidence for binding of an unsaturated heparin disaccharide to CXCL12 attained through solution NMR spectroscopy and x-ray crystallography. Increasing concentrations of the disaccharide altered the two-dimensional 1H-15N-HSQC spectra of CXCL12, which identified two clusters of residues. One cluster corresponds to {beta}-strands in the dimer interface. The second includes the amino-terminal loop and the a-helix. In the x-ray structure two unsaturated disaccharides are present. One is in the dimer interface with direct contacts between residues His25, Lys27, and Arg41 of CXCL12 and the heparin disaccharide. The second disaccharide contacts Ala20, Arg21, Asn30, and Lys64. This is the first x-ray structure of a CXC class chemokine in complex with glycosaminoglycans. Based on the observation of two heparin binding sites, we propose a mechanism in which GAGs bind around CXCL12 dimers as they sequester and present CXCL12 to CXCR4.« less

Natural products with hypoglycemic, hypotensive, hypocholesterolemic, antiatherosclerotic and antithrombotic activities.

PubMed

Wang, H X; Ng, T B

1999-01-01

This article reviews compounds of botanical origin which are capable of lowering plasma levels of glucose and cholesterol and blood pressure, as well as compounds inhibiting atherosclerosis and thrombosis. Hypoglycemic natural products comprise flavonoids, xanthones, triterpenoids, alkaloids, glycosides, alkyldisulfides, aminobutyric acid derivatives, guanidine, polysaccharides and peptides. Hypotensive compounds include flavonoids, diterpenes, alkaloids, glycosides, polysaccharides and proteins. Among natural products with hypocholesterolemic activity are beta-carotene, lycopene, cycloartenol, beta-sitosterol, sitostanol, saponin, soybean protein, indoles, dietary fiber, propionate, mevinolin (beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitor) and polysaccharides. Heparins, flavonoids, tocotrienols, beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins), garlic compounds and fungal proteases exert antithrombotic action. Statins and garlic compounds also possess antiatherosclerotic activity.

Structural Characterization of the E2 Domain of APL-1, a C. Elegans Homolog of Human Amyloid Precursor Protein, and its Heparin Binding Site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoopes, J.; Liu, X; Xu, X

2010-01-01

The amyloid {beta}-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able tomore » map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.« less

Immune complexes formed following the binding of anti-platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion.

PubMed

Xiao, Zhihua; Visentin, Gian P; Dayananda, Kannayakanahalli M; Neelamegham, Sriram

2008-08-15

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcgammaRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.

Polymeric Coatings that Mimic the Endothelium: Combining Nitric Oxide Release with Surface-Bound Active Thrombomodulin and Heparin

PubMed Central

Wu, Biyun; Gerlitz, Bruce; Grinnell, Brian W.; Meyerhoff, Mark E.

2007-01-01

Multi-functional bilayer polymeric coatings are prepared with both controlled nitric oxide (NO) release and surface-bound active thrombomodulin (TM) alone or in combination with immobilized heparin. The outer-layer is made of CarboSil, a commercially available copolymer of silicone rubber (SR) and polyurethane (PU). The CarboSil is either carboxylated or aminated via an allophanate reaction with a diisocyanate compound followed by a urea-forming reaction between the generated isocyanate group of the polymer and the amine group of an amino acid (glycine), an oligopeptide (triglycine) or a diamine. The carboxylated CarboSil can then be used to immobilize TM through the formation of an amide bond between the surface carboxylic acid groups and the lysine residues of TM. Aminated CarboSil can also be employed to initially couple heparin to the surface, and then the carboxylic acid groups on heparin can be further used to anchor TM. Both surface-bound TM and heparin’s activity are evaluated by chromogenic assays and found to be at clinically significant levels. The underlying NO release layer is made with another commercial SR-PU copolymer (PurSil) mixed with a lipophilic NO donor (N-diazeniumdiolated dibutylhexanediamine (DBHD/N2O2)). The NO release rate can be tuned by changing the thickness of top coatings, and the duration of NO release at physiologically relevant levels can be as long as 2 weeks. The combination of controlled NO release as well as immobilized active TM and heparin from/on the same polymeric surface mimics the highly thromboresistant endothelium layer. Hence, such multifunctional polymer coatings should provide more blood-compatible surfaces for biomedical devices. PMID:17597201

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence.

PubMed

Zeng, Hai-tao; Ren, Zi; Guzman, Luis; Wang, Xiaoqian; Sutton-McDowall, Melanie L; Ritter, Lesley J; De Vos, Michel; Smitz, Johan; Thompson, Jeremy G; Gilchrist, Robert B

2013-06-01

Does heparin ablate the advantageous effects of cyclic adenosine mono-phosphate (cAMP) modulators during pre-in vitro maturation (IVM) and have a deleterious effect in standard oocyte IVM? Heparin interrupts energy metabolism and meiotic progression and adversely affects subsequent development of oocytes under conditions of elevated cAMP levels in cumulus-oocyte complexes (COCs) after pre-IVM treatment with forskolin. In animal IVM studies, artificial regulation of meiotic resumption by cAMP-elevating agents improves subsequent oocyte developmental competence. Heparin has no effect on spontaneous, FSH- or epidermal growth factor (EGF)-stimulated meiotic maturation. An in vitro cross-sectional study was conducted using immature mouse and human COCs. Depending on individual experimental design, COCs were treated during pre-IVM with or without heparin, in the presence or absence of forskolin and/or 3-isobutyl-1-methylxanthine (IBMX), and then COC function was assessed by various means. Forty-two women with polycystic ovaries (PCOs) or polycystic ovarian syndrome (PCOS) donated COCs after oocyte retrieval in a non-hCG-triggered IVM cycle. COCs were collected in pre-IVM treatments and then cultured for 40 h and meiotic progression was assessed. COCs from 21- to 24-day-old female CBA F1 mice were collected 46 h after stimulation with equine chorionic gonadotrophin. Following treatments, COCs were checked for meiotic progression. Effects on mouse oocyte metabolism were measured by assessing oocyte mitochondrial membrane potential using JC-1 staining and oocyte ATP content. Post-IVM mouse oocyte developmental competence was assessed by in vitro fertilization and embryo production. Blastocyst quality was evaluated by differential staining of inner cell mass (ICM) and trophectoderm (TE) layers. In the absence of heparin in pre-IVM culture, the addition of cAMP modulators did not affect human oocyte MII competence after 40 h. In standard IVM, heparin supplementation in pre-IVM did not affect MII competence; however, when heparin was combined with cAMP modulators, MII competence was significantly reduced from 65 to 15% (P < 0.05). In mouse experiments, heparin alone in pre-IVM significantly delayed germinal vesicle breakdown (GVBD) so that fewer GVBDs were observed at 0 and 1 h of IVM (P < 0.05), but not by 2 or 3 h of IVM. Combined treatment with IBMX and forskolin in the pre-IVM medium produced a large delay in GVBD such that no COCs exhibited GVBD in the first 1 h of IVM, and the addition of heparin in pre-IVM further significantly delayed the progression of GVBD (P < 0.05), in a dose-dependent manner (P < 0.01). Combined IBMX and forskolin treatment of mouse COCs during pre-IVM significantly increased mitochondrial membrane potential and ATP production in the oocyte at the end of pre-IVM (P < 0.05), and significantly improved fertilization, embryo development and quality (P < 0.05). However, heparin abolished the IBMX + forskolin-stimulated increase in mitochondrial membrane potential and ATP production (P < 0.05), and adversely affected embryonic cleavage, development rates and embryo quality (P < 0.05). This latter adverse combinational effect was negated when mouse COCs were collected in heparin and IBMX for 15 min, washed and then cultured for 45 min in IBMX and forskolin without heparin. Experiments in mice found that heparin ablation of the advantageous effects of cAMP modulators during pre-IVM was associated with altered oocyte metabolism, but the mechanism by which heparin affects metabolism remains unclear. This study has revealed a novel and unexpected interaction between heparin and cAMP modulators in pre-IVM in immature mouse and human oocytes, and established a means to collect oocytes using heparin while modulating oocyte cAMP to improve developmental potential.

Immune complexes formed following the binding of anti–platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion

PubMed Central

Xiao, Zhihua; Visentin, Gian P.; Dayananda, Kannayakanahalli M.

2008-01-01

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcγRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin. PMID:18539895

Inhibition of B16-BL6 melanoma lung colonies by semisynthetic sulfaminoheparosan sulfates from E. coli K5 polysaccharide.

PubMed

Poggi, Andreina; Rossi, Cosmo; Casella, Nicola; Bruno, Cristiana; Sturiale, Luisella; Dossi, Carla; Naggi, Annamaria

2002-08-01

Heparin (H), heparan sulfate (HS), and related glycosaminoglycans can inhibit cancer cell invasion, possibly due to their ability to interact with vascular growth factors, adhesion molecules, endoglycosidases, and signaling proteins, in addition to the well-known effects on the clotting system. We evaluated the antitumor activity of a series of semisynthetic sulfaminoheparosan sulfates (SAHSs) with different degree and distribution of sulfates, obtained by chemical modifications of the E. coli K5 polysaccharide, namely type A, B, and C compounds. B16-BL6 melanoma cells (10 5 cells/mouse) were injected intravenously (i.v.) in a lateral tail vein of C57BL6 mice at a dose of 0.5 mg/ mouse together with test compounds. Tumor lung nodules were significantly reduced as compared with controls only by H (95.5 +/- 1.0% inhibition), SAHS-2 (84.2 +/- 5.0% inhibition), and SAHS-4 (91.1 +/- 4.2% inhibition), among compounds tested. SAHS-2 and SAHS-4 are type B compounds, with a sulfate/carboxylate ratio similar to that of H. A typical mammalian HS showed only 54.8% inhibition. Supersulfated low-molecular-weight heparin and heparan sulfate (ssLMWH and ssLMWHS) showed an activity similar to that of unfractionated compounds. H and SAHS-4 inhibited dose dependently B16-BL6 lung colonies, with IC-50 values of 0.05 and 0.1 mg/mouse, respectively. The relationship with ex vivo anticoagulant potency was evaluated by activated partial thromboplastin time (aPTT) on mouse plasma at different time intervals after i.v. injection (0.1 to 0.5 mg/mouse) of the compound. H showed a dose-dependent anticoagulant activity lasting up to 2 hours, whereas SAHS-4 showed a potent anticoagulant effect only at a dose of 0.5 mg/mouse. Accordingly, H but not SAHS-4 consistently inhibited B16-BL6 lung colonies when given 1 hour before tumor cells. SAHS-4 derivatives, with different size and/or affinity depleted of AT binding sites, showed an inhibitory effect on B16-BL6 melanoma similar to that of SAHS-4, suggesting that the greater antitumor effect of H was not due to AT-mediated inhibition of blood clotting. Interactions with other blood inhibitors, such as heparin cofactor II or tissue factor pathway inhibitory protein cannot be ruled out. The better effect of H may be due to persistence in the circulation and/or ability to inhibit tumor neoangiogenesis.

The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheffield, William P., E-mail: sheffiel@mcmaster.ca; Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario; Bhakta, Varsha

The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assaysmore » of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.« less

GlycCompSoft: Software for Automated Comparison of Low Molecular Weight Heparins Using Top-Down LC/MS Data

PubMed Central

Li, Lingyun; Zhang, Fuming; Hu, Min; Ren, Fuji; Chi, Lianli; Linhardt, Robert J.

2016-01-01

Low molecular weight heparins are complex polycomponent drugs that have recently become amenable to top-down analysis using liquid chromatography-mass spectrometry. Even using open source deconvolution software, DeconTools, and automatic structural assignment software, GlycReSoft, the comparison of two or more low molecular weight heparins is extremely time-consuming, taking about a week for an expert analyst and provides no guarantee of accuracy. Efficient data processing tools are required to improve analysis. This study uses the programming language of Microsoft Excel™ Visual Basic for Applications to extend its standard functionality for macro functions and specific mathematical modules for mass spectrometric data processing. The program developed enables the comparison of top-down analytical glycomics data on two or more low molecular weight heparins. The current study describes a new program, GlycCompSoft, which has a low error rate with good time efficiency in the automatic processing of large data sets. The experimental results based on three lots of Lovenox®, Clexane® and three generic enoxaparin samples show that the run time of GlycCompSoft decreases from 11 to 2 seconds when the data processed decreases from 18000 to 1500 rows. PMID:27942011

Heparin-mimicking multilayer coating on polymeric membrane via LbL assembly of cyclodextrin-based supramolecules.

PubMed

Deng, Jie; Liu, Xinyue; Ma, Lang; Cheng, Chong; Shi, Wenbin; Nie, Chuanxiong; Zhao, Changsheng

2014-12-10

In this study, multifunctional and heparin-mimicking star-shaped supramolecules-deposited 3D porous multilayer films with improved biocompatibility were fabricated via a layer-by-layer (LbL) self-assembly method on polymeric membrane substrates. Star-shaped heparin-mimicking polyanions (including poly(styrenesulfonate-co-sodium acrylate; Star-PSS-AANa) and poly(styrenesulfonate-co-poly(ethylene glycol)methyl ether methacrylate; Star-PSS-EGMA)) and polycations (poly(methyl chloride-quaternized 2-(dimethylamino)ethyl methacrylate; Star-PMeDMA) were first synthesized by atom transfer radical polymerization (ATRP) from β-cyclodextrin (β-CD) based cores. Then assembly of 3D porous multilayers onto polymeric membrane surfaces was carried out by alternating deposition of the polyanions and polycations via electrostatic interaction. The surface morphology and composition, water contact angle, blood activation, and thrombotic potential as well as cell viability for the coated heparin-mimicking films were systematically investigated. The results of surface ATR-FTIR spectra and XPS spectra verified successful deposition of the star-shaped supramolecules onto the biomedical membrane surfaces; scanning electron microscopy (SEM) and atomic force microscopy (AFM) observations revealed that the modified substrate had 3D porous surface morphology, which might have a great biological influence on the biointerface. Furthermore, systematic in vitro investigation of protein adsorption, platelet adhesion, human platelet factor 4 (PF4, indicates platelet activation), activate partial thromboplastin time (APTT), thrombin time (TT), coagulation activation (thrombin-antithrombin III complex (TAT, indicates blood coagulant)), and blood-related complement activation (C3a and C5a, indicates inflammation potential) confirmed that the heparin-mimicking multilayer coated membranes exhibited ultralow blood component activations and excellent hemocompatibility. Meanwhile, after surface coating, endothelial cell viability was also promoted, which indicated that the heparin-mimicking multilayer coating might extend the application fields of polymeric membranes in biomedical fields.

Naked eye detection of infertility based on sperm protamine-induced aggregation of heparin gold nanoparticles.

PubMed

Vidya, Raj; Saji, Alex

2018-05-01

The development of an easy to use, one-pot, environmentally friendly, non-invasive and label-free colorimetric probe for the determination of semen protamines, the biochemical marker of male fertility, using heparin gold nanoparticles (HAuNPs) is presented. The affinity of HAuNPs for protamines was due to the electrostatic interactions between polycationic protamine and polyanionic heparin. The binding of HAuNPs to protamine was characterized by variation in the plasmon absorption spectra followed by a visibly observable colour change of the solution from red to blue. We observed a red shift in the plasmon peak and the method exhibited linearity in the range of 10-70 ng/mL with a detection limit of 5 ng/mL, which is much lower than that reported for colorimetric sensors of protamine. The colour change and the variation in the absorbance of HAuNPs were highly specific for protamines in the presence of different interfering compounds and the method was successfully applied for determining protamine in real samples of semen and serum. Rather than a quantitative estimation, it seems that the method provides a quick screening between a large array of positive and negative samples and, moreover, it maintains the privacy of the user. The method appears to be simple and would be very useful in third-world countries where high-tech diagnostic aids are inaccessible to the majority of the population. Graphical Abstract Heparin gold nanoparticles aided visual detection of infertility.

Structure Determination of Natural Products by Mass Spectrometry.

PubMed

Biemann, Klaus

2015-01-01

I review laboratory research on the development of mass spectrometric methodology for the determination of the structure of natural products of biological and medical interest, which I conducted from 1958 to the end of the twentieth century. The methodology was developed by converting small peptides to their corresponding polyamino alcohols to make them amenable to mass spectrometry, thereby making it applicable to whole proteins. The structures of alkaloids were determined by analyzing the fragmentation of a known alkaloid and then using the results to deduce the structures of related compounds. Heparin-like structures were investigated by determining their molecular weights from the mass of protonated molecular ions of complexes with highly basic, synthetic peptides. Mass spectrometry was also employed in the analysis of lunar material returned by the Apollo missions. A miniaturized gas chromatograph mass spectrometer was sent to Mars on board of the two Viking 1976 spacecrafts.

Interactions between nattokinase and heparin/GAGs.

PubMed

Zhang, Fuming; Zhang, Jianhua; Linhardt, Robert J

2015-12-01

Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK's potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin's interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin's interaction with antithrombin and fibroblast growth factors.

Notes from the Field: Fungal Bloodstream Infections Associated with a Compounded Intravenous Medication at an Outpatient Oncology Clinic - New York City, 2016.

PubMed

Vasquez, Amber M; Lake, Jason; Ngai, Stephanie; Halbrook, Megan; Vallabhaneni, Snigdha; Keckler, M Shannon; Moulton-Meissner, Heather; Lockhart, Shawn R; Lee, Christopher T; Perkins, Kiran; Perz, Joseph F; Antwi, Mike; Moore, Miranda S; Greenko, Jane; Adams, Eleanor; Haas, Janet; Elkind, Sandra; Berman, Marjorie; Zavasky, Dani; Chiller, Tom; Ackelsberg, Joel

2016-11-18

On May 24, 2016, the New York City Department of Health and Mental Hygiene notified CDC of two cases of Exophiala dermatitidis bloodstream infections among patients with malignancies who had received care from a single physician at an outpatient oncology facility (clinic A). Review of January 1-May 31, 2016 microbiology records identified E. dermatitidis bloodstream infections in two additional patients who also had received care at clinic A. All four patients had implanted vascular access ports and had received intravenous (IV) medications, including a compounded IV flush solution containing saline, heparin, vancomycin, and ceftazidime, compounded and administered at clinic A.

Thromboembolic Prophylaxis with Heparin in Patients with Blunt Solid Organ Injuries Undergoing Non-operative Treatment.

PubMed

Khatsilouskaya, Tatsiana; Haltmeier, Tobias; Cathomas, Marionna; Eberle, Barbara; Candinas, Daniel; Schnüriger, Beat

2017-05-01

Patients with blunt solid organ injuries (SOI) are at risk for venous thromboembolism (VTE), and VTE prophylaxis is crucial. However, little is known about the safety of early prophylactic administration of heparin in these patients. This is a retrospective study including adult trauma patients with SOI (liver, spleen, kidney) undergoing non-operative management (NOM) from 01/01/2009 to 31/12/2014. Three groups were distinguished: prophylactic heparin (low molecular weight heparin or low-dose unfractionated heparin) ≤72 h after admission ('early heparin group'), >72 h after admission ('late heparin group'), and no heparin ('no heparin group'). Patient and injury characteristics, transfusion requirements, and outcomes (failed NOM, VTE, and mortality) were compared between the three groups. Overall, 179 patients were included; 44.7% in the 'early heparin group,' 34.6% in the 'late heparin group,' and 20.8% in the 'no heparin group.' In the 'late heparin group,' the ISS was significantly higher than in the 'early' and 'no heparin groups' (median 29.0 vs. 17.0 vs. 19.0; p < 0.001). The overall NOM failure rate was 3.9%. Failed NOM was significantly more frequent in the 'no heparin group' compared to the 'early' and 'late heparin groups' (10.8 vs. 3.2 vs. 1.3%; p = 0.043). In the 'early heparin group' 27.5% patients suffered from a high-grade SOI; none of these patients failed NOM. Mortality did not differ significantly. Although not statistically significant, VTE were more frequent in the 'no heparin group' compared to the 'early' and 'late heparin groups' (10.8 vs. 4.8 vs. 1.3%; p = 0.066). In patients with SOI, heparin was administered early in a high percentage of patients and was not associated with an increased NOM failure rate or higher in-hospital mortality.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

Free energy calculations of glycosaminoglycan-protein interactions.

PubMed

Gandhi, Neha S; Mancera, Ricardo L

2009-10-01

Glycosaminoglycans (GAGs) are complex highly charged linear polysaccharides that have a variety of roles in biological processes. We report the first use of molecular dynamics (MD) free energy calculations using the MM/PBSA method to investigate the binding of GAGs to protein molecules, namely the platelet endothelial cell adhesion molecule 1 (PECAM-1) and annexin A2. Calculations of the free energy of the binding of heparin fragments of different sizes reveal the existence of a region of low GAG-binding affinity in domains 5-6 of PECAM-1 and a region of high affinity in domains 2-3, consistent with experimental data and ligand-protein docking studies. A conformational hinge movement between domains 2 and 3 was observed, which allows the binding of heparin fragments of increasing size (pentasaccharides to octasaccharides) with an increasingly higher binding affinity. Similar simulations of the binding of a heparin fragment to annexin A2 reveal the optimization of electrostatic and hydrogen bonding interactions with the protein and protein-bound calcium ions. In general, these free energy calculations reveal that the binding of heparin to protein surfaces is dominated by strong electrostatic interactions for longer fragments, with equally important contributions from van der Waals interactions and vibrational entropy changes, against a large unfavorable desolvation penalty due to the high charge density of these molecules.

A gravimetric analysis of protein-oligosaccharide interactions.

PubMed

Rudd, T; Gallagher, J T; Ron, D; Nichols, R J; Fernig, D G

2003-04-01

Interactions between an immobilized, heparin-derived octasaccharide and growth factors have been observed using a quartz crystal microbalance-dissipation (QCM-D). This device can measure the amount of growth factors binding to the octasaccharide surface and also the change of dissipation of the surface. Dissipation is a measure of how the adhered material 'damps' the surface vibrations. The octasaccharides were anchored through their reducing ends by the intermediary of the alkanethiol molecule, which covalently binds to the crystal surface through the thiol group. As expected, heparin sulphate binding growth factors bound to the octasaccharide, but the change in mass of growth factor bound per unit change in dissipation is different for the different growth factors. Suggesting that the structures of the various growth factor-octasaccharide complexes are different, therefore, indicates that the change in dissipation can give insights into the structure, orientation and packing of the oligosaccharide-growth factor complexes.

Enantiomeric and Diastereomeric Self-Assembled Multivalent (SAMul) Nanostructures - Understanding the Effects of Chirality on Binding to Polyanionic Heparin and DNA.

PubMed

Thornalley, Kiri; Laurini, Erik; Pricl, Sabrina; Smith, David K

2018-05-15

A family of four self-assembling lipopeptides containing Ala-Lys peptides attached to a C16 aliphatic chain was synthesised. These compounds form two enantiomeric pairs that bear a diastereomeric relationship to one another (C16-L-Ala-L-Lys/C16-D-Ala-D-Lys) and (C16-D-Ala-L-Lys/C16-L-Ala-D-Lys). These diastereomeric pairs have very different critical micelle concentrations (CMCs), with LL/DD < DL/LD suggesting more effective assembly of the former. The self-assembled multivalent (SAMul) systems bind biological polyanions as result of the cationic lysine groups on their surfaces. Polyanion binding was investigated using dye displacement assays and isothermal calorimetry (ITC). On heparin binding, there was no significant enantioselectivity, but there was a binding preference for the diastereomeric assemblies with lower CMCs. Conversely, on binding DNA, there was a significant enantioselective preference for systems displaying D-lysine ligands, with a further slight preference for attachment to L-alanine, with the CMC being irrelevant. Binding to adaptive, ill-defined heparin has a large favourable entropic term, suggesting it depends primarily on the cationic SAMul nanostructure maximising surface contact with heparin, which can adapt, displacing solvent and other ions. Conversely, binding to well-defined, shape-persistent DNA has a larger favourable enthalpic term, and combined with the enantioselectivity, this allows us to suggest that its SAMul binding is based on optimised individual electrostatic interactions at the molecular level, with a preference for binding to D-lysine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

NASA Astrophysics Data System (ADS)

Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

2013-11-01

Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

Effects on infarct size and left ventricular function of early intravenous injection of anistreplase in acute myocardial infarction. The APSIM Study Investigators.

PubMed

Bassand, J P; Bernard, Y; Lusson, J R; Machecourt, J; Cassagnes, J; Borel, E

1990-03-01

A total of 231 patients suffering from a first acute myocardial infarction were randomly allocated within 4 hours following the onset of symptoms either to anistreplase or anisoylated plasminogen streptokinase activator complex (APSAC), 30 U over 5 minutes, or to conventional heparin therapy, 5000 IU in bolus injection. Heparin was reintroduced in both groups 4 h after initial therapy at a dosage of 500 IU/kg per day. A total of 112 patients received anistreplase and 119 received heparin within a mean period of 188 +/- 62 min following the onset of symptoms. Infarct size was estimated from single photon emission computerized tomography and expressed in percentage of the total myocardial volume. The patency rate of the infarct-related artery was 77% in the anistreplase group and 36% in the heparin group (p less than 0.001). Left ventricular ejection fraction determined from contrast angiography was significantly higher in the anistreplase group than in the heparin group (6 absolute percentage point difference). A significant 31% reduction in infarct size was found in the anistreplase group (33% for the anterior wall infarction subgroup [p less than 0.05] and 16% for the inferior wall infarction subgroup, NS). A close inverse relation was found between the values of left ventricular ejection fraction and infarct size (r = -.73, p less than 0.01). In conclusion, early infusion of anistreplase in acute myocardial infarction produced a high early patency rate, a significant limitation of infarct size, and a significant preservation of left ventricular systolic function, mainly in the anterior wall infarctions.

Heparin allergy: delayed-type non-IgE-mediated allergic hypersensitivity to subcutaneous heparin injection.

PubMed

Trautmann, Axel; Seitz, Cornelia S

2009-08-01

Itching erythematous or eczematous plaques around injection sites are quite frequent side effects of heparin treatment and clinical symptoms of delayed-type non-IgE-mediated allergic hypersensitivity (DTH) to heparin. For diagnosis, intradermal, patch, and subcutaneous challenge tests with heparins are suitable. In most cases, changing the subcutaneous therapy from unfractionated to low molecular weight heparin or treatment with heparinoids does not provide improvement because of extensive cross-reactivity. Hirudin polypeptides, which exhibit a different chemical structure, are a safe therapeutic alternative for subcutaneous application, however. Importantly, despite DTH to subcutaneously injected heparins, most patients tolerate heparin intravenously. Moreover, in case of therapeutic necessity and DTH to heparins, the simple shift from subcutaneous to intravenous heparin administration without prior testing may be justified.

Heparins from porcine and bovine intestinal mucosa: Are they similar drugs?

PubMed

Aquino, Rafael S; Pereira, Mariana S; Vairo, Bruno C; Cinelli, Leonardo P; Santos, Gustavo R C; Fonseca, Roberto J C; Mourão, Paulo A S

2010-05-01

Increasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides -->4-alpha-IdoA2S-1-->4-alpha-GlcNS6S-1-->. Heparin from bovine intestine is also composed of highly 2-sulfated alpha-iduronic acid residues, but the sulfation of the alpha-glucosamine units vary significantly: approximately 50% are 6- and N -disulfated, as in porcine heparin, while approximately 36% are 6-desulfated and approximately 14% N -acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.

Interactions between nattokinase and heparin/GAGs

PubMed Central

Zhang, Fuming

2015-01-01

Nattokinase (NK) is a serine protease extracted from a traditional Japanese food called natto. Due to its strong fibrinolytic and thrombolytic activity, NK is regarded as a valuable dietary supplement or nutraceutical for the oral thrombolytic therapy. In addition, NK has been investigated for some other medical applications including treatment of hypertension, Alzheimer’s disease, and vitreoretinal disorders. The most widely used clinical anticoagulants are heparin and low molecular weight heparins. The interactions between heparin and proteins modulate a diverse patho-physiological processes and heparin modifies the activity of serine proteases. Indeed, heparin plays important roles in almost all of NK’s potential therapeutically applications. The current report relies on surface plasmon resonance spectroscopy to examine NK interacting with heparin as well as other glycosaminoglycans (GAGs). These studies showed that NK is a heparin binding protein with an affinity of ~250 nM. Examination with differently sized heparin oligosaccharides indicated that the interaction between NK and heparin is chain-length dependent and the minimum size for heparin binding is a hexasaccharide. Studies using chemically modified heparin showed the 6-O-sulfo as well as the N-sulfo groups but not the 2-O-sulfo groups within heparin, are essential for heparin’s interaction with NK. Other GAGs (including HS, DS, and CSE) displayed modest binding affinity to NK. NK also interfered with other heparin-protein interactions, including heparin’s interaction with antithrombin and fibroblast growth factors. PMID:26412225

Horseradish peroxidase-nanoclay hybrid particles of high functional and colloidal stability.

PubMed

Pavlovic, Marko; Rouster, Paul; Somosi, Zoltan; Szilagyi, Istvan

2018-08-15

Highly stable dispersions of enzyme-clay nanohybrids of excellent horseradish peroxidase activity were developed. Layered double hydroxide nanoclay was synthesized and functionalized with heparin polyelectrolyte to immobilize the horseradish peroxidase enzyme. The formation of a saturated heparin layer on the platelets led to charge inversion of the positively charged bare nanoclay and to highly stable aqueous dispersions. Great affinity of the enzyme to the surface modified platelets resulted in strong horseradish peroxidase adsorption through electrostatic and hydrophobic interactions as well as hydrogen bonding network and prevented enzyme leakage from the obtained material. The enzyme kept its functional integrity upon immobilization and showed excellent activity in decomposition of hydrogen peroxide and oxidation of an aromatic compound in the test reactions. In addition, remarkable long term functional stability of the enzyme-nanoclay hybrid was observed making the developed colloidal system a promising antioxidant candidate in biomedical treatments and industrial processes. Copyright © 2018 Elsevier Inc. All rights reserved.

[Neutralization of anticoagulant activity of heparin by N-[(2-hydroxy-3-trimethylammonium) propyl] chloride derivatives of chitosan].

PubMed

Shagdarova, B Ts; Drozd, N N; Il'ina, A V; Logviniva, Yu S; Varlamov, V P

2016-01-01

Alkylated derivatives of low molecular weight chitosan with different substitution degrees of 98, 40, and 9% (I, II, and III respectively) have been synthesized. The structure of the obtained derivatives was defined by spectral assays (IR-spectroscopy and proton magnetic resonance). Chitosan derivatives were characterized with positive zeta-potential (33–51 mV) and solubility from 2 to 100 mg/mL in pH 7.4 and 25°C. It was shown that, at a concentration of 0.0014–0.0029 mg/mL, derivative I, as well as protamine sulfate, could be used to neutralize the anticoagulant activity of unfractionated or low molecular weight heparin. At a concentration of 0.0029–0.58 mg/mL, derivative I enhanced platelet aggregation, which would be necessary when hemostatic compounds or materials were used. Derivatives II and III enhanced platelet aggregation to a lesser extent.

Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

PubMed

Ceriello, A; Marchi, E; Barbanti, M; Milani, M R; Giugliano, D; Quatraro, A; Lefebvre, P

1990-04-01

The effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfate-dependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

Preclinical Validation of the Heparin-Reactive Peptide p5+14 as a Molecular Imaging Agent for Visceral Amyloidosis

DOE PAGES

Wall, Jonathan; Martin, Emily B.; Richey, Tina; ...

2015-04-27

Amyloid is a complex pathologic matrix comprised principally of para-crystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloidoses are rare (~3500 new cases per year in the US); thus, routine diagnosis is often challenging, and effective treatment options are limited, resulting in high morbidity and mortality rates. Glycosaminoglycans contribute inextricably to the formation of amyloid fibrils and foster the deposition of amyloid in tissues. Those present in amyloid deposits are biochemically and electrochemically distinct from glycosaminoglycans found in the plasma membrane and extracellular matrices of healthy tissues due to the presence of a high degree of heparin-like hypersulfation. We havemore » exploited this unique property and evaluated heparin-reactive peptides, such as p5+14. Herein we show efficacious detection of murine systemic amyloid in vivo by using molecular imaging, and the specific targeting of the peptide to major forms of human amyloid in tissue sections. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients; thus, providing a novel technique for prognostication, patient stratification, and monitoring response to therapy.« less

Clinical picture of heparin-induced thrombocytopenia (HIT) and its differentiation from non-HIT thrombocytopenia.

PubMed

Warkentin, Theodore E

2016-10-28

HIT is an acquired antibody-mediated disorder strongly associated with thrombosis, including microthrombosis secondary to disseminated intravascular dissemination (DIC). The clinical features of HIT are reviewed from the perspective of the 4Ts scoring system for HIT, which emphasises its characteristic timing of onset of thrombocytopenia. HIT antibodies recognize multimolecular complexes of platelet factor 4 (PF4)/heparin. However, a subset of HIT sera recognise PF4 bound to platelet chondroitin sulfate; these antibodies activate platelets in vitro and in vivo even in the absence of heparin, thus explaining: delayed-onset HIT (where HIT begins or worsens after stopping heparin); persisting HIT (where HIT takes several weeks to recover); spontaneous HIT syndrome (a disorder clinically and serologically resembling HIT but without proximate heparin exposure); and fondaparinux-associated HIT (four distinct syndromes featuring thrombocytopenia that begins or worsens during treatment with fondaparinux), with a new patient case presented with ongoing thrombocytopenia (and fatal haemorrhage) during treatment of HIT with fondaparinux, with fondaparinux-dependent platelet activation induced by patient serum ("fondaparinux cross-reactivity"). Ironically, despite existence of fondaparinux-associated HIT, this pentasaccharide anticoagulant is a frequent treatment for HIT (including one used by the author). HIT can be confused with other disorders, including those with a) timing similar to HIT (e. g. abciximab-associated thrombocytopenia of delayed-onset); b) combined thrombocytopenia/thrombosis (e. g. symmetrical peripheral gangrene secondary to acute DIC and shock liver); and c) both timing of onset and thrombosis (e. g. warfarin-associated venous limb gangrene complicating cancer-associated DIC). By understanding clinical and pathophysiological similarities and differences between HIT and non-HIT mimicking disorders, the clinician is better able to make the correct diagnosis.

Interaction of antithrombin with sulfated, low molecular weight lignins: opportunities for potent, selective modulation of antithrombin function.

PubMed

Henry, Brian L; Connell, Justin; Liang, Aiye; Krishnasamy, Chandravel; Desai, Umesh R

2009-07-31

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891-31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80-87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

[Perioperative coagulation management in microsurgery: report of the consensus workshops in the course of the 31st and 32nd Annual Meeting of the German-language Working Group for microsurgery of the peripheral nerves and vessels (DAM) November 2009 in Erlangen and November 2010 in Basel].

PubMed

Schmitz, M; Riss, R; Kneser, U; Jokuszies, A; Harder, Y; Beier, J P; Schäfer, D J; Vogt, P M; Fansa, H; Andree, C; Pierer, G; Horch, R E

2011-12-01

Microsurgery is a very relevant component of reconstructive surgery. In this context anticoagulation plays an increasing role. At the moment there are no unanimously accepted prospective studies or generally accepted regimes available that could serve as evidence-based guidelines for the prevention of thrombosis in microsurgery. With regard to this problem the aim of a series of workshops during the annual meetings of the German-speaking group for microsurgery in 2009 and 2010 was to establish a first possible consensus. This article reflects the main aspects of the ongoing development of a generally acceptable guideline for anticoagulation in microsurgery as interim report of these consensus workshops. Basically there are 3 main agents in thromboprophylaxis available: antiplatelet drugs, dextran and heparin. In the course of the workshops no general use of aspirin or dextran for anticoagulation in microsurgery was recommended. The use of heparin as anticoagulation agent is advisable for different indications. Low molecular heparins (LMH) have certain advantages in comparison to unfractionated heparins (UFH) and are therefore preferred by most participants. Indications for UFH are still complex microsurgical revisions, renal failure and some specific constellations in patients undergoing reconstruction of the lower extremity, where the continuous administration of heparin is recommended. At the moment of clamp release a single-shot of UFH is still given by many microsurgeons, despite a lack of scientific evidence. Future prospective clinical trials and the establishment of a generally accepted evidence-based guideline regarding anticoagulation treatment in microsurgery are deemed necessary. © Georg Thieme Verlag KG Stuttgart · New York.

The AN69 ST haemodialysis membrane under conditions of two different extracorporeal circuit rinse protocols a comparison of thrombogenicity parameters.

PubMed

Richtrova, Pavlina; Opatrny, Karel; Vit, Ladislav; Sefrna, Frantisek; Perlik, Radek

2007-10-01

Thrombogenicity is an important parameter of haemodialysis (HD) membrane biocompatibility. The surface of the polyacrylonitrile AN69 ST membrane is coated with a polyethylenimine. This modification allows heparin adsorption. The binding of heparin to the membrane surface occurs during priming of the extracorporeal circuit (ECC) by rinsing it with saline and heparin. Our aims were to assess and compare the thrombogenicity of the AN69 ST membrane under conditions of two extracorporeal circuit (ECC) rinse protocols-with and without unfractionated heparin (UFH). In a prospective, crossover and randomized study, we examined 10 patients during HD after ECC preparation with either rinse protocols. Prior to HD and at 15, 60 and 240 min, we determined plasma levels of the thrombin-antithrombin complexes (TAT), platelet factor 4 (PF4), heparin concentration (antiXa) and thrombocyte count. Systemic anticoagulation was performed using UFH. During HD after ECC rinse without UFH, there was a significantly earlier and more marked increase in TAT compared with UFH-containing rinse (P <0.05). Using Spearman coefficient, we demonstrated a significant correlation between TAT and antiXa at 60 min (r = -0.534) and 240 min (r = -0.538). A comparison of the TAT/antiXa ratios between rinses at 60 min revealed a significantly higher increase in TAT following UFH-free rinse (P <0.05). There was no difference in PF4 between the rinses. Platelet count did not change significantly during HD using either rinse protocol. Based on plasma TAT levels, ECC priming with an UFH-containing solution reduces the thrombogenicity of the AN69 ST membrane. There is no significant difference between both types of priming concerning PF4 and thrombocyte count.

Obstacles in the diagnostics and therapy of heparin-induced thrombocytopenia.

PubMed

Antonijević, Nebojsa M; Radovanović, Nebojsa; Obradović, Slobodan; Vucelić, Dragica; Stojanović, Bojan; Miković, Danijela; Kovac, Mirjana; Kocica, Tina; Tadić, Svetlana; Antonijević, Irina; Drasković, Snezana; Djordjević, Valentina; Calija, Branko; Perunicić, Jovan; Vasiljević, Zorana

2010-01-01

An immune-mediated, severe, acquired prothrombotic disorder, heparin-induced thrombocytopenia type II (HIT II) occurs in 0.5-5% of patients exposed to unfractionated heparin longer than 5-7 days. Arterial and venous thromboses are induced by HIT II in about 35-50% of patients. Typical death rate for HIT is about 29%, while 21% of HIT patients result in amputation of a limb. The trend towards the occurrence of HIT due to the administration of low molecular weight heparins (LMWH) taking ever conspicuous place in the standard venous thromboembolism (VTE) prophylaxis has been more frequently observed recently. It is considered that LMWH may cause HIT II in about 0.25-1%. The need for further modification of HIPA assays with LMWH has been imposed in the HIT laboratory diagnostics, heretofore overburdened with complexity. There are several constantly opposing problems arising in HIT laboratory diagnostics, one of which is that in a certain number of patients immunologic assays detect nonpathogenic antibodies (mainly IgM or IgA heparin-PF4 antibodies) while, on the other hand, the occurrence of HIT pathogenetically mediated by minor antigens (neutrophil-activating peptide 2 or interleukin 8) may be neglected in certain cases. The following factors play an important role in the interpretation of each laboratory HIT assays performed: 1. correlation with HIT clinical probability test, the best known of which is 4T'score, 2. the interpretation of the laboratory findings dependent on the time of the thrombocytopenia onset, as well as 3. the sensitivity and specificity of each test respectively. The HIT diagnostics in the presence of other comorbid states which may also induce thrombocytopenia, more precisely known as pseudo HIT (cancer, sepsis, disseminated intravascular coagulation, pulmonary embolism, antiphospholipid syndrome, etc), represents a specific clinical problem.

Managing the therapeutic dilemma: patients with spontaneous intracerebral hemorrhage and urgent need for anticoagulation.

PubMed

Bertram, M; Bonsanto, M; Hacke, W; Schwab, S

2000-03-01

Physicians face a therapeutic dilemma in patients with acute hemorrhagic stroke requiring long-term, high-intensity anticoagulants because this treatment increases the risk of intracranial hemorrhage (ICH) 8- to 11-fold. We retrospectively studied 15 patients with ICH which occurred under anticoagulation with phenprocoumon, with an international norrmalized ratio (INR) of 2.5-6.5 on admission. Hemispheric, thalamic, cerebellar, intraventricular, or subarachnoid hemorrhage without aneurysm occurred. Absolute indications for anticoagulation were double, mitral, or aortic valve replacement, combined mitral valve failure with atrial fibrillation and atrial enlargement, internal carotid artery-jugular vein graft, frequently recurring deep vein thrombosis with risk of pulmonary embolism, and severe nontreatable ischemic heart disease. As soon as the diagnosis of ICH was established, INR normalization was attempted in all patients by administration of prothrombin complex, fresh frozen plasma, or vitamin K. After giving phenprocoumon antagonists (and neurosurgical therapy in four patients) heparin administration was started. Nine patients received full-dose intravenous and six low-dose subcutaneous heparin. The following observations were made: (a) All patients with effective, full-dose heparin treatment with a 1.5- to 2-fold elevation in partial thromboplastin time after normalization of the INR were discharged without complication. (b) Three of four of the patients with only incomplete correction of the INR (> 1.35) experienced relevant rebleeding within 3 days (all patients with an INR higher than 1.5), two of whom were on full-dose heparin. (c) Three of seven of the patients with normalized INR and without significant PTT elevation developed severe cerebral embolism. Although our data are based on a retrospective analysis, they support treatment with intravenous heparin (partial thromboplastin time 1.5-2 times baseline value) after normalization of the INR in patients with an ICH and an urgent need for anticoagulation.

Qualitative and quantitative analysis of heparin and low molecular weight heparins using size exclusion chromatography with multiple angle laser scattering/refractive index and inductively coupled plasma/mass spectrometry detectors.

PubMed

Ouyang, Yilan; Zeng, Yangyang; Yi, Lin; Tang, Hong; Li, Duxin; Linhardt, Robert J; Zhang, Zhenqing

2017-11-03

Heparin, a highly sulfated glycosaminoglycan, has been used as a clinical anticoagulant over 80 years. Low molecular weight heparins (LMWHs), heparins partially depolymerized using different processes, are widely used as clinical anticoagulants. Qualitative molecular weight (MW) and quantitative mass content analysis are two important factors that contribute to LMWH quality control. Size exclusion chromatography (SEC), relying on multiple angle laser scattering (MALS)/refractive index (RI) detectors, has been developed for accurate analysis of heparin MW in the absence of standards. However, the cations, which ion-pair with the anionic polysaccharide chains of heparin and LMWHs, had not been considered in previous reports. In this study, SEC with MALS/RI and inductively coupled plasma/mass spectrometry detectors were used in a comprehensive analytical approach taking both anionic polysaccharide and ion-paired cations heparin products. This approach was also applied to quantitative analysis of heparin and LMWHs. Full profiles of MWs and mass recoveries for three commercial heparin/LMWH products, heparin sodium, enoxaparin sodium and nadroparin calcium, were obtained and all showed higher MWs than previously reported. This important improvement more precisely characterized the MW properties of heparin/LMWHs and potentially many other anionic polysaccharides. Copyright © 2017 Elsevier B.V. All rights reserved.

New synthetic peptide with efficacy for heparin reversal and low toxicity and immunogenicity in comparison to protamine sulfate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Tong; Meng, Zhiyun; Zhu, Xiaoxia

Protamine sulfate (PS), the only clinically approved antidote to unfractionated heparin (UFH), is widely used for cardiopulmonary surgery or other extracorporeal circulation situations. However, the applications of PS have accompanied various severe side-effects. In this study, we presented a novel synthesized peptide compound (RRRRR-RRRRR-RRRRR-sulfate, R15S) served as a safer UFH antidote. Comparison studies were conducted between PS and R15S on efficacy and safety, aided by heparin neutralization studies, drug toxicity studies and anaphylactic analysis. Our research demonstrated that R15S contained comparable UFH neutralization activity in vitro and in rats in vivo as to active partial thromboplastin time (APTT) and anti-FXa assays. Theremore » was no cytotoxicity for R15S at 60 μg mg{sup −1} or below and the median lethal dose (LD{sub 50}) of R15S in mice was 35.4 mg kg{sup −1}, both of which were similar to that of PS. Furthermore, R15S exhibited no immunogenicity while it was obvious in guinea pigs immunized with PS. The level of cross-reactivity to anti-PS antibodies of R15S was about 30%. Both of which indicated much safer properties of R15S than PS. In conclusion, we presented a promising candidate R15S for UFH reversal, which is effective in neutralizing UFH and potentially safer in use. - Highlights: • R15 sulfate has a comparable unfractionated heparin neutralization activity to PS. • R15 sulfate avoided severe anaphylactic responses which happened to PS in clinical use. • R15 sulfate could provide a stricter quality control compared to PS which may vary with different batches.« less

Results of the HepZero study comparing heparin-grafted membrane and standard care show that heparin-grafted dialyzer is safe and easy to use for heparin-free dialysis.

PubMed

Laville, Maurice; Dorval, Marc; Fort Ros, Joan; Fay, Renaud; Cridlig, Joëlle; Nortier, Joëlle L; Juillard, Laurent; Dębska-Ślizień, Alicja; Fernández Lorente, Loreto; Thibaudin, Damien; Franssen, Casper; Schulz, Michael; Moureau, Frédérique; Loughraieb, Nathalie; Rossignol, Patrick

2014-12-01

Heparin is used to prevent clotting during hemodialysis, but heparin-free hemodialysis is sometimes needed to decrease the risk of bleeding. The HepZero study is a randomized, multicenter international controlled open-label trial comparing no-heparin hemodialysis strategies designed to assess non-inferiority of a heparin grafted dialyzer (NCT01318486). A total of 251 maintenance hemodialysis patients at increased risk of hemorrhage were randomly allocated for up to three heparin-free hemodialysis sessions using a heparin-grafted dialyzer or the center standard-of-care consisting of regular saline flushes or pre-dilution. The first heparin-free hemodialysis session was considered successful when there was neither complete occlusion of air traps or dialyzer, nor additional saline flushes, changes of dialyzer or bloodlines, or premature termination. The current standard-of-care resulted in high failure rates (50%). The success rate in the heparin-grafted membrane arm was significantly higher than in the control group (68.5% versus 50.4%), which was consistent for both standard-of-care modalities. The absolute difference between the heparin-grafted membrane and the controls was 18.2%, with a lower bound of the 90% confidence interval equal to plus 7.9%. The hypothesis of the non-inferiority at the minus 15% level was accepted, although superiority at the plus 15% level was not reached. Thus, use of a heparin-grafted membrane is a safe, helpful, and easy-to-use method for heparin-free hemodialysis in patients at increased risk of hemorrhage.

Structural characterization of pharmaceutical heparins prepared from different animal tissues.

PubMed

Fu, Li; Li, Guoyun; Yang, Bo; Onishi, Akihiro; Li, Lingyun; Sun, Peilong; Zhang, Fuming; Linhardt, Robert J

2013-05-01

Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007-2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin III-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient. Copyright © 2013 Wiley Periodicals, Inc.

PROFILING GLYCOL-SPLIT HEPARINS BY HPLC/MS ANALYSIS OF THEIR HEPARINASE-GENERATED OLIGOSACCHARIDES1

PubMed Central

Alekseeva, Anna; Casu, Benito; Torri, Giangiacomo; Pierro, Sabrina; Naggi, Annamaria

2012-01-01

Glycol-split (gs) heparins, obtained by periodate oxidation / borohydride reduction of heparin currently used as anticoagulant and antithrombotic drug, are arousing increasing interest in anti-cancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs-heparins has not been studied yet in detail. In this work, an ion-pair reversed-phase chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs-heparins usually prepared from porcine mucosal heparin. The method has been also found very effective in analyzing glycol-split derivatives obtained from heparins of different animal and tissue origin. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs-heparins mainly contain tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs-heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues has been shown to be a marker of the gs-modified AT binding site within heparin chains. PMID:23201389

In vivo distribution and antitumor activity of heparin-stabilized doxorubicin-loaded liposomes.

PubMed

Han, Hee Dong; Lee, Aeri; Song, Chung Kil; Hwang, Taewon; Seong, Hasoo; Lee, Chong Ock; Shin, Byung Cheol

2006-04-26

The purpose of this study was to investigate the effect of heparin conjugation to the surface of doxorubicin (DOX)-loaded liposomes on the circulation time, biodistribution and antitumor activity after intravenous injection in murine B16F10 melanoma tumor-bearing mice. The heparin-conjugated liposomes (heparin-liposomes) were prepared by fixation of the negatively charged heparin to the positively charged liposomes. The existence of heparin on the liposomal surface was confirmed by measuring the changes in the particle size, zeta potential and heparin amount of the liposomes. The stability of the heparin-liposomes in serum was higher than that of the control liposomes, due to the heparin-liposomes being better protected from the adsorption of serum proteins. The DOX-loaded heparin-liposomes showed high drug levels for up to 64 h after the intravenous injection and the half-life of DOX was approximately 8.4- or 1.5-fold higher than that of the control liposomes or polyethyleneglycol-fixed liposomes (PEG-liposomes), respectively. The heparin-liposomes accumulated to a greater extent in the tumor than the control or PEG-liposomes as a result of their lower uptake by the reticuloendothelial system cells in the liver and spleen. In addition, the DOX-loaded heparin-liposomes retarded the growth of the tumor effectively compared with the control or PEG-liposomes. These results indicate the promising potential of heparin-liposomes as a new sterically stabilized liposomal delivery system for the enhancement of the therapeutic efficacy of chemotherapeutic agents.

Antidyslipidemic and antioxidant effects of novel Lupeol-derived chalcones.

PubMed

Srivastava, Shishir; Sonkar, Ravi; Mishra, Sunil Kumar; Tiwari, Avinash; Balaramnavar, Vishal M; Balramnavar, Vishal; Mir, Snober; Bhatia, Gitika; Saxena, Anil K; Lakshmi, Vijai

2013-10-01

A series of Lupeol-based chalcones have been synthesized aiming to enhance the therapeutic efficacy of parent compound, the novel compounds were evaluated for their antidyslipidemic activity in triton-WR 1339 induced hyperlipidemic rats. Among the ten synthesized chalcones, the most active K4, K8, and K9 reversed the plasma levels of TC by (24, 25, 27 %), phospholipid by (25, 26, 25 %) and triacylglycerol by (27, 24, 24 %) respectively. In addition, the compounds showed significant in vitro antioxidant activity. The lipid lowering activity of these compounds were mediated through lipoprotein lipase activation (12-21 %) and enhanced post-heparin lipolytic activity (15-16 %). The compounds also displayed noteworthy inhibitory effect on 3-hydroxy-3-methyl-glutaryl reductase activity (in vitro). The in vitro effect of the most active compounds on MDI-induced adipogenesis using 3T3-L1 preadipocytes at 10 and 20 μM concentrations showed significant inhibition (20-32 %) of adipogenesis.

Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

PubMed Central

Mellor, P; Harvey, J R; Murphy, K J; Pye, D; O'Boyle, G; Lennard, T W J; Kirby, J A; Ali, S

2007-01-01

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I125 CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases. PMID:17726466

Bovine and porcine heparins: different drugs with similar effects on human haemodialysis

PubMed Central

2013-01-01

Background Heparins from porcine and bovine intestinal mucosa differ in their structure and also in their effects on coagulation, thrombosis and bleeding. However, they are used as undistinguishable drugs. Methods We compared bovine and porcine intestinal heparin administered to patients undergoing a particular protocol of haemodialysis. We compared plasma concentrations of these two drugs and also evaluated how they affect patients and the dialyzer used. Results Compared with porcine heparin, bovine heparin achieved only 76% of the maximum plasma concentration as IU mL-1. This observation is consistent with the activities observed in the respective pharmaceutical preparations. When the plasma concentrations were expressed on weight basis, bovine heparin achieved a maximum concentration 1.5 fold higher than porcine heparin. The reduced anticoagulant activity and higher concentration, on weight basis, achieved in the plasma of patients under dialysis using bovine instead of porcine heparin did not affect significantly the patients or the dialyzer used. The heparin dose is still in a range, which confers security and safety to the patients. Discussion Despite no apparent difference between bovine and porcine intestinal heparins in the haemodialysis practice, these two types of heparins should be used as distinct drugs due to their differences in structure and biological effects. Conclusions The reduced anticoagulant activity achieved in the plasma of patients under dialysis using bovine instead of porcine heparin did not affect significantly the patients or the dialyzer. PMID:23763719

Pathway of oral absorption of heparin with sodium N-[8-(2-hydroxybenzoyl)amino] caprylate.

PubMed

Malkov, Dmitry; Wang, Huai-Zhen; Dinh, Steven; Gomez-Orellana, Isabel

2002-08-01

The oral bioavailability of heparin is negligible. Recent studies, however, have shown that sodium N-[8-(2-hydroxybenzoyl) amino]caprylate (SNAC) and other N-acylated amino acids enable oral heparin absorption. To investigate the mechanism by which heparin crosses the intestinal epithelium in the presence of SNAC, we have used fluorescence microscopy to follow the transport of heparin across Caco-2 cell monolayers. The experiments were carried out on Caco-2 monolayers and Caco-2 cells grown to confluence on culture dishes, using different concentrations of SNAC. The localization of fluorescently labeled heparin was determined using epi-fluorescence and confocal microscopy. DNA dyes were used to determine the effect of SNAC on the plasma membrane integrity. F-actin was labeled with fluorescent phalloidin to investigate the stability of perijunctional actin rings in the presence of SNAC. Heparin was detected in the cytoplasm only after incubation of the cells with heparin and SNAC. No DNA staining was observed in cells incubated with a DNA dye in the presence of SNAC concentrations at which heparin transport occurred. In addition, no signs of actin redistribution or perijunctional ring disbandment were observed during the transport of heparin. The results indicate that SNAC enables heparin transport across Caco-2 monolayers via the transcellular pathway. Heparin transport in the presence of SNAC is selective and does not involve permeabilization of the plasma membrane or tight junction disruption.

78 FR 36786 - Linking Marketplace Heparin Product Attributes and Manufacturing Processes to Bioactivity and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-19

... heparin. The condition leads to formation of abnormal blood clots and concomitant complications associated... antibody formation although these smaller chain length heparins are much less likely to lead to clinical... components of heparin that lead to the pathogenesis of HIT is the lack of pure component heparin standards...

Analysis and characterization of heparin impurities.

PubMed

Beni, Szabolcs; Limtiaco, John F K; Larive, Cynthia K

2011-01-01

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.

Isolation and characterization of a heparin with low antithrombin activity from the body of Styela plicata (Chordata-Tunicata). Distinct effects in venous and arterial models of thrombosis

PubMed Central

Santos, Joana C.; Mesquita, Juliana M. F.; Belmiro, Celso L.R.; da Silveira, Carolina B.M.; Viskov, Christian; Mourier, Pierre A.; Pavão., Mauro S.G.

2008-01-01

Introduction a heparin preparation with low antithrombin activity and different disaccharide composition than mammalian heparin was isolated from the body of the ascidian Styela plicata (Chordata-Tunicata). The disaccharide composition and the effect of the invertebrate glycan on venous and arterial models of thrombosis was investigated. Methods and Results High performance liquid chromatography of the products formed by a mixture of heparin-lyases showed that the ascidian heparin is composed mainly by ΔUA(2SO4)-1→4-β-D-GlcN(SO4) (47.5%), ΔUA(2SO4)-1→4-β-D-GlcN(SO4)(6SO4) (38.3%) disaccharides. Smaller amounts of the disaccharides ΔUA(2SO4)-1→4-β-D-GlcN(SO4)(3SO4)(6SO4) (2.8%) and ΔUA(2SO4)-1→4-β-D-GlcN(SO4)(3SO4) (8.0%). The invertebrate heparin has an aPTT activity of 18 IU/mg and an antithrombin-mediated anti-thrombin and anti-factor Xa activities 10-fold lower than that of mammalian heparin. In a venous model of thrombosis in the vena cava, S.plicata heparin inhibits only 80% of thrombosis at a dose 10-fold higher than that of the mammalian heparin that inhibits 100% of thrombosis. However, in an arterio-shunt model of arterial thrombosis, both S.plicata and mammalian heparin possess equivalent antithrombotic activity. It is also shown that at equivalent doses, ascidian heparin has a lower bleeding effect than mammalian heparin. Conclusion the antithrombin-mediated anticoagulant activity of heparin polymers is not directly related to antithrombotic potency in the arterio-venous shunt. The results of the present work suggest that heparin preparations obtained from the body of S.plicata may have a safer therapeutic action in the treatment of arterial thrombosis than mammalian heparin. PMID:17482241

Structure of the Complex between a Heparan Sulfate Octasaccharide and Mycobacterial Heparin-Binding Hemagglutinin.

PubMed

Huang, Teng-Yi; Irene, Deli; Zulueta, Medel Manuel L; Tai, Tzu-Jui; Lain, Shih-Han; Cheng, Cheng-Po; Tsai, Ping-Xi; Lin, Shu-Yi; Chen, Zhi-Geng; Ku, Chiao-Chu; Hsiao, Chwan-Deng; Chyan, Chia-Lin; Hung, Shang-Cheng

2017-04-03

Heparin-binding hemagglutinin (HBHA) is a 199 amino acid virulence factor at the envelope of Mycobacterium tuberculosis that contributes to latent tuberculosis. The binding of HBHA to respiratory epithelial cells, which leads to extrapulmonary dissemination of the pathogen, is mediated by cell-surface heparan sulfate (HS). We report the structural characterization of the HBHA/HS complex by NMR spectroscopy. To develop a model for the molecular recognition, the first chemically synthesized uniformly 13 C- and 15 N-labeled HS octasaccharide and a uniformly 13 C- and 15 N-labeled form of HBHA were prepared. Residues 180-195 at the C-terminal region of HBHA show large chemical shift perturbation upon association with the octasaccharide. Molecular dynamics simulations conforming to the multidimensional NMR data revealed key electrostatic and even hydrophobic interactions between the binding partners that may aid in the development of agents targeting the binding event. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The regulatory role of heparin on c-Met signaling in hepatocellular carcinoma cells.

PubMed

İşcan, Evin; Güneş, Aysim; Korhan, Peyda; Yılmaz, Yeliz; Erdal, Esra; Atabey, Neşe

2017-06-01

The role of heparin as an anticoagulant is well defined; however, its role in tumorigenesis and tumor progression is not clear yet. Some studies have shown that anticoagulant treatment in cancer patients improve overall survival, however, recent clinical trials have not shown a survival benefit in cancer patients receiving heparin treatment. In our previous studies we have shown the inhibitory effects of heparin on Hepatocyte Growth Factor (HGF)-induced invasion and migration in hepatocellular carcinoma (HCC) cells. In this study, we showed the differential effects of heparin on the behaviors of HCC cells based on the presence or absence of HGF. In the absence of HGF, heparin activated HGF/c-Met signaling and promoted motility and invasion in HCC cells. Heparin treatment led to c-Met receptor dimerization and activated c-Met signaling in an HGF independent manner. Heparin-induced c-Met activation increased migration and invasion through ERK1/2, early growth response factor 1 (EGR1) and Matrix Metalloproteinases (MMP) axis. Interestingly, heparin modestly decreased the proliferation of HCC cells by inhibiting activatory phosphorylation of Akt. The inhibition of c-Met signaling reversed heparin-induced increase in motility and invasion and, proliferation inhibition. Our study provides a new perspective into the role of heparin on c-Met signaling in HCC.

Differentiating low-molecular-weight heparins based on chemical, biological, and pharmacologic properties: implications for the development of generic versions of low-molecular-weight heparins.

PubMed

Jeske, Walter P; Walenga, Jeanine M; Hoppensteadt, Debra A; Vandenberg, Curtis; Brubaker, Aleah; Adiguzel, Cafer; Bakhos, Mamdouh; Fareed, Jawed

2008-02-01

Low-molecular-weight heparins (LMWHs) are polypharmacologic drugs used to treat thrombotic and cardiovascular disorders. These drugs are manufactured using different chemical and enzymatic methods, resulting in products with distinct chemical and pharmacologic profiles. Generic LMWHs have been introduced in Asia and South America, and several generic suppliers are seeking regulatory approval in the United States and the European Union. For simple small-molecule drugs, generic drugs have the same chemical structure, potency, and bioavailability as the innovator drug. Applying this definition to complex biological products such as the LMWHs has proved difficult. One major issue is defining appropriate criteria to demonstrate bioequivalence; pharmacopoeial specifications alone appear to be inadequate. Whereas available generic versions of LMWHs exhibit similar molecular and pharmacopoeial profiles, marked differences in their biological and pharmacologic behavior have been noted. Preliminary studies have demonstrated differences in terms of anti-Xa activity and tissue factor pathway inhibitor release after subcutaneous administration, as well as antiplatelet and profibrinolytic effects. The current data emphasize the need to consider multiple functional parameters when defining bioequivalence of biologic drugs with complex structures and activities and also underscore the importance of further pharmacologic studies involving animal models and human clinical trials. The U.S. Food and Drug Administration and the European Medicine Evaluation Agency are currently developing guidelines for the acceptance of biosimilar agents including LMWHs. Until such guidelines are complete, generic interchange may not be feasible.

Heparin-like activity in uterine fluid.

PubMed Central

Foley, M E; Griffin, B D; Zuzel, M; Aparicio, S R; Bradbury, K; Bird, C C; Clayton, J K; Jenkins, D M; Scott, J S; Rajah, S M; McNichol, G P

1978-01-01

Uterine fluid was collected from a group of normal patients and a group of patients with menorrhagia. Heparin-like activity was detected in 34 out of 38 samples using an anti-Xa heparin assay. The heparin-like activity in uterine fluid was inhibited by adding the heparin antagonist hexadimethrine bromide to the assay. Concentrations of fibrinogen-fibrin degradation products (FDPs) were measured in five samples of uterine fluid. FDPs in the concentration detected had no effect on the anti-Xa assay. Heparin-like activity was higher in the group with menorrhagia, although the differences were not significant. Heparin-like activity increased throughout the menstrual cycle and decreased during menstruation, suggesting a possible cyclical variation in activity. There was no correlation between mast cell numbers in the endometrium and myometrium and heparin-like activity in uterine fluid and no correlation between the numbers and the stage in the menstrual cycle. In a few patients with intrauterine contraceptive devices (IUCDs) heparin-like activity was increased. PMID:687899

Heparin-induced thrombocytopenia: real-world issues.

PubMed

Linkins, Lori-Ann; Warkentin, Theodore E

2011-09-01

Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating antibodies. HIT sera often activate platelets without needing heparin-such heparin-"independent" platelet activation can be associated with HIT beginning or worsening despite stopping heparin ("delayed-onset HIT"). We address important issues in HIT diagnosis and therapy, using a recent cohort of HIT patients to illustrate influences of heparin type; triggers for HIT investigation; serological features of heparin-independent platelet activation; and treatment. In our cohort of recent HIT cases ( N = 13), low-molecular-weight heparin (dalteparin) was a common causative agent ( N = 8, 62%); most patients were diagnosed after HIT-thrombosis had occurred; and danaparoid was the most frequently selected treatment. Heparin-independent platelet activation was common (7/13 [54%]) and predicted slower platelet count recovery (>1 week) among evaluable patients (5/5 vs 1/6; P = 0.015). In our experience with argatroban-treated patients, HIT-associated consumptive coagulopathy confounds anticoagulant monitoring. Our observations provide guidance on practical aspects of HIT diagnosis and management. Thieme Medical Publishers.

Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe.

PubMed

Mehta, Pramod Kumar; Lee, Hyeri; Lee, Keun-Hyeung

2017-05-15

The selective and sensitive detection of heparin, an anticoagulant in clinics as well as its contaminant oversulfated chondroitin sulfate (OSCS) is of great importance. We first reported a ratiometric sensing method for heparin as well as OSCS contaminants in heparin using a fluorescent peptidyl probe (Pep1, pyrene-GSRKR) and heparin-digestive enzyme. Pep1 exhibited a highly sensitive ratiometric response to nanomolar concentration of heparin in aqueous solution over a wide pH range (2~11) and showed highly selective ratiometric response to heparin among biological competitors such as hyaluronic acid and chondroitin sulfate. Pep1 showed a linear ratiometric response to nanomolar concentrations of heparin in aqueous solutions and in human serum samples. The detection limit for heparin was calculated to be 2.46nM (R 2 =0.99) in aqueous solutions, 2.98nM (R 2 =0.98) in 1% serum samples, and 3.43nM (R 2 =0.99) in 5% serum samples. Pep1 was applied to detect the contaminated OSCS in heparin with heparinase I, II, and III, respectively. The ratiometric sensing method using Pep1 and heparinase II was highly sensitive, fast, and efficient for the detection of OSCS contaminant in heparin. Pep1 with heparinase II could detect as low as 0.0001% (w/w) of OSCS in heparin by a ratiometric response. Copyright © 2017 Elsevier B.V. All rights reserved.

Sensitive detection of oversulfated chondroitin sulfate in heparin sodium or crude heparin with a colorimetric microplate based assay.

PubMed

Sommers, Cynthia D; Mans, Daniel J; Mecker, Laura C; Keire, David A

2011-05-01

In this work we describe a 96-well microplate assay for oversulfated chondroitin sulfate A (OSCS) in heparin, based on a water-soluble cationic polythiophene polymer (3-(2-(N-(N'-methylimidazole))ethoxy)-4-methylthiophene (LPTP)) and heparinase digestion of heparin. The assay takes advantage of several unique properties of heparin, OSCS, and LPTP, including OSCS inhibition of heparinase I and II activity, the molecular weight dependence of heparin-LPTP spectral shifts, and the distinct association of heparin fragments and OSCS to LPTP. These factors combine to enable detection of the presence of 0.003% w/w spiked OSCS in 10 μg of heparin sodium active pharmaceutical ingredient (API) using a plate reader and with visual detection to 0.1% levels. The same detection limit for OSCS was observed in the presence of 10% levels of dermatan sulfate (DS) or chondroitin sulfate A (CSA) impurities. In addition, we surveyed a selection of crude heparin samples received by the agency in 2008 and 2009 to determine average and extreme DS, CSA, and galactosamine weight percent levels. In the presence of these impurities and the variable heparin content in the crude heparin samples, spiked OSCS was reliably detected to the 0.1% w/w level using a plate reader. Finally, authentically OSCS contaminated heparin sodium API and crude samples were distinguished visually by color from control samples using the LPTP/heparinase test.

Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles.

PubMed

Tellier, Liane E; Miller, Tobias; McDevitt, Todd C; Temenoff, Johnna S

2015-10-28

Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

The clinical evaluation of low-dose heparin in haemodialysis: a prospective study using the heparin-coated AN69 ST membrane.

PubMed

Chanard, Jacques; Lavaud, Sylvie; Maheut, Hervé; Kazes, Isabelle; Vitry, Fabien; Rieu, Philippe

2008-06-01

The AN69 ST haemodialysis membrane, a new membrane resulting from coating polyethyleneimine upon the polyacrylonitrile surface, binds heparin. In patients at risk of bleeding, a pilot study has demonstrated the efficient anticoagulant effect of this heparin-coated membrane. Study design. In chronic haemodialyzed patients, we evaluated whether this anticoagulant effect can be validated in a controlled, prospective, open study. Pragmatically, we tested the hypothesis of no difference of the massive clotting rate in two groups of patients haemodialyzed either with 50% reduced standard doses of nonfractionated heparin using the heparin-coated AN69 ST or with a full dose of heparin (100%) using another type of dialysis membrane that does not bind heparin. Secondary objectives included evaluation of partial clotting, changes in haemoglobin levels, erythropoietin consumption and dialyzer performances. One hundred and eighty-four patients were elected and 170 finally included in an 18-month follow-up study. They were allocated to one of the two arms of the study. In the heparin-reduced group (n = 85, mean age: 73 +/- 11 years), 12 472 sessions were performed after priming the AN69 ST dialyzer with 2 L of heparinized saline (5000 IU/L heparin) and using 50% reduced doses of previously administered heparin. In the control group with standard heparin (n = 85, mean age: 74 +/- 13 years), 14 154 sessions were analysed (NS), and mean heparin doses were 2718 +/- 1388 and 4800 +/- 1564 IU per session, respectively (P < 0.001). In the heparin-reduced group, massive clotting occurred in 1.4 per 1000 sessions, whereas it occurred in 1.6 per 1000 sessions in the standard heparin group (P < 0.05). Mild to moderate partial clotting in the venous drip chamber and in the dialyzer was evaluated in a subset of patients, on a visual scale. It was more frequent in the experimental group than in the control group (P < 0.001). Platelets, haemoglobin levels, erythropoietin needs and dialyzer performances remained unchanged in both groups. The global mean death rate was 16.8% per year and did not differ significantly between groups. The use of the heparin-coated AN69 ST membrane allows a 50% reduction of standard doses of nonfractionated heparin administration for routine haemo- dialysis without increasing the risk of massive clotting of the extracorporeal circuit. This result needs confirmation since massive clotting questions clinical practice and is team dependent.

Synthetic heparin-binding growth factor analogs

DOEpatents

Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

2007-01-23

The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

Effect of oral administration of unfractionated heparin (UFH) on coagulation parameters in plasma and levels of urine and fecal heparin in dogs

PubMed Central

Erickson, Malathi; Hiebert, Linda M.; Carr, Anthony P.; Stickney, Jocelyn D.

2014-01-01

The effects of heparin administration, by the oral route, were evaluated in dogs. In single and multiple dose studies (single 7.5 mg/kg, multiple 3 × 7.5 mg/kg per 48 h), plasma, urine, and fecal samples were collected at various times up to 120 h after oral administration of unfractionated heparin. Changes in plasma and urine anti-Xa activity, plasma and urine anti-IIa activity, plasma activated partial thromboplastin time (APTT) and antithrombin (ATIII), and chemical heparin in urine and feces were examined with time. There was support for heparin absorption, with significant differences in APTT, heparin in plasma as determined by anti-Xa activity (Heptest) in the single dose study and plasma anti-Xa activity, anti-IIa activity and ATIII; and chemical heparin in urine in the multiple dose study. No clinical evidence of bleeding was detected in any dog during the studies. Oral heparin therapy may be applicable for thromboembolic disease in animals. Further studies are warranted to determine the effects of oral heparin at the endothelial level in the dog. PMID:24982550

Evidence-based algorithm for heparin dosing before cardiopulmonary bypass. Part 1: Development of the algorithm.

PubMed

McKinney, Mark C; Riley, Jeffrey B

2007-12-01

The incidence of heparin resistance during adult cardiac surgery with cardiopulmonary bypass has been reported at 15%-20%. The consistent use of a clinical decision-making algorithm may increase the consistency of patient care and likely reduce the total required heparin dose and other problems associated with heparin dosing. After a directed survey of practicing perfusionists regarding treatment of heparin resistance and a literature search for high-level evidence regarding the diagnosis and treatment of heparin resistance, an evidence-based decision-making algorithm was constructed. The face validity of the algorithm decisive steps and logic was confirmed by a second survey of practicing perfusionists. The algorithm begins with review of the patient history to identify predictors for heparin resistance. The definition for heparin resistance contained in the algorithm is an activated clotting time < 450 seconds with > 450 IU/kg heparin loading dose. Based on the literature, the treatment for heparin resistance used in the algorithm is anti-thrombin III supplement. The algorithm seems to be valid and is supported by high-level evidence and clinician opinion. The next step is a human randomized clinical trial to test the clinical procedure guideline algorithm vs. current standard clinical practice.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Lung Heparin and Porcine Intestine Heparin.

PubMed

Guan, Yudong; Xu, Xiaohui; Liu, Xinyue; Sheng, Anran; Jin, Lan; Linhardt, Robert J; Chi, Lianli

2016-06-01

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin-derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin-derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Outbreak of Adverse Reactions Associated with Contaminated Heparin

PubMed Central

Blossom, David B.; Kallen, Alexander J.; Patel, Priti R.; Elward, Alexis; Robinson, Luke; Gao, Ganpan; Langer, Robert; Perkins, Kiran M.; Jaeger, Jennifer L.; Kurkjian, Katie M.; Jones, Marilyn; Schillie, Sarah F.; Shehab, Nadine; Ketterer, Daniel; Venkataraman, Ganesh; Kishimoto, Takashi Kei; Shriver, Zachary; McMahon, Ann W.; Austen, K. Frank; Kozlowski, Steven; Srinivasan, Arjun; Turabelidze, George; Gould, Carolyn V.; Arduino, Matthew J.; Sasisekharan, Ram

2013-01-01

BACKGROUND In January 2008, the Centers for Disease Control and Prevention began a nationwide investigation of severe adverse reactions that were first detected in a single hemodialysis facility. Preliminary findings suggested that heparin was a possible cause of the reactions. METHODS Information on clinical manifestations and on exposure was collected for patients who had signs and symptoms that were consistent with an allergic-type reaction after November 1, 2007. Twenty-one dialysis facilities that reported reactions and 23 facilities that reported no reactions were included in a case–control study to identify facility-level risk factors. Unopened heparin vials from facilities that reported reactions were tested for contaminants. RESULTS A total of 152 adverse reactions associated with heparin were identified in 113 patients from 13 states from November 19, 2007, through January 31, 2008. The use of heparin manufactured by Baxter Healthcare was the factor most strongly associated with reactions (present in 100.0% of case facilities vs. 4.3% of control facilities, P<0.001). Vials of heparin manufactured by Baxter from facilities that reported reactions contained a contaminant identified as oversulfated chondroitin sulfate (OSCS). Adverse reactions to the OSCS-contaminated heparin were often characterized by hypotension, nausea, and shortness of breath occurring within 30 minutes after administration. Of 130 reactions for which information on the heparin lot was available, 128 (98.5%) occurred in a facility that had OSCS-contaminated heparin on the premises. Of 54 reactions for which the lot number of administered heparin was known, 52 (96.3%) occurred after the administration of OSCS-contaminated heparin. CONCLUSIONS Heparin contaminated with OSCS was epidemiologically linked to adverse reactions in this nationwide outbreak. The reported clinical features of many of the cases further support the conclusion that contamination of heparin with OSCS was the cause of the outbreak. PMID:19052120

Hydrolysis of bovine and caprine milk fat globules by lipoprotein lipase. Effects of heparin and skim milk on lipase distribution and on lipolysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundheim, G.; Bengtsson-Olivecrona, G.

1987-12-01

Heparin can dissociate lipoprotein lipase from casein micelles, and addition of heparin enhances lipolysis in bovine but not in caprine milk. Heparin shortened the lag-time for binding of lipoprotein lipase to milk fat globules and for lipolysis. Heparin counteracted the inhibitory effects of skim milk on binding of lipase and on lipolysis. Heparin stimulated lipolysis in all bovine milk samples when added before cooling and in spontaneously lipolytic milk samples also when added after cooling. Heparin enhanced lipolysis of isolated milk fat globules. Hence, its effect is not solely due to dissociation of lipoprotein lipase from the casein micelles. Coolingmore » of goat milk caused more marked changes in the distribution of lipase than cooling of bovine milk; the fraction of added /sup 125/I-labeled lipase that bound to cream increased from about 8 to 60%. In addition, caprine skim milk caused less inhibition of lipolysis than bovine skim milk. These observations provide an explanation for the high degree of cold storage lipolysis in goat milk. Heparin had only small effects on the distribution of lipoprotein lipase in caprine milk, which explains why heparin has so little effect on lipolysis in caprine milk. The distribution of /sup 35/S-labeled heparin in bovine milk was studied. In warm milk less than 10% bound to the cream fraction, but when milk was cooled, binding of heparin to cream increased to 45%. These results suggest that there exists in the skim fraction a relatively small amount of a heparin-binding protein, which on cooling of milk adsorbs to the milk fat, or suggests that cooling induces a conformational change in a membrane protein such that its affinity for heparin increases.« less

Inappropriate documentation of heparin allergy in the medical record because of misdiagnosis of heparin-induced thrombocytopenia: frequency and consequences.

PubMed

McMahon, C M; Tanhehco, Y C; Cuker, A

2017-02-01

Essentials Misdiagnosis of heparin-induced thrombocytopenia (HIT) may be associated with adverse outcomes. We conducted a study of patients with a heparin allergy in the chart due to misdiagnosis of HIT. 42% of patients with a heparin allergy due to suspected HIT were clearly HIT-negative. 68% were unnecessarily treated with an alternative anticoagulant, 66% of whom had major bleeding. Background It is recommended that heparin be added to the allergy list of patients with heparin-induced thrombocytopenia (HIT). Misdiagnosis of HIT could lead to inappropriate documentation of a heparin allergy and adverse outcomes. Objectives To determine the frequency and consequences of inappropriate documentation of a heparin allergy because of misdiagnosis of HIT. Methods We conducted a cohort study of patients with an inappropriate heparin allergy listed in the electronic medical record (EMR) because of misdiagnosis of HIT. We searched the EMR for patients with a new heparin allergy. Patients were eligible if the reason for allergy listing was suspected acute HIT and laboratory testing for HIT was performed within 60 days. Subjects were defined as 'HIT-negative' if they had a 4Ts score of ≤ 3 or negative laboratory test results. Results Of 239 subjects with a new heparin allergy documented because of concern regarding HIT, 100 (42%) met the prespecified definition of HIT-negative. Sixty-eight (68%) HIT-negative subjects unnecessarily received an alternative parenteral anticoagulant for a median duration of 10.5 days. Among these 68 patients, 45 (66%) met criteria for major bleeding. Sixty-eight (68%) of the 100 HIT-negative subjects had an inappropriate allergy to heparin documented that persisted in the EMR for > 3 years beyond the index hospitalization. Conclusions Inappropriate listing of heparin as an allergy in the EMR because of misdiagnosis of HIT is common, is associated with substantial rates of unnecessary alternative anticoagulant use and major bleeding, and tends to persist beyond the index admission. © 2016 International Society on Thrombosis and Haemostasis.

Structural and binding studies of SAP-1 protein with heparin.

PubMed

Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

2015-03-01

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

Click-coated, heparinized, decellularized vascular grafts

PubMed Central

Dimitrievska, Sashka; Cai, Chao; Weyers, Amanda; Balestrini, Jenna L.; Lin, Tylee; Sundaram, Sumati; Hatachi, Go; Spiegel, David A.; Kyriakides, Themis R.; Miao, Jianjun; Li, Guoyun; Niklason, Laura; Linhardt, Robert J.

2014-01-01

A novel method enabling the engineering of a dense and appropriately oriented heparin-containing layer on decellularized aortas has been developed. Amino groups of decellularized aortas were first modified to azido groups using 3-azidobenzoic acid. Azide-clickable dendrons were attached onto the azido groups through “alkyne-azide” click chemistry, affording a ten-fold amplification of adhesions sites. Dendron end groups were finally decorated with end-on modified heparin chains. Heparin chains were oriented like heparan sulfate groups on native endothelial cells surface. XPS, NMR, MS and FTIR were used to characterize the synthesis steps, building the final heparin layered coatings. Continuity of the heparin coating was verified using fluorescent microscopy and histological analysis. Efficacy of heparin linkage was demonstrated with factor Xa antithrombogenic assay and platelet adhesion studies. The results suggest that oriented heparin immobilization to decellularized aortas may improve the in vivo blood compatibility of decellularized aortas and vessels. PMID:25463496

Effect of switching unfractionated heparin to low-molecular-weight heparin on serum potassium in hemodialysis patients.

PubMed

Ezzatzadegan Jahromi, Shahrokh; Mahmoodi, Mohammad Saleh; Behroozi, Fatemeh; Roozbeh, Jamshid; Emamghoreishi, Fatemeh

2014-11-01

Unfractionated (UF) heparin is the most common anticoagulant used during hemodialysis. Failure of the kidneys to excrete potassium as well as heparin-induced reduction of aldosterone synthesis put hemodialysis patients at risk of hyperkalemia. It has not yet been clearly known whether hyperkalemia is also induced by low-molecular-weight (LMW) heparins. This study aimed to evaluate the effect of switching UF heparin to LMW heparin enoxaparin, as an anticoagulant during hemodialysis, on serum potassium level in patients on hemodialysis. In two hemodialysis units, 58 patients were randomly assigned into two groups, to receive two different anticoagulation protocols for 3 weeks; one group continued to receive their routine dose of UF heparin, 5000 units, and the other received enoxaparin, 0.5 mg/kg, at the beginning of each hemodialysis session. While there was no significant difference between baseline blood measurements of the two groups in terms of kidney function tests and electrolytes, following 3 weeks of the study, the mean serum potassium level decreased from 4.9 ± 0.8 mEq/L to 4.5 ± 0.5 mEq/L in the LMW heparin group (P = .001); however, there was no change in the mean serum potassium level in those who continued to receive their usual dose of UF heparin. In a subgroup analysis, diabetic patients in the enoxaparin group did not experience significant reduction in serum potassium levels. Our study revealed the role of LMW heparins as a potential alternative to UF heparins in the hemodialysis patients with hyperkalemia.

An enzyme-linked immunosorbent assay for the evaluation of thrombocytopenia induced by heparin.

PubMed

Howe, S E; Lynch, D M

1985-05-01

Five patients with heparin-associated thrombocytopenia (HAT) were evaluated by platelet aggregation and quantitation of immunoglobulin binding to intact target platelets in both the presence and absence of heparin. These patients developed thrombocytopenia (12,000 to 70,000 platelets/microliter) 7 to 15 days and embolic and hemorrhagic complications 9 to 15 days after the initiation of heparin therapy. Platelet aggregation after the addition of heparin was demonstrated in two of four HAT serum samples, whereas normal serum samples showed no significant platelet aggregation. The five HAT serum samples showed normal to elevated baseline serum platelet-bindable immunoglobulin (SPBIg) with a range of 4.3 to 11.4 fg/platelet (normal less than or equal to 1.0 to 6.5 fg/platelet). When HAT sera were incubated with target platelets and heparin (5 U/ml), the SPBIg increased to 8.5 to 37.5 fg/platelet, a mean increase of 148% in the presence of heparin. Normal and control serum samples (from 10 normal laboratory volunteers, nine patients without thrombocytopenia receiving heparin, nine patients with autoimmune thrombocytopenic purpura, and nine patients with nonimmune thrombocytopenia not receiving heparin) showed only a slight increase in SPBIg of 0 to 2.8 fg/platelet above baseline, a mean increase of 15% after heparin incubation with the serum samples. The measurement of SPBIg of washed platelets incubated with test serum samples in the presence and absence of heparin is potentially a specific and sensitive in vitro test for the diagnosis of HAT and may prove more sensitive than platelet aggregation studies with heparin.

Heparin: Past, Present, and Future.

PubMed

Oduah, Eziafa I; Linhardt, Robert J; Sharfstein, Susan T

2016-07-04

Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007-2008 increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious disease. A variety of strategies have been proposed to produce a bioengineered heparin. In this review, we discuss several of these strategies including microbial production, mammalian cell production, and chemoenzymatic modification. We also propose strategies for creating "designer" heparins and heparan-sulfates with various biochemical and physiological properties.

Heparin-Induced Thrombocytopenia with Associated Thrombosis in Children after the Fontan Operation

PubMed Central

Porcelli, Rosalia; Moskowitz, Bonnie C.; Cetta, Frank; Graham, Lynn C.; Godwin, John E.; Eidem, Benjamin W.; Prechel, M. Margaret; Walenga, Jeanine M.

2003-01-01

Heparin-induced thrombocytopenia is a widely recognized clinical disorder. The spectrum of disease ranges from clinically insignificant to severe thrombosis (heparin-induced thrombocytopenia with associated thrombosis). Overall, thrombosis occurs in approximately 33% of adults diagnosed with heparin-induced thrombocytopenia and has been associated with high morbidity and mortality rates. Diagnostic testing for this disorder is not standard in children with thrombocytopenia who are receiving heparin, despite the fact that children with congenital heart disease may be exposed to heparin frequently. There are few reported cases of heparin-induced thrombocytopenia with associated thrombosis in children; herein, we describe the cases of 2 children who developed this disorder after undergoing a Fontan operation. (Tex Heart Inst J 2003;30:58–61) PMID:12638673

[The percutaneous action of a heparin-allantoin-dexpanthenol combination in a specific ointment base. Thrombolytic action on the rabbit ear].

PubMed

Tauschel, H D; Bonacina, F; Galetti, F

1984-01-01

Experimentally induced thrombi of ear veins in albino rabbits have been treated locally with heparin-containing ointments in presence or absence of allantoin and dexpanthenol, the heparin concentration varying. While the ointments, containing heparin only, induce no or only minor thrombolytic activity, the combination ointments Hepathrombin Adenylchemie containing heparin, allantoin and dexpanthenol show significant thrombolytic activity. This effect is dependent upon the heparin concentration, yet, heparin doses above 50 000 IU per 100 g of ointment do not enhance the thrombolysis furthermore. Further, the studies show that the effective components of Hepathrombin do penetrate into and through the skin, allantoin and dexpanthenol being important components of the ointment probably supporting the transdermal penetration of heparin. The studies also demonstrate the only local thrombolytic effect of the Hepathrombin ointments because the thrombus of the right ear, always treated with ointment base only, did not show any change in length as contrasted to that of the left ear of the same animal treated with the Hepathrombin ointments. Mechanisms of the locally by Hepathrombin/heparin induced thrombolysis will be discussed.

Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues.

PubMed

Jiang, Bin; Suen, Rachel; Wertheim, Jason A; Ameer, Guillermo A

2016-12-12

Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.

Preparation of Low Molecular Weight Heparin by Microwave Discharge Electrodeless Lamp/TiO2 Photo-Catalytic Reaction.

PubMed

Lee, Do-Jin; Kim, Byung Hoon; Kim, Sun-Jae; Kim, Jung-Sik; Lee, Heon; Jung, Sang-Chul

2015-01-01

An MDEL/TiO2 photo-catalyst hybrid system was applied, for the first time, for the production of low molecular weight heparin. The molecular weight of produed heparin decreased with increasing microwave intensity and treatment time. The abscission of the chemical bonds between the constituents of heparin by photo-catalytic reaction did not alter the characteristics of heparin. Formation of by-products due to side reaction was not observed. It is suggested that heparin was depolymerized by active oxygen radicals produced during the MDEL/TiO2 photo-chemical reaction.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattori, Masaki; Sugino, Etsuko; Minoura, Katsuhiko

One of the priorities in Alzheimer research is to develop a compound that inhibits the filament formation of tau protein. Since the three- or four-repeat microtubule-binding domain (MBD) in tau protein plays an essential role in filament formation, the inhibitory behavior of cyanidin (Cy) and methylene blue (MB) with respect to heparin-induced filament formation of MBD in a neutral solution (pH 7.6) was characterized by fluorescence, circular dichroism, and electron microscopy measurements. The planar aromatic ring of Cy and the N-unsubstituted phenothiazine ring of MB were shown to be necessary for the inhibition. However, the inhibitory responses with respect tomore » heparin-induced filament formation to the second and third repeat peptides of MBD were different: Cy suppresses the formation and MB does not prevent the formation. This suggests the importance of the first and fourth repeat peptides in the inhibitory activity of MB for MBD filament formation. In this study, we showed that the decrease of thioflavin S fluorescence intensity is not always linked to inhibition of filament formation.« less

High Sulfation and a High Molecular Weight Are Important for Anti-hepcidin Activity of Heparin

PubMed Central

Asperti, Michela; Naggi, Annamaria; Esposito, Emiliano; Ruzzenenti, Paola; Di Somma, Margherita; Gryzik, Magdalena; Arosio, Paolo; Poli, Maura

2016-01-01

Heparins are efficient inhibitors of hepcidin expression even in vivo, where they induce an increase of systemic iron availability. Heparins seem to act by interfering with BMP6 signaling pathways that control the expression of liver hepcidin, causing the suppression of SMAD1/5/8 phosphorylation. The anti-hepcidin activity persists also when the heparin anticoagulant property is abolished or reduced by chemical reactions of oxidation/reduction (glycol-split, Gs-Heparins) or by high sulfation (SS-Heparins), but the structural characteristics needed to optimize this inhibitory activity have not been studied in detail. To this aim we analyzed three different heparins (Mucosal Heparin, the Glycol split RO-82, the partially desulfated glycol-split RO-68 and the oversulfated SSLMWH) and separated them in fractions of molecular weight in the range 4–16 kD. Since the distribution of the negative charges in heparins contributes to the activity, we produced 2-O- and 6-O-desulfated heparins. These derivatives were analyzed for the capacity to inhibit hepcidin expression in hepatic HepG2 cells and in mice. The two approaches produced consistent results and showed that the anti-hepcidin activity strongly decreases with molecular weight below 7 kD, with high N-acetylation and after 2-O and 6-O desulfation. The high sulfation and high molecular weight properties for efficient anti-hepcidin activity suggest that heparin is involved in multiple binding sites. PMID:26955355

Heparin-induced hyperkalemia.

PubMed

Thomas, C M; Thomas, J; Smeeton, F; Leatherdale, B A

2008-05-01

An 85-year-old lady with type 2 diabetes mellitus of 32 years duration with peripheral neuropathy was admitted under the vascular surgeons with extensive gangrene of her lower limb. She was on insulin for the last 7 years. Initial investigations showed normal serum electrolytes. She was started on antibiotics and unfractionated heparin, and her electrolytes showed hyperkalemia, which persisted on active treatment. Her short synacthen test showed good response, renin was normal with low aldosterone, urinary pH, sodium, potassium and osmolality was normal. On stopping heparin serum, potassium became normal. On restarting heparin (low molecular weight) during a suspected episode of pulmonary embolism, she developed hyperkalemia and heparin was stopped. Her potassium and aldosterone became normal on discontinuation of heparin. She developed hyperkalemia with both unfractionated and low molecular weight heparin.

Low incidence of heparin-induced skin lesions in orthopedic surgery patients with low-molecular-weight heparins.

PubMed

Schindewolf, M; Paulik, M; Kroll, H; Kaufmann, R; Wolter, M; Boehncke, W-H; Lindhoff-Last, E; Recke, A; Ludwig, R J

2018-04-23

Heparins are widely prescribed for prevention and therapy of arterial and venous thromboembolic diseases. Heparin-induced skin lesions are the most frequent adverse effects of subcutaneous heparin treatment in non-surgical patients (7.5-39.8%); no data exist on surgical patients. Commonly, they are due to a delayed-type hypersensitivity reaction (DTH), but may also be a manifestation of life-threatening heparin-induced thrombocytopenia (HIT). Lesions of both entities resemble initially. The risk of HIT is highest among heparin-anticoagulated orthopedic surgery patients. To determine incidence and causes of heparin-induced skin lesions in major orthopedic surgery patients. In a prospective cohort study consecutive patients with subcutaneous low-molecular-weight heparin (LMWH) treatment were examined for cutaneous adverse effects. Further diagnostics (skin biopsy, clinical/laboratory assessment for thrombosis, bleeding, HIT, cross-allergies) were performed. Six of 316 enrolled patients (1.9%; 95% CI 0.4%-3.4%) developed heparin-induced skin lesions. All were caused by a DTH reaction, none was due to HIT or other rare heparin-associated skin diseases. Therapeutic use (dosage) of LMWH was identified as only risk factor (odds ratio: 3.1, 95%CI: 1.4-4.9; p=0.00141). In addition to DTH, 5 thromboembolic, 4 major bleeding complications but no cases of HIT or cross-allergies were observed. Orthopedic surgery patients have - unlike non-surgical patients - a low risk for heparin-induced skin lesions during LMWH treatment; all lesions were due to a DTH reaction. The risk for DTH differs considerably between individual patient cohorts. No association with HIT was observed. These data help to tailor anticoagulatory treatment individually and to increase patient safety. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Haemostatic agent etamsylate in vitro and in vivo antagonizes anti-coagulant activity of heparin.

PubMed

Cobo-Nuñez, M Yolanda; El Assar, Mariam; Cuevas, Pedro; Sánchez-Ferrer, Alberto; Martínez-González, Jennifer; Rodríguez-Mañas, Leocadio; Angulo, Javier

2018-05-15

Etamsylate is indicated for several anti-hemorrhagic indications in human and veterinary medicine. However, etamsylate has been shown to be effective only in specific hemorrhagic situations. Furthermore, mechanism of action of etamsylate is not known but recent research has shown its ability to inhibit heparin binding to several growth factors. We have evaluated the ability of etamsylate to interfere with the activities of heparin. Effects of etamsylate on vasodilatory activity of heparin were evaluated in rat aortic segments. Influence of etamsylate on anticoagulant activity of heparin was evaluated in vitro by determining prothrombin (PT) time and activated partial thromboplastin time (aPTT) in dog blood and in vivo by determining the interference of systemic and topical etamsylate on heparin-induced extension in bleeding time (BT) in rats. Despite failing to inhibit heparin-induced vasodilation of rat aorta, etamsylate significantly reduced the increase in aPTT caused by heparin (+30.4 ± 6.7% vs. +15.0 ± 2.8% for etamsylate at 100 µM, P < 0.05). Etamsylate also antagonized the anticoagulant effects driven by heparin in vivo since prevented the heparin-induced increase in BT when systemically (i.p.) administered (+94.6 ± 7.5% vs. +57.9 ± 9.2% at 10 mg/kg, P < 0.05, vs. +22.2 ± 16.8% at 30 mg/kg, P < 0.01). Additionally, topically applied etamsylate (125 mg/ml) significantly reduced heparin-induced BT increase (+102.5 ± 3.2% vs. +54.0 ± 5.8%, P < 0.01). These evidences show a pharmacological interference by etamsylate on heparin activities antagonizing pro-hemorrhagic effects of heparin in vitro and in vivo without inhibiting its vasodilatory properties. This ability could help to explain pharmacological effects of etamsylate and proposes its role for reversing pro-hemorrhagic states. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of heparin oligosaccharides by capillary electrophoresis-negative-ion electrospray ionization mass spectrometry.

PubMed

Lin, Lei; Liu, Xinyue; Zhang, Fuming; Chi, Lianli; Amster, I Jonathan; Leach, Franklyn E; Xia, Qiangwei; Linhardt, Robert J

2017-01-01

Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins. Graphical abstract Most hyphenated analytical approaches that rely on liquid chromatography-MS require relatively long separation times, produce incomplete resolution of oligosaccharide mixtures, use eluents that are incompatible with electrospray ionization, or require oligosaccharide derivatization. Here we demonstrate the analysis of heparin oligosaccharides, including disaccharides, ultralow molecular weight heparin, and a low molecular weight heparin, using a novel electrokinetic pump-based CE-MS coupling eletrospray ion source. Reverse polarity CE separation and negative-mode electrospray ionization were optimized using a volatile methanolic ammonium acetate electrolyte and sheath fluid. The online CE hyphenated negative-ion electrospray ionization MS on an LTQ Orbitrap mass spectrometer was useful in disaccharide compositional analysis and bottom-up and top-down analysis of low molecular weight heparin. The application of this CE-MS method to ultralow molecular heparin suggests that a charge state distribution and the low level of sulfate group loss that is achieved make this method useful for online tandem MS analysis of heparins.

Heparin increases food intake through AgRP neurons

USDA-ARS?s Scientific Manuscript database

Although the widely used anticoagulant drug heparin has been shown to have many other biological functions independent of its anticoagulant role, its effects on energy homeostasis are unknown. Here, we demonstrate that heparin level is negatively associated with nutritional states and that heparin t...

Layer-by-Layer Heparinization of the Cell Surface by Using Heparin-Binding Peptide Functionalized Human Serum Albumin.

PubMed

Song, Guowei; Hu, Yaning; Liu, Yusheng; Jiang, Rui

2018-05-20

Layer-by-layer heparinization of therapeutic cells prior to transplantation is an effective way to inhibit the instant blood-mediated inflammatory reactions (IBMIRs), which are the major cause of early cell graft loss during post-transplantation. Here, a conjugate of heparin-binding peptide (HBP) and human serum albumin (HSA), HBP-HSA, was synthesized by using heterobifunctional crosslinker. After the first heparin layer was coated on human umbilical vein endothelial cells (HUVECs) by means of the HBP-polyethylene glycol-phospholipid conjugate, HBP-HSA and heparin were then applied to the cell surface sequentially to form multiple layers. The immobilization and retention of heparin were analyzed by confocal microscopy and flow cytometry, respectively, and the cytotoxity of HBP-HSA was further evaluated by cell viability assay. Results indicated that heparin was successfully introduced to the cell surface in a layer-by-layer way and retained for at least 24 h, while the cytotoxity of HBP-HSA was negligible at the working concentration. Accordingly, this conjugate provides a promising method for co-immobilization of heparin and HSA to the cell surface under physiological conditions with improved biocompatibility.

Analysis of the Spectroscopic Aspects of Cationic Dye Basic Orange 21.

PubMed

Eizig, Zehavit; Major, Dan T; Kasdan, Harvey L; Afrimzon, Elena; Zurgil, Naomi; Sobolev, Maria; Deutsch, Mordechai

2015-09-24

Spectroscopic properties of cationic dye basic orange 21 (BO21) in solutions, in solids, and within leukocytes were examined. Results obtained with solutions indicate that influence of variables such as pH, viscosity, salt composition, and various proteins on the absorption spectrum of BO21 is negligible. However, in the presence of heparin, a blue shift (484-465 nm) is observed, which is attributed to the aggregation of BO21 on the polyanion. Applying density functional theory demonstrates that in aqueous solutions (a) the formation of BO21 oligomers is thermodynamically favorable, they are oriented in an antiparallel dipolar arrangement, and their binding energies are lower than those of parallel dipolar arrangements, (b) association between BO21 aggregates and heparin is highly favorable, and (c) the blue shift is due to the mixing of π → π* transitions caused by BO21 molecule stacking. However, when embedded in basophils, the absorption spectra of intracellular BO21 is extremely red-shifted, with two peaks (at 505 and 550 nm) found to be attributed to BO21 and the BO21-heparin complex, respectively, which are intracellularly hosted in nonaqueous environments. Initial evidence of the ability to differentiate between leukocyte types by BO21 is presented.

Profiling analysis of low molecular weight heparins by multiple heart-cutting two dimensional chromatography with quadruple time-of-flight mass spectrometry.

PubMed

Ouyang, Yilan; Zeng, Yangyang; Rong, Yinxiu; Song, Yue; Shi, Lv; Chen, Bo; Yang, Xinlei; Xu, Naiyu; Linhardt, Robert J; Zhang, Zhenqing

2015-09-01

Low molecular weight heparins (LMWHs) are polydisperse and microheterogenous mixtures of polysaccharides used as anticoagulant drugs. Profiling analysis is important for obtaining deeper insights into the structure of LMWHs. Previous oligosaccharide mapping methods are relatively low resolution and are unable to show an entire picture of the structural complexity of LMWHs. In the current study a profiling method was developed relying on multiple heart-cutting, two-dimensional, ultrahigh performance liquid chromatography with quadruple time-of-flight mass spectrometry. This represents an efficient, automated, and robust approach for profiling LMWHs. Using size-exclusion chromatography and ion-pairing reversed-phase chromatography in a two-dimensional separation, LMW components of different sizes and LMW components of the same size but with different charges and polarities can be resolved, providing a more complete picture of a LMWH. Structural information on each component was then obtained with quadrupole time-of-flight mass spectrometry. More than 80 and 120 oligosaccharides were observed and unambiguously assigned from the LMWHs, nadroparin and enoxaparin, respectively. This method might be useful for quality control of LMWHs and as a powerful tool for heparin-related glycomics.

NMR characterization of the interaction between the C-terminal domain of interferon-γ and heparin-derived oligosaccharides

PubMed Central

Vanhaverbeke, Cécile; Simorre, Jean-Pierre; Sadir, Rabia; Gans, Pierre; Lortat-Jacob, Hugues

2004-01-01

Interferons are cytokines that play a complex role in the resistance of mammalian hosts to pathogens. IFNγ (interferon-γ) is secreted by activated T-cells and natural killer cells. IFNγ is involved in a wide range of physiological processes, including antiviral activity, immune response, cell proliferation and apoptosis, as well as the stimulation and repression of a variety of genes. IFNγ activity is modulated by the binding of its C-terminal domain to HS (heparan sulphate), a glycosaminoglycan found in the extracellular matrix and at the cell surface. In the present study, we analysed the interaction of isolated heparin-derived oligosaccharides with the C-terminal peptide of IFNγ by NMR, in aqueous solution. We observed marked changes in the chemical shifts of both peptide and oligosaccharide compared with the free state. Our results provide evidence of a binding through electrostatic interactions between the charged side chains of the protein and the sulphate groups of heparin that does not induce specific conformation of the C-terminal part of IFNγ. Our data also indicate that an oligosaccharide size of at least eight residues displays the most efficient binding. PMID:15270718

Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps.

PubMed

Hayakawa, Y; Hayashi, T; Hayashi, T; Niiya, K; Sakuragawa, N

1995-10-01

While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.

Functionalization of chitosan/poly(lactic acid-glycolic acid) sintered microsphere scaffolds via surface heparinization for bone tissue engineering.

PubMed

Jiang, Tao; Khan, Yusuf; Nair, Lakshmi S; Abdel-Fattah, Wafa I; Laurencin, Cato T

2010-06-01

Scaffolds exhibiting biological recognition and specificity play an important role in tissue engineering and regenerative medicine. The bioactivity of scaffolds in turn influences, directs, or manipulates cellular responses. In this study, chitosan/poly(lactic acid-co-glycolic acid) (chitosan/PLAGA) sintered microsphere scaffolds were functionalized via heparin immobilization. Heparin was successfully immobilized on chitosan/PLAGA scaffolds with controllable loading efficiency. Mechanical testing showed that heparinization of chitosan/PLAGA scaffolds did not significantly alter the mechanical properties and porous structures. In addition, the heparinized chitosan/PLAGA scaffolds possessed a compressive modulus of 403.98 +/- 19.53 MPa and a compressive strength of 9.83 +/- 0.94 MPa, which are in the range of human trabecular bone. Furthermore, the heparinized chitosan/PLAGA scaffolds had an interconnected porous structure with a total pore volume of 30.93 +/- 0.90% and a median pore size of 172.33 +/- 5.89 mum. The effect of immobilized heparin on osteoblast-like MC3T3-E1 cell growth was investigated. MC3T3-E1 cells proliferated three dimensionally throughout the porous structure of the scaffolds. Heparinized chitosan/PLAGA scaffolds with low heparin loading (1.7 microg/scaffold) were shown to be capable of stimulating MC3T3-E1 cell proliferation by MTS assay and cell differentiation as evidenced by elevated osteocalcin expression when compared with nonheparinized chitosan/PLAGA scaffold and chitosan/PLAGA scaffold with high heparin loading (14.1 microg/scaffold). This study demonstrated the potential of functionalizing chitosan/PLAGA scaffolds via heparinization with improved cell functions for bone tissue engineering applications.

Sodium citrate 4% versus heparin as a lock solution in hemodialysis patients with central venous catheters.

PubMed

Yon, Calantha K; Low, Chai L

2013-01-15

The effects of heparin versus sodium citrate 4% as a lock solution on catheter-related infections (CRIs), catheter patency, and hospitalizations in long-term hemodialysis patients with central venous catheters (CVCs) were compared. Data for patients receiving heparin lock solutions were collected from July 2008 to July 2009. Data on patients receiving sodium citrate 4% lock solution were collected from September 2009 through December 2010. Patients who were receiving the heparin lock solution who continued to have a CVC in September 2009 were transitioned from heparin to sodium citrate catheter 4% lock solution. New patients with CVCs placed after September 2009 received sodium citrate 4% without a period of using heparin lock solution. Pertinent information on patient medical history, bleeding or clotting events, infections, and hospitalization was collected. Data were collected retrospectively for the heparin group and prospectively for the sodium citrate group. Data were collected from 360 patient-months among 60 patients during the heparin treatment period and from 451 patient-months among 58 patients during the sodium citrate period. Thirty-three patients were common to both study groups. There were significantly more CRIs and CRIs per 1000 catheter-days in the heparin than the sodium citrate treatment group. Secondary outcomes of hospitalizations and catheter thrombosis were comparable. CRIs and thrombosis led to significantly more catheter exchanges or removals in the heparin group than the sodium citrate group. In patients with long-term hemodialysis catheters, a lock solution of sodium citrate 4% was associated with fewer CRIs and similar effectiveness when compared with heparin 5000 units/mL.

Insulin and heparin co-immobilized 3D polyester fabrics for the cultivation of fibroblasts in low-serum media.

PubMed

Türkoğlu Saşmazel, Hilal; Aday, Sezin; Gümüşderelioğlu, Menemşe

2007-08-01

Insulin and/or heparin immobilized/co-immobilized non-woven polyester fabric (NWPF) discs were developed for the cultivation of L929 mouse fibroblasts in low-serum media. At first, NWPF discs were hydrolyzed to obtain a carboxylic acid group-introduced matrix (NWPF-hydrolyzed). Insulin and heparin co-immobilized NWPF (NWPF-insulin-heparin) was prepared by the grafting of PEO onto NWPF-hydrolyzed disc (NWPF-PEO), followed by the reaction first with insulin and then heparin. In the presence of spacer arm, PEO, the amount of immobilized insulin molecules significantly increased from 6.96 to 84.45 microg/cm(2). The amount of heparin bound to the NWPF-PEO (5.93 microg/cm(2)) was higher than that of the insulin immobilized surface (4.59 microg/cm(2)). Insulin and heparin immobilized NWPF discs were observed with fluorescence microscopy by labeling the insulin and heparin with 8-anilino-1-naphthalene sulfonic acid (ANS) or fluorescein isothiocyanate (FITC), respectively. L929 fibroblasts were used to check the cell adhesion and cell growth capabilities of modified NWPF discs in low-serum media (containing 5% fetal bovine serum). Optical photographs showed that after 2nd day of the culture, fibroblastic cells spread along the length of modified fibers, eventually filling the interfiber space. At the end of 6-day growth period, cell yield in the presence of immobilized heparin was a little bit higher than that of the immobilized insulin. Co-immobilized (insulin/heparin) NWPF discs did not accelerate the cell growth as well as insulin or heparin immobilized discs.

Basic Residues of β-Sheet A Contribute to Heparin Binding and Activation of Vaspin (Serpin A12).

PubMed

Ulbricht, David; Oertwig, Kathrin; Arnsburg, Kristin; Saalbach, Anja; Pippel, Jan; Sträter, Norbert; Heiker, John T

2017-01-20

Many members of the serine protease inhibitor (serpin) family are activated by glycosaminoglycans (GAGs). Visceral adipose tissue-derived serpin (vaspin), serpin A12 of the serpin family, and its target protease kallikrein 7 (KLK7) are heparin-binding proteins, and inhibition of KLK7 by vaspin is accelerated by heparin. However, the nature of GAG binding to vaspin is not known. Here, we measured vaspin binding of various glycosaminoglycans and low molecular weight heparins by microscale thermophoresis and analyzed acceleration of protease inhibition by these molecules. In addition, basic residues contributing to heparin binding and heparin activation were identified by a selective labeling approach. Together, these data show that vaspin binds heparin with high affinity (K D = 21 ± 2 nm) and that binding takes place at a basic patch on top of β-sheet A and is different from other heparin-binding serpins. Mutation of basic residues decreased heparin binding and activation of vaspin. Similarly, reactive center loop insertion into sheet A decreased heparin binding because it disturbs the basic cluster. Finally, using vaspin-overexpressing keratinocyte cells, we show that a significant part of secreted vaspin is bound in the extracellular matrix on the cell surface. Together, basic residues of central β-sheet A contribute to heparin binding and activation of vaspin. Thus, binding to GAGs in the extracellular matrix can direct and regulate vaspin interaction with target proteases or other proteins and may play an important role in the various beneficial functions of vaspin in different tissues. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Heparin-functionalized polymeric biomaterials in tissue engineering and drug delivery applications

PubMed Central

Liang, Yingkai; Kiick, Kristi L.

2014-01-01

Heparin plays an important role in many biological processes, via its interaction with various proteins, and hydrogels and nanoparticles comprising heparin exhibit attractive properties such as anticoagulant activity, growth factor binding, as well as antiangiogenic and apoptotic effects, making them great candidates for emerging applications. Accordingly, this review summarizes recent efforts in the preparation of heparin-based hydrogels and formation of nanoparticles, as well as the characterization of their properties and applications. The challenges and future perspectives for heparin-based materials are also discussed. Prospects are promising for heparin-containing polymeric biomaterials in diverse applications ranging from cell carriers for promoting cell differentiation to nanoparticle therapeutics for cancer treatment. PMID:23911941

Heparin functionalization increases retention of TGF-β2 and GDF5 on biphasic silk fibroin scaffolds for tendon/ligament-to-bone tissue engineering.

PubMed

Font Tellado, Sònia; Chiera, Silvia; Bonani, Walter; Poh, Patrina S P; Migliaresi, Claudio; Motta, Antonella; Balmayor, Elizabeth R; van Griensven, Martijn

2018-05-01

The tendon/ligament-to-bone transition (enthesis) is a highly specialized interphase tissue with structural gradients of extracellular matrix composition, collagen molecule alignment and mineralization. These structural features are essential for enthesis function, but are often not regenerated after injury. Tissue engineering is a promising strategy for enthesis repair. Engineering of complex tissue interphases such as the enthesis is likely to require a combination of biophysical, biological and chemical cues to achieve functional tissue regeneration. In this study, we cultured human primary adipose-derived mesenchymal stem cells (AdMCs) on biphasic silk fibroin scaffolds with integrated anisotropic (tendon/ligament-like) and isotropic (bone/cartilage like) pore alignment. We functionalized those scaffolds with heparin and explored their ability to deliver transforming growth factor β2 (TGF-β2) and growth/differentiation factor 5 (GDF5). Heparin functionalization increased the amount of TGF-β2 and GDF5 remaining attached to the scaffold matrix and resulted in biological effects at low growth factor doses. We analyzed the combined impact of pore alignment and growth factors on AdMSCs. TGF-β2 and pore anisotropy synergistically increased the expression of tendon/ligament markers and collagen I protein content. In addition, the combined delivery of TGF-β2 and GDF5 enhanced the expression of cartilage markers and collagen II protein content on substrates with isotropic porosity, whereas enthesis markers were enhanced in areas of mixed anisotropic/isotropic porosity. Altogether, the data obtained in this study improves current understanding on the combined effects of biological and structural cues on stem cell fate and presents a promising strategy for tendon/ligament-to-bone regeneration. Regeneration of the tendon/ligament-to-bone interphase (enthesis) is of significance in the repair of ruptured tendons/ligaments to bone to improve implant integration and clinical outcome. This study proposes a novel approach for enthesis regeneration based on a biomimetic and integrated tendon/ligament-to-bone construct, stem cells and heparin-based delivery of growth factors. We show that heparin can keep growth factors local and biologically active at low doses, which is critical to avoid supraphysiological doses and associated side effects. In addition, we identify synergistic effects of biological (growth factors) and structural (pore alignment) cues on stem cells. These results improve current understanding on the combined impact of biological and structural cues on the multi-lineage differentiation capacity of stem cells for regenerating complex tissue interphases. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Increase in the growth inhibition of bovine pulmonary artery smooth muscle cells by an O-hexanoyl low-molecular-weight heparin derivative.

PubMed

Garg, Hari G; Hales, Charles A; Yu, Lunyin; Butler, Melissa; Islam, Tasneem; Xie, Jin; Linhardt, Robert J

2006-11-06

Proliferation of pulmonary artery smooth muscle cells (PASMCs) appears to play a significant role in chronic pulmonary hypertension. The proliferation of PASMCs is strongly inhibited by some commercial heparin preparations. Heparin fragments were prepared by periodate treatment, followed by sodium borohydride reduction, to enhance potency. The tributylammonium salt of this fragmented heparin was O-acylated with hexanoic anhydride. Gradient polyacrylamide gel electrophoresis showed that the major heparin fragment contained eight disaccharide units. NMR analysis showed that approximately one hexanoyl group per disaccharide residue was present. The O-hexanoyl heparin fragments were assayed for growth inhibitory effect on bovine PASMCs in culture. This derivative was found to be more effective in growth inhibition of bovine PASMCs in culture than the heparin from which it was derived. In the future, it is envisioned that this or similar derivatives may be an effective treatment for pulmonary hypertension.

Electrophoresis for the analysis of heparin purity and quality.

PubMed

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J

2012-06-01

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of Low-Molecular-Weight Heparins Prepared From Bovine Heparins With Enoxaparin.

PubMed

Liu, Xinyue; St Ange, Kalib; Fareed, Jawed; Hoppensteadt, Debra; Jeske, Walter; Kouta, Ahmed; Chi, Lianli; Jin, Caijuan; Jin, Yongsheng; Yao, Yiming; Linhardt, Robert J

2017-09-01

Heparin and its low-molecular-weight heparin (LMWH) derivatives are widely used clinical anticoagulants. These drugs are critical for the practice of medicine in applications including kidney dialysis, cardiopulmonary bypass, and in the management of venous thromboembolism. Currently, these drugs are derived from livestock, primarily porcine intestine. The worldwide dependence on a single animal species has made the supply chain for this critical drug quite fragile, leading to the search for other sources of these drugs, including bovine tissues such as bovine intestine or lung. A number of laboratories are currently examining the similarities and differences between heparins prepared from porcine and bovine tissues. The current study is designed to compare LMWH prepared from bovine heparins through chemical β-elimination, a process currently used to prepare the LMWH, enoxaparin, from porcine heparin. Using top-down, bottom-up, compositional analysis and bioassays, LMWHs, derived from bovine lung and intestine, are shown to closely resemble enoxaparin.

Electrophoresis for the analysis of heparin purity and quality

PubMed Central

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J.

2012-01-01

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. PMID:22736353

PF4/heparin antibody testing and treatment of heparin-induced thrombocytopenia in the intensive care unit.

PubMed

Wanat, Matthew; Fitousis, Kalliopi; Hall, Jeff; Rice, Lawrence

2013-06-01

The diagnosis of heparin-induced thrombocytopenia (HIT) may be challenging in critically ill patients, as heparin exposures are ubiquitous, and thrombocytopenia is common. Unwarranted ordering and incorrect interpretation of heparin antibody tests can expose a patient to adverse drug events and imposes a significant economic burden on our health care system. A prospective, observational study was performed over 4 months on all adult patients located in 5 intensive care units, with a heparin antibody test ordered. A platelet factor 4/heparin enzyme-linked immunosorbent assay (ELISA) test was ordered in 131 patients. In total, 110 patients had a low 4Ts score (0-3), and of these 103 had a negative ELISA result. In patients with a low 4Ts score, 0 (0%) of 110 had an optical density value >1.0. One hundred twenty-nine patients (98%) had another possible cause of thrombocytopenia identified. In critically ill patients, low 4Ts scores indicate a low probability of HIT, and heparin antibody testing in these patients is not useful.

Anticoagulant effects of inhaled unfractionated heparin in the dog as determined by partial thromboplastin time and factor Xa activity.

PubMed

Manion, Jill S; Thomason, John M; Langston, Vernon C; Claude, Andrew K; Brooks, Marjory B; Mackin, Andrew J; Lunsford, Kari V

2016-01-01

To evaluate the anticoagulant effects of inhaled heparin in dogs. This study was conducted in 3 phases. In phase 1, bronchoalveolar lavage fluid (BALf) was collected to generate an in vitro calibration curve to relate heparin concentration to the activated partial thromboplastin time (aPTT). In phase 2, heparin was administered via nebulization to determine the threshold dose needed to prolong systemic aPTT. In phase 3, the local anticoagulant activity of inhaled heparin was determined by measurement of BALf anti-Xa activity and aPTT. University teaching hospital. Six healthy intact female Walker Hounds were used in this study. Two dogs were used for each phase. Inhaled unfractionated sodium heparin was administered in doses ranging from 50,000 to 200,000 IU. In vitro addition of heparin to BALf caused a prolongation in aPTT. Inhaled heparin at doses as high as 200,000 IU failed to prolong systemic aPTT, and a threshold dose could not be determined. No significant local anticoagulant effects were detected. Even at doses higher than those known to be effective in people, inhaled heparin appears to have no detectable local or systemic anticoagulant effects in dogs with the current delivery method. © Veterinary Emergency and Critical Care Society 2015.

The non-anticoagulant heparin-like K5 polysaccharide derivative K5-N,OSepi attenuates myocardial ischaemia/reperfusion injury

PubMed Central

Collino, Massimo; Pini, Alessandro; Mastroianni, Rosanna; Benetti, Elisa; Lanzi, Cecilia; Bani, Daniele; Chini, Jacopo; Manoni, Marco; Fantozzi, Roberto; Masini, Emanuela

2012-01-01

Heparin and low molecular weight heparins have been demonstrated to reduce myocardial ischaemia/reperfusion (I/R) injury, although their use is hampered by the risk of haemorrhagic and thrombotic complications. Chemical and enzymatic modifications of K5 polysaccharide have shown the possibility of producing heparin-like compounds with low anticoagulant activity and strong anti-inflammatory effects. Using a rat model of regional myocardial I/R, we investigated the effects of an epimerized N-,O-sulphated K5 polysaccharide derivative, K5-N,OSepi, on infarct size and histological signs of myocardial injury caused by 30 min. ligature of the left anterior descending coronary artery followed by 1 or 24 h reperfusion. K5-N,OSepi (0.1–1 mg/kg given i.v. 15 min. before reperfusion) significantly reduced the extent of myocardial damage in a dose-dependent manner. Furthermore, we investigated the potential mechanism(s) of the cardioprotective effect(s) afforded by K5-N,OSepi. In left ventricular samples, I/R induced mast cell degranulation and a robust increase in lipid peroxidation, free radical-induced DNA damage and calcium overload. Markers of neutrophil infiltration and activation were also induced by I/R in rat hearts, specifically myeloperoxidase activity, intercellular-adhesion-molecule-1 expression, prostaglandin-E2 and tumour-necrosis-factor-α production. The robust increase in oxidative stress and inflammatory markers was blunted by K5-N,OSepi, in a dose-dependent manner, with maximum at 1 mg/kg. Furthermore, K5-N,OSepi administration attenuated the increase in caspase 3 activity, Bid and Bax activation and ameliorated the decrease in expression of Bcl-2 within the ischaemic myocardium. In conclusion, we demonstrate that the cardioprotective effect of the non-anticoagulant K5 derivative K5-N,OSepi is secondary to a combination of anti-apoptotic and anti-inflammatory effects. PMID:22248092

Nebulized heparin and N-acetylcysteine for smoke inhalational injury: A case report.

PubMed

Ashraf, Umair; Bajantri, Bharat; Roa-Gomez, Gabriella; Venkatram, Sindhaghatta; Cantin, Amanda; Diaz-Fuentes, Gilda

2018-05-01

Every year, ∼40,000 people suffer burn-related injuries in the United States. Despite recent advances, the odds of dying from exposure to fire, flames, or smoke are one in ∼1500. Smoke inhalation causes injury to the airways via a complex physiological process, and the treatment is mainly supportive. Many recent interventions aim to decrease the formation of fibrin casts, the main cause of airway damage in these patients. Among these, treatment with a combination of nebulized heparin and N-acetylcysteine (NAC) has shown benefit. We describe the case of a 58-year-old man who presented after smoke inhalation during a fire. Soot was found in the nostrils when he was admitted to our hospital, and after he began coughing up carbonaceous material, he was electively intubated and placed on volume assist control ventilation. Bronchoscopy on the first day of intensive care confirmed the injury from smoke inhalation and revealed mucosal edema and soot involving the tracheobronchial tree. Inhaled unfractionated heparin of 10,000 IU in 3 mL of 0.9% normal saline alternating every 2 hours with 3 mL of 20% NAC was started 48 hours after admission and continued for 7 days. Bronchoscopy on the fifth day of intensive care showed significant improvement in airway edema and a resolution of soot. On the basis of our experience with this case and limited literature, we posit that nebulized heparin and NAC may be of benefit in patients with inhalational smoke-induced lung injury and mild-to-severe lung injury scores.

An international survey of current practice in the laboratory assessment of anticoagulant therapy with heparin.

PubMed

Favaloro, Emmanuel J; Bonar, Roslyn; Sioufi, John; Wheeler, Michael; Low, Joyce; Aboud, Margaret; Lloyd, John; Street, Alison; Marsden, Katherine

2005-06-01

We conducted a survey of laboratory practice for assessment of heparin anticoagulant therapy by participants of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP). A questionnaire was sent to 646 laboratories enrolled in the Haematology component of the QAP, requesting details of tests used for monitoring heparin therapy. Seventy laboratories (10.8%) returned results that indicated that they performed laboratory monitoring of heparin therapy. Most laboratories (69/70 = 98.6%) use the activated partial thromboplastin time (APTT) to monitor unfractionated heparin, with eight (11.4%) also using the APTT for monitoring low molecular weight (LMW) heparin. Five (7.1%) laboratories use the thrombin time (TT) test to help monitor heparin therapy and 37 (52.9%) laboratories use an anti-Xa assay to monitor heparin (either LMW or unfractionated). Normal reference ranges (NRR) for APTT differed considerably between laboratories, even those using the same reagent. Therapeutic ranges (TR) also differed considerably between laboratories, for both APTT and the anti-Xa assay. Laboratory differences in NRR and TR using the same reagents could only be partly explained by the use of different instrumentation. There is a large variation in current laboratory practice relating to monitoring of heparin anticoagulant therapy. This finding is similar to that of a similar survey conducted by the RCPA QAP almost a decade ago. This study suggests that better standardisation is still required for laboratory monitoring of heparin therapy.

Molecular Weights of Bovine and Porcine Heparin Samples: Comparison of Chromatographic Methods and Results of a Collaborative Survey.

PubMed

Bertini, Sabrina; Risi, Giulia; Guerrini, Marco; Carrick, Kevin; Szajek, Anita Y; Mulloy, Barbara

2017-07-19

In a collaborative study involving six laboratories in the USA, Europe, and India the molecular weight distributions of a panel of heparin sodium samples were determined, in order to compare heparin sodium of bovine intestinal origin with that of bovine lung and porcine intestinal origin. Porcine samples met the current criteria as laid out in the USP Heparin Sodium monograph. Bovine lung heparin samples had consistently lower average molecular weights. Bovine intestinal heparin was variable in molecular weight; some samples fell below the USP limits, some fell within these limits and others fell above the upper limits. These data will inform the establishment of pharmacopeial acceptance criteria for heparin sodium derived from bovine intestinal mucosa. The method for MW determination as described in the USP monograph uses a single, broad standard calibrant to characterize the chromatographic profile of heparin sodium on high-resolution silica-based GPC columns. These columns may be short-lived in some laboratories. Using the panel of samples described above, methods based on the use of robust polymer-based columns have been developed. In addition to the use of the USP's broad standard calibrant for heparin sodium with these columns, a set of conditions have been devised that allow light-scattering detected molecular weight characterization of heparin sodium, giving results that agree well with the monograph method. These findings may facilitate the validation of variant chromatographic methods with some practical advantages over the USP monograph method.

In vivo antithrombotic properties of a heparin from the oocyte test cells of the sea squirt Styela plicata(Chordata-Tunicata).

PubMed

Cardilo-Reis, L; Cavalcante, M C M; Silveira, C B M; Pavão, M S G

2006-11-01

In the ascidian Styela plicata, the oocytes are surrounded by two types of accessory cells named follicle cells and test cells. A heparin-like substance with an anticoagulant activity equivalent to 10% of mammalian heparin and about 5% as potent as the mammalian counterpart for the inhibition of thrombin by antithrombin was isolated from the oocyte test cells. In the present study, we compared the antithrombotic and hemorrhagic effects of sea squirt oocyte test cell heparin with those of porcine heparin in rat models of venous thrombosis and blood loss. Intravenous administration of the oocyte test cell heparin to Wistar rats (both sexes, weighing approximately 300 g, N = 4 in each group) at a dose of 5.0 mg/kg body weight, which produced a 1.8-fold increase in plasma activated partial thromboplastin time, inhibited thrombosis by 45 +/- 13.5% (mean +/- SD) without any bleeding effect. The same dose of porcine heparin inhibited thrombosis by 100 +/- 1.4%, but produced a blood loss three times greater than that of the saline-treated control. However, 10-fold reduction of the dose of porcine heparin to 0.5 mg/kg body weight, which produced a 5-fold increase in plasma-activated partial thromboplastin time, inhibited thrombosis by 70 +/- 13% without any bleeding effect. The antithrombotic properties of a new heparin isolated from test cells of the sea squirt S. plicata, reported here for the first time, indicate that, although sea squirt oocyte test cell heparin was a poor anticoagulant compared to porcine heparin, it had a significant antithrombotic effect without causing bleeding.

Heparin and structurally related polymers attenuate eotaxin-1 (CCL11) release from human airway smooth muscle.

PubMed

Kanabar, V; Page, C P; Simcock, D E; Karner, C; Mahn, K; O'Connor, B J; Hirst, S J

2008-06-01

The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

PubMed Central

Zhao, Wenjing; McCallum, Scott A.; Xiao, Zhongping; Zhang, Fuming; Linhardt, Robert J.

2011-01-01

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs. PMID:21658003

Synthetic heparin-binding factor analogs

DOEpatents

Pena, Louis A [Poquott, NY; Zamora, Paul O [Gaithersburg, MD; Lin, Xinhua [Plainview, NY; Glass, John D [Shoreham, NY

2010-04-20

The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Neutralization of heparin activity.

PubMed

Pai, Menaka; Crowther, Mark A

2012-01-01

Heparin is the mainstay in the treatment and prevention of thrombosis in such diverse clinical settings as venous thromboembolism, acute coronary syndrome, cardiopulmonary bypass, and hemodialysis. However, the major complication of heparin - like that of all anticoagulants - is bleeding. Heparin may need to be reversed in the following settings: clinically significant bleeding; prior to an invasive procedure; at the conclusion of a procedure involving extracorporeal circulation (e.g., cardiopulmonary bypass, dialysis). This chapter discusses protamine sulfate, as well as several other agents that are able to neutralize heparin, including their pharmacological properties, indications, dosing, and efficacy.

The localisation of the heparin binding sites of human and murine interleukin-12 within the carboxyterminal domain of the P40 subunit.

PubMed

Garnier, Pascale; Mummery, Rosemary; Forster, Mark J; Mulloy, Barbara; Gibbs, Roslyn V; Rider, Christopher C

2018-05-09

We have previously shown that the heterodimeric cytokine interleukin-12, and the homodimer of its larger subunit p40, both bind to heparin and heparan sulfate with relatively high affinity. In the present study we characterised these interactions using a series of chemically modified heparins as competitive inhibitors. Human interleukin-12 and p40 homodimer show indistinguishable binding profiles with a panel of heparin derivatives, but that of murine interleukin-12 is distinct. Heparin markedly protects the human and murine p40 subunits, but not the p35 subunits, from cleavage by the bacterial endoprotease LysC, further implicating the larger subunit as the location of the heparin binding site. Moreover the essential role of the carboxyterminal D3 domain in heparin binding is established by the failure of a truncated construct of the p40 subunit lacking this domain to bind. Predictive docking calculations indicate that a cluster of basic residues at the tip of the exposed C'D' loop within D3 is important in heparin binding. However since the human and murine C'D' loops differ considerably in length, the mode and three dimensional orientation of heparin binding are likely to differ substantially between the human and murine p40s. Thus overall the binding of IL-12 via its p40 subunit to heparin-related polysaccharides of the extracellular matrix appears to be functionally important since it has been conserved across mammalian species despite this structural divergence. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

The anticoagulant effect of heparin during radiofrequency ablation (RFA) in patients taking apixaban or rivaroxaban.

PubMed

Brendel, L C; Dobler, F; Hessling, G; Michel, J; Braun, S L; Steinsiek, A L; Groha, P; Eckl, R; Deisenhofer, I; Hyseni, A; Roest, M; Ott, I; Steppich, B

2017-09-01

Measuring the anticoagulant effect of heparin during radiofrequency ablation (RFA) in patients taking apixaban and rivaroxaban is challenging, since the activated coagulation time (ACT) does not seem to reflect the true anticoagulant activity of these drugs. We therefore evaluated coagulation properties of apixaban and rivaroxaban during RFA by different coagulation assays to better monitor periprocedural hemostasis. The study included 90 patients (61 ± 12 years) with atrial fibrillation who underwent RFA procedures. Patients received 20 mg rivaroxaban (n = 73) once or 5 mg apixaban (n = 17) twice daily 4 weeks prior to the procedure. During RFA, unfractionated heparin i.v. was given to maintain an ACT of 250-300 s. Blood samples were taken before and 10, 60, and 360 min after heparin administration. Heparin displayed a lower anti-Xa activity in rivaroxaban-treated patients compared to apixaban-treated patients. In contrast, D-dimer and prothrombin fragment F1+2 plasma levels indicated a higher activation of the coagulation cascade in apixaban/heparin than in rivaroxaban/heparin patients. This discordant coagulative state measured in vitro had no clinical impact in terms of bleeding or thromboembolic complications. We found different biochemical responses to rivaroxaban/heparin and apixaban/heparin during RFA. Precaution is necessary when monitoring periprocedural hemostasis in DOAC patients to avoid mismanagement.

LMW Heparin Prevents Increased Kidney Expression of Proinflammatory Mediators in (NZBxNZW)F1 Mice

PubMed Central

Kanapathippillai, Premasany; Rekvig, Ole Petter; Fenton, Kristin Andreassen

2013-01-01

We have previously demonstrated that continuous infusion of low molecular weight (LMW) heparin delays autoantibody production and development of lupus nephritis in (NZBxNZW)F1 (B/W) mice. In this study we investigated the effect of LMW heparin on renal cytokine and chemokine expression and on nucleosome-mediated activation of nucleosome-specific splenocytes. Total mRNA extracted from kidneys of heparin-treated or -untreated B/W mice was analysed by qPCR for the expression of several cytokines, chemokines, and Toll-like receptors. Splenocytes taken from B/W mice were stimulated with nucleosomes with or without the presence of heparin. Splenocyte cell proliferation as thymidine incorporation and the expression of costimulatory molecules and cell activation markers were measured. Heparin treatment of B/W mice reduced the in vivo expression of CCR2, IL1β, and TLR7 compared to untreated B/W mice. Nucleosome-induced cell proliferation of splenocytes was not influenced by heparin. The expression of CD80, CD86, CD69, CD25, CTLA-4, and TLR 2, 7, 8, and 9 was upregulated upon stimulation by nucleosomes, irrespective of whether heparin was added to the cell culture or not. In conclusion, treatment with heparin lowers the kidney expression of proinflammatory mediators in B/W mice but does not affect nucleosomal activation of splenocytes. PMID:24151519

Platelet count recovery and seroreversion in immune HIT despite continuation of heparin: further observations and literature review.

PubMed

Shih, Andrew W; Sheppard, Jo-Ann I; Warkentin, Theodore E

2017-10-05

One of the standard distinctions between type 1 (non-immune) and type 2 (immune-mediated) heparin-induced thrombocytopenia (HIT) is the transience of thrombocytopenia: type 1 HIT is viewed as early-onset and transient thrombocytopenia, with platelet count recovery despite continuing heparin administration. In contrast, type 2 HIT is viewed as later-onset (i. e., 5 days or later) thrombocytopenia in which it is generally believed that platelet count recovery will not occur unless heparin is discontinued. However, older reports of type 2 HIT sometimes did include the unexpected observation that platelet counts could recover despite continued heparin administration, although without information provided regarding changes in HIT antibody levels in association with platelet count recovery. In recent years, some reports of type 2 HIT have confirmed the observation that platelet count recovery can occur despite continuing heparin administration, with serological evidence of waning levels of HIT antibodies ("seroreversion"). We now report two additional patient cases of type 2 HIT with platelet count recovery despite ongoing therapeutic-dose (1 case) or prophylactic-dose (1 case) heparin administration, in which we demonstrate concomitant waning of HIT antibody levels. We further review the literature describing this phenomenon of HIT antibody seroreversion and platelet count recovery despite continuing heparin administration. Our observations add to the concept that HIT represents a remarkably transient immune response, including sometimes even when heparin is continued.

Heparin induced alterations in clearance and distribution of blood-borne microparticles following operative trauma.

PubMed

Saba, T M; Antikatzides, T G

1979-04-01

The influence of systemic heparin administration on the vascular clearance and tissue distribution of blood-borne microparticles was evaluated in normal rats and rats after operation (laparotomy plus intestinal manipulation) utilizing an (131)I- colloid which is phagocytized by the reticuloendothelial system (RES). Intravenous heparin administration (100 USP/100g body weight) into normal animals three minutes prior to colloid injection (50 mg/lOOg) induced a significant increase in pulmonary localization of the microparticles as compared to nonheparinized control rats, while hepatic and splenic uptake were decreased. Surgical trauma decreased hepatic RE uptake and increased pulmonary localization of the microparticles when injected systemically at 60 minutes postsurgery. Heparin administration 60 minutes after surgery and three minutes prior to colloid injection, magnified the increased pulmonary localization response with an associated further depression of the RES. The ability of heparin to alter both RE clearance function and lung localization of microparticles was dose dependent and a function of the interval between heparin administration and systemic particulate infusion. Thus, low dose heparin administration was capable of stimulating RE activity while heparin in doses of excess of 50 USP units/lOOg body weight decreased RE function. These findings suggest that the functional state of the hepatic RE system can be greatly affected in a dose-dependent manner by systemic heparin administration which may influence distribution of blood-borne microparticles.

Heparin pharmacovigilance in Brazil.

PubMed

Junqueira, Daniela Rezende Garcia; Viana, Thércia Guedes; Peixoto, Eliane R de M; Barros, Fabiana C R de; Carvalho, Maria das Graças; Perini, Edson

2011-01-01

To investigate the biological origin of injectable unfractioned heparin available in Brazilian market by discussing the impact of the profile of commercial products and the changes in heparin monograph on the drug safety. The Anvisa data base for the Registered Products of Pharmaceutical Companies and the Dictionary of Pharmaceutical Specialties (DEF 2008/2009) were searched. A survey with industries having an active permission for marketing the drug in Brazil was conducted. Five companies were granted a permission to market unfractioned heparin in Brazil. Three of them are porcine in origin and two of them are bovine in origin, with only one explicitly showing this information in the package insert. The effectiveness and safety of heparin studied in non-Brazilian populations may not represent the Brazilian reality, since most countries no longer produce bovine heparin. The currently marketed heparin has approximately 10% less anticoagulant activity than that previously produced and this change may have clinical implications. Evidence about the lack of dose interchangeability between bovine and porcine heparins and the unique safety profile of these drugs indicates the need to follow the treatment and the patients' response. Events threatening the patient's safety must be reported to the pharmacovigilance system in each particular country.

A photoacoustic tool for therapeutic drug monitoring of heparin (Conference Presentation)

NASA Astrophysics Data System (ADS)

Wang, Junxin; Hartanto, James; Jokerst, Jesse V.

2017-03-01

Heparin is used broadly in cardiac, pulmonary, surgical, and vascular medicine to treat thrombotic disorders with over 500 million doses per year globally. Despite this widespread use, it has a narrow therapeutic window and is one of the top three medication errors. The active partial thromboplastin time (PTT) monitors heparin, but this blood test suffers from long turnaround times, a variable reference range, and limited utility with low molecular weight heparin. Here, we describe an imaging technique that can monitor heparin concentration and activity in real time using photoacoustic spectroscopy via methylene blue as a simple and Federal Drug Agency-approved contrast agent. We found a strong correlation between heparin concentration and photoacoustic signal measured in phosphate buffered saline (PBS) and blood (R2>0.90). Clinically relevant concentrations were detected in blood with a heparin detection limit of 0.28 U/mL and a low molecular weight heparin (enoxaparin) detection limit of 72 μg/mL. We validated this imaging approach by correlation to the PTT (Pearson's r = 0.86; p<0.05) as well as with protamine sulfate treatment. To the best of our knowledge, this is the first report to use imaging data to monitor anticoagulation.

Platelet factor 4/heparin antibodies in blood bank donors.

PubMed

Hursting, Marcie J; Pai, Poulomi J; McCracken, Julianna E; Hwang, Fred; Suvarna, Shayela; Lokhnygina, Yuliya; Bandarenko, Nicholas; Arepally, Gowthami M

2010-11-01

Platelet factor 4 (PF4)/heparin antibody, typically associated with heparin therapy, is reported in some heparin-naive people. Seroprevalence in the general population, however, remains unclear. We prospectively evaluated PF4/heparin antibody in approximately 4,000 blood bank donors using a commercial enzyme-linked immunosorbent assay for initial and then repeated (confirmatory) testing. Antibody was detected initially in 249 (6.6%; 95% confidence interval [CI], 5.8%-7.4%) of 3,795 donors and repeatedly in 163 (4.3%; 95% CI, 3.7%-5.0%) of 3,789 evaluable donors. "Unconfirmed" positives were mostly (93%) low positives (optical density [OD] = 0.40-0.59). Of 163 repeatedly positive samples, 116 (71.2%) were low positives, and 124 (76.1%) exhibited heparin-dependent binding. Predominant isotypes of intermediate to high seropositive samples (OD >0.6) were IgG (20/39 [51%]), IgM (9/39 [23%]), and indeterminate (10/39 [26%]). The marked background seroprevalence of PF4/heparin antibody (4.3%-6.6%) with the preponderance of low (and frequently nonreproducible) positives in blood donors suggests the need for further assay calibration, categorization of antibody level, and studies evaluating clinical relevance of "naturally occurring" PF4/heparin antibodies.

Controllable production of low molecular weight heparins by combinations of heparinase I/II/III.

PubMed

Wu, Jingjun; Zhang, Chong; Mei, Xiang; Li, Ye; Xing, Xin-Hui

2014-01-30

Enzymatic depolymerization of heparin by heparinases is promising for production of low molecular weight heparins (LMWHs) as anticoagulants, due to its mild reaction conditions and high selectivity. Here, different heparinase combinations were used to depolymerize heparin. Heparinase I and heparinase II can depolymerize heparin more efficiently than heparinase III, respectively, but heparinase III was the best able to protect the anticoagulant activities of LMWHs. Heparinase III and heparinase I/II combinations were able to efficiently depolymerize heparin to LMWHs with higher anticoagulant activity than the LMWHs produced by the respective heparinase I and heparinase II. HepIII and HepI is the best combination for maintaining high anti-IIa activity (75.7 ± 4.21 IU/mg) at the same Mw value. Furthermore, considering both the changes in molecular weight and anticoagulant activity, the action patterns of heparinase I and heparinase II were found not to follow the exolytic and processive depolymerizing mechanism from the reducing end of heparin. Copyright © 2013 Elsevier Ltd. All rights reserved.

Authentication of animal origin of heparin and low molecular weight heparin including ovine, porcine and bovine species using 1D NMR spectroscopy and chemometric tools.

PubMed

Monakhova, Yulia B; Diehl, Bernd W K; Fareed, Jawed

2018-02-05

High resolution (600MHz) nuclear magnetic resonance (NMR) spectroscopy is used to distinguish heparin and low-molecular weight heparins (LMWHs) produced from porcine, bovine and ovine mucosal tissues as well as their blends. For multivariate analysis several statistical methods such as principal component analysis (PCA), factor discriminant analysis (FDA), partial least squares - discriminant analysis (PLS-DA), linear discriminant analysis (LDA) were utilized for the modeling of NMR data of more than 100 authentic samples. Heparin and LMWH samples from the independent test set (n=15) were 100% correctly classified according to its animal origin. Moreover, by using 1 H NMR coupled with chemometrics and several batches of bovine heparins from two producers were differentiated. Thus, NMR spectroscopy combined with chemometrics is an efficient tool for simultaneous identification of animal origin and process based manufacturing difference in heparin products. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Young-Hyun; Garron, Marie-Line; Kim, Hye-Yeon

2010-01-12

Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a {beta}-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with anmore » ({alpha}/{alpha}){sub 6} fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity.« less

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2012-04-24

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

Dual chain synthetic heparin-binding growth factor analogs

DOEpatents

Zamora, Paul O [Gaithersburg, MD; Pena, Louis A [Poquott, NY; Lin, Xinhua [Plainview, NY

2009-10-06

The invention provides synthetic heparin-binding growth factor analogs having two peptide chains each branched from a branch moiety, such as trifunctional amino acid residues, the branch moieties separated by a first linker of from 3 to about 20 backbone atoms, which peptide chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a second linker, which may be a hydrophobic second linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

From Farm to Pharma: An Overview of Industrial Heparin Manufacturing Methods.

PubMed

van der Meer, Jan-Ytzen; Kellenbach, Edwin; van den Bos, Leendert J

2017-06-21

The purification of heparin from offal is an old industrial process for which commercial recipes date back to 1922. Although chemical, chemoenzymatic, and biotechnological alternatives for this production method have been published in the academic literature, animal-tissue is still the sole source for commercial heparin production in industry. Heparin purification methods are closely guarded industrial secrets which are not available to the general (scientific) public. However by reviewing the academic and patent literature, we aim to provide a comprehensive overview of the general methods used in industry for the extraction of heparin from animal tissue.

Addressing endotoxin issues in bioengineered heparin.

PubMed

Suwan, Jiraporn; Torelli, Amanda; Onishi, Akihiro; Dordick, Jonathan S; Linhardt, Robert J

2012-01-01

Heparin is a widely used clinical anticoagulant that is prepared from pig intestine. A contamination of heparin in 2008 has led to a reexamination of animal-derived pharmaceuticals. A bioengineered heparin prepared by bacterial fermentation and chemical and enzymatic processing is currently under development. This study examines the challenges of reducing or removing endotoxins associated with this process that are necessary to proceed with preclinical in vivo evaluation of bioengineered heparin. The current process is assessed for endotoxin levels, and strategies are examined for endotoxin removal from polysaccharides and enzymes involved in this process. © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Apoptosis of lymphocytes in the presence of Cr(V) complexes: role in Cr(VI)-induced toxicity.

PubMed

Vasant, C; Balamurugan, K; Rajaram, R; Ramasami, T

2001-08-03

Cr(VI) compounds have been declared as a potent occupational carcinogen by IARC (1990) through epidemiological studies among workers in chrome plating, stainless-steel, and pigment industries. Studies relating to the role of intermediate oxidation states such as Cr(V) and Cr(IV) in Cr(VI)-induced carcinogenicity are gaining importance. In this study, issues relating to toxicity elicited by Cr(V) have been addressed and comparisons made with those relating to Cr(VI) employing human peripheral blood lymphocytes. Lymphocytes have been isolated from heparinized blood by Ficoll-Hypaque density gradient centrifugation and exposed to Cr(V) complexes viz. sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[Cr(V)O(ehba)(2)], 1 and sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[Cr(V)O(hmba)(2)], 2 and Cr(VI). The phytohemagglutinin (PHA)-induced proliferation of lymphocytes has been found to be inhibited by the two complexes of Cr(V) and chromate Cr(VI) in a time- and concentration-dependent manner. Viability of cells decreases in the presence of Cr(V). Apoptosis appears to be the mode of cell death in the presence of both Cr(V) and Cr(VI). Pretreatment of cells with antioxidants before exposure to chromium(V) complexes reverse apoptosis partially. Possibility for the formation and implication of reactive oxygen species in Cr(V)-induced apoptosis of human lymphocyte cells has been indicated in this investigation. The intermediates of Cr(V) and radical species in the biotoxic pathways elicited by Cr(VI) seems feasible. Copyright 2001 Academic Press.

Benefit of heparin in peripheral venous and arterial catheters: systematic review and meta-analysis of randomised controlled trials

PubMed Central

Randolph, Adrienne G; Cook, Deborah J; Gonzales, Calle A; Andrew, Maureen

1998-01-01

Objective: To evaluate the effect of heparin on duration of catheter patency and on prevention of complications associated with use of peripheral venous and arterial catheters. Design: Critical appraisal and meta-analysis of 26 randomised controlled trials that evaluated infusion of heparin intermittently or continuously. Thirteen trials of peripheral venous catheters and two of peripheral arterial catheters met criteria for inclusion. Main outcome measures: Data on the populations, interventions, outcomes, and methodological quality. Results: For peripheral venous catheters locked between use flushing with 10 U/ml of heparin instead of normal saline did not reduce the incidence of catheter clotting and phlebitis or improve catheter patency. When heparin was given as a continuous infusion at 1 U/ml the risk of phlebitis decreased (relative risk 0.55; 95% confidence interval 0.39 to 0.77), the duration of patency increased, and infusion failure was reduced (0.88; 0.72 to 1.07). Heparin significantly prolonged duration of patency of radial artery catheters and decreased the risk of clot formation (0.51; 0.42 to 0.61). Conclusions: Use of intermittent heparin flushes at doses of 10 U/ml in peripheral venous catheters locked between use had no benefit over normal saline flush. Infusion of low dose heparin through a peripheral arterial catheter prolonged the duration of patency but further study is needed to establish its benefit for peripheral venous catheters. Key messages Despite almost universal use, agreement has not been reached on the need to administer heparin through peripheral intravascular catheters The results of 13 trials on peripheral venous catheters and two trials on peripheral arterial catheters were critically appraised to clarify what evidence supports the use of heparin Flushing peripheral venous catheters locked between use with heparinised saline at 10 U/ml is no more beneficial than flushing with normal saline Heparin significantly prolongs the duration of peripheral arterial catheter patency and decreases the risk of clot formation In peripheral venous catheters heparin added to the infusion at 1 U/ml decreases phlebitis and may prolong duration of catheter patency and decrease infusion failure PMID:9550955

Adequacy of Fixed-Dose Heparin Infusions for Venous Thromboembolism Prevention after Microsurgical Procedures.

PubMed

Bertolaccini, Corinne M; Prazak, Ann Marie B; Agarwal, Jayant; Goodwin, Isak A; Rockwell, W Bradford; Pannucci, Christopher J

2018-05-22

 In microvascular surgery, patients often receive unfractionated heparin infusions to minimize risk for microvascular thrombosis. Patients who receive intravenous (IV) heparin are believed to have adequate prophylaxis against venous thromboembolism (VTE). Whether a fixed dose of IV heparin provides detectable levels of anticoagulation, or whether the "one size fits all" approach provides adequate prophylaxis against VTE remains unknown. This study examined the pharmacodynamics of fixed-dose heparin infusions and the effects of real-time, anti-factor Xa (aFXa) level driven heparin dose adjustments.  This prospective clinical trial recruited adult microvascular surgery patients placed on a fixed-dose (500 units/h) unfractionated heparin infusion during their initial microsurgical procedure. Steady-state aFXa levels, a marker of unfractionated heparin efficacy and safety, were monitored. Patients with out-of-range aFXa levels received protocol-driven real-time dose adjustments. Outcomes of interest included aFXa levels in response to heparin 500 units/h, number of dose adjustments required to achieve goal aFXa levels, time to reach goal aFXa level, and 90-day clinically relevant bleeding and VTE.  Twenty patients were recruited prospectively. None of 20 patients had any detectable level of anticoagulation in response to heparin infusions at 500 units/h. The median number of dose adjustments required to reach goal level was five, and median weight-based dose to reach goal level was 11.8 units/kg/h. Real-time dose adjustments significantly increased the proportion of patients with in-range levels (60 vs. 0%, p  = 0.0001). The 90-day VTE rate was 5% and 90-day clinically relevant bleeding rate was 5%.  Fixed-dose heparin infusions at a rate of 500 units/h do not provide a detectable level of anticoagulation after microsurgical procedures and are insufficient for the majority of patients who require VTE prophylaxis. Weight-based heparin infusions at 10 to 12 units/kg/h deserve future study in patients undergoing microsurgical procedures to increase the proportion of patients receiving adequate VTE prophylaxis. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.

Enoxaparin versus unfractionated heparin with fibrinolysis for ST-elevation myocardial infarction.

PubMed

Antman, Elliott M; Morrow, David A; McCabe, Carolyn H; Murphy, Sabina A; Ruda, Mikhail; Sadowski, Zygmunt; Budaj, Andrzej; López-Sendón, Jose L; Guneri, Sema; Jiang, Frank; White, Harvey D; Fox, Keith A A; Braunwald, Eugene

2006-04-06

Unfractionated heparin is often used as adjunctive therapy with fibrinolysis in patients with ST-elevation myocardial infarction. We compared a low-molecular-weight heparin, enoxaparin, with unfractionated heparin for this purpose. We randomly assigned 20,506 patients with ST-elevation myocardial infarction who were scheduled to undergo fibrinolysis to receive enoxaparin throughout the index hospitalization or weight-based unfractionated heparin for at least 48 hours. The primary efficacy end point was death or nonfatal recurrent myocardial infarction through 30 days. The primary end point occurred in 12.0 percent of patients in the unfractionated heparin group and 9.9 percent of those in the enoxaparin group (17 percent reduction in relative risk, P<0.001). Nonfatal reinfarction occurred in 4.5 percent of the patients receiving unfractionated heparin and 3.0 percent of those receiving enoxaparin (33 percent reduction in relative risk, P<0.001); 7.5 percent of patients given unfractionated heparin died, as did 6.9 percent of those given enoxaparin (P=0.11). The composite of death, nonfatal reinfarction, or urgent revascularization occurred in 14.5 percent of patients given unfractionated heparin and 11.7 percent of those given enoxaparin (P<0.001); major bleeding occurred in 1.4 percent and 2.1 percent, respectively (P<0.001). The composite of death, nonfatal reinfarction, or nonfatal intracranial hemorrhage (a measure of net clinical benefit) occurred in 12.2 percent of patients given unfractionated heparin and 10.1 percent of those given enoxaparin (P<0.001). In patients receiving fibrinolysis for ST-elevation myocardial infarction, treatment with enoxaparin throughout the index hospitalization is superior to treatment with unfractionated heparin for 48 hours but is associated with an increase in major bleeding episodes. These findings should be interpreted in the context of net clinical benefit. (ClinicalTrials.gov number, NCT00077792.). Copyright 2006 Massachusetts Medical Society.

[Thrombocytopenia induced by type II heparin and myocardial infarct: 2 case reports].

PubMed

Antonijević, Nabojsa; Stanojević, Milica; Perunicić, Jovan; Djokić, Milan; Miković, Danijla; Kovac, Mirjana; Miljić, Predrag; Milosević, Rajko; Terzić, Branka; Vasiljević, Zorana

2004-01-01

Heparin-induced thrombocytopenia (HIT) type II is an acquired thrombophylic state and life-threatening immune complication of a heparin treatment mainly clinically manifested by marked thrombocytopenia, frequently by arterial and venous thrombosis, and sometimes by skin changes. Functional assay as heparin aggregation test and 14C-serotonin release assays are used in diagnostics as well as antigen assays of which detection tests for heparin-platelet factor 4 antibodies are most frequently used. Considering the fact that there is no single reliable assays for HIT II detection available, sometimes it is necessary to combine both of the above-mentioned types of assays. We present the case of a 57-year-old patient with an acute anterior myocardial infarction with cardiac insufficiency of III and IV degree according to Killip, recurrent ventricular fibrillation and diabetes mellitus type II developing thrombocytopenia to 37 x 10(9)/l accompanied with typical skin changes. The diagnosis was confirmed by the heparin aggregation test. The second patient aged 70 undergoing the treatment for anteroseptal myocardial infarction and reinfarction of the inferior wall complicated by a cardiogenic shock and acute right bundle branch block developed thrombocytopenia 59 x 10(9)/l on the third day of the heparin therapy, with the remark that he had received a heparin therapy during the first infarction as well. Antibodies against heparin-platelet factor 4 were detected by particle gel ID-HPF4 immuno-assay. In both patients, the disease had a lethal outcome despite all then available therapeutic measures applied. Further on we discuss advantages of certain types of tests, a therapy doctrine, need for urgent therapeutic measures, inclusive of the administration of antithrombins, avoidance of harmful procedures like low-molecular-weight heparins administration and prophylactic platelet transfusion as well as preventive measures.

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

PubMed

Zhao, Gaofeng; Shaik, Rahamthulla S; Zhao, Hang; Beagle, John; Kuo, Shuennwen; Hales, Charles A

2011-05-01

The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression. Copyright © 2011. Published by Mosby, Inc.

The oxidative mechanism of heparin interferes with radical production by glucose and reduces the degree of glycooxidative modifications on human serum albumin.

PubMed

Finotti, P; Pagetta, A; Ashton, T

2001-04-01

Among substances which may prove useful in preventing or reducing the progression of glycooxidative modifications of proteins, heparin plays a unique role. To elucidate the mechanism whereby heparin may favourably influence the protein structure during glycation, human serum albumin (HSA) was glycated with both 25 and 50 mM glucose in the absence and presence of 12 microg.mL(-1) low-molecular-mass heparin. Glycation caused: (a) modifications of fluorescence emission and excitation spectra consistent with the covalent attachment of glucose to protein; (b) a significant increase in the esterase activity of HSA on p-nitrophenyl acetate; (c) a reduced susceptibility to tryptic digestion and (d) enhanced formation of high-molecular mass aggregates of HSA. These alterations were accompanied by oxidative reactions, as the EPR spectra showed a clear-cut radical signal, dependent on glucose concentration, further confirmed by measurement of the carbonyl content of HSA, as an indirect proof of oxidative damage. In the presence of heparin all the above alterations, especially at 25 mM glucose, turned out to be antagonized. The effects of heparin were dependent on its specific binding to HSA, which triggered an oxidative mechanism strikingly different from that caused by glucose. In the presence of heparin, only the radical species catalyzed by heparin was detected across all samples of glycated HSA, irrespective of glucose concentration. In addition, at 25 mM glucose, enhancement of the oxidative capacity of heparin was also observed. The results demonstrate that the oxidative mechanism sustained by heparin mediates biological effects that may be beneficial in reducing the extent of glycooxidative damage on HSA.

Heparin Resistance and Anticoagulation Failure in a Challenging Case of Cerebral Venous Sinus Thrombosis.

PubMed

King, Adam B; O'Duffy, Anne E; Kumar, Avinash B

2016-07-01

We report a challenging case of cerebral venous sinus thrombosis (multiple etiologic factors) that was complicated by heparin resistance secondary to suspected antithrombin III (ATIII) deficiency. A 20-year-old female previously healthy and currently 8 weeks pregnant presented with worsening headaches, nausea, and decreasing Glasgow Coma Scale/Score (GCS), necessitating mechanical ventilatory support. Imaging showed extensive clots in multiple cerebral venous sinuses including the superior sagittal sinus, transverse, sigmoid, jugular veins, and the straight sinus. She was started on systemic anticoagulation and underwent mechanical clot removal and catheter-directed endovascular thrombolysis with limited success. Complicating the intensive care unit care was the development of heparin resistance, with an inability to reach the target partial thomboplastin time (PTT) of 60 to 80 seconds. At her peak heparin dose, she was receiving >35 000 units/24 h, and her PTT was subtherapeutic at <50 seconds. Deficiency of ATIII was suspected as a possible etiology of her heparin resistance. Fresh frozen plasma was administered for ATIII level repletion. Given her high thrombogenic risk and challenges with conventional anticoagulation regimens, we transitioned to argatroban for systemic anticoagulation. Heparin produces its major anticoagulant effect by inactivating thrombin and factor X through an AT-dependent mechanism. For inhibition of thrombin, heparin must bind to both the coagulation enzyme and the AT. A deficiency of AT leads to a hypercoagulable state and decreased efficacy of heparin that places patients at high risk of thromboembolism. Heparin resistance, especially in the setting of critical illness, should raise the index of suspicion for AT deficiency. Argatroban is an alternate agent for systemic anticoagulation in the setting of heparin resistance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wall, Jonathan; Martin, Emily B.; Richey, Tina

Amyloid is a complex pathologic matrix comprised principally of para-crystalline protein fibrils and heparan sulfate proteoglycans. Systemic amyloidoses are rare (~3500 new cases per year in the US); thus, routine diagnosis is often challenging, and effective treatment options are limited, resulting in high morbidity and mortality rates. Glycosaminoglycans contribute inextricably to the formation of amyloid fibrils and foster the deposition of amyloid in tissues. Those present in amyloid deposits are biochemically and electrochemically distinct from glycosaminoglycans found in the plasma membrane and extracellular matrices of healthy tissues due to the presence of a high degree of heparin-like hypersulfation. We havemore » exploited this unique property and evaluated heparin-reactive peptides, such as p5+14. Herein we show efficacious detection of murine systemic amyloid in vivo by using molecular imaging, and the specific targeting of the peptide to major forms of human amyloid in tissue sections. Furthermore, we have demonstrated that the peptide also binds synthetic amyloid fibrils that lack glycosaminoglycans implying that the dense anionic motif present on heparin is mimicked by the amyloid protein fibril itself. These biochemical and functional data support the translation of radiolabeled peptide p5+14 for the clinical imaging of amyloid in patients; thus, providing a novel technique for prognostication, patient stratification, and monitoring response to therapy.« less

Low molecular weight protamine (LMWP): a nontoxic protamine substitute and an effective cell-penetrating peptide.

PubMed

He, Huining; Ye, Junxiao; Liu, Ergang; Liang, Qiuling; Liu, Quan; Yang, Victor C

2014-11-10

Low molecular weight protamine (LMWP) is a peptide fragment produced in our laboratory from enzymatic digestion of native protamine. More than 30 papers studying the properties and applications of LMWP have been published by our group in various journals since its initial discovery in 1999. Results have shown that LMWP could completely neutralize the anticoagulant functions of both heparin and low molecular weight heparin (LMWH), with reduced antigenicity and cross-reactivity toward the mice-derived anti-protamine antibodies. Aside from its potential as a heparin/LMWH antagonist, LMWP also shows the ability to retard insulin adsorption by the formation of an insoluble complex, making it a less toxic long-lasting insulin product than the conventional neutral protamine Hagedorn (NPH) insulin for diabetic control. Importantly, LMWP (Sequence: VSRRRRRRGGRRRR), with 10 arginine residues in its structure, could function as a cell-penetrating peptide (CPP), also termed protein transduction domain (PTD), to achieve effective intracellular protein or gene delivery in clinical practice. In this paper, we present a thorough review of our work related to LMWP, with the aim of providing readers an insight into its potential to be a clinical protamine substitute as well as a non-toxic cell penetrating peptide applicable to achieve intracellular protein and gene delivery. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification of foot-and-mouth disease virus by heparin as ligand for certain strains.

PubMed

Du, Ping; Sun, Shiqi; Dong, Jinjie; Zhi, Xiaoying; Chang, Yanyan; Teng, Zhidong; Guo, Huichen; Liu, Zaixin

2017-04-01

The goal of this project was to develop an easily operable and scalable process for the recovery and purification of foot-and-mouth disease virus (FMDV) from cell culture. Heparin resins HipTrap Heparin HP and AF-Heparin HC-650 were utilized to purify FMDV O/HN/CHA/93. Results showed that the purity of AF-Heparin HC-650 was ideal. Then, the O/HN/CHA/93, O/Tibet/CHA/99, Asia I/HN/06, and A/CHA/HB/2009 strains were purified by AF-Heparin HC-650. Their affinity/virus recoveries were approximately 51.2%/45.8%, 71.5%/70.9%, 96.4%/73.5, and 59.5%/42.1%, respectively. During a stepwise elution strategy, the viral particles were mainly eluted at 300mM ionic strength peaks. The heparin affinity chromatography process removed more than 94% of cellular and medium proteins. Anion exchange resin Capto Q captured four FMD virus particles; 40% of binding proteins and 80%-90% of viral particles were eluted at 450mM NaCl. Moreover, ionic strength varied from 30 to 450mM had no effect on the immunity to FMDV. The results revealed that heparin sulfate may be the main receptor for CHA/99 strain attachment-susceptible cells. Heparin affinity chromatography can reach perfect results, especially when used as a ligand of the virus. Anion exchange is useful only as previous step for further purification. Copyright © 2016. Published by Elsevier B.V.

The risk for cross-reactions after a cutaneous delayed-type hypersensitivity reaction to heparin preparations is independent of their molecular weight: a systematic review.

PubMed

Weberschock, Tobias; Meister, Anna Christina; Bohrt, Kevin; Schmitt, Jochen; Boehncke, Wolf-Henning; Ludwig, Ralf J

2011-10-01

Heparins are a widely used class of drugs known to cause delayed-type hypersensitivity (DTH) reactions. Recent publications indicate that the incidence of these may be higher than previously thought. To date, patient-related but no drug-related risk factors for the development of DTH reactions to heparins have been identified, although molecular weight is discussed as a potentially relevant parameter. To address this, a systematic review was conducted on the frequency of cross-reactions after DTH reactions to heparin preparations. We electronically searched MEDLINE and EMBASE, hand-searched selected journals and references, and contacted experts for unpublished data. Sixty-six publications and unpublished data of 14 patients resulted in 198 patients with 1084 tests for cross-reactivity. The primary causative agents were mostly unfractionated heparin (50%) and low molecular weight heparins (49.5%). Cross-reactions were more likely after an initial DTH reaction to unfractionated heparin than after an initial DTH reaction to low molecular weight heparin. Our findings also indicate that molecular weight does not correlate with the risk for cross-reactivity, which is in line with recent observations, indicating that different heparins have to be individually considered. The available data demonstrated the lowest overall risk for cross-reactions for pentosan polysulfate (36.4%) and fondaparinux (10.4%). In the clinical context, fondaparinux is recommended as the current best alternative when a DTH reaction occurs. © 2011 John Wiley & Sons A/S.

Dual-functional composite with anticoagulant and antibacterial properties based on heparinized silk fibroin and chitosan.

PubMed

Wang, Jianglin; Hu, Wei; Liu, Qun; Zhang, Shengmin

2011-07-01

Heparinized biomaterials exhibit great anticoagulant properties. However, they promote proliferation of Staphylococcus aureus (S. aureus) and therefore cause infection within the bloodstream upon implantation in vivo. In the present study, an interesting dual-functional composite with anticoagulant and antibacterial properties based on heparinized silk fibroin and chitosan was synthesized. First, heparin was grafted onto the silk fibroin by covalent immobilization with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). All data gathered from Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and elemental analysis (EA) indicated that the heparin was successfully immobilized onto the silk fibroin. The dual-functional composite of heparinized silk fibroin and chitosan was then fabricated by a blending method. The anticoagulant activity of the heparinized materials was evaluated using the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT). The results showed that both heparinized silk fibroin and the composite material exhibited better hemocompatibility in comparison with single silk fibroin or chitosan. The antibacterial property of the materials was investigated by the pour-plate method. Results further suggested that the composite antibacterial property with respect to S. aureus was significantly enhanced. The dual-functionality of the composite material may supply a potential choice in blood contact devices. Copyright © 2011 Elsevier B.V. All rights reserved.

Nerve growth factor loaded heparin/chitosan scaffolds for accelerating peripheral nerve regeneration.

PubMed

Li, Guicai; Xiao, Qinzhi; Zhang, Luzhong; Zhao, Yahong; Yang, Yumin

2017-09-01

Artificial chitosan scaffolds have been widely investigated for peripheral nerve regeneration. However, the effect was not as good as that of autologous grafts and therefore could not meet the clinical requirement. In the present study, the nerve growth factor (NGF) loaded heparin/chitosan scaffolds were fabricated via electrostatic interaction for further improving nerve regeneration. The physicochemical properties including morphology, wettability and composition were measured. The heparin immobilization, NGF loading and release were quantitatively and qualitatively characterized, respectively. The effect of NGF loaded heparin/chitosan scaffolds on nerve regeneration was evaluated by Schwann cells culture for different periods. The results showed that the heparin immobilization and NGF loading did not cause the change of bulk properties of chitosan scaffolds except for morphology and wettability. The pre-immobilization of heparin in chitosan scaffolds could enhance the stability of subsequently loaded NGF. The NGF loaded heparin/chitosan scaffolds could obviously improve the attachment and proliferation of Schwann cells in vitro. More importantly, the NGF loaded heparin/chitosan scaffolds could effectively promote the morphology development of Schwann cells. The study may provide a useful experimental basis to design and develop artificial implants for peripheral nerve regeneration and other tissue regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Spontaneous heparin-induced thrombocytopenia (HIT) syndrome: HIT without any heparin exposure.

PubMed

Miyata, Shigeki

2016-01-01

Heparin-induced thrombocytopenia (HIT) is a pro-thrombotic side effect of heparin therapy caused by HIT antibodies with platelet-activating properties. Recent advances in understanding of spontaneous HIT syndrome, which can occur even without any heparin exposure despite its clinical and serological characteristics being similar to those of HIT, reveal the following HIT clinical features atypical for an immune-mediated disease. Heparin-naïve patients can develop IgG antibodies as early as day 4, as in a secondary immune response. Evidence for an anamnestic response upon heparin re-exposure is lacking. In addition, HIT antibodies are relatively short-lived, unlike those in a secondary immune response. Antigen immunoassays are commonly used worldwide for serological diagnosis of HIT. However, such assays do not indicate whether HIT antibodies have platelet-activating properties, leading to low diagnostic specificity for HIT. The detection of platelet-activating antibodies using a washed platelet activation assay is crucial for making a HIT diagnosis. These atypical clinical and serological features should be carefully considered while appropriately diagnosing HIT, which leads to appropriate therapy such as immediate administration of an alternative anticoagulant for preventing thromboembolic events and re-administration of heparin during surgery involving cardiopulmonary bypass when HIT antibodies are no longer detectable.

Limitation of myocardial infarct size and preservation of left ventricular function by early administration of APSAC in myocardial infarction.

PubMed

Bassand, J P; Machecourt, J; Cassagnes, J; Lusson, J R; Borel, E; Schiele, F

1989-07-05

In cases of acute myocardial infarction (MI), it has been shown that preserving left ventricular function and limiting infarct size with early reperfusion of the occluded artery by means of a thrombolytic agent could eventually result in a reduced mortality rate. The aim of the APSIM study (anisoylated plasminogen streptokinase activator complex [APSAC] dans l'infarctus du Myocarde) was to demonstrate that early administration of APSAC in patients with recent acute MI could limit the infarct size and preserve left ventricular systolic function. In all, 231 patients with a first acute MI were randomly allocated to either APSAC (30 U over 5 minutes) or to conventional heparin therapy (5,000 IU in bolus injection) within 5 hours of the onset of symptoms. Of these patients, 112 received APSAC and 119 received heparin within a mean period of 188 +/- 62 minutes after the onset of symptoms. The patency rate of the infarct-related artery was 77% in the APSAC group and 36% in the heparin group (p less than 0.001). Left ventricular ejection fraction determined from contrast angiography was significantly higher in the APSAC than in the heparin group. This was true for the entire population (0.53 +/- 0.13 vs 0.47 +/- 0.13, p = 0.002) as well as for the subgroups of anterior and inferior wall infarctions (0.47 +/- 0.13 vs 0.4 +/- 0.16, p = 0.004 and 0.56 +/- 0.11 vs 0.51 +/- 0.09, p = 0.02). At 3 weeks, the difference remained significant for patients with anterior MI.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a new nanoparticle-based lateral-flow immunoassay for the exclusion of heparin-induced thrombocytopenia (HIT).

PubMed

Sachs, Ulrich J; von Hesberg, Jakob; Santoso, Sentot; Bein, Gregor; Bakchoul, Tamam

2011-12-01

Heparin-induced thrombocytopenia (HIT) is an adverse complication of heparin caused by HIT antibodies (abs) that recognise platelet factor 4-heparin (PF4/hep) complexes. Several laboratory tests are available for the confirmation and/or refutation of HIT. A reliable and rapid single-sample test is still pending. It was the objective of this study to evaluate a new lateral-flow immunoassay based on nanoparticle technology. A cohort of 452 surgical and medical patients suspected of having HIT was evaluated. All samples were tested in two IgG-specific ELISAs, in a particle gel immunoassay (PaGIA) and in a newly developed lateral-flow immunoassay (LFI-HIT) as well as in a functional test (HIPA). Clinical pre-test probability was determined using 4T's score. Platelet-activating antibodies were present in 34/452 patients, all of whom had intermediate to high clinical probability. PF4/hep abs were detected in 79, 87, 86, and 63 sera using the four different immunoassays. The negative predictive values (NPV) were 100% for both ELISA tests and LFI-HIT but only 99.2% for PaGIA. There were less false positives (n=29) in the LFI-HIT compared to any other test. Additionally, significantly less time was required to perform LFI-HIT than to perform the other immunoassays. In conclusion, a newly developed lateral-flow assay, LFI-HIT, was capable of identifying all HIT patients in a cohort in a short period of time. Beside an NPV of 100%, the rate of false-positive signals is significantly lower with LFI-HIT than with other immunoassay(s). These performance characteristics suggest a high potency in reducing the risk and costs in patients suspected of having HIT.

Can oral vitamin K before elective surgery substitute for preoperative heparin bridging in patients on vitamin K antagonists?

PubMed

Steib, A; Barre, J; Mertes, M; Morel, M H; Nathan, N; Ozier, Y; Treger, M; Samama, C M

2010-03-01

After a vitamin K antagonist (VKA) overdose, 1-2 mg of oral vitamin K can lower the International Normalized Ratio (INR) to the therapeutic range. To establish whether oral vitamin K can substitute for heparin bridging and decrease the INR to < or = 1.5 before elective surgery. Patients on long-term VKAs were randomized either to heparin bridging after the last VKA dose on day -5 before surgery (group H) or to VKA treatment until day -2, followed by 1 mg of oral vitamin K on the day before surgery (group K). Blood clotting variables were assessed on days -5/-2, 1 and 0, and postoperatively. If the target INR was not achieved 2 h before incision, surgery was deferred or performed after injection of prothrombin complex concentrate (PCC). In 30 of 94 included patients, baseline INR was outside the chosen range (18, INR < 2; 12, INR > 3.5), leaving 34 eligible patients in group H and 30 in group K. The groups were balanced in terms of body mass index, VKA treatment duration and indication, scheduled surgery, preoperative and postoperative hemoglobin, and blood loss. The INR was significantly higher in group K on days -1 and 0 than in group H. An INR < or = 1.5 was not achieved in 20 group K patients (66%). Surgery was postponed or performed after PCC injection in 12 of these 20 patients. Oral vitamin K (1 mg) cannot substitute for heparin bridging before surgery. In addition, one-third of patients on VKAs were exposed to a risk of bleeding (overdose) or thrombosis (underdose), thus highlighting the need for new oral anticoagulants.

Novel Family of Insect Salivary Inhibitors Blocks Contact Pathway Activation by Binding to Polyphosphate, Heparin, and Dextran Sulfate

PubMed Central

Alvarenga, Patricia H.; Xu, Xueqing; Oliveira, Fabiano; Chagas, Andrezza C.; Nascimento, Clarissa R.; Francischetti, Ivo M.B.; Juliano, Maria A.; Juliano, Luiz; Scharfstein, Julio; Valenzuela, Jesus G.; Ribeiro, José M.C.; Andersen, John F.

2014-01-01

Objective Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. Approach and Results Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. Conclusions The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism. PMID:24092749

Determination of Oversulphated Chondroitin Sulphate and Dermatan Sulphate in unfractionated heparin by (1)H-NMR - Collaborative study for quantification and analytical determination of LoD.

PubMed

McEwen, I; Mulloy, B; Hellwig, E; Kozerski, L; Beyer, T; Holzgrabe, U; Wanko, R; Spieser, J-M; Rodomonte, A

2008-12-01

Oversulphated Chondroitin Sulphate (OSCS) and Dermatan Sulphate (DS) in unfractionated heparins can be identified by nuclear magnetic resonance spectrometry (NMR). The limit of detection (LoD) of OSCS is 0.1% relative to the heparin content. This LoD is obtained at a signal-to-noise ratio (S/N) of 2000:1 of the heparin methyl signal. Quantification is best obtained by comparing peak heights of the OSCS and heparin methyl signals. Reproducibility of less than 10% relative standard deviation (RSD) has been obtained. The accuracy of quantification was good.

Chromatographic molecular weight measurements for heparin, its fragments and fractions, and other glycosaminoglycans.

PubMed

Mulloy, Barbara; Hogwood, John

2015-01-01

Glycosaminoglycan samples are usually polydisperse, consisting of molecules with differing length and differing sequence. Methods for measuring the molecular weight of heparin have been developed to assure the quality and consistency of heparin products for medicinal use, and these methods can be applied in other laboratory contexts. In the method described here, high-performance gel permeation chromatography is calibrated using appropriate heparin molecular weight markers or a single broad standard calibrant, and used to characterize the molecular weight distribution of polydisperse samples or the peak molecular weight of monodisperse, or approximately monodisperse, heparin fractions. The same technology can be adapted for use with other glycosaminoglycans.

Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes.

PubMed

Makino-Okamura, Chieko; Niki, Yoko; Takeuchi, Seiji; Nishigori, Chikako; Declercq, Lieve; Yaroch, Daniel B; Saito, Naoaki

2014-11-01

To gain insight for the role of mast cell-produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin-treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2 ) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti-inflammation role in epidermis of human skin. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Immobilized enzymes to convert N-sulfo, N-acetyl heparosan to a critical intermediate in the production of bioengineered heparin.

PubMed

Xiong, Jian; Bhaskar, Ujjwal; Li, Guoyun; Fu, Li; Li, Lingyun; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J

2013-09-10

Heparin is a critically important anticoagulant drug that is prepared from pig intestine. In 2007-2008, there was a crisis in the heparin market when the raw material was adulterated with the toxic polysaccharide, oversulfated chondroitin sulfate, which was associated with 100 deaths in the U.S. alone. As the result of this crisis, our laboratory and others have been actively pursuing alternative sources for this critical drug, including synthetic heparins and bioengineered heparin. In assessing the bioengineering processing costs it has become clear that the use of both enzyme-catalyzed cofactor recycling and enzyme immobilization will be needed for commercialization. In the current study, we examine the use of immobilization of C₅-epimerase and 2-O-sulfotransferase involved in the first enzymatic step in the bioengineered heparin process, as well as arylsulfotransferase-IV involved in cofactor recycling in all three enzymatic steps. We report the successful immobilization of all three enzymes and their use in converting N-sulfo, N-acetyl heparosan into N-sulfo, N-acetyl 2-O-sulfo heparin. Copyright © 2013 Elsevier B.V. All rights reserved.

Heparin-Mimicking Polymers: Synthesis and Biological Applications

PubMed Central

2016-01-01

Heparin is a naturally occurring, highly sulfated polysaccharide that plays a critical role in a range of different biological processes. Therapeutically, it is mostly commonly used as an injectable solution as an anticoagulant for a variety of indications, although it has also been employed in other forms such as coatings on various biomedical devices. Due to the diverse functions of this polysaccharide in the body, including anticoagulation, tissue regeneration, anti-inflammation, and protein stabilization, and drawbacks of its use, analogous heparin-mimicking materials are also widely studied for therapeutic applications. This review focuses on one type of these materials, namely, synthetic heparin-mimicking polymers. Utilization of these polymers provides significant benefits compared to heparin, including enhancing therapeutic efficacy and reducing side effects as a result of fine-tuning heparin-binding motifs and other molecular characteristics. The major types of the various polymers are summarized, as well as their applications. Because development of a broader range of heparin-mimicking materials would further expand the impact of these polymers in the treatment of various diseases, future directions are also discussed. PMID:27739666

Heparin-Mimicking Polymers: Synthesis and Biological Applications.

PubMed

Paluck, Samantha J; Nguyen, Thi H; Maynard, Heather D

2016-11-14

Heparin is a naturally occurring, highly sulfated polysaccharide that plays a critical role in a range of different biological processes. Therapeutically, it is mostly commonly used as an injectable solution as an anticoagulant for a variety of indications, although it has also been employed in other forms such as coatings on various biomedical devices. Due to the diverse functions of this polysaccharide in the body, including anticoagulation, tissue regeneration, anti-inflammation, and protein stabilization, and drawbacks of its use, analogous heparin-mimicking materials are also widely studied for therapeutic applications. This review focuses on one type of these materials, namely, synthetic heparin-mimicking polymers. Utilization of these polymers provides significant benefits compared to heparin, including enhancing therapeutic efficacy and reducing side effects as a result of fine-tuning heparin-binding motifs and other molecular characteristics. The major types of the various polymers are summarized, as well as their applications. Because development of a broader range of heparin-mimicking materials would further expand the impact of these polymers in the treatment of various diseases, future directions are also discussed.

[Heparin-induced thrombocytopenia developed during the acute phase after left upper lobectomy for lung cancer].

PubMed

Mitomo, Hideki; Miyamoto, Akira; Tabata, Toshiharu; Sugawara, Takafumi; Yabuki, Hiroshi; Fujimura, Shigefumi

2014-12-01

Heparin-induced thrombocytopenia (HIT) is a serious adverse effect of heparin administration. This must not be rarely encountered but is not often reported in Japan compared to Western countries. A 68-year-old woman underwent left upper lobectomy for lung cancer. Low-dose unfractionated heparin was administrated to prevent thromboembolism after the operation. Two days later, sudden dyspnea appeared and ultracardiosonography showing an extensive thromboembolus from the main trunk to both main branches of pulmonary artery indicated pulmonary embolization. After the establishment of percutaneous cardiopulmonary support (PCPS) support, the embolus was removed by emergent open heart surgery. However, despite further unfractionated heparin administration following embolization surgery, other thrombus was identified in both the bi-lateral internal jagular veins and inferior vena cava by ultrasonography and contrast computed tomography( CT). Her platelet count was decreased gradually despite platelet transfusion. Plate factor 4( PF4) antibody against heparin in her blood examination was found, and HIT II was diagnosed. Discontinuation of unfractionated heparin and administration of antithrombin agent improved platelet count, and no additional embolization was identified.

Bivalirudin vs heparin with or without tirofiban during primary percutaneous coronary intervention in acute myocardial infarction: the BRIGHT randomized clinical trial.

PubMed

Han, Yaling; Guo, Jincheng; Zheng, Yang; Zang, Hongyun; Su, Xi; Wang, Yu; Chen, Shaoliang; Jiang, Tiemin; Yang, Ping; Chen, Jiyan; Jiang, Dongju; Jing, Quanmin; Liang, Zhenyang; Liu, Haiwei; Zhao, Xin; Li, Jing; Li, Yi; Xu, Bo; Stone, Gregg W

2015-04-07

The safety and efficacy of bivalirudin compared with heparin with or without glycoprotein IIb/IIIa inhibitors in patients with acute myocardial infarction (AMI) undergoing primary percutaneous coronary intervention (PCI) are uncertain. To determine if bivalirudin is superior to heparin alone and to heparin plus tirofiban during primary PCI. Multicenter, open-label trial involving 2194 patients with AMI undergoing primary PCI at 82 centers in China between August 2012 and June 2013. Patients were randomly assigned to receive bivalirudin with a post-PCI infusion (n = 735), heparin alone (n = 729), or heparin plus tirofiban with a post-PCI infusion (n = 730). Among patients treated with bivalirudin, a postprocedure 1.75 mg/kg/h infusion was administered for a median of 180 minutes (IQR, 148-240 minutes). The primary end point was 30-day net adverse clinical events, a composite of major adverse cardiac or cerebral events (all-cause death, reinfarction, ischemia-driven target vessel revascularization, or stroke) or bleeding. Additional prespecified safety end points included the rates of acquired thrombocytopenia at 30 days, and stent thrombosis at 30 days and 1 year. Net adverse clinical events at 30 days occurred in 65 patients (8.8%) of 735 who were treated with bivalirudin compared with 96 patients (13.2%) of 729 treated with heparin (relative risk [RR], 0.67; 95% CI, 0.50-0.90; difference, -4.3%, 95% CI, -7.5% to -1.1%; P = .008); and 124 patients (17.0%) of 730 treated with heparin plus tirofiban (RR for bivalirudin vs heparin plus tirofiban, 0.52; 95% CI, 0.39-0.69; difference, -8.1%, 95% CI, -11.6% to -4.7%; P < .001). The 30-day bleeding rate was 4.1% for bivalirudin, 7.5% for heparin, and 12.3% for heparin plus tirofiban (P < .001). There were no statistically significant differences between treatments in the 30-day rates of major adverse cardiac or cerebral events (5.0% for bivalirudin, 5.8% for heparin, and 4.9% for heparin plus tirofiban, P = .74), stent thrombosis (0.6% vs 0.9% vs 0.7%, respectively, P = .77), acquired thrombocytopenia (0.1% vs 0.7% vs 1.1%; P = .07), or in acute (<24-hour) stent thrombosis (0.3% in each group). At the 1-year follow-up, the results remained similar. Among patients with AMI undergoing primary PCI, the use of bivalirudin with a median 3-hour postprocedure PCI-dose infusion resulted in a decrease in net adverse clinical events compared with both heparin alone and heparin plus tirofiban. This finding was primarily due to a reduction in bleeding events with bivalirudin, without significant differences in major adverse cardiac or cerebral events or stent thrombosis. clinicaltrials.gov Identifier: NCT01696110.

Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marks, J.L.; Ax, R.L.

1985-08-01

The binding of the glycosaminoglycan (3H) heparin to bull spermatozoa was compared with nonreturn rates of dairy bulls. Semen samples from five bulls above and five below an average 71% nonreturn rate were used. Samples consisted of first and second ejaculates on a single day collected 1 d/wk for up to 5 consecutive wk. Saturation binding assays using (TH) heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for (TH) heparin between bulls of high and low fertility. Dissociation constants were 69.0 and 119.3 pmol for bullsmore » of high and low fertility, respectively. In contrast, the number of binding sites for (TH) heparin did not differ significantly among bulls. Differences in binding affinity of (TH) heparin to bull sperm might be used to predict relative fertility of dairy bulls.« less

Surface immobilization of heparin on functional polyisobutylene-based thermoplastic elastomer as a potential artificial vascular graft

NASA Astrophysics Data System (ADS)

Wu, Yi-Bo; Li, Kang; Xiang, Dong; Zhang, Min; Yang, Dan; Zhang, Jin-Han; Mao, Jing; Wang, Hao; Guo, Wen-Li

2018-07-01

Polyisobutylene-based thermoplastic elastomer (TPE) is a new soft biomaterial. Hydroxyl functional dendritic polyisobutylene-based TPEs (arb-SIBS-OH), which satisfy the design requirements for small-diameter vascular substitutes, were synthesized by controlled carbocationic polymerization. Creep property, which is the destructive weakness of polyisobutylene-based TPEs, was significantly improved with the formation of a "double network" promoted by branched structure and microphase separation. Compatibility of arb-SIBS-OH with rabbit blood was markedly enhanced by modifying heparin grafted from these hydroxyl functional groups. Application of "click chemistry" to immobilize heparin on arb-SIBS-OH surface was apparently effective in enhancing the bioactivity of heparin. Immobilized heparin, which directly bonded by ester bonds, was more likely to form multi-point binding on arb-SIBS-OH surface. This process hindered the accessibility of the heparin active sequence to antithrombin.

Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation.

PubMed

Li, Bojun; Lin, Zhe; Mitsi, Maria; Zhang, Yang; Vogel, Viola

2015-01-01

An increasing body of evidence suggests important roles of extracellular matrix (ECM) in regulating stem cell fate. This knowledge can be exploited in tissue engineering applications for the design of ECM scaffolds appropriate to direct stem cell differentiation. By probing the conformation of fibronectin (Fn) using fluorescence resonance energy transfer (FRET), we show here that heparin treatment of the fibroblast-derived ECM scaffolds resulted in more extended conformations of fibrillar Fn in ECM. Since heparin is a highly negatively charged molecule while fibronectin contains segments of positively charged modules, including FnIII13, electrostatic interactions between Fn and heparin might interfere with residual quaternary structure in relaxed fibronectin fibers thereby opening up buried sites. The conformation of modules FnIII12-14 in particular, which contain one of the heparin binding sites as well as binding sites for many growth factors, may be activated by heparin, resulting in alterations in growth factor binding to Fn. Indeed, upregulated osteogenic differentiation was observed when hMSCs were seeded on ECM scaffolds that had been treated with heparin and were subsequently chemically fixed. In contrast, either rigidifying relaxed fibers by fixation alone, or heparin treatment without fixation had no effect. We hypothesize that fibronectin's conformations within the ECM are activated by heparin such as to coordinate with other factors to upregulate hMSC osteogenic differentiation. Thus, the conformational changes of fibronectin within the ECM could serve as a 'converter' to tune hMSC differentiation in extracellular matrices. This knowledge could also be exploited to promote osteogenic stem cell differentiation on biomedical surfaces.

Safety and potential anticoagulant effects of nebulised heparin in burns patients with inhalational injury at Singapore General Hospital Burns Centre.

PubMed

Yip, Lian Yee; Lim, Yen Fang; Chan, Hong Ngee

2011-11-01

Nebulised heparin, N-acetylcysteine (NAC) and salbutamol were shown to decrease reintubation rates, incidence of atelectasis and mortality in paediatric patients and reduce lung injury scores in adult burns patients with inhalational lung injury (ILI). Nebulised heparin, NAC and salbutamol treatment protocol was introduced in Singapore General Hospital (SGH) Burns Centre in 2006. However, safety data on the use of nebulised heparin and NAC for burns patients with ILI is not well established. In this study, we investigated the safety and potential anticoagulant effects of nebulised heparin in burns patients with ILI. A retrospective study with historical control was conducted. The treatment group consisted of 52 mechanically ventilated adult patients, with a diagnosis of ILI as confirmed by bronchoscopy, admitted to burn intensive care unit (BICU) from the year 2006 to 2009. The group was treated with nebulised heparin, NAC and salbutamol. The control group consists of 11 mechanically ventilated BICU ILI patients treated from year 2001 to 2005 before protocol initiation. Blood coagulation indices (prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet count) were monitored and bleeding incidences were assessed. Blood coagulation indices did not suggest an increase risk of bleeding with nebulised heparin. The APTT, PT and platelet count followed a similar trend for both groups over 7 days. No clinically significant increase in bleeding risk was found to be associated with nebulised heparin. Nebulised heparin was not found to potentiate the risk of bleeding in burns patients with ILI. Copyright © 2011 Elsevier Ltd and ISBI. All rights reserved.

Prospective multicentre cohort study of heparin-induced thrombocytopenia in acute ischaemic stroke patients

PubMed Central

Kawano, Hiroyuki; Yamamoto, Haruko; Miyata, Shigeki; Izumi, Manabu; Hirano, Teruyuki; Toratani, Naomi; Kakutani, Isami; Sheppard, Jo-Ann I; Warkentin, Theodore E; Kada, Akiko; Sato, Shoichiro; Okamoto, Sadahisa; Nagatsuka, Kazuyuki; Naritomi, Hiroaki; Toyoda, Kazunori; Uchino, Makoto; Minematsu, Kazuo

2011-01-01

Acute ischaemic stroke patients sometimes receive heparin for treatment and/or prophylaxis of thromboembolic complications. This study was designed to elucidate the incidence and clinical features of heparin-induced thrombocytopenia (HIT) in acute stroke patients treated with heparin. We conducted a prospective multicentre cohort study of 267 patients who were admitted to three stroke centres within 7 d after stroke onset. We examined clinical data until discharge and collected blood samples on days 1 and 14 of hospitalization to test anti-platelet factor 4/heparin antibodies (anti-PF4/H Abs) using an enzyme-linked immunosorbent assay (ELISA); platelet-activating antibodies were identified by serotonin-release assay (SRA). Patients with a 4Ts score ≥4 points, positive-ELISA, and positive-SRA were diagnosed as definite HIT. Heparin was administered to 172 patients (64·4%: heparin group). Anti-PF4/H Abs were detected by ELISA in 22 cases (12·8%) in the heparin group. Seven patients had 4Ts ≥ 4 points. Among them, three patients (1·7% overall) were also positive by both ELISA and SRA. National Institutes of Health Stroke Scale score on admission was high (range, 16–23) and in-hospital mortality was very high (66·7%) in definite HIT patients. In this study, the incidence of definite HIT in acute ischaemic stroke patients treated with heparin was 1·7% (95% confidence interval: 0·4–5·0). The clinical severity and outcome of definite HIT were unfavourable. PMID:21671895

Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

PubMed

Stokol, Tracy; Serpa, Priscila Beatriz da Silva; Zahid, Muhammad N; Brooks, Marjory B

2016-01-01

Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo . We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1-0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation. These drugs have potential as adjunctive therapy to reduce the serious complications associated with EHV-1-induced thrombosis. Treatment trials are warranted to determine whether these drugs yield clinical benefit when administered to horses infected with EHV-1.

Atomic force microscopy of adsorbed proteoglycan mimetic nanoparticles: Toward new glycocalyx-mimetic model surfaces.

PubMed

Hedayati, Mohammadhasan; Kipper, Matt J

2018-06-15

Blood vessels present a dense, non-uniform, polysaccharide-rich layer, called the endothelial glycocalyx. The polysaccharides in the glycocalyx include polyanionic glycosaminoglycans (GAGs). This polysaccharide-rich surface has excellent and unique blood compatibility. We report new methods for preparing and characterizing dense GAG surfaces that can serve as models of the vascular endothelial glycocalyx. The GAG-rich surfaces are prepared by adsorbing heparin or chondroitin sulfate-containing polyelectrolyte complex nanoparticles (PCNs) to chitosan-hyaluronan polyelectrolyte multilayers (PEMs). The surfaces are characterized by PeakForce tapping atomic force microscopy, both in air and in aqueous pH 7.4 buffer, and by PeakForce quantitative nanomechanics (PF-QNM) mode with high spatial resolution. These new surfaces provide access to heparin-rich or chondroitin sulfate-rich coatings that mimic both composition and nanoscale structural features of the vascular endothelial glycocalyx. Copyright © 2018. Published by Elsevier Ltd.

Recent developments in separation of low molecular weight heparin anticoagulants.

PubMed

Sadowski, Radosław; Gadzała-Kopciuch, Renata; Buszewski, Bogusław

2017-10-05

The general function of anticoagulants is to prevent blood clotting and growing of the existing clots in blood vessels. In recent years, there has been a significant improvement in developing methods of prevention as well as pharmacologic and surgical treatment of thrombosis. For over the last two decades, low molecular weight heparins (LMWHs) have found their application in the antithrombotic diseases treatment. These types of drugs are widely used in clinical therapy. Despite the biological and medical importance of LMWHs, they have not been completely characterized in terms of their chemical structure. Due to both, the structural complexity of these anticoagulants and the presence of impurities, their structural characterization requires the employment of advanced analytical techniques. Since separation techniques play the key role in these endeavors, this review will focus on the presentation of recent developments in the separation of LMWH anticoagulants. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Study of five cell salvage machines in coronary artery surgery.

PubMed

Burman, J F; Westlake, A S; Davidson, S J; Rutherford, L C; Rayner, A S; Wright, A M; Morgan, C J; Pepper, J R

2002-06-01

We evaluated the effectiveness, ease of use and safety of five machines for blood salvage during coronary artery surgery. All were equally effective in concentrating red cells. We measured haemoglobin, packed cell volume, free haemoglobin, white cells, neutrophil elastase, platelets, thrombin-antithrombin complex (TAT), prothrombin activation peptide F1.2, fibrin degradation product (d-dimers), tissue plasminogen activator (tPA) and heparin in wound blood, in washed cell suspensions and in a unit of bank blood prepared for each patient. All machines were equally safe and easy to use and were equally effective in removing heparin and the physiological components measured. There were no adverse effects on patients. Clotting factors are severely depleted both in salvaged blood, even before washing, and in bank blood. Cell savers are a valuable adjunct to coronary artery surgery, but careful monitoring of coagulation is required when the volumes of either bank blood or salvaged blood are large.

Heparin and insulin in the management of hypertriglyceridemia-associated pancreatitis: case series and literature review.

PubMed

Kuchay, Mohammad Shafi; Farooqui, Khalid J; Bano, Tarannum; Khandelwal, Manoj; Gill, Harmandeep; Mithal, Ambrish

2017-01-01

Severe hypertriglyceridemia accounts for up to 7% of all cases of acute pancreatitis. Heparin and insulin activate lipoprotein lipase (LPL), thereby reducing plasma triglyceride levels. However, the safety and efficacy of heparin and insulin in the treatment of hypertriglyceridemia-associated acute pancreatitis have not been well established yet. We successfully used heparin and insulin as first-line therapy in four consecutive patients with acute pancreatitis secondary to hypertriglyceridemia. In a literature search, we revised almost all reports published to date of patients managed successfully with this combination. Heparin and insulin appear to be a safe, effective, and inexpensive first-line therapy for hypertriglyceridemia-associated acute pancreatitis.

P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins.

PubMed

Hostettler, Nina; Naggi, Annamaria; Torri, Giangiacomo; Ishai-Michaeli, Riva; Casu, Benito; Vlodavsky, Israel; Borsig, Lubor

2007-11-01

Vascular cell adhesion molecules, P- and L-selectins, facilitate metastasis of cancer cells in mice by mediating interactions with platelets, endothelium, and leukocytes. Heparanase is an endoglycosidase that degrades heparan sulfate of extracellular matrix, thereby promoting tumor invasion and metastasis. Heparin is known to efficiently attenuate metastasis in different tumor models. Here we identified modified, nonanticoagulant species of heparin that specifically inhibit selectin-mediated cell-cell interactions, heparanase enzymatic activity, or both. We show that selective inhibition of selectin interactions or heparanase with specific heparin derivatives in mouse models of MC-38 colon carcinoma and B16-BL6 melanoma attenuates metastasis. Selectin-specific heparin derivatives attenuated metastasis of MC-38 carcinoma, but heparanase-specific derivatives had no effect, in accordance with the virtual absence of heparanase activity in these cells. Heparin derivatives had no further effect on metastasis in mice deficient in P- and L-selectin, indicating that selectins are the primary targets of heparin antimetastatic activity. Selectin-specific and heparanase-specific derivatives attenuated metastasis of B16-BL6 melanomas to a similar extent. When mice were injected with a derivative containing both heparanase and selectin inhibitory activity, no additional attenuation of metastasis could be observed. Thus, selectin-specific heparin derivatives efficiently attenuated metastasis of both tumor cell types whereas inhibition of heparanase led to reduction of metastasis only in tumor cells producing heparanase.

Brief communication: Preoperative anticoagulant activity after bridging low-molecular-weight heparin for temporary interruption of warfarin.

PubMed

O'Donnell, Martin J; Kearon, Clive; Johnson, Judy; Robinson, Marlene; Zondag, Michelle; Turpie, Irene; Turpie, Alexander G

2007-02-06

Preoperative low-molecular-weight heparin (LMWH) is often used when warfarin therapy is interrupted for surgery. To determine the preoperative anticoagulant activity of LMWH following a standardized "bridging" regimen. Prospective cohort study. Single university hospital. Consecutive patients who had warfarin therapy interrupted before an invasive procedure. Enoxaparin, 1 mg/kg of body weight, twice daily. The last dose was administered the evening before surgery. Blood anti-factor Xa heparin levels measured shortly before surgery. Preoperative anti-Xa heparin levels were obtained in 80 patients at an average of 14 hours after the last dose of enoxaparin was administered. The average anti-Xa heparin level was 0.6 U/mL. The anti-Xa heparin level, measured shortly before surgery, was 0.5 U/mL or greater in 54 (68%) patients and 1.0 U/mL or greater in 13 (16%) patients. A shorter interval since the last dose (P < 0.001) and a higher body mass index (P = 0.001) were associated with higher preoperative anti-Xa heparin levels. The small sample size limits accurate estimates of the frequency of the clinical outcomes. A single regimen of LMWH was evaluated. Anti-Xa heparin levels often remain high at the time of surgery if a last dose of a twice-daily regimen of LMWH is given the evening before surgery.

Characterization of currently marketed heparin products: key tests for LMWH quality assurance.

PubMed

Ye, Hongping; Toby, Timothy K; Sommers, Cynthia D; Ghasriani, Houman; Trehy, Michael L; Ye, Wei; Kolinski, Richard E; Buhse, Lucinda F; Al-Hakim, Ali; Keire, David A

2013-11-01

During the 2007-2008 heparin crisis it was found that the United States Pharmacopeia (USP) testing monograph for heparin sodium or low molecular weight heparins did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS). In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to detect not only the contaminant OSCS, but also to improve assurance of quality and purity of these drug products. The USP monographs for the low molecular weight heparins (LMWHs) approved for use in the United States (dalteparin, tinzaparin and enoxaparin) are also undergoing revision to include many of the same tests used for heparin sodium, including; one-dimensional (1D) 500 MHz (1)H NMR, SAX-HPLC, percent galactosamine in total hexosamine and anticoagulation time assays with purified Factor IIa or Factor Xa. These tests represent orthogonal approaches for heparin identification, measurement of bioactivity and for detection of process impurities or contaminants in these drug products. Here we describe results from a survey of multiple lots from three types of LMWHs in the US market which were collected after the 2009 heparin sodium monograph revision. In addition, innovator and generic versions of formulated enoxaparin products purchased in 2011 are compared using these tests and found to be highly similar within the discriminating power of the assays applied. Published by Elsevier B.V.

The effect of controlled release of PDGF-BB from heparin-conjugated electrospun PCL/gelatin scaffolds on cellular bioactivity and infiltration

PubMed Central

Lee, Jongman; Yoo, James J.; Atala, Anthony; Lee, Sang Jin

2013-01-01

Heparin-conjugated electrospun poly(ε-caprolactone) (PCL)/gelatin scaffolds were developed to provide controlled release of platelet-derived growth factor-BB (PDGF-BB) and allow prolonged bioactivity of this molecule. A mixture of PCL and gelatin was electrospun into three different morphologies. Next, heparin molecules were conjugated to the reactive surface of the scaffolds. This heparin-conjugated scaffold allowed the immobilization of PDGF-BB via electrostatic interaction. In vitro PDGF-BB release profiles indicated that passive physical adsorption of PDGF-BB to non-heparinized scaffolds resulted in an initial burst release of PDGF-BB within 5 days, which then leveled off. However, electrostatic interaction between PDGF-BB and the heparin-conjugated scaffolds gave rise to a sustained release of PDGF-BB over the course of 20 days without an initial burst. Moreover, PDGF-BB that was strongly bound to the heparin-conjugated scaffolds enhanced smooth muscle cell (SMC) proliferation. In addition, scaffolds composed of 3.0 µm diameter fibers that were immobilized with PDGF-BB accelerated SMC infiltration into the scaffold when compared to scaffolds composed of smaller diameter fibers or scaffolds that did not release PDGF-BB. We concluded that the combination of the large pore structure in the scaffolds and the heparin-mediated delivery of PDGF-BB provided the most effective cellular interactions through synergistic physical and chemical cues. PMID:22770570

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis.

PubMed

Waern, Ida; Karlsson, Iulia; Thorpe, Michael; Schlenner, Susan M; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Åbrink, Magnus; Hellman, Lars; Pejler, Gunnar; Wernersson, Sara

2012-12-01

Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

NASA Astrophysics Data System (ADS)

Minsky, Burcu Baykal; Dubin, Paul L.; Kaltashov, Igor A.

2017-04-01

The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions.

Rationale and design of the HepZero study: a prospective, multicenter, international, open, randomized, controlled clinical study with parallel groups comparing heparin-free dialysis with heparin-coated dialysis membrane (Evodial) versus standard care: study protocol for a randomized controlled trial.

PubMed

Rossignol, Patrick; Dorval, Marc; Fay, Renaud; Ros, Joan Fort; Loughraieb, Nathalie; Moureau, Frédérique; Laville, Maurice

2013-06-01

Anticoagulation for chronic dialysis patients with contraindications to heparin administration is challenging. Current guidelines state that in patients with increased bleeding risks, strategies that can induce systemic anticoagulation should be avoided. Heparin-free dialysis using intermittent saline flushes is widely adopted as the method of choice for patients at risk of bleeding, although on-line blood predilution may also be used. A new dialyzer, Evodial (Gambro, Lund, Sweden), is grafted with unfractionated heparin during the manufacturing process and may allow safe and efficient heparin-free hemodialysis sessions. In the present trial, Evodial was compared to standard care with either saline flushes or blood predilution. The HepZero study is the first international (seven countries), multicenter (10 centers), randomized, controlled, open-label, non-inferiority (and if applicable subsequently, superiority) trial with two parallel groups, comprising 252 end-stage renal disease patients treated by maintenance hemodialysis for at least 3 months and requiring heparin-free dialysis treatments. Patients will be treated during a maximum of three heparin-free dialysis treatments with either saline flushes or blood predilution (control group), or Evodial. The first heparin-free dialysis treatment will be considered successful when there is: no complete occlusion of air traps or dialyzer rendering dialysis impossible; no additional saline flushes to prevent clotting; no change of dialyzer or blood lines because of clotting; and no premature termination (early rinse-back) because of clotting.The primary objectives of the study are to determine the effectiveness of the Evodial dialyzer, compared with standard care in terms of successful treatments during the first heparin-free dialysis. If the non-inferiority of Evodial is demonstrated then the superiority of Evodial over standard care will be tested. The HepZero study results may have major clinical implications for patient care. ClinicalTrials.gov NCT01318486.

Bivalirudin is associated with improved clinical and economic outcomes in heart failure patients undergoing percutaneous coronary intervention: Results from an observational database.

PubMed

Pinto, Duane S; Kohli, Payal; Fan, Weihong; Kirtane, Ajay J; Kociol, Robert D; Meduri, Christopher; Deliargyris, Efthymios N; Prats, Jayne; Reynolds, Matthew R; Stone, Gregg W; Michael Gibson, C

2016-02-15

Outcomes with bivalirudin compare favorably with heparin ± GPIIb/IIIa receptor inhibition (heparin ± GPI) during percutaneous coronary intervention (PCI). Patients with congestive heart failure (CHF) have increased risk for complications. The objective was to investigate clinical and economic outcomes for bivalirudin ± GPI vs. heparin ± GPI among PCI patients with CHF. Using the Premier Hospital Database, PCI patients with CHF were stratified by anticoagulant: bivalirudin, bivalirudin ± GPI, heparin and heparin ± GPI. The probability of receiving bivalirudin ± GPI was estimated using individual and hospital variables. Using propensity scores, each bivalirudin ± GPI patient was matched to a heparin ± GPI patient. The primary outcome was in-hospital death. Bleeding rates, transfusion, length of stay and in-hospital cost were ascertained. Overall, 116,313 patients at 315 hospitals received bivalirudin (n = 45,559) bivalirudin + GPI (n = 8,115), heparin (n = 27,972) or heparin + GPI (n = 34,667). Patients had STEMI (21.2%), NSTEMI (29.1%), unstable angina (16.6%), stable angina (5.7%) or other ischemic heart disease (24.2%). Of these, 79.1% of bivalirudin patients matched, resulting in 84,948 analyzed patients. Compared with heparin ± GPI patients, bivalirudin ± GPI patients had fewer deaths (3.3% vs. 3.9%; p < 0.0001), less clinically apparent bleeding (10.2% vs. 11.4%; p < 0.0001), clinically apparent bleeding with transfusion (2.7% vs. 3.2%, p <0.0001), and transfusion (8.5% vs. 9.8%, p < 0.0001). Patients receiving bivalirudin had shorter length of stay (6.3 vs. 6.8 days; p < 0.0001) and lower in-hospital cost (mean $26,706 vs. $27,166 [median $19,414 vs. $19,798]; p < 0.0001). In conclusion, this is the largest retrospective analysis of PCI patients with CHF and demonstrates bivalirudin ± GPI compared with heparin ± GPI is associated with lower inpatient rates of death, bleeding, and cost. © 2015 Wiley Periodicals, Inc.

Necessity of heparin for maintaining peripheral venous catheters: A systematic review and meta-analysis

PubMed Central

You, Tao; Jiang, Jianliang; Chen, Jianchang; Xu, Weiting; Xiang, Li; Jiao, Yang

2017-01-01

Heparin has typically been used as a flushing or infusion solution for vascular lines in daily practice. However, several clinical trials have yielded controversial results about the benefits of heparin in maintaining peripheral venous catheters. The present meta-analysis was conducted to evaluate the efficacy of heparin on the patency profiles and complications in peripheral intravenous catheters. PubMed, Embase and Cochrane Central Register of Controlled Trials were searched up to February 2016 for randomized controlled trials comparing heparin with placebo in maintaining peripheral intravenous catheters. Additional studies were retrieved from the reference lists of identified articles. In total 32 eligible studies were included, from which the pooled standard mean difference (SMD), relative risk (RR) and corresponding 95% confidence interval (CI) were calculated. The use of heparin as a continuous infusion significantly prolonged the duration of patency (SMD, 0.90; 95% CI, 0.48–1.32; P<0.001), reduced rates of infusion failure (RR, 0.83; 95% CI, 0.76–0.92; P<0.001) and occlusion (RR, 0.82; 95% CI, 0.69–0.98; P<0.05) in a peripheral intravenous catheter. However, there were no significant changes in the duration of patency and infusion failure when heparin was used intermittently as a flushing solution, although a significantly decreased risk of occlusion was observed in this setting (RR, 0.80; 95% CI, 0.66–0.98; P<0.05). Furthermore, the risk of phlebitis was significantly decreased by both continuous infusion (RR, 0.66; 95% CI, 0.58–0.75; P<0.01) and intermittent flushing (RR, 0.70; 95% CI, 0.56–0.86; P<0.01) of heparin in peripheral venous catheters. In conclusion, the use of heparin as continuous infusion in peripheral intravenous catheters improved the duration of patency, reduced infusion failure and phlebitis, whereas heparin as intermittent flushing showed more benefits in ameliorating phlebitis rather than in patency profiles. PMID:28810636

Aspirin or heparin or both in the treatment of recurrent spontaneous abortion in women with antiphospholipid antibody syndrome: a meta-analysis of randomized controlled trials.

PubMed

Lu, Chang; Liu, Yong; Jiang, Hai-Li

2018-01-10

This study was designed to evaluate the efficacy of aspirin or heparin or both in the treatment for recurrent spontaneous abortion (RSA) in women with antiphospholipid antibody syndrome (APS). Systematic searches for randomized clinical trials (RCTs) evaluating on live birth and preterm delivery, preeclampsia, intrauterine growth restriction, gestational diabetes, bleeding of RSA with APS patients receiving aspirin, and heparin therapy were carried out, from PubMed, EMBASE, ScienceDirect, and CNKI. Related data were extracted from eligible studies and then subjected to Reviewer Manage 5.3 for analysis. Relative risk (RR) and its 95% confidence interval were calculated. Nineteen publications with randomized controlled trials were selected for this study, which included a total of 1251 pregnant patients with diagnosis of RSA with APS. With respect to live birth, it was remarkably improved in aspirin plus heparin or heparin alone group [RR =1.23, 95% CI (1.12-1.36), p < .0001; RR = 1.18, 95% CI (1.03-1.35), p = .02]; aspirin alone group, however, there was no statistically significant difference compare to placebo [RR = 0.97, 95% CI (0.80-1.16), p = .71]. Meanwhile, aspirin plus heparin therapy did not significantly reduce the risk of recurrent placenta-mediated pregnancy complications including preterm birth, intrauterine growth retardation (IUGR), gestational diabetes, and minor bleeding. A beneficial therapeutic effect of heparin alone therapy was found on preventing preterm birth and low-dose aspirin plus heparin therapy was significant reduce the risk of preeclampsia. An improvement of pregnancy outcomes in women with RSA and APS can be achieved by treatment strategies combining low-dose aspirin plus heparin or heparin alone. Aspirin alone, by contrast, seemed inferior to other treatments in achieving more live birth.

Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration.

PubMed

Fu, Li; Zhang, Fuming; Li, Guoyun; Onishi, Akihiro; Bhaskar, Ujjwal; Sun, Peilong; Linhardt, Robert J

2014-05-01

The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Heparin Stimulates Elastogenesis: Application to Silk-Based Vascular Grafts

PubMed Central

Baughman, Cassandra; Kaplan, David L.; Castellot, John J.

2013-01-01

With over 500,000 coronary artery bypass grafts (CABG) performed annually in the United States alone, there is a significant clinical need for a small diameter tissue engineered vascular graft. A principle goal in tissue engineering is to develop materials and growth conditions that encourage appropriate re-cellularization and extracellular matrix formation in vivo. A particular challenge in vascular tissue engineering results from the inability of adult cells to produce elastin, as its expression is developmentally limited. We investigated factors to stimulate elastogenesis in vitro, and found that heparin treatment of adult human vascular smooth muscle cells promoted the formation of elastic fibers. This effect was heparin-specific, and dependent on cell density and growth state. We then applied this information to a silk-based construct, and found that immobilized heparin showed essentially identical biological effects to that of soluble heparin. These findings indicate that heparinized vascular grafts may promote elastin formation and regulate restenosis, in addition to heparin’s well-established antithrombotic properties. Given the increase in elastin mRNA level and the increase in extracellular elastin present, our data suggests that there may be multiple levels of elastin regulation that are mediated by heparin treatment. PMID:21600981

Preparation and in vitro evaluation of heparin-loaded polymeric nanoparticles.

PubMed

Jiao, Y Y; Ubrich, N; Marchand-Arvier, M; Vigneron, C; Hoffman, M; Maincent, P

2001-01-01

Nanoparticles of a highly soluble macromolecular drug, heparin, were formulated with two biodegradable polymers (poly-E-caprolactone [PCL] and poly (D, L-lactic-co-glycolic-acid) 50/50 [PLAGA]) and two nonbiodegradable positively charged polymers (Eudragit RS and RL) by the double emulsion and solvent evaporation method, using a high-pressure homogenization device. The encapsulation efficiency and heparin release profiles were studied as a function of the type of polymers employed (alone or in combination) and the concentration of heparin. Optimal encapsulation efficiency was observed when 5000 IU of heparin were incorporated in the first emulsion. High drug entrapment efficiency was observed in both Eudragit RS and RL nanoparticles (60% and 98%, respectively), compared with PLAGA and PCL nanoparticles (<14%). The use of the two types of Eudragit in combination with PCL and PLAGA increased the encapsulation efficiency compared with these two biodegradable polymers used alone; however, the in vitro drug release was not modified and remained low. On the other hand, the addition of esterase to the dissolution medium resulted in a significant increase in heparin release. The in vitro biological activity of released heparin, evaluated by measuring the anti-Xa activity by a colorimetric assay, was conserved after the encapsulation process.

Heparin-based hydrogels with tunable sulfation & degradation for anti-inflammatory small molecule delivery.

PubMed

Peng, Yifeng; Tellier, Liane E; Temenoff, Johnna S

2016-08-16

Sustained release of anti-inflammatory agents remains challenging for small molecule drugs due to their low molecular weight and hydrophobicity. Therefore, the goal of this study was to control the release of a small molecule anti-inflammatory agent, crystal violet (CV), from hydrogels fabricated with heparin, a highly sulfated glycosaminoglycan capable of binding positively-charged molecules such as CV. In this system, both electrostatic interactions between heparin and CV and hydrogel degradation were tuned simultaneously by varying the level of heparin sulfation and varying the amount of dithiothreitol within hydrogels, respectively. It was found that heparin sulfation significantly affected CV release, whereby more sulfated heparin hydrogels (Hep and Hep(-N)) released CV with near zero-order release kinetics (R-squared values between 0.96-0.99). Furthermore, CV was released more quickly from fast-degrading hydrogels than slow-degrading hydrogels, providing a method to tune total CV release between 5-15 days while maintaining linear release kinetics. In particular, N-desulfated heparin hydrogels exhibited efficient CV loading (∼90% of originally included CV), near zero-order CV release kinetics, and maintenance of CV bioactivity after release, making this hydrogel formulation a promising CV delivery vehicle for a wide range of inflammatory diseases.

NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

PubMed

Higai, Koji; Imaizumi, Yuzo; Suzuki, Chiho; Azuma, Yutaro; Matsumoto, Kojiro

2009-09-04

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

[The percutaneous effect of a heparin-allantoin-dexpanthenol combination in a specific ointment base. Anti-allergic, anti-inflammatory effect in the PCA test in the rat].

PubMed

Rudolph, C; Tauschel, H D

1984-01-01

Local application of a heparin-allantoin-dexpanthenol (Hepathrombin-Adenylchemie) ointment to rats 15 min prior to induction of a passive cutaneous anaphylaxis (PCA) reaction inhibits the anaphylactic reaction as compared to the ointment base. Also, the Evans Blue content as a measure for vascular permeability and the oedema weights are reduced under the heparin containing ointment. The antiallergic/antiinflammatory effect is probably due to heparin.

Low-molecular-weight heparins: differential characterization/physical characterization.

PubMed

Guerrini, Marco; Bisio, Antonella

2012-01-01

Low-molecular-weight heparins (LMWHs), derived from unfractionated heparin (UFH) through different depolymerization processes, have advantages with respect to the parent heparin in terms of pharmacokinetics, convenience of administration, and reduced side effects. Each LMWH can be considered as an independent drug with its own activity profile, placing significance on their biophysical characterization, which will also enable a better understanding of their structure-function relationship. Several chemical and physical methods, some involving sample modification, are now available and are reviewed.

[Intensive care of children with DIC syndrome based on the use of low molecular weight heparins].

PubMed

Chuprova, A V; Shmakov, A N; Solov'ev, O N; Anokhina, T Iu; Loskutova, S A; Pinegina, Iu S

2002-01-01

The first section of this paper presents data on low-molecular-weight heparins: pharmacokinetics and pharmacodynamics, advantages in comparison with common heparin. The second section presents the results of fraxiparin and clivarin use in 43 children aged 9 months to 14 years with acute/subacute DIC syndrome of infectious origin. Therapeutic and maintenance doses, the mode of injection of low-molecular-weight heparins, and methods for laboratory monitoring of their efficiency and safety are presented.

A comparative analysis of warfarin and low-dose heparin as thromboembolism prophylaxis in total hip replacement patinets.

PubMed Central

Ritter, M A; HamiltonCW

1975-01-01

Warfarin, low-dose heparin, or a combination of low-dose heparin and hydrocortisone was administered to 300 patients undergoing total hip replacement. The lowest incidence of thromboembolic (5 per cent) was attained with Warfarin. Further investigation into the method of administration of low-dose heparin is necessary before it can be used effectively as thromboembolism prophylaxis in total hip replacement patients. The addition of hydrocortisone was not found useful. PMID:1138642

Ultrasensitive colorimetric detection of heparin based on self-assembly of gold nanoparticles on graphene oxide.

PubMed

Fu, Xiuli; Chen, Lingxin; Li, Jinhua

2012-08-21

A novel colorimetric method was developed for ultrasensitive detection of heparin based on self-assembly of gold nanoparticles (AuNPs) onto the surface of graphene oxide (GO). Polycationic protamine was used as a medium for inducing the self-assembly of citrate-capped AuNPs on GO through electrostatic interaction, resulting in a shift in the surface plasmon resonance (SPR) absorption of AuNPs and exhibiting a blue color. Addition of polyanionic heparin disturbed the self-assemble of AuNPs due to its strong affinity to protamine. With the increase of heparin concentration, the amounts of self-assembly AuNPs decreased and the color changed from blue to red in solution. Therefore, a "blue-to-red" colorimetric sensing strategy based on self-assembly of AuNPs could be established for heparin detection. Compared with the commonly reported aggregation-based methods ("red-to-blue"), the color change from blue to red was more eye-sensitive, especially in low concentration of target. Moreover, stronger interaction between protamine and heparin led to distinguish heparin from its analogues as well as various potentially coexistent physiological species. The strategy was simply achieved by the self-assembly nature of AuNPs and the application of two types of polyionic media, showing it to be label-free, simple, rapid and visual. This method could selectively detect heparin with a detection limit of 3.0 ng mL(-1) in standard aqueous solution and good linearity was obtained over the range 0.06-0.36 μg mL(-1) (R = 0.9936). It was successfully applied to determination of heparin in fetal bovine serum samples as low as 1.7 ng mL(-1) with a linear range of 0-0.8 μg mL(-1).

Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineered heparin

PubMed Central

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A.; Esko, Jeffrey D.; Linhardt, Robert J.; Sharfstein, Susan T.

2012-01-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary. PMID:22326251

Improvement in hemocompatibility of chitosan/soy protein composite membranes by heparinization.

PubMed

Wang, Xiaomei; Shi, Na; Chen, Yan; Li, Chen; Du, Xinshen; Jin, Weihua; Chen, Yun; Chang, Peter R

2012-01-01

To improve the hemocompatibility of chitosan/soy protein isolate composite membranes by heparinization. Chitosan/soy protein isolate membranes (ChS-n, n=0, 10 and 30, corresponding to the soy protein isolate content in the membranes) and heparinized ChS-n membranes (HChS-n) were prepared by blending in dilute HAc/NaAc solution. The hemocompatibility of ChS-n and HChS-n membranes were comparatively evaluated by measuring surface heparin density, blood platelet adhesion, plasma recalcification time (PRT), thrombus formation and hemolysis assay. The surface heparin density analysis showed that heparinized chitosan/SPI soy protein isolate membranes have been successfully prepared by blending. The density of heparin on the surface of HChS-n membranes was in the range of 0.67-1.29 μg/cm2. The results of platelet adhesion measurement showed that the platelet adhesion numbers of HChS-n membranes were lower than those of the corresponding ChS-n membranes. The PRT of the HChS-0, HChS-10 and HChS-30 membranes were around 292, 306 and 295 s, respectively, which were longer than the corresponding ChS-0 (152 s), ChS-10 (204 s) and ChS-30 (273 s) membranes. The hemolysis rate of HChS-n membranes was lower than 1%. The hemocompatibility of ChS membranes could be improved by blending with heparin. Compared with ChS membranes, HChS membranes showed lower platelet adhesion, longer PRT, higher BCI, significant thromboresistivity and a lower hemolysis rate due to the heparinization. This widens the application of chitosan and soy protein-based biomaterials that may come in contact with blood.

Human IGF-I propeptide A promotes articular chondrocyte biosynthesis and employs glycosylation-dependent heparin binding.

PubMed

Shi, Shuiliang; Kelly, Brian J; Wang, Congrong; Klingler, Ken; Chan, Albert; Eckert, George J; Trippel, Stephen B

2018-03-01

Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis. Copyright © 2017 Elsevier B.V. All rights reserved.

Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin.

PubMed

Baik, Jong Youn; Gasimli, Leyla; Yang, Bo; Datta, Payel; Zhang, Fuming; Glass, Charles A; Esko, Jeffrey D; Linhardt, Robert J; Sharfstein, Susan T

2012-03-01

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary. Copyright © 2012 Elsevier Inc. All rights reserved.

Glycosaminoglycan-functionalized poly-lactide-co-glycolide nanoparticles: synthesis, characterization, cytocompatibility, and cellular uptake

PubMed Central

Lamichhane, Surya P; Arya, Neha; Ojha, Nirdesh; Kohler, Esther; Shastri, V Prasad

2015-01-01

The efficient delivery of chemotherapeutics to the tumor via nanoparticle (NP)-based delivery systems remains a significant challenge. This is compounded by the fact that the tumor is highly dynamic and complex environment composed of a plurality of cell types and extracellular matrix. Since glycosaminoglycan (GAG) production is altered in many diseases (or pathologies), NPs bearing GAG moieties on the surface may confer some unique advantages in interrogating the tumor microenvironment. In order to explore this premise, in the study reported here poly-lactide-co-glycolide (PLGA) NPs in the range of 100–150 nm bearing various proteoglycans were synthesized by a single-step nanoprecipitation and characterized. The surface functionalization of the NPs with GAG moieties was verified using zeta potential measurements and X-ray photoelectron spectroscopy. To establish these GAG-bearing NPs as carriers of therapeutics, cellular toxicity assays were undertaken in lung epithelial adenocarcinoma (A549) cells, human pulmonary microvascular endothelial cells (HPMEC), and renal proximal tubular epithelial cells. In general NPs were well tolerated over a wide concentration range (100–600 μg/mL) by all cell types and were taken up to appreciable extents without any adverse cell response in A549 cells and HPMEC. Further, GAG-functionalized PLGA NPs were taken up to different extents in A459 cells and HPMEC. In both cell systems, the uptake of heparin-modified NPs was diminished by 50%–65% in comparison to that of unmodified PLGA. Interestingly, the uptake of chondroitin sulfate NPs was the highest in both cell systems with 40%–60% higher uptake when compared with that of PLGA, and this represented an almost twofold difference over heparin-modified NPs. These findings suggest that GAG modification can be explored as means of changing the uptake behavior of PLGA NPs and these NP systems have potential in cancer therapy. PMID:25632234

Delayed-onset heparin-induced skin necrosis: a rare complication of perioperative heparin therapy.

PubMed

Gan, Weh Kiat

2017-11-03

An uncommon case of delayed-onset dalteparin-induced skin necrosis in an 83-year-old Caucasian female patient associated with heparin-induced thrombocytopaenia (HIT) presenting on day 30 following dalteparin therapy is reported. Investigations revealed mild thrombocytopaenia with normal protein C, protein S, coagulation screen and positive test for heparin-platelet factor-4 antibody. Clinical diagnosis of heparin-induced skin necrosis with HIT was made. Dalteparin injection was discontinued promptly and substituted with fondaparinux therapy. The patient achieved good recovery following cessation of dalteparin therapy and was subsequently discharged. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

History of drugs for thrombotic disease. Discovery, development, and directions for the future.

PubMed

Mueller, R L; Scheidt, S

1994-01-01

The history of the antithrombotic agents--aspirin, heparin, warfarin, and the thrombolytics--is a rich and lively odyssey of serendipity, perseverance, vision, and conflict involving a number of striking personalities. The history of aspirin spans ages and continents from Hippocrates' analgesic for women in labor to the rediscovery of the white willow bark by English country scholar Reverend Edward Stone. Bayer chemist Felix Hoffmann reinvented aspirin for his ailing father; suburban physician L.L. Craven pioneered the prophylactic antithrombotic uses of aspirin; and Sir John Vane elucidated aspirin's mechanism of action as the inhibition of prostaglandin synthetase. Heparin was discovered by McLean, working as a medical student in 1915 in search of a pure procoagulant in dog liver. His original impure material differed somewhat from today's heparin, but purified heparin was rapidly accepted for a myriad of clinical uses; to this day, diverse new properties of this complex glycosaminoglycan continue to be elucidated. The oral anticoagulants emerged from veterinary research in the 1920s on a hemorrhagic disorder afflicting cattle that consumed spoiled sweet clover hay. Several chance encounters led Karl Link and his University of Wisconsin team to the identification of dicumarol as the offending agent in 1939 and its widespread therapeutic use by Wright and others in the 1940s. Link later developed warfarin as a rodenticide, but its use in humans soon followed in the 1950s. Vitamin K was discovered in the 1930s; its involvement in the mechanism of the anticoagulant agents was not delineated until the 1970s. The intrinsic ability of clotted blood to liquify and the fibrinolytic properties of normal urine were noted in the 1800s. Tillett and Sherry's group stumbled on the fibrinolytic properties of streptokinase in the 1930s and pioneered the therapeutic use of streptokinase in the 1940s and of urokinase in the 1960s. Several teams found tissue-type plasminogen activator in various body sites beginning in the 1940s, leading to its cloning and widespread use in the 1980s; anisoylated plasminogen-streptokinase activator complex is an example of rational drug design. The discoverers of these diverse agents have not only provided physicians with a potent armamentarium of antithrombotic drugs but also helped elucidate much basic science and vividly demonstrated the merits of perseverance, independent thought, and adherance to the scientific method.

Can we produce heparin/heparan sulfate biomimetics using "mother-nature" as the gold standard?

PubMed

Farrugia, Brooke L; Lord, Megan S; Melrose, James; Whitelock, John M

2015-03-05

Heparan sulfate (HS) and heparin are glycosaminoglycans (GAGs) that are heterogeneous in nature, not only due to differing disaccharide combinations, but also their sulfate modifications. HS is well known for its interactions with various growth factors and cytokines; and heparin for its clinical use as an anticoagulant. Due to their potential use in tissue regeneration; and the recent adverse events due to contamination of heparin; there is an increased surge to produce these GAGs on a commercial scale. The production of HS from natural sources is limited so strategies are being explored to be biomimetically produced via chemical; chemoenzymatic synthesis methods and through the recombinant expression of proteoglycans. This review details the most recent advances in the field of HS/heparin synthesis for the production of low molecular weight heparin (LMWH) and as a tool further our understanding of the interactions that occur between GAGs and growth factors and cytokines involved in tissue development and repair.

Heparin-coated extracorporeal circulation systems in heart surgery.

PubMed

Tagarakis, Georgios I; Tsilimingas, Nikolaos B

2009-11-01

Despite the progress accomplished in the field of off-pump heart surgery, the vast majority of cardiac operations are still performed with the use of extracorporeal circulation, otherwise known as "heart-lung machine." This valuable tool, however, is connected with various complications, partly deriving from the application of intravenous heparin, necessary for the extracorporeal circuits to function. In order to deal with these complications, which among others include postoperative hemorrhage and systemic inflammatory response, several extracorporeal circulation systems, which contain a heparin-coating on their blood-contacting surfaces, have been developed with patents. The philosophy behind the creation of these systems is that with the controlled absorption and interaction of this heparin with the blood elements, adequate intraoperative anticoagulation with lower doses of systemic heparin and fewer systemic complications can be achieved. The idea of the use of heparin coatings has also been applied in other settings, such as in renal dialysis catheters, ECMO (extracorporeal membrane oxygenation), MECC (minimized extracorporeal circulation) and left ventricle assist devices.

Interactions of oversulfated chondroitin sulfate (OSCS) from different sources with unfractionated heparin.

PubMed

Gray, Angel; Litinas, Evangelos; Jeske, Walter; Fareed, Jawed; Hoppensteadt, Debra

2012-01-01

In 2008, oversulfated chondroitin sulfate (OSCS) was identified as the main contaminant in recalled heparin. Oversulfated chondroitin sulfate can be prepared from bovine (B), porcine (P), shark (Sh), or skate (S) origin and may produce changes in the antithrombotic, bleeding, and hemodynamic profile of heparins. This study examines the interactions of various OSCSs on heparin in animal models of thrombosis and bleeding, as well as on the anticoagulant and antiprotease effects in in vitro assays. Mixtures of 70% unfractionated heparin (UFH) with 30% OSCS from different sources were tested. In the in vitro activated partial thromboplastin time (aPTT) assay, all contaminant mixtures showed a decrease in clotting times. In addition, a significant increase in bleeding time compared to the control (UFH/saline) was observed. In the thrombosis model, no significant differences were observed. The OSCSs significantly increased anti-Xa activity in ex vivo blood samples. These results indicate that various sources of OSCS affect the hemostatic properties of heparin.

Monitoring of heparin concentration in serum by Raman spectroscopy within hollow core photonic crystal fiber

NASA Astrophysics Data System (ADS)

Khetani, Altaf; Tiwari, Vidhu S.; Harb, Alaa; Anis, Hanan

2011-08-01

The feasibility of using hollow core photonic crystal fiber (HC-PCF) in conjunction with Raman spectroscopy has been explored for real time monitoring of heparin concentration in serum. Heparin is an important blood anti-coagulant whose precise monitoring and controlling in patients undergoing cardiac surgery and dialysis is of utmost importance. Our method of heparin monitoring offers a novel alternative to existing clinical procedures in terms of accuracy, response time and sample volume. The optical design configuration simply involves a 785-nm laser diode whose light is coupled into HC-PCF filled with heparin-serum mixtures. By non-selectively filling HC-PCF, a strong modal field overlap is obtained. Consequently, an enhanced Raman signal (>90 times) is obtained from various heparin-serum mixtures filled HC-PCFs compared to its bulk counterpart (cuvette). The present scheme has the potential to serve as a `generic biosensing tool' for diagnosing a wide range of biological samples.

Comparison of hematologic values in blood samples with lithium heparin or dipotassium ethylenediaminetetraacetic acid anticoagulants in Hispaniolan Amazon parrots (Amazona ventralis).

PubMed

Guzman, David Sanchez-Migallon; Mitchell, Mark A; Gaunt, Stephen D; Beaufrère, Hugues; Tully, Thomas N

2008-06-01

Blood samples were collected from 20 Hispaniolan Amazon parrots (Amazona ventralis) and were divided into tubes that contained dipotassium ethylenediaminetetraacetic acid (K2EDTA) and lithium heparin. Complete blood cell counts were determined in each sample within 2 hours of collection. The level of agreement in results was moderate for plasma protein, packed cell volume (PCV), and leukocyte, monocyte, and lymphocyte counts between the anticoagulants. Plasma protein and PCV values were significantly lower in samples with lithium heparin than in those with K2EDTA, whereas lymphocyte numbers were significantly higher in lithium heparin samples than in K2EDTA samples. The level of agreement was good for the other cell types (heterophils, eosinophils, and basophils) when comparing the different anticoagulants. The poor level of agreement between anticoagulants with the increase in thrombocyte clumping in lithium heparin samples indicates that the use of lithium heparin as anticoagulant may affect thrombocyte count. No negative effects on morphology and staining of blood cells were apparent in smears from heparin samples compared with K2EDTA samples. Within the different values compared, the limits of agreement are small enough to be confident that lithium heparin can be used for routine CBC counts in a clinical setting. The use of the same anticoagulant should be recommended to follow trends within the same patient, especially when considering plasma protein concentration, PCV, and lymphocyte count.

The economic impact of enoxaparin versus unfractionated heparin for prevention of venous thromboembolism in acute ischemic stroke patients

PubMed Central

Pineo, Graham F; Lin, Jay; Annemans, Lieven

2012-01-01

Venous thromboembolism (VTE) is a common complication after acute ischemic stroke that can be prevented by the use of anticoagulants. Current guidelines from the American College of Chest Physicians recommend that patients with acute ischemic stroke and restricted mobility receive prophylactic low-dose unfractionated heparin or a low-molecular-weight heparin. Results from clinical studies, most recently from PREVAIL (PREvention of Venous Thromboembolism After Acute Ischemic Stroke with LMWH and unfractionated heparin), suggest that the low-molecular-weight heparin, enoxaparin, is preferable to unfractionated heparin for VTE prophylaxis in patients with acute ischemic stroke and restricted mobility. This is due to a better clinical benefit-to-risk ratio, with the added convenience of once-daily administration. In line with findings from modeling studies and real-world data in acutely ill medical patients, recent economic data indicate that the higher drug cost of enoxaparin is offset by the reduction in clinical events as compared with the use of unfractionated heparin for the prevention of VTE after acute ischemic stroke, particularly in patients with severe stroke. With national performance measures highlighting the need for hospitals to examine their VTE practices, the relative costs of different regimens are of particular importance to health care decision-makers. The data reviewed here suggest that preferential use of enoxaparin over unfractionated heparin for the prevention of VTE after acute ischemic stroke may lead to reduced VTE rates and concomitant cost savings in clinical practice. PMID:22570556

Membrane type 1-matrix metalloproteinase cleaves off the NH2-terminal portion of heparin-binding epidermal growth factor and converts it into a heparin-independent growth factor.

PubMed

Koshikawa, Naohiko; Mizushima, Hiroto; Minegishi, Tomoko; Iwamoto, Ryo; Mekada, Eisuke; Seiki, Motoharu

2010-07-15

Epidermal growth factor (EGF) receptors (ErbB) and EGF family members represent promising targets for cancer therapy. Heparin-binding EGF (HB-EGF) is a member of the EGF family and is an important target for therapy in some types of human cancers. Processing of HB-EGF by proprotein convertases, and successively, by ADAM family proteases, generates a soluble growth factor that requires heparin as a cofactor. Although heparin potentiates HB-EGF activity in vitro, it is not clear how the heparin-binding activity of HB-EGF is regulated. Here, we show that membrane type 1-matrix metalloproteinase (MT1-MMP; MMP14), a potent invasion-promoting protease, markedly enhances HB-EGF-dependent tumor formation in mice. MT1-MMP additionally cleaves HB-EGF and removes the NH(2)-terminal 20 amino acids that are important for binding heparin. Consequently, the processing of HB-EGF by MT1-MMP converts HB-EGF into a heparin-independent growth factor with enhanced mitogenic activity, and thereby, expression of both proteins costimulates tumor cell growth in vitro and in vivo. The ErbB family of receptors expressed in human gastric carcinoma cells play a role in mediating enhanced HB-EGF activity by MT1-MMP during invasive cell growth in collagen. Thus, we shed light on a new mechanism whereby HB-EGF activity is regulated that should be considered when designing HB-EGF-targeted cancer therapy. (c)2010 AACR.

Cell-surface prion protein interacts with glycosaminoglycans.

PubMed Central

Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

2002-01-01

We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

Improving reliability of chemometric models for authentication of species origin of heparin by switching from 1D to 2D NMR experiments.

PubMed

Monakhova, Yulia B; Fareed, Jawed; Yao, Yiming; Diehl, Bernd W K

2018-05-10

Nuclear magnetic resonance (NMR) spectroscopy is regarded as one of the most powerful and versatile analytical approaches to assure the quality of heparin preparations. In particular, it was recently demonstrated that by using 1 H NMR coupled with chemometrics heparin and low molecular weight heparin (LMWH) samples derived from three major animal species (porcine, ovine and bovine) can be differentiated [Y.B. Monakhova et al. J. Pharm. Anal. 149 (2018) 114-119]. In this study, significant improvement of existing chemometric models was achieved by switching to 2D NMR experiments (heteronuclear multiple-quantum correlation (HMQC) and diffusion-ordered spectroscopy (DOSY)). Two representative data sets (sixty-nine heparin and twenty-two LMWH) belonged to different batches and distributed by different commercial companies were investigated. A trend for animal species differentiation was observed in the principal component analysis (PCA) score plot built based on the DOSY data. A superior model was constructed using HMQC experiments, where individual heparin (LMWH) clusters as well as their blends were clearly differentiated. The predictive power of different classification methods as well as unsupervised techniques (independent components analysis, ICA) clearly proved applicability of the model for routine heparin and LMWH analysis. The switch from 1D to 2D NMR techniques provides a wealth of additional information, which is beneficial for multivariate modeling of NMR spectroscopic data for heparin preparations. Copyright © 2018 Elsevier B.V. All rights reserved.

Effects of heparin fractions on the prevention of skin necrosis resulting from adriamycin extravasation: an experimental study.

PubMed

Askar, Ibrahim; Erbas, M Kemal; Gurlek, Ali

2002-09-01

Extravasation of a chemotherapeutic agent is one of the most frequent complications in cancer patients. Full-thickness skin necrosis often occurs after extravasation. Alternative approaches to treatment are local wound care, elevation, and hypothermia. It was shown that heparin prevents skin necrosis. In this experimental study, the effects of heparin fractions on the prevention of skin necrosis were compared by applying an extravasation model of Adriamycin in rats. Forty Sprague-Dawley male rats weighing 250 to 300 g were used. A total of 0.3 ml doxorubicin hydrochloride was administered subcutaneously to all rats. Ten minutes later, in the control group (group I), 1 ml normal saline was administered subcutaneously. In the first experimental group (group II), 100 U per day heparin sodium was administered in a volume of 1 ml subcutaneously. In the second experimental group (group III), nadroparin calcium (5 anti-Xa U per kilogram per day) was administered. In the third and last experimental group (group IV), dalteparin sodium (5 anti-Xa U per kilogram per day) was administered. All drugs were administered for 2 weeks. Necrotic areas were measured 4 weeks later. Statistical analysis was performed using the Kruskal-Wallis analysis of variance and the Mann-Whitney test. Heparin fractions caused a decreased ulcer rate and size than controls ( < 0.05). There was no superiority among heparin fractions. The authors think that low-molecular weight heparins are preferred, considering the higher risk of bleeding with unfractionated heparin.

Distorted secretory granule composition in mast cells with multiple protease deficiency.

PubMed

Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

2013-10-01

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

The effect of coimmobilizing heparin and fibronectin on titanium on hemocompatibility and endothelialization.

PubMed

Li, Guicai; Yang, Ping; Qin, Wei; Maitz, Manfred F; Zhou, Shuo; Huang, Nan

2011-07-01

Currently available cardiovascular implants, such as heart valves and stents, exhibit suboptimal biocompatibility because of the incomplete endothelialization and sequential thrombosis formation especially after a long-term implantation. To improve the blood compatibility and endothelialization simultaneously and ensure the long-term effect of the cardiovascular implants, a technique of combining electrostatic interaction and coimmobilization was developed to form heparin and fibronectin (Hep/Fn) films on aminosilanized titanium (Ti) surfaces. The Hep/Fn coimmobilized films were stable after immersion in PBS for five days, probed by wettability studies and by the release kinetics of heparin and fibronectin. Blood compatibility tests showed that the coimmobilized Hep/Fn films displayed lower hemolysis rate, prolonged blood coagulation time, higher AT III binding density, less platelets activation and aggregation, and less fibrinogen conformational change compared with Ti surface. Endothelial cells (ECs) seeding and fibronectin bioactivity results showed more attached and proliferated ECs and exposed cell-binding sites on the Hep/Fn immobilized samples than that on Ti surfaces. Thus, the Hep/Fn coimmobilized films kept excellent bioactivity even after immersion in PBS for five days. Systemic evaluation suggests that the coimmobilization of Hep/Fn complex improves the blood compatibility and promotes the endothelialization simultaneously. We envisage that this method will provide a potential and effective selection for biomaterials surface modification of cardiovascular implants. Copyright © 2011 Elsevier Ltd. All rights reserved.

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

21 CFR 864.7525 - Heparin assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells.

PubMed

Feyerabend, Thorsten B; Li, Jin-Ping; Lindahl, Ulf; Rodewald, Hans-Reimer

2006-04-01

Biosynthesis of heparin, a mast cell-derived glycosaminoglycan with widespread importance in medicine, has not been fully elucidated. In biosynthesis of heparan sulfate (HS), a structurally related polysaccharide, HS glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) to L-iduronic acid (IdoA) residues. We have generated Hsepi-null mouse mutant mast cells, and we show that the same enzyme catalyzes the generation of IdoA in heparin and that 'heparin' lacking IdoA shows a distorted O-sulfation pattern.

Heparin Characterization: Challenges and Solutions

NASA Astrophysics Data System (ADS)

Jones, Christopher J.; Beni, Szabolcs; Limtiaco, John F. K.; Langeslay, Derek J.; Larive, Cynthia K.

2011-07-01

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.

Further purification of human pituitary-derived chondrocyte growth factor: heparin-binding and cross-reactivity with antiserum to basic FGF.

PubMed

Too, C K; Murphy, P R; Hamel, A M; Friesen, H G

1987-05-14

The previously described human pituitary-derived chondrocyte growth factor (CGF), mitogenic for rabbit fetal chondrocytes, was found to bind to heparin-Sepharose and was eluted with 1.5M NaCl. Further characterization of CGF demonstrated a molecular weight of 18-20 kD and cross-reactivity with antiserum to synthetic bovine basic fibroblast growth factor (FGF1-24). When human pituitaries were homogenized in 0.15 ammonium sulfate (pH 5.5) and the extract chromatographed on heparin-Sepharose, 98% of the mitogenic activity was adsorbed to heparin and eluted with 3M NaCl. These findings indicate that CGF is closely related or identical to basic FGF and that the bulk of mitogenic activity in the human pituitary extracts binds to heparin.

Biophysical investigations on the interaction of the major bovine seminal plasma protein, PDC-109, with heparin.

PubMed

Sankhala, Rajeshwer S; Damai, Rajani S; Anbazhagan, V; Kumar, C Sudheer; Bulusu, Gopalakrishnan; Swamy, Musti J

2011-11-10

PDC-109, the major bovine seminal plasma protein, binds to sperm plasma membrane and modulates capacitation in the presence of heparin. In view of this, the PDC-109/heparin interaction has been investigated employing various biophysical approaches. Isothermal titration calorimetric studies yielded the association constant and changes in enthalpy and entropy for the interaction at 25 °C (pH 7.4) as 1.92 (±0.2) × 10(5) M(-1), 18.6 (±1.6) kcal M(-1), and 86.5 (±5.1) cal M(-1) K(-1), respectively, whereas differential scanning calorimetric studies indicated that heparin binding results in a significant increase in the thermal stability of PDC-109. The affinity decreases with increase in pH and ionic strength, consistent with the involvement of electrostatic forces in this interaction. Circular dichroism spectroscopic studies indicated that PDC-109 retains its conformational features even up to 70-75 °C in the presence of heparin, whereas the native protein unfolds at about 55 °C. Atomic force microscopic studies demonstrated that large oligomeric structures are formed upon binding of PDC-109 to heparin, indicating an increase in the local density of the protein, which may be relevant to the ability of heparin to potentiate PDC-109 induced sperm capacitation.

Comparison of a priori versus provisional heparin therapy on radial artery occlusion after transradial coronary angiography and patent hemostasis (from the PHARAOH Study).

PubMed

Pancholy, Samir B; Bertrand, Olivier F; Patel, Tejas

2012-07-15

Systemic anticoagulation decreases the risk of radial artery occlusion (RAO) after transradial catheterization and standard occlusive hemostasis. We compared the efficacy and safety of provisional heparin use only when the technique of patent hemostasis was not achievable to standard a priori heparin administration after radial sheath introduction. Patients referred for coronary angiography were randomized in 2 groups. In the a priori group, 200 patients received intravenous heparin (50 IU/kg) immediately after sheath insertion. In the provisional group, 200 patients did not receive heparin during the procedure. After sheath removal, hemostasis was obtained using a TR band (Terumo corporation, Tokyo, Japan) with a plethysmography-guided patent hemostasis technique. In the provisional group, no heparin was given if radial artery patency could be obtained and maintained. If radial patency was not achieved, a bolus of heparin (50 IU/kg) was given. Radial artery patency was evaluated at 24 hours (early RAO) and 30 days after the procedure (late RAO) by plethysmography. Patent hemostasis was obtained in 67% in the a priori group and 74% in the provisional group (p = 0.10). Incidence of RAO remained similar in the 2 groups at the early (7.5% vs 7.0%, p = 0.84) and late (4.5% vs 5.0%, p = 0.83) evaluations. Women, patients with diabetes, patients having not received heparin, and patients without radial artery patency during hemostasis had more RAO. By multivariate analysis, patent radial artery during hemostasis (odds ratio [OR] 0.03, 95% confidence interval [CI] 0.004 to 0.28, p = 0.002) and diabetes (OR 11, 95% CI 3 to 38,p <0.0001) were independent predictors of late RAO, whereas heparin was not (OR 0.45 95% CI 0.13 to 1.54, p = 0.20). In conclusion, our results suggest that maintenance of radial artery patency during hemostasis is the most important parameter to decrease the risk of RAO. In selected cases, provisional use of heparin appears feasible and safe when patent hemostasis is maintained. Copyright © 2012 Elsevier Inc. All rights reserved.

Heparin use in a rat hemorrhagic shock model induces biologic activity in mesenteric lymph separate from shock

PubMed Central

Qin, Yong; Prescott, Lauriston M.; Deitch, Edwin A.; Kaiser, Vicki L.

2011-01-01

Experimental data has shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock (TSS) induces neutrophil activation, cytotoxicity, decreased red blood cell deformability and bone marrow colony growth suppression. These data have lead to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of Multiple Organ Failure (MOF) following THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). HUVEC toxicity was not induced by lymph when alternate anti-coagulants (citrate and EDTA) were used in THS. HUVEC toxicity was induced by lymph after heparin but not saline or citrate injection into TSS and naïve animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein (LPL) and hepatic (HL)) were detected in the plasma and lymph from THS and naïve animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified LPL added to naïve lymph induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models thus necessitating a review of previous work in this field. PMID:21063238

Heparin use in a rat hemorrhagic shock model induces biologic activity in mesenteric lymph separate from shock.

PubMed

Qin, Yong; Prescott, Lauriston M; Deitch, Edwin A; Kaiser, Vicki L

2011-04-01

Experimental data have shown that mesenteric lymph from rats subjected to trauma-hemorrhagic shock (THS) but not trauma-sham shock induces neutrophil activation, cytotoxicity, decreased red blood cell (RBC) deformability, and bone marrow colony growth suppression. These data have led to the hypothesis that gut factors produced from THS enter the systemic circulation via the mesenteric lymphatics and contribute to the progression of multiple organ failure after THS. Ongoing studies designed to identify bioactive lymph agents implicated factors associated with the heparin use in the THS procedure. We investigated if heparin itself was responsible for reported toxicity to human umbilical vein endothelial cells (HUVECs). Human umbilical vein endothelial cell toxicity was not induced by lymph when alternate anticoagulants (citrate and EDTA) were used in THS. Human umbilical vein endothelial cell toxicity was induced by lymph after heparin but not saline or citrate injection into trauma-sham shock and naive animals and was dose dependent. Activities of both heparin-releasable lipases (lipoprotein and hepatic) were detected in the plasma and lymph from THS and naive animals receiving heparin but not citrate or saline. Lymph-induced HUVEC toxicity correlated with lymph lipase activities. Finally, incubation of HUVECs with purified lipoprotein lipase added to naive lymph-induced toxicity in vitro. These data show that heparin, not THS, is responsible for the reported lymph-mediated HUVEC toxicity through its release of lipases into the lymph. These findings can provide alternative explanations for several of the THS effects reported in the literature using heparin models, thus necessitating a review of previous work in this field.

Cost-utility of enoxaparin compared with unfractionated heparin in unstable coronary artery disease

PubMed Central

Nicholson, Tricia; McGuire, Alistair; Milne, Ruairidh

2001-01-01

Background Low molecular weight heparins hold several advantages over unfractionated heparin including convenience of administration. Enoxaparin is one such heparin licensed in the UK for use in unstable coronary artery disease (unstable stable angina and non-Q wave myocardial infarction). In these patients, two large randomised controlled trials and their meta-analysis showed small benefits for enoxaparin over unfractionated heparin at 30–43 days and potentially at one year. We found no relevant published full economic evaluations, only cost studies, one of which was conducted in the UK. The other studies, from the US, Canada and France, are difficult to interpret since their resource use and costs may not reflect UK practice. Methods We aimed to compare the benefits and costs of short-term treatment (two to eight days) with enoxaparin and unfractionated heparin in unstable coronary artery disease. We used published data sources to estimate the incremental cost per quality adjusted life year (QALY), adopting a NHS perspective and using 1998 prices. Results The base case was a 0.013 QALY gain and net cost saving of £317 per person treated with enoxaparin instead of unfractionated heparin. All but one sensitivity analysis showed net savings and QALY gains, the exception (the worst case) being a cost per QALY of £3,305. Best cases were a £495 saving and 0.013 QALY gain, or a £317 saving and 0.014 QALY gain per person. Conclusions Enoxaparin appears cost saving compared with unfractionated heparin in patients with unstable coronary artery disease. However, cost implications depend on local revascularisation practice. PMID:11701090

SPATHOLOBUS SUBERECTUS STEM EXTRACT IMPROVES THE PROTECTIVE EFFECT OF HEPARIN ON CERULEIN-INDUCED PANCREATITIS.

PubMed

Shao, Zhengyi

2017-01-01

The present study evaluates the effect of Spatholobus suberectus stem extract (SS) in the management of pancreatitis alone and in combination with heparin. Pancreatitis was induced pancreatitis by cerulean (50μg/kg, i.p.) five times at an interval of 1 h without any pretreatment of drug. Rats were treated with SS (100 and 200 mg/kg, p. o.) and heparin (150 U/kg, i.p.) alone and in combination for the duration of a week. Later pancreatic weight and blood flow was estimated and different biochemical parameters like concentration of D-dimer and Interleukin 1β (IL-Ιβ) and activity of amylase and lipase were determined in blood of pancreatitis rats. Moreover effect of drug treatment on DNA synthesis and histopathology was also estimated on cerulean induced pancreatitis rats. Results of this study suggest that treatment with SS alone and in combination with heparin significantly increase in prothrombin time and pancreatic blood flow than negative control group. There was significant decrease in concentration of IL-Ιβ and D-dimer and activity of amylase and lipase in SS and heparin treated group than negative control group. Pancreatic DNA synthesis was also found to be reduced in SS and heparin alone and in combination treated group. Histopathology study also reveals that treatment with SS and heparin alone and in combination reduces edema, hemorrhages, leukocyte infiltration in the TS of pancreatic tissues. Present study concludes that treatment with SS alone effectively manages the pancreatitis by ceasing the inflammatory pathway and potentiates the effect of heparin in the management of pancreatitis.

Polyguluronate sulfate, polymannuronate sulfate, and their oligosaccharides have antithrombin III- and heparin cofactor II-independent anticoagulant activity

NASA Astrophysics Data System (ADS)

Zeng, Xuan; Lan, Ying; Zeng, Pengjiao; Guo, Zhihua; Hao, Cui; Zhang, Lijuan

2017-04-01

Cardiovascular disease is the leading causes of death. However, the complications can be treated with heparin and heparinoids, such as heparin pentasaccharide Fondaparinux, dermatan sulfate, and PSS made from alginate extracted from brown seaweeds by chemical sulfation. Alginate is composed of a linear backbone of polymannuronate (PM), polyguluronate (PG), and alternate residues of mannuronic acid and guluronic acid. It is unknown if heparin and sulfated PG (PGS)/PM (PMS) have the same or different anticoagulant molecular targets. In the current study, the anticoagulant activities of PGS, PMS, and their oligosaccharides were directly compared to that of heparin, Fondaparinux, and dermatan sulfate by the activated partial thrombinplastin time (aPTT) assay using normal, antithrombin III (ATIII)-deficient, heparin co-factor II (HCII)-deficient, and ATIII- and HCII-double deficient human plasmas. Our results showed that PGS, PMS, and their oligosaccharides had better anticoagulant activity than that of Fondaparinux in all four human plasmas tested. As expected, heparin was the best anticoagulant in normal plasma. Moreover, PGS, PGS6, PGS12, PGS25, PMS6, PMS12, and PMS25 were better anticoagulants than dermatan sulfate in HCII-deficient plasma. Most strikingly, PGS, PGS12, PGS25, PMS6, PMS12, and PMS25 were better anticoagulants than that of heparin in ATIII- and HCII-double deficient human plasma. The results revealed for the first time that sulfated alginate had ATIII- and HCII-independent anticoagulant activities. Therefore, developing PGS and PMS-based anticoagulants might require to discover their major molecular targets and to develop target-specific anticoagulant assays.

Sulfated Low Molecular Weight Lignins, Allosteric Inhibitors of Coagulation Proteinases via the Heparin Binding Site, Significantly Alter the Active Site of Thrombin and Factor Xa Compared to Heparin

PubMed Central

Henry, Brian L.; Desai, Umesh R.

2014-01-01

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. PMID:25242245

Sulfated low molecular weight lignins, allosteric inhibitors of coagulation proteinases via the heparin binding site, significantly alter the active site of thrombin and factor xa compared to heparin.

PubMed

Henry, Brian L; Desai, Umesh R

2014-11-01

Sulfated low molecular weight lignins (LMWLs) have been found to bind in the heparin binding sites of coagulation proteinases. LMWLs represent a library of diverse non-carbohydrate, aromatic molecules which are structures different from heparin, but still potently inhibit thrombin and factor Xa. To better understand their mechanism of action, we studied the effects of three sulfated LMWLs (CDSO3, FDSO3, and SDSO3) on the active sites of thrombin and factor Xa. LMWLs were found to uniformly inhibit the catalytic activity of thrombin and factor Xa, regardless of the substrate used. Michaelis-Menten kinetic studies indicate that maximal velocity of hydrolysis of each chromogenic substrate decreases significantly in the presence of sulfated LMWLs, while the effect on Michaelis constant is dependent on the nature of the substrate. These studies indicate that LMWLs inhibit thrombin and factor Xa through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. As opposed to heparin, LMWLs significantly alter the binding of the active site fluorescent ligand p-aminobenzamidine. LMWLs also had a greater effect on the molecular orientation of fluorescein-labeled His 57 than heparin. The molecular geometry surrounding the most important catalytic amino acid, Ser 195, was significantly altered by the binding of LMWLs while heparin had no measurable effect on Ser 195. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold. Copyright © 2014 Elsevier Ltd. All rights reserved.

Heparin bridge therapy and post-polypectomy bleeding.

PubMed

Kubo, Toshiyuki; Yamashita, Kentaro; Onodera, Kei; Iida, Tomoya; Arimura, Yoshiaki; Nojima, Masanori; Nakase, Hiroshi

2016-12-07

To identify risk factors for post-polypectomy bleeding (PPB), focusing on antithrombotic agents. This was a case-control study based on medical records at a single center. PPB was defined as bleeding that occurred 6 h to 10 d after colonoscopic polypectomy and required endoscopic hemostasis. As risk factors for PPB, patient-related factors including anticoagulants, antiplatelets and heparin bridge therapy as well as polyp- and procedure-related factors were evaluated. All colonoscopic hot polypectomies, endoscopic mucosal resections and endoscopic submucosal dissections performed between January 2011 and December 2014 were reviewed. PPB occurred in 29 (3.7%) of 788 polypectomies performed during the study period. Antiplatelet or anticoagulant agents were prescribed for 210 (26.6%) patients and were ceased before polypectomy except for aspirin and cilostazol in 19 cases. Bridging therapy using intravenous unfractionated heparin was adopted for 73 patients. The univariate analysis revealed that anticoagulants, heparin bridge, and anticoagulants plus heparin bridge were significantly associated with PPB ( P < 0.0001) whereas antiplatelets and antiplatelets plus heparin were not. None of the other factors including age, gender, location, size, shape, number of resected polyps, prophylactic clipping and resection method were correlated with PPB. The multivariate analysis demonstrated that anticoagulants and anticoagulants plus heparin bridge therapy were significant risk factors for PPB ( P < 0.0001). Of the 29 PPB cases, 4 required transfusions and none required surgery. A thromboembolic event occurred in a patient who took anticoagulant. Patients taking anticoagulants have an increased risk of PPB, even if the anticoagulants are interrupted before polypectomy. Heparin-bridge therapy might be responsible for the increased PPB in patients taking anticoagulants.

Long-term low-molecular-weight heparin and the post-thrombotic syndrome: a systematic review.

PubMed

Hull, Russell D; Liang, Jane; Townshend, Grace

2011-08-01

Post-thrombotic syndrome causes considerable morbidity. The Home-LITE study showed a lower incidence of post-thrombotic syndrome and venous ulcers after 3 months of treating deep vein thrombosis with the low-molecular-weight heparin tinzaparin versus oral anticoagulation. This systematic review examined whether long-term treatment of deep vein thrombosis using low-molecular-weight heparin, rather than oral anticoagulation, reduces development of post-thrombotic syndrome. We identified 9 articles comparing treatment of deep vein thrombosis using long-term low-molecular-weight heparin with any comparator, which reported outcomes relevant to the post-thrombotic syndrome assessed ≥ 3 months post-deep vein thrombosis. Pooled analysis of 2 studies yielded an 87% risk reduction with low-molecular-weight heparin in the incidence of venous ulcers at ≥ 3 months (P = .019). One study showed an overall odds ratio of 0.77 (P = .001) favoring low-molecular-weight heparin for the presence of 8 patient-reported post-thrombotic syndrome signs and symptoms. Pooled analysis of 5 studies showed a risk ratio for low-molecular-weight heparin versus oral anticoagulation of 0.66 (P < .0001) for complete recanalization of thrombosed veins. These results support the lower incidence of post-thrombotic syndrome and venous ulcers observed in Home-LITE. Long-term treatment with low-molecular-weight heparin rather than oral anticoagulation after a deep vein thrombosis may reduce or prevent development of signs and symptoms associated with post-thrombotic syndrome. Post-thrombotic syndrome and associated acute ulcers may develop more rapidly after deep vein thrombosis than previously recognized. Copyright © 2011 Elsevier Inc. All rights reserved.

Structure and Activity of a New Low Molecular Weight Heparin Produced by Enzymatic Ultrafiltration

PubMed Central

FU, LI; ZHANG, FUMING; LI, GUOYUN; ONISHI, AKIHIRO; BHASKAR, UJJWAL; SUN, PEILONG; LINHARDT, ROBERT J.

2014-01-01

The standard process for preparing the low molecular weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield due to the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin’s core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. PMID:24634007

The role of heparin in sepsis: much more than just an anticoagulant.

PubMed

Li, Xu; Ma, Xiaochun

2017-11-01

Despite progress in antibiotic treatment, mechanical ventilation, fluid resuscitation and blood glucose maintenance, sepsis remains a cause of high mortality in the intensive care unit to date, there are no proven treatment strategies for the routine management of septic patients. The extensive interaction between inflammation and coagulation contributes to the basic pathophysiology of sepsis. Thus, the agents that attenuate the activation of both inflammation and coagulation may improve the outcome in sepsis. Apart from the well-known anticoagulant effects of heparin, it also possesses various immunomodulatory properties and protects glycocalyx from shedding. Hence, heparin seems to be such an agent. Immunothrombosis plays an important role in early host defence against bacterial dissemination, thus the proper timing for anticoagulant therapy should be determined. We review the available experimental and clinical data supporting the use of heparin in sepsis. At this time the use of heparin in the treatment of sepsis is conflicting. Future trials of heparin therapy for sepsis should concentrate on the very severely ill patients, in whom benefit is most likely to be demonstrated. © 2017 John Wiley & Sons Ltd.

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Heparin affinity purification of extracellular vesicles

PubMed Central

Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

2015-01-01

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

Inhibitory effect of heparin on neutrophil phagocytosis and burst production using a new whole-blood cytofluorometric method for determination.

PubMed

Salih, H; Husfeld, L; Adam, D

1997-12-31

The influence of heparin on Polymorphonuclear (PMN s) leukocytes was investigated using a new whole-blood cytofluorometric method (patent granted for the test with the number P 4334935.8-41) with Candida albicans and Staphylococcus aureus as test microorganisms. After comparing the effect of equal volumes of two widely used heparins we examined the influence of 5 different heparin-concentrations. Using both yeasts and bacteria, we found a significant, dose-depending decrease of the percentage of phagocyting PMN's and of phagocytized microorganisms as well as of the resulting percentage of PMN s producing respiratory burst along the kinetics. Furthermore we could demonstrate that heparin independently of phagocytosis produces a dose-dependent decrease of burst production of PMN's. Our results indicate that the use of heparins as anticoagulant for immunological investigations as well as clinically with patients under immunosuppressive therapy should be critically reconsidered. This applies even more because due to the evaluated dose-dependent decrease of phagocyte function no boundary for the inhibiting effect can be declared.

The effectiveness of heparin, platelet-rich plasma (PRP), and silver nanoparticles on prevention of postoperative peritoneal adhesion formation in rats.

PubMed

Makarchian, Hamid Reza; Kasraianfard, Amir; Ghaderzadeh, Pezhman; Javadi, Seyed Mohammad Reza; Ghorbanpoor, Manoochehr

2017-01-01

To assess the effectiveness of heparin, platelet-rich plasma (PRP), and silver nanoparticles on prevention of postoperative adhesion in animal models. Sixty males Albino Wistar rats aged 5 to 6 weeks were classified into five groups receiving none, heparin, PRP, silver nanoparticles, PRP plus silver nanoparticles intraperitoneally. After 2 weeks, the animals underwent laparotomy and the damaged site was assessed for peritoneal adhesions severity. The mean severity scores were 2.5 ± 0.9, 2.16 ± 0.7, 1.5 ± 0.5, 2.66 ± 0.88, and 2.25 ± 0.62 in the control, heparin, PRP, silver and PRP plus silver groups, respectively with significant intergroup difference (p = 0.004). The highest effective material for preventing adhesion formation was PRP followed by heparin and PRP plus silver. Moreover, compared to the controls, only use of PRP was significantly effective, in terms of adhesion severity (p = 0.01) . Platelet-rich plasma alone may have the highest efficacy for preventing postoperative peritoneal adhesions in comparison with heparin, silver nanoparticles and PRP plus silver nanoparticles.

Facilitation of learning and modulation of frontal cortex acetylcholine by ventral pallidal injection of heparin glucosaminoglycan.

PubMed

De Souza Silva, M A; Jezek, K; Weth, K; Müller, H W; Huston, J P; Brandao, M L; Hasenöhrl, R U

2002-01-01

We examined the effects of heparin on learning and frontal cortex acetylcholine parameters following injection of the glucosaminoglycan into the ventral pallidum. In Experiment 1, possible mnemoactive effects of intrapallidal heparin injection were assessed. Rats with chronically implanted cannulae were administered heparin (0.1, 1.0, 10 ng) or vehicle (0.5 microl) and were tested on a one-trial step-through avoidance task. Two retention tests were carried out in each animal, one at 1.5 h after training to measure short-term memory and another at 24 h to measure long-term memory. Post-trial intrapallidal injection of 1.0 ng heparin improved both short- and long-term retention of the task, whereas the lower and the higher dose of the glucosaminoglycan had no effect. When the effective dose of heparin was injected 5 h, rather than immediately after training, it no longer facilitated long-term retention of the conditioned avoidance response. In Experiment 2, the effects of ventral pallidal heparin injection on frontal cortex acetylcholine and choline concentrations were investigated with in vivo microdialysis in anaesthetized rats. Heparin, administered in the dose of 1.0 ng, which was effective in facilitating avoidance performance, produced a delayed increase in cortical acetylcholine levels ipsi- and contralaterally to the side of intrabasalis injection, resembling the known neurochemical effects obtained for another glycosaminoglycan, chondroitin sulfate, which recently was shown to facilitate inhibitory avoidance learning and to increase frontal cortex acetylcholine. The present findings indicate that heparin, like other extracellular matrix proteoglycans, can exert beneficial effects on memory and strengthen the presumptive relationship between such promnestic effects of proteoglycans and basal forebrain cholinergic mechanisms. The data are discussed with respect to the presumed roles of matrix molecules in extrasynaptic volume transmission and in the 'cross-talk' between synapses.

Synergism of aspirin and heparin with a low-frequency non-invasive ultrasound system for augmentation of in-vitro clot lysis.

PubMed

Atar, Shaul; Neuman, Yoram; Miyamoto, Takashi; Chen, Ming; Birnbaum, Yochai; Luo, Huai; Kobal, Sergio; Siegel, Robert J

2003-06-01

Aspirin, glycoprotein IIb/IIIa inhibitors and heparin are routinely used in acute coronary syndromes. Previously we showed that there is synergism between ultrasound and heparin and tirofiban in augmenting blood clot disruption. However, there is a little data on a possible synergism of low-frequency ultrasound with aspirin for in-vitro clot dissolution, and especially on the combination of aspirin with heparin and/or glycoprotein IIb/IIIa inhibitors. Human blood clots (n = 320) were incubated for 10 or 20 minutes in saline containing aspirin alone or combined with heparin and/or tirofiban and/or eptifibatide. Clots were randomly treated with low-frequency ultrasound (27.3 kHz) or incubation only. The percent clot weight loss and the incremental effect of ultrasound were calculated. The most significant incremental effect of ultrasound on clot weight reduction was detected with aspirin alone (5.2 +/- 2.3% and 5.2 +/- 2.6% after 10' and 20', p = 0.04 and p = 0.06, respectively) and in combination with heparin (8.8 +/- 2.5% and 11.5 +/- 2.7% after 10' and 20', p = 0.01 and p = 0.0001, respectively). The greatest absolute magnitude of clot weight reduction was observed with ultrasound combined with aspirin and heparin (48.5 +/- 9.5% after 20'). The addition of tirofiban or eptifibatide to aspirin, heparin and ultrasound did not increase clot lysis. However, eptifibatide had significantly better synergism than tirofiban (p = 0.025 and p = 0.015, after 10 and 20 minutes, respectively). Aspirin alone or in combination with heparin results in significant augmentation of clot lysis and is synergistic with application of low-frequency ultrasound for 10 and 20 minutes only. These results may have important implications for a possible use of low-frequency ultrasound in treatment algorithms of acute coronary syndromes.

Heparin concentration is critical for cell culture with human platelet lysate.

PubMed

Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

2013-09-01

Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

PubMed

Ueda, H; Saitoh, T; Kojima, K; Ogawa, H

1999-09-01

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.

Nebulized heparin is associated with fewer days of mechanical ventilation in critically ill patients: a randomized controlled trial.

PubMed

Dixon, Barry; Schultz, Marcus J; Smith, Roger; Fink, James B; Santamaria, John D; Campbell, Duncan J

2010-01-01

Prolonged mechanical ventilation has the potential to aggravate or initiate pulmonary inflammation and cause lung damage through fibrin deposition. Heparin may reduce pulmonary inflammation and fibrin deposition. We therefore assessed whether nebulized heparin improved lung function in patients expected to require prolonged mechanical ventilation. Fifty patients expected to require mechanical ventilation for more than 48 hours were enrolled in a double-blind randomized placebo-controlled trial of nebulized heparin (25,000 U) or placebo (normal saline) 4 or 6 hourly, depending on patient height. The study drug was continued while the patient remained ventilated to a maximum of 14 days from randomization. Nebulized heparin was not associated with a significant improvement in the primary end-point, the average daily partial pressure of oxygen to inspired fraction of oxygen ratio while mechanically ventilated, but was associated with improvement in the secondary end-point, ventilator-free days amongst survivors at day 28 (22.6 ± 4.0 versus 18.0 ± 7.1, treatment difference 4.6 days, 95% CI 0.9 to 8.3, P = 0.02). Heparin administration was not associated with any increase in adverse events. Nebulized heparin was associated with fewer days of mechanical ventilation in critically ill patients expected to require prolonged mechanical ventilation. Further trials are required to confirm these findings. The Australian Clinical Trials Registry (ACTR-12608000121369).

Modulation of human endothelial cell proliferation and migration by fucoidan and heparin.

PubMed

Giraux, J L; Matou, S; Bros, A; Tapon-Bretaudière, J; Letourneur, D; Fischer, A M

1998-12-01

Fucoidan is a sulfated polysaccharide extracted from brown seaweeds. It has anticoagulant and antithrombotic properties and inhibits, as well as heparin, vascular smooth muscle cell growth. In this study, we investigated, in the presence of serum and human recombinant growth factors, the effects of fucoidan and heparin on the growth and migration of human umbilical vein endothelial cells (HUVEC) in culture. We found that fucoidan stimulated fetal bovine serum-induced HUVEC proliferation, whereas heparin inhibited it. In the presence of fibroblast growth factor-1 (FGF-1), both fucoidan and heparin potentiated HUVEC growth. In contrast, fucoidan and heparin inhibited HUVEC proliferation induced by FGF-2, but did not influence the mitogenic activity of vascular endothelial growth factor (VEGF). In the in vitro migration assay from a denuded area of confluent cells, the two sulfated polysaccharides markedly enhanced the migration of endothelial cells in the presence of FGF-1. Finally, a weak inhibitory effect on cell migration was found only with the two polysaccharides at high concentrations (> or = 100 micro/ml) in presence of serum or combined with FGF-2. All together, the results indicated that heparin and fucoidan can be used as tools to further investigate the cellular mechanisms regulating the proliferation and migration of human vascular cells. Moreover, the data already suggest a potential role of fucoidan as a new therapeutic agent of vegetal origin in the vascular endothelium wound repair.

Association between Activated Partial Thromboplastin Time and the Amount of Infused Heparin at Bone Marrow Transplantation.

PubMed

Kusuda, Machiko; Kimura, Shun-Ichi; Misaki, Yukiko; Yoshimura, Kazuki; Gomyo, Ayumi; Hayakawa, Jin; Tamaki, Masaharu; Akahoshi, Yu; Ugai, Tomotaka; Kameda, Kazuaki; Wada, Hidenori; Ishihara, Yuko; Kawamura, Koji; Sakamoto, Kana; Sato, Miki; Terasako-Saito, Kiriko; Kikuchi, Misato; Nakasone, Hideki; Kako, Shinichi; Tanihara, Aki; Kanda, Yoshinobu

2018-03-27

The actual heparin concentration of harvested allogeneic bone marrow varies among harvest centers. We monitor the activated partial thromboplastin time (APTT) of the patient during bone marrow infusion and administer prophylactic protamine according to the APTT. We retrospectively reviewed the charts of consecutive patients who underwent bone marrow transplantation without bone marrow processing at our center between April 2007 and March 2016 (n = 94). APTT was monitored during marrow transfusion in 52 patients. We analyzed the relationship between the APTT ratio and several parameters related to heparin administration. As a result, the weight-based heparin administration rate (U/kg/hour) seemed to be more closely related to the APTT ratio (r = .38, P = .005) than to the total amount of heparin. There was no significant correlation between the APTT ratio and renal or liver function. Bleeding complications during and early after infusion were seen in 3 of 52 patients, and included intracranial, nasal, and punctured-skin bleeding. The APTT ratio during transfusion was over 5.88 in the former 2 patients and 2.14 in the latter. All of these patients recovered without sequelae. In conclusion, slow bone marrow infusion is recommended to decrease the weight-based heparin administration rate when the heparin concentration per patient body weight is high. Copyright © 2018 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

[Diagnosis and treatment of heparin-induced thrombocytopenia (HIT) based on its atypical immunological features].

PubMed

Miyata, Shigeki; Maeda, Takuma

2016-03-01

Heparin-induced thrombocytopenia (HIT) is a prothrombotic side effect of heparin therapy caused by HIT antibodies, i.e., anti-platelet factor 4 (PF4)/heparin IgG with platelet-activating properties. For serological diagnosis, antigen immunoassays are commonly used worldwide. However, such assays do not indicate their platelet-activating properties, leading to low specificity for the HIT diagnosis. Therefore, over-diagnosis is currently the most serious problem associated with HIT. The detection of platelet-activating antibodies using a washed platelet activation assay is crucial for appropriate HIT diagnosis. Recent advances in our understanding of the pathogenesis of HIT include it having several clinical features atypical for an immune-mediated disease. Heparin-naïve patients can develop IgG antibodies as early as day 4, as in a secondary immune response. Evidence for an anamnestic response on heparin re-exposure is lacking. In addition, HIT antibodies are relatively short-lived, unlike those in a secondary immune response. These lines of evidence suggest that the mechanisms underlying HIT antibody formation may be compatible with a non-T cell-dependent immune reaction. These atypical clinical and serological features should be carefully considered while endeavoring to accurately diagnose HIT, which leads to appropriate therapies such as immediate administration of an alternative anticoagulant to prevent thromboembolic events and re-administration of heparin during surgery involving cardiopulmonary bypass when HIT antibodies are no longer detectable.

Acute intraoperative heparin-induced thrombocytopenia (HIT) and thrombosis during coronary artery bypass grafting: Two case reports providing evidence for the role of preoperative LMWH in triggering sensitization.

PubMed

Khoury, M; Pitsis, A; Poumpouridou-Kioura, H; Soufla, G; Kanthou, C; Matoula, N; Angelidis, A; Melissari, E

2016-10-01

Systemic anticoagulation is necessary during cardiac surgery. To date, the only well established anticoagulation protocol involves the use of heparin. However, heparin can cause heparin-induced thrombocytopenia (HIT) a potentially life threatening immune-mediated thromboembolic syndrome. Until now, devastating consequences of HIT syndrome in patients undergoing heart surgery have been described, but only postoperatively. Here we report the development of HIT syndrome during cardiac revascularization by intra-operative heparin administration in two patients previously exposed to LMWH. We report on two patients who developed rapid and profound intravascular coagulation with severe thrombocytopenia (platelet count decreased from ≥250×10 9 /L to 50×10 9 /L) due to HIT development caused by heparin administration during coronary artery bypass graft surgery. In addition we report that fondaparinux, given intra-operatively in association with antithrombin, may be a suitable alternative anticoagulant for successfully preventing the devastating consequences of intra-operative HIT development. To our knowledge, this is the first report describing the development of acute intra-operative HIT, secondary to high-dose UFH administered for coronary revascularization, in which the unexpected presence of platelet-activating anti-PF4/heparin antibodies at surgery was explained by preoperative administration of a one-week course of LMWH but without any preoperative evidence for HIT. Copyright © 2016 Elsevier Ltd. All rights reserved.

The role of heparins and nano-heparins as therapeutic tool in breast cancer.

PubMed

Afratis, Nikos A; Karamanou, Konstantina; Piperigkou, Zoi; Vynios, Demitrios H; Theocharis, Achilleas D

2017-06-01

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.

Heparin conjugated quantum dots for in vitro imaging applications.

PubMed

Maguire, Ciaran Manus; Mahfoud, Omar Kazem; Rakovich, Tatsiana; Gerard, Valerie Anne; Prina-Mello, Adriele; Gun'ko, Yurii; Volkov, Yuri

2014-11-01

In this work heparin-gelatine multi-layered cadmium telluride quantum dots (QDgel/hep) were synthesised using a novel 'one-pot' method. The QDs produced were characterised using various spectroscopic and physiochemical techniques. Suitable QDs were then selected and compared to thioglycolic acid stabilised quantum dots (QDTGA) and gelatine coated quantum dots (QDgel) for utilisation in in vitro imaging experiments on live and fixed permeabilised THP-1, A549 and Caco-2 cell lines. Exposure of live THP-1 cells to QDgel/hep resulted in localisation of the QDs to the nucleus of the cells. QDgel/hep show affinity for the nuclear compartment of fixed permeabilised THP-1 and A549 cells but remain confined to cytoplasm of fixed permeabilised Caco-2 cells. It is postulated that heparin binding to the CD11b receptor facilitates the internalisation of the QDs into the nucleus of THP-1 cells. In addition, the heparin layer may reduce the unfavourable thrombogenic nature of quantum dots observed in vivo. In this study, heparin conjugated quantum dots were found to have superior imaging properties compared to its native counterparts. The authors postulate that heparin binding to the CD11b receptor facilitates QD internalization to the nucleus, and the heparin layer may reduce the in vivo thrombogenic properties of quantum dots. Copyright © 2014 Elsevier Inc. All rights reserved.

Recombinant viral RdRps can initiate RNA synthesis from circular templates

PubMed Central

RANJITH-KUMAR, C.T.; KAO, C.C.

2006-01-01

The crystal structure of the recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) revealed extensive interactions between the fingers and the thumb subdomains, resulting in a closed conformation with an established template channel that should specifically accept single-stranded templates. We made circularized RNA templates and found that they were efficiently used by the HCV RdRp to synthesize product RNAs that are significantly longer than the template, suggesting that RdRp could exist in an open conformation prior to template binding. RNA synthesis using circular RNA templates had properties similar to those previously documented for linear RNA, including a need for higher GTP concentration for initiation, usage of GTP analogs, sensitivity to salt, and involvement of active-site residues for product formation. Some products were resistant to challenge with the template competitor heparin, indicating that the elongation complexes remain bound to template and are competent for RNA synthesis. Other products were not elongated in the presence of heparin, indicating that the elongation complex was terminated. Lastly, recombinant RdRps from two other flaviviruses and from the Pseudomonas phage φ6 also could use circular RNA templates for RNA-dependent RNA synthesis, although the φ6 RdRp could only use circular RNAs made from the 3′-terminal sequence of the φ6 genome. PMID:16373481

Mechanisms of Staphylococcus epidermidis adhesion to model biomaterial surfaces: Establising a link between thrombosis and infection

NASA Astrophysics Data System (ADS)

Higashi, Julie Miyo

Infections involving Staphylococcus epidermidis remain a life threatening complication associated with the use of polymer based cardiovascular devices. One of the critical steps in infection pathogenesis is the adhesion of the bacteria to the device surface. Currently, mechanisms of S. epidermidis adhesion are incompletely understood, but are thought to involve interactions between bacteria, device surface, and host blood elements in the form of adsorbed plasma proteins and surface adherent platelets. Our central hypothesis is that elements participating in thrombosis also promote S. epidermidis adhesion by specifically binding to the bacterial surface. The adhesion kinetics of S. epidermidis RP62A to host modified model biomaterial surface octadecyltrichlorosilane (OTS) under hydrodynamic shear conditions were characterized. Steady state adhesion to adsorbed proteins and surface adherent platelets was achieved at 90-120 minutes and 60-90 minutes, respectively. A dose response curve of S. epidermidis adhesion in the concentration range of 10sp7{-}10sp9 bac/mL resembled a multilayer adsorption isotherm. Increasing shear stress was found to LTA, and other LTA blocking agents significantly decreased S. epidermidis adhesion to the fibrin-platelet clots, suggesting that this interaction between S. epidermidis and fibrin-platelet clots is specific. Studies evaluated the adhesion of S. epidermidis to polymer immobilized heparin report conflicting results. Paulsson et al., showed that coagulase negative staphylococci adhered in comparable numbers to both immobilized heparin and nonheparinized surfaces, while exhibiting significantly greater adhesion to both surfaces than S. aureus. Preadsorption of the surfaces with specific heparin binding plasma proteins vitronectin, fibronectin, laminin, and collagen significantly increased adhesion. It was postulated that immobilized heparin contained binding sites for the plasma proteins, exposing bacteria binding domains of the protein. Aggregation of heparin coated (adsorbed) polystyrene beads by the same S. epidermidis strains did not correlate with the adherence assays on the immobilized heparin surfaces and staphylococcal binding was dependent on media ionic strength and pH, suggesting that the conformation of the heparin polysaccharide chains was crucial to the interaction (132). Another study by Arciola et al., showed that heparin modified PMMA intraocular lenses sustained significantly less adherent bacteria than unmodified PMMA lenses, and that the chromatogram of structural fatty acids of only the S. epidermidis adherent to heparin modified PMMA changed (133). Because neither of these investigators thoroughly characterized their heparin modified surfaces, the difference in their results could easily be explained by the notorious heterogeneity of the heparin molecular weight, bioactivity, or surface coverage on these polymers.

Gender-based outcomes of bivalirudin versus heparin in patients undergoing percutaneous coronary interventions: Meta-analysis of randomized controlled trials.

PubMed

Mina, George S; Firouzbakht, Tina; Modi, Kalgi; Dominic, Paari

2017-11-01

We aimed to perform a gender-based meta-analysis of the outcome of bivalirudin versus heparin in patients undergoing percutaneous coronary interventions (PCI). Bivalirudin has been shown to decrease major bleeding when compared to heparin ± glycoprotein IIb/IIIa inhibitors (GPI) in patients undergoing PCI. It is unclear, however, if those differences in outcomes are the same for men and women. We included randomized controlled trials (RCTs) that compared bivalirudin to heparin with or without GPI in patients undergoing PCI and reported outcome data that were stratified by gender. Random effect model was used to pool odds ratio (OR) and 95% confidence intervals (CI). We included 9 trials with 33,224 patients. Bivalirudin decreased major bleeding when compared to heparin plus routine GPI in both men (OR: 0.51, P < 0.001) and women (OR: 0.55, P < 0.001). However, when GPI were used selectively with heparin, the bleeding lowering effect of bivalirudin was statistically significant in men (OR: 0.69, P = 0.02) but not in women (OR: 0.71, P = 0.21). When compared to heparin ± GPI, there was a nonstatistically significant trend toward lower all-cause mortality with bivalirudin in both men (OR: 0.76, P = 0.055) and women (OR: 0.79, P = 0.21). There were no significant differences in major adverse cardiovascular events between heparin and bivalirudin in both men and women. Bivalirudin decreases major bleeding in both men and women when compared to heparin plus routine GPI. However, when compared to heparin alone, the bleeding lowering benefit of bivalirudin is less evident in women. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Vascular access site complication in transfemoral coronary angiography between uninterrupted warfarin and heparin bridging.

PubMed

Wongcharoen, Wanwarang; Pinyosamosorn, Kittipong; Gunaparn, Siriluck; Boonnayhun, Suchada; Thonghong, Tasalak; Suwannasom, Pannipa; Phrommintikul, Arintaya

2017-08-01

Warfarin discontinuation with heparin bridging is a common practice in patients receiving warfarin prior to elective coronary angiography (CAG). The uninterrupted warfarin strategy has been suggested to be alternative option for patients with high thromboembolic risk. Therefore, we aimed to assess the safety of elective transfemoral CAG during uninterrupted warfarin therapy compared to heparin bridging. This study was a randomized open-label design with blinded event evaluation. The 110 consecutive patients (age ≥ 18 years) receiving warfarin before the planned transfemoral CAG were randomly assigned to either heparin bridging or uninterrupted warfarin with targeted INR (2.0-3.0). The primary outcome was the incidence of major vascular access site complications. The baseline characteristics were comparable between two groups (mean age was 60.1 ± 7.8 years, 49 males). The mean INR on the day of CAG of heparin bridging and uninterrupted warfarin groups was 1.2 ± 0.3 and 2.2 ± 0.5 (P < 0.001). The major vascular access site complications occurred in 3 of 55 (5.5%) heparin-bridging patients and in none of 55 uninterrupted warfarin patients (P = 0.243). The total vascular access site complications occurred in 6 (10.9%) heparin-bridging and one (1.8%) uninterrupted warfarin patients (P = 0.113). No patient developed either other bleeding or thromboembolic events during 7 days after CAG. We demonstrated that an uninterrupted warfarin strategy did not increase vascular access site complications in patients undergoing transfemoral CAG compared to heparin bridging therapy. Due to the safety and the ease of uninterrupted warfarin strategy, this approach should be encouraged in patients receiving long-term warfarin who undergo elective transfemoral CAG. © 2017, Wiley Periodicals, Inc.

Profiling Heparin-Chemokine Interactions Using Synthetic Tools

PubMed Central

de Paz, Jose L.; Moseman, E. Ashley; Noti, Christian; Polito, Laura; von Andrian, Ulrich H.; Seeberger, Peter H.

2009-01-01

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity. PMID:18030990

Heparin bridge therapy and post-polypectomy bleeding

PubMed Central

Kubo, Toshiyuki; Yamashita, Kentaro; Onodera, Kei; Iida, Tomoya; Arimura, Yoshiaki; Nojima, Masanori; Nakase, Hiroshi

2016-01-01

AIM To identify risk factors for post-polypectomy bleeding (PPB), focusing on antithrombotic agents. METHODS This was a case-control study based on medical records at a single center. PPB was defined as bleeding that occurred 6 h to 10 d after colonoscopic polypectomy and required endoscopic hemostasis. As risk factors for PPB, patient-related factors including anticoagulants, antiplatelets and heparin bridge therapy as well as polyp- and procedure-related factors were evaluated. All colonoscopic hot polypectomies, endoscopic mucosal resections and endoscopic submucosal dissections performed between January 2011 and December 2014 were reviewed. RESULTS PPB occurred in 29 (3.7%) of 788 polypectomies performed during the study period. Antiplatelet or anticoagulant agents were prescribed for 210 (26.6%) patients and were ceased before polypectomy except for aspirin and cilostazol in 19 cases. Bridging therapy using intravenous unfractionated heparin was adopted for 73 patients. The univariate analysis revealed that anticoagulants, heparin bridge, and anticoagulants plus heparin bridge were significantly associated with PPB (P < 0.0001) whereas antiplatelets and antiplatelets plus heparin were not. None of the other factors including age, gender, location, size, shape, number of resected polyps, prophylactic clipping and resection method were correlated with PPB. The multivariate analysis demonstrated that anticoagulants and anticoagulants plus heparin bridge therapy were significant risk factors for PPB (P < 0.0001). Of the 29 PPB cases, 4 required transfusions and none required surgery. A thromboembolic event occurred in a patient who took anticoagulant. CONCLUSION Patients taking anticoagulants have an increased risk of PPB, even if the anticoagulants are interrupted before polypectomy. Heparin-bridge therapy might be responsible for the increased PPB in patients taking anticoagulants. PMID:28018108

Influence of apixaban on antifactor Xa levels in a patient with acute kidney injury.

PubMed

Wendte, Jodi; Voss, Glenn; VanOverschelde, Beau

2016-04-15

The case of a patient requiring conversion from apixaban to heparin in the setting of acute kidney injury is reported. A 70-year-old man was initiated on apixaban 5 mg twice daily for new-onset, nonvalvular atrial fibrillation with a CHA2DS2-VASc score of 4, indicating a high risk of stroke. Soon after starting apixaban, he experienced pulmonary edema with pneumonia requiring hospitalization. During the course of hospitalization, the patient developed acute kidney injury requiring hemodialysis, and apixaban was stopped due to concerns about altered pharmacokinetics and impaired drug elimination in this setting. A heparin infusion was started 36 hours after the last dose of apixaban was administered. Antifactor Xa levels were monitored consistent with the hospital's standard practice protocols. The initial and repeat antifactor Xa concentrations were elevated (1.8-4.4 IU/mL) for up 72 hours after stopping the heparin infusion. Given the suspected interference of apixaban with standard antifactor Xa level monitoring, the heparin protocol was modified to reflect drip-rate adjustments based on activated partial thromboplastin times (aPTTs). The hospital protocol for heparin infusions was reinstituted on hospital day 7, with dosage adjustments based on antifactor Xa levels. The patient remained on a continuous heparin infusion for atrial fibrillation for the remainder of his hospitalization without complications or bleeding events. A 70-year-old man with new-onset nonvalvular atrial fibrillation and receiving apixaban discontinued this therapy and was given heparin instead due to acute kidney injury. His heparin dosage was successfully adjusted based on antifactor Xa levels and aPPTs. Copyright © 2016 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Safety of low dose heparin in elective coronary angioplasty.

PubMed Central

Koch, K. T.; Piek, J. J.; de Winter, R. J.; David, G. K.; Mulder, K.; Tijssen, J. G.; Lie, K. I.

1997-01-01

OBJECTIVES: To evaluate the safety of a low dose of heparin in consecutive stable patients undergoing elective percutaneous transluminal coronary angioplasty (PTCA). DESIGN: Open prospective study in a single centre. PATIENTS: 1375 consecutive patients had elective PTCA (1952 lesions: type A 11%, B1 34%, B2 36%, and C 19%). There were no angiographic exclusion criteria. INTERVENTIONS: A bolus of 5000 IU heparin was used as the standard anticoagulation regimen during PTCA. The sheaths were removed immediately after successful completion of the procedure. Prolongation of heparin treatment was left to the operator's discretion. MAIN OUTCOME MEASURES: Procedural success was defined as < 50% residual stenosis without death from any cause, acute myocardial infarction, urgent coronary bypass surgery, or repeat angioplasty within 48 hours for acute recurrent ischaemia; the need for prolonged heparinisation; and the occurrence of puncture site complications. RESULTS: Procedural success without clinical events was achieved in 90% of patients. Mortality was 0.3%; coronary bypass surgery was performed in 1.7% of the procedures. The rate of myocardial infarction was 3.3%; repeat angioplasty within 48 hours was carried out in 0.7% of patients. A total of 89.1% of the patients were treated according to the protocol. Prolonged treatment with heparin was considered necessary in 123 patients (8.9%). Repeat angioplasty for abrupt closure was performed in two patients shortly after sheath removal and in two during prolonged heparinisation. Puncture site complications occurred in 2.1% of patients (low dose heparin 1.9% and prolonged heparinisation 4.9%). CONCLUSION: Elective PTCA can be safely performed using a low dose of heparin, with a negligible risk for subacute closure. Low dose heparin may reduce the incidence of puncture site complications, shorten hospitalisation, and enable out-patient angioplasty. PMID:9227294

Perioperative use of iloprost in cardiac surgery patients diagnosed with heparin-induced thrombocytopenia-reactive antibodies or with true HIT (HIT-reactive antibodies plus thrombocytopenia): An 11-year experience.

PubMed

Palatianos, George; Michalis, Alkiviadis; Alivizatos, Petros; Lacoumenda, Stavroula; Geroulanos, Stefanos; Karabinis, Andreas; Iliopoulou, Eugenia; Soufla, Giannoula; Kanthou, Chryso; Khoury, Mazen; Sfyrakis, Petros; Stavridis, George; Astras, George; Vassili, Maria; Antzaka, Christina; Marathias, Katerina; Kriaras, Ioannis; Tasouli, Androniki; Papadopoulos, Kyrillos; Katafygioti, Marina; Matoula, Nikoletta; Angelidis, Antonios; Melissari, Euthemia

2015-07-01

Thrombocytopenia and thromboembolism(s) may develop in heparin immune-mediated thrombocytopenia (HIT) patients after reexposure to heparin. At the Onassis Cardiac Surgery Center, 530 out of 17,000 patients requiring heart surgery over an 11-year period underwent preoperative HIT assessment by ELISA and a three-point heparin-induced platelet aggregation assay (HIPAG). The screening identified 110 patients with HIT-reactive antibodies, out of which 46 were also thrombocytopenic (true HIT). Cardiac surgery was performed in HIT-positive patients under heparin anticoagulation and iloprost infusion. A control group of 118 HIT-negative patients received heparin but no iloprost during surgery. For the first 20 patients, the dose of iloprost diminishing the HIPAG test to ≤5% was determined prior to surgery by in vitro titration using the patients' own plasma and donor platelets. In parallel, the iloprost "target dose" was also established for each patient intraoperatively, but before heparin administration. Iloprost was infused initially at 3 ng/kg/mL and further adjusted intraoperatively, until ex vivo aggregation reached ≤5%. As a close correlation was observed between the "target dose" identified before surgery and that established intraoperatively, the remaining 90 patients were administered iloprost starting at the presurgery identified "target dose." This process significantly reduced the number of intraoperative HIPAG reassessments needed to determine the iloprost target dose, and reduced surgical time, while maintaining similar primary clinical outcomes to controls. Therefore, infusion of iloprost throughout surgery, under continuous titration, allows cardiac surgery to be undertaken safely using heparin, while avoiding life-threatening iloprost-induced hypotension in patients diagnosed with HIT-reactive antibodies or true HIT. © 2015 Wiley Periodicals, Inc.

Anticoagulation in pregnant women with mechanical heart valve prostheses

PubMed Central

Meschengieser, S; Fondevila, C; Santarelli, M; Lazzari, M

1999-01-01

OBJECTIVE—To evaluate the outcome of pregnancy in women with mechanical heart valve prostheses in relation to the anticoagulant treatment used in the first trimester and the incidence of thrombotic and bleeding complications.
METHODS—92 pregnancies in 59 women were followed between 1986 and 1997. In 31 pregnancies, oral anticoagulants were discontinued when pregnancy was diagnosed and subcutaneous heparin was started (12 500 U every 12 hours) adjusted to prolong the adjusted partial thromboplastin time to twice the control level. In the second trimester oral anticoagulants were resumed but changed to heparin again 15 days before the expected delivery date. In 61 pregnancies oral anticoagulants were continued during the first trimester. The same regimen of heparin was used for delivery.
RESULTS—Abortion or fetal losses were similar (p = 0.5717) in women exposed to oral anticoagulants in the first trimester (13/61; 25%) compared with those who received adjusted subcutaneous heparin (6/31; 19%). Embolic episodes were more common (p = 0.0029) in women who received heparin (4.92%) compared with those on oral anticoagulants (0.33%). Embolic episodes were cerebral and transient. No valve thromboses were observed. No malformations appeared in the 71 newborns, except for one case of hydrocephalus. There were no maternal deaths secondary to thrombotic complications. The only death was the result of major bleeding after the delivery of a premature stillborn.
CONCLUSIONS—Oral anticoagulants seem to be safer for the mother than adjusted subcutaneous heparin. Heparin does not offer a clear advantage over oral anticoagulation in the pregnancy outcome.


Keywords: pregnancy; oral anticoagulants; heparin; prosthetic valves PMID:10377303

3-O sulfation of heparin leads to hepatotropism and longer circulatory half-life.

PubMed

Miller, Colton M; Xu, Yongmei; Kudrna, Katrina M; Hass, Blake E; Kellar, Brianna M; Egger, Andrew W; Liu, Jian; Harris, Edward N

2018-05-17

Heparins are common blood anticoagulants that are critical for many surgical and biomedical procedures used in modern medicine. In contrast to natural heparin derived from porcine gut mucosa, synthetic heparins are homogenous by mass, polymer length, and chemistry. Stable cell lines expressing the human and mouse Stabilin receptors were used to evaluate endocytosis of natural and synthetic heparin. We chemoenzymatically produced synthetic heparin consisting of 12 sugars (dodecamers) containing 14 sulfate groups resulting in a non-3-O sulfated structure (n12mer). Half of the n12mer was modified with a 3-O sulfate on a single GlcNS sugar producing the 3-O sulfated heparin (12mer). Wildtype (WT), Stabilin-1 knock-out (KO), and Stabilin-2 KO C57BL/6 mice were developed and used for metabolic studies and provided as a source for primary liver sinusoidal endothelial cells. Human and mouse Stabilin-2 receptors had very similar endocytosis rates of both the 12mer and n12mer, suggesting that they are functionally similar in primary cells. Subcutaneous injections of the n12mer and 12mer revealed that the 12mer had a much longer half-life in circulation and a higher accumulation in liver. The n12mer never accumulated in circulation and was readily excreted by the kidneys before liver accumulation could occur. Liver sinusoidal endothelial cells from the Stabilin-2 KO mice had lower uptake rates for both dodecamers, whereas, the Stabilin-1 KO mice had lower endocytosis rates for the 12mer than the n12mer. 3-O sulfation of heparin is correlated to both a longer circulatory half-life and hepatotropism which is largely performed by the Stabilin receptors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Heparin removal by ecteola-cellulose pre-treatment enables the use of plasma samples for accurate measurement of anti-Yellow fever virus neutralizing antibodies.

PubMed

Campi-Azevedo, Ana Carolina; Peruhype-Magalhães, Vanessa; Coelho-Dos-Reis, Jordana Grazziela; Costa-Pereira, Christiane; Yamamura, Anna Yoshida; Lima, Sheila Maria Barbosa de; Simões, Marisol; Campos, Fernanda Magalhães Freire; de Castro Zacche Tonini, Aline; Lemos, Elenice Moreira; Brum, Ricardo Cristiano; de Noronha, Tatiana Guimarães; Freire, Marcos Silva; Maia, Maria de Lourdes Sousa; Camacho, Luiz Antônio Bastos; Rios, Maria; Chancey, Caren; Romano, Alessandro; Domingues, Carla Magda; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis

2017-09-01

Technological innovations in vaccinology have recently contributed to bring about novel insights for the vaccine-induced immune response. While the current protocols that use peripheral blood samples may provide abundant data, a range of distinct components of whole blood samples are required and the different anticoagulant systems employed may impair some properties of the biological sample and interfere with functional assays. Although the interference of heparin in functional assays for viral neutralizing antibodies such as the functional plaque-reduction neutralization test (PRNT), considered the gold-standard method to assess and monitor the protective immunity induced by the Yellow fever virus (YFV) vaccine, has been well characterized, the development of pre-analytical treatments is still required for the establishment of optimized protocols. The present study intended to optimize and evaluate the performance of pre-analytical treatment of heparin-collected blood samples with ecteola-cellulose (ECT) to provide accurate measurement of anti-YFV neutralizing antibodies, by PRNT. The study was designed in three steps, including: I. Problem statement; II. Pre-analytical steps; III. Analytical steps. Data confirmed the interference of heparin on PRNT reactivity in a dose-responsive fashion. Distinct sets of conditions for ECT pre-treatment were tested to optimize the heparin removal. The optimized protocol was pre-validated to determine the effectiveness of heparin plasma:ECT treatment to restore the PRNT titers as compared to serum samples. The validation and comparative performance was carried out by using a large range of serum vs heparin plasma:ECT 1:2 paired samples obtained from unvaccinated and 17DD-YFV primary vaccinated subjects. Altogether, the findings support the use of heparin plasma:ECT samples for accurate measurement of anti-YFV neutralizing antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

The use of heparin in infertility and recurrent pregnancy loss: Are its antiviral properties at play?

PubMed

Eliassen, Eva; Marci, Roberto; Di Luca, Dario; Rizzo, Roberta

2017-05-01

Recently, human herpesvirus 6 (HHV-6) has been implicated in cases of poor pregnancy outcomes. The ability of HHV-6 to disrupt endothelial cell functioning may inhibit the creation of an appropriate uterine environment for implantation and fetal development, resulting in infertility and pregnancy loss, among other complications. Heparin has been used to treat pregnant women who are predisposed to thrombosis, and it has also been used in some women with infertility or recurrent pregnancy loss without associated thrombotic events. Positive pregnancy outcomes after heparin treatment in these groups indicate that heparin may alter the uterine environment to make it more suitable for implantation and fetal growth. In this paper, we propose that in some cases, heparin may accomplish this by inhibiting HHV-6 transfer to the endometrium and/or by offsetting the virally-mediated effects of endothelial cell injury. Copyright © 2017 Elsevier Ltd. All rights reserved.

Thromboelastography during coronary artery bypass grafting surgery of severe hemophilia A patient - the effect of heparin and protamine on factor VIII activity.

PubMed

Misgav, Mudi; Mandelbaum, Tal; Kassif, Yigal; Berkenstadt, Haim; Tamarin, Ilia; Kenet, Gili

2017-06-01

: Coronary artery bypass grafting surgery (CABG) in hemophilia patients is challenging. Thromboelastography (TEG) is useful to assess hemostasis perioperatively. A patient with severe hemophilia A underwent CABG with TEG studies. After factor VIII (FVIII) bolus dose, TEG was normalized. Following 'on-pump' heparinization, protamine administration revealed prolonged TEG-R and TEG-R with heparinase confirming it, whereas the activated clotting time was normal, suggesting low FVIII activity rather than excess of heparin. Another FVIII bolus yielded complete normalization of all TEG parameters. Data are compatible with in-vitro assays performed in our laboratory, showing that both heparin and protamine may impair measurable FVIII activity. The rational use of TEG measurements enabled more accurate hemostatic therapy application with regard to FVIII, heparin and protamine administration. Adopting this approach may lead to a better therapy tailoring for hemophilia patients undergoing CABG surgery.

Efficacy of aprotinin with various anticoagulant agents in cardiopulmonary bypass.

PubMed

Terrell, M R; Walenga, J M; Koza, M J; Pifarré, R

1996-08-01

Aprotinin has recently been approved for clinical use in cardiopulmonary bypass. Although unfractionated heparin has been the only anticoagulant widely used for cardiopulmonary bypass, disadvantages involving heparin have led to ongoing investigations of alternative anticoagulant agents. The objective of this study was to evaluate the efficacy of aprotinin in combination with other anticoagulant agents, specifically low molecular weight heparin and recombinant hirudin, using a dog model of cardiopulmonary bypass. The blood conservation resulting from the use of aprotinin was observed only with unfractionated heparin. Efficacy of anticoagulation as measured by protein deposits in the bypass circuit filter revealed an unexpected reduction in the quantity of deposits when aprotinin was used in combination with low molecular weight heparin. As alternative anticoagulant agents are sought, the potential benefits of aprotinin in the reduction of operative blood loss must be evaluated independently for each anticoagulant agent.

Low molecular weight heparin restores antithrombin III activity from hyperglycemia induced alterations.

PubMed

Ceriello, A; Marchi, E; Palazzni, E; Quatraro, A; Giugliano, D

1990-01-01

Alteration of antithrombin III (ATIII) activity, glycemia level dependent, exists in diabetes mellitus. In this study the ability of a low molecular weight heparin (LMWH) (Fluxum, Alfa-Wassermann S.p.A., Bologna, Italy), as well as unfractioned héparin, to preserve ATIII activity from glucose-induced alterations, both in vitro and in vivo, is reported. The subcutaneous and intravenous LMWH and heparin administration increases basal depressed ATIII activity in diabetic patients. Heparin shows an equivalent effect on both anti-IIa and anti-Xa activity of ATIII, while LMWH is more effective in preserving the anti-Xa activity. Similarity, heparin preserves ATIII activity from hyperglycemia-induced alterations, during hyperglycemic clamp, and LMWH infusion is able to preserve a significant amount of anti-Xa activity from glucose-induced alterations. Since diabetic patients show a high incidence of thrombotic accidents, LMWH appears to be a promising innovation for the prevention of diabetic thrombophylia.

Unsuitability of evacuated tubes for monitoring heparin therapy by activated partial thromboplastin time.

PubMed Central

Heyns, A D; van den Berg, D J; Kleynhans, P H; du Toit, P W

1981-01-01

Activated partial thromboplastin times (APTT) for monitoring heparin therapy for venous thromboembolism tended to be inappropriately short if blood was collected in commercially available evacuated glass tubes. Five types of evacuated tubes marketed under the trade names Vacutainer and Venoject were examined. The APTT of heparinized blood collected in these tubes correlated poorly (r = 0.04 to 4 = 0.25) with that of blood samples from the same patients collected in plastic tubes. Most of the evacuated tube APTT were shorter than that of blood collected in plastic or siliconised glass tubes, but the results were unpredictable and varied from tube to tube and from batch to batch. This effect on heparin is apparently due to an unidentified substances which is eluted from the rubber stoppers of the tubes. Heparin control according to the APTT blood collected in these evacuated tubes is hazardous. PMID:7462439

Heparin: new life for an old drug.

PubMed

Aláez-Versón, Carlos Raúl; Lantero, Elena; Fernàndez-Busquets, Xavier

2017-07-01

Heparin is one of the oldest drugs, which nevertheless remains in widespread clinical use as an inhibitor of blood coagulation. The history of its identification a century ago unfolded amid one of the most fascinating scientific controversies turning around the distribution of credit for its discovery. The composition, purification and structure-function relationship of this naturally occurring glycosaminoglycan regarding its classical role as anticoagulant will be dealt with before proceeding to discuss its therapeutic potential in, among other, inflammatory and infectious disease, cancer treatment, cystic fibrosis and Alzheimer's disease. The first bibliographic reference hit using the words 'nanomedicine' and 'heparin' is as recent as 2008. Since then, nanomedical applications of heparin have experienced an exponential growth that will be discussed in detail, with particular emphasis on its antimalarial activity. Some of the most intriguing potential applications of heparin nanomedicines will be exposed, such as those contemplating the delivery of drugs to the mosquito stages of malaria parasites.

Structural Insights into Ail-Mediated Adhesion in Yersinia pestis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamashita, Satoshi; Lukacik, Petra; Barnard, Travis J.

2012-01-30

Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin,more » and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.« less

The History of Antithrombotic Therapy: The Discovery of Heparin, the Vitamin K Antagonists, and the Utility of Aspirin.

PubMed

Handin, Robert I

2016-10-01

The administration of intravenous heparin to postoperative patients by Barritt and Jordan reduced the incidence of fatal and nonfatal pulmonary embolism and established heparin as the standard for parenteral anticoagulation. The coumarin family of vitamin K antagonists quickly became the standard for long-term oral anticoagulation. Aspirin became a widely used antithrombotic agent after the discovery that chronic oral administration reduced the incidence of secondary strokes and myocardial infarction. This article gives a brief history of antithrombotic therapy, including the discovery of heparin, the vitamin k antagonists, and the utility of aspirin. Copyright © 2016 Elsevier Inc. All rights reserved.

[Intraoperative administration of low-molecular-weight heparins in reconstructive vascular operations].

PubMed

Pokrovskiĭ, A V; Demidova, V S; Titova, M I; Gontarenko, V N; Burtseva, E A

2008-01-01

The article deals with analysing the outcomes of administering low-molecular-weight heparins (LMWH) by the example of nadroparin ("Fraxiparin") during the intraoperative period in patients diagnosed with atherosclerotic lesions of femoropoplietal-crural segment of the lower-limb arteries as compared with non-fractionated heparin (NFH). Studied were the alterations in the parameters of the plasmatic and thrombocytic links of haemostasis on the background of administering various molecular-weight fractions of heparin. A conclusion was drawn on advantageous use of LMWH in the cohort of the patients involved. Also presented herein is an analysis of the literature data concerning appropriate usage of LMWH during the intraoperative period.

Use of prestudy heparin did not influence the efficacy and safety of rivaroxaban in patients treated for symptomatic venous thromboem-bolism in the EINSTEIN DVT and EINSTEIN PE studies.

PubMed

Prandoni, Paolo; Prins, Martin H; Cohen, Alexander T; Müller, Katharina; Pap, Ákos F; Tewes, Miriam C; Lensing, Anthonie W A

2015-02-01

In the EINSTEIN DVT and EINSTEIN PE studies, the majority of patients received heparins to bridge the period during venous thromboembolism (VTE) diagnosis confirmation and the start of the study. In contrast to vitamin K antagonists (VKAs), rivaroxaban may not require initial heparin treatment. To evaluate the effect of prestudy heparin on the efficacy and safety of rivaroxaban relative to enoxaparin/VKA, the 3-month incidence of recurrent VTE, and the 14-day incidence of major and nonmajor clinically relevant bleeding were compared in patients who did and did not receive prestudy heparin. Of the 8,281 patients randomized, 6,937 (83.8%) received prestudy heparin (mean ± SD duration = rivaroxaban: 1.04 [± 0.74] days; enoxaparin 1.03 [± 0.42] days), and 1,344 (16.2%) did not. In patients who did not receive prestudy heparin, the incidences of recurrent VTE were similar in rivaroxaban (15 of 649, 2.3%) and enoxaparin/VKA (13 of 695, 1.9%) patients (adjusted hazard ratio [HR] = 1.11; 95% confidence interval [CI] = 0.52 to 2.37). The incidences of recurrent VTE were also similar in rivaroxaban (54 of 3,501, 1.5%) and enoxaparin/VKA (69 of 3,436, 2.0%) patients who did receive prestudy heparin (adjusted HR = 0.74; 95% CI = 0.52 to 1.06; pinteraction  = 0.32). The incidences of major or nonmajor clinically relevant bleeding with rivaroxaban were not significantly different from those with enoxaparin/VKA, either with (105 of 3,485, 3.0% vs. 104 of 3,428, 3.0%; adjusted HR = 0.98; 95% CI = 0.75 to 1.29) or without (24 of 645, 3.7% vs. 30 of 688, 4.4%; adjusted HR = 0.81; 95% CI = 0.46 to 1.40; pinteraction  = 0.68) prestudy heparin. Although the majority of patients in the EINSTEIN studies received prestudy heparin, there were no notable differences in treatment effect of rivaroxaban versus enoxaparin/VKA in those who did and did not receive it. © 2015 by the Society for Academic Emergency Medicine.

Effect of pre-procedural interrupted apixaban on heparin anticoagulation during catheter ablation for atrial fibrillation: a prospective observational study.

PubMed

Bin Abdulhak, Aref A; Kennedy, Kevin F; Gupta, Sanjaya; Giocondo, Michael; Ramza, Brian; Wimmer, Alan P

2015-11-01

Effective intraprocedural anticoagulation is considered essential to minimize the risk of thromboembolism in catheter ablation (CA) of atrial fibrillation (AF). The effect of interrupted apixaban on intraprocedural heparin dosing requirements and levels of achieved anticoagulation with heparin has not been well studied. The purpose of the present study was to compare heparin administration and activated clotted times (ACTs) for patients undergoing CA for AF treated with interrupted apixaban before the procedure with patients on uninterrupted warfarin. Consecutive patients undergoing CA for AF treated with interrupted apixaban or uninterrupted warfarin were prospectively enrolled. Heparin administration determined by a standard protocol and normalized to patient weight and procedure duration, as well as rapidity, and degree of anticoagulation with heparin (as measured by mean ACT, peak ACT, time to ACT ≥300 s, and time to ACT ≥350 s) were compared between the groups. Forty-eight patients were enrolled (25 apixaban and 23 warfarin). Heparin administered by bolus (51.3 ± 21.5 vs 27.8 ± 9.6 units/kg/h; p < 0.001) and mean heparin drip rate (25.3 ± 3.6 vs 20.7 ± 2.4 units/kg/h; p < 0.001) were significantly higher in the apixaban group compared to the warfarin group. Despite greater heparin administration, apixaban patients achieved a significantly lower mean ACT (332.3 ± 17.0 vs 384.5 ± 53.9; p < 0.001) and peak ACT (369.5 ± 22.6 vs 432.3 ± 75.8, p < 0.001) compared to the warfarin group. The time to ACT ≥350 s (66.7 ± 35.8 vs 26.9 ± 34.0; p < 0.001) was significantly longer for apixaban-treated patients. Outcome differences persisted after analysis using linear models and Cox proportional hazard regression with adjustment for propensity scores. A standard intraprocedural heparin protocol results in delayed and lower levels of anticoagulation as measured by the ACT for interrupted apixaban-treated patients in comparison to those on uninterrupted warfarin during CA of AF.

Heparin Reduced Mortality and Sepsis in Severely Burned Children

PubMed Central

Zayas, G.J.; Bonilla, A.M.; Saliba, M.J

2007-01-01

Summary Objectives. In El Salvador, before 1999, morbidity and mortality in severely burned children were high. In 1998, all children with burns of 40% or larger size died and sepsis was found. With heparin use in 1999, some similarly burned children survived, and sepsis, pain, procedures, and scars were noted to be less. This retrospective study presents the details. Methods. A study was conducted at the National Children's Hospital in El Salvador of all children with burns over 20% size treated in 1998, when no heparin was used, and in 1999, when heparin was added to burns treatment, using an ethics committee approved protocol in use in twelve other countries. Sodium aqueous heparin solution USP from an intestinal source was infused intravenously and applied topically onto burn surfaces and within blisters for the first 1-3 days post-burn. Then heparin, in diminishing doses, was continued only topically until healing. The treatments in 1998 and 1999 were otherwise the same, except that fewer procedures were needed in 1999. Results. There were no significant differences in gender, age, weight, burn aetiology, or burn size between the burned children in 1998 and those in 1999. Burn pain was relieved and pain medicine was not needed in children treated with heparin in 1999. In 1998, one child survived who had a 35% size burn, and the eight children died who had burns of 40% and over. The survival rate was one out of nine (11%). The average burn size was 51.7%. With heparin use in 1999, six of the ten children survived burns of 50.7% average size. The increase in survival with heparin from 11% to 60% and, therefore, the decrease in mortality from 89% to 40% were significant (p < 0.04). Clinical symptoms and positive blood cultures documented bacterial sepsis in the nine children in 1998. In 1999, the blood cultures for sepsis were positive in the four children who died and negative in the six who survived. The nine versus four differences in the incidence of sepsis between 1998 and 1999 was significant (p < 0.008). The survivors had notably smooth skin. Conclusions. The use of heparin in this study relieved burn pain, significantly reduced mortality and sepsis with fewer procedures, and discernibly improved cosmetic results. PMID:21991064

Pentadecapeptide BPC 157 reduces bleeding time and thrombocytopenia after amputation in rats treated with heparin, warfarin or aspirin.

PubMed

Stupnisek, Mirjana; Franjic, Sandra; Drmic, Domagoj; Hrelec, Masa; Kolenc, Danijela; Radic, Bozo; Bojic, Davor; Vcev, Aleksandar; Seiwerth, Sven; Sikiric, Predrag

2012-05-01

Recently, in rat abdominal aorta terminoterminal-anastomosis the stable gastric pentadecapeptide BPC 157 prevents obstructive thrombus formation and rapidly destroys already formed obstructive thrombus. Also, BPC 157 wound healing may signify the clot as conductive matrix or "scaffold" to speed up wound healing process, and decrease bleeding. Here, in rats, BPC 157 (10 μg/kg, 10 ng/kg) improved always reduced bleeding time and amount of bleeding after (tail) amputation only, heparin (250 mg/kg, 25mg/kg, 10mg/kg i.v.), warfarin (1.5mg/kg i.g. once daily for 3 consecutive days), aspirin (0.1g/kg i.g. (once daily/3 consecutive days) or 1.0 g/kg i.p. once), and amputation associated with those agents application. BPC 157 counteracting regimens (i.v., i.p., i.g. (immediately after any challenge)) correspondingly follow the route of bleeding-agents application. All heparin-, warfarin-, and aspirin-rats and normal-rats that received BPC 157 exhibited lesser fall in platelets count. BPC 157 attenuated over-increased APTT-, TT-values in 10mg/kg heparin-rats, but did not influence heparin activity (anti-Xa test). Indicatively, unless counteracted in BPC 157 rats, excessive bleeding-acute thrombocytopenia (<20% of initial values in heparin-rats) approaches substantial fall in platelets count known in type II HIT. Also, BPC 157 markedly prolongs the survival time (heparin-rats, 25mg/kg, right foot amputation). Copyright © 2011 Elsevier Ltd. All rights reserved.

Systematic Evaluation of the Use of Human Plasma and Serum for Mass-Spectrometry-Based Shotgun Proteomics.

PubMed

Lan, Jiayi; Núñez Galindo, Antonio; Doecke, James; Fowler, Christopher; Martins, Ralph N; Rainey-Smith, Stephanie R; Cominetti, Ornella; Dayon, Loïc

2018-04-06

Over the last two decades, EDTA-plasma has been used as the preferred sample matrix for human blood proteomic profiling. Serum has also been employed widely. Only a few studies have assessed the difference and relevance of the proteome profiles obtained from plasma samples, such as EDTA-plasma or lithium-heparin-plasma, and serum. A more complete evaluation of the use of EDTA-plasma, heparin-plasma, and serum would greatly expand the comprehensiveness of shotgun proteomics of blood samples. In this study, we evaluated the use of heparin-plasma with respect to EDTA-plasma and serum to profile blood proteomes using a scalable automated proteomic pipeline (ASAP 2 ). The use of plasma and serum for mass-spectrometry-based shotgun proteomics was first tested with commercial pooled samples. The proteome coverage consistency and the quantitative performance were compared. Furthermore, protein measurements in EDTA-plasma and heparin-plasma samples were comparatively studied using matched sample pairs from 20 individuals from the Australian Imaging, Biomarkers and Lifestyle (AIBL) Study. We identified 442 proteins in common between EDTA-plasma and heparin-plasma samples. Overall agreement of the relative protein quantification between the sample pairs demonstrated that shotgun proteomics using workflows such as the ASAP 2 is suitable in analyzing heparin-plasma and that such sample type may be considered in large-scale clinical research studies. Moreover, the partial proteome coverage overlaps (e.g., ∼70%) showed that measures from heparin-plasma could be complementary to those obtained from EDTA-plasma.

Comparative effectiveness of 30 % trisodium citrate and heparin lock solution in preventing infection and dysfunction of hemodialysis catheters: a randomized controlled trial (CITRIM trial).

PubMed

Correa Barcellos, Franklin; Pereira Nunes, Bruno; Jorge Valle, Luciana; Lopes, Thiago; Orlando, Bianca; Scherer, Cintia; Nunes, Marcia; Araújo Duarte, Gabriela; Böhlke, Maristela

2017-04-01

Central venous catheters (CVC) are the only option when hemodialysis is needed for patients without definitive vascular access. However, CVC is associated with complications, such as infection, thrombosis, and dysfunction, leading to higher mortality and expenditures. The aim of this study was to compare the effectiveness of 30 % trisodium citrate (TSC30 %) with heparin as CVC lock solutions in preventing catheter-related bloodstream infections (CRBSI) and dysfunction in hemodialysis patients. Randomized, double-blind controlled trial comparing the event-free survival of non-tunneled CVC locked with heparin or TSC30 % in adult hemodialysis patients. The study included 464 catheters, 233 in heparin group, and 231 in TSC30 % group. The CRBSI-free survival of TSC30 % group was significantly shorter than that of heparin group. When stratified by insertion site, heparin was better than TSC30 % only in subclavian CVC. The dysfunction-free survival was not different between groups in the main analysis, but there is also a shorter survival among subclavian CVC locked with TSC30 % in stratified analysis. There was no difference on CRBSI-free or dysfunction-free survival between jugular vein CVC locked with heparin or 30 % citrate. However, subclavian CVC locked with 30 % citrate presented shorter event-free survival. This difference may be related to anatomical and positional effects, CVC design, and hydraulic aspects of the lock solution. CLINICALTRIALS. NCT02563041.

Heparin sodium compliance to the new proposed USP monograph: elucidation of a minor structural modification responsible for a process dependent 2.10 ppm NMR signal.

PubMed

Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Viskov, Christian

2011-01-25

Heparin is a highly sulfated hetero polysaccharide mixture found and extracted from mammalian tissues. It has been widely used as an anticoagulant drug during the past decades. In the new proposed USP heparin monograph, the ¹H NMR acceptance criteria to prevent contamination by over sulfated chondroitin sulfate (OSCS), or other persulfated glycosaminoglycans, specifies that no unidentified signals greater than 4% of the mean of signal height of 1 and 2 should be present in the following ranges: 0.10-2.00, 2.10-3.20, and 5.70-8.00 ppm. However, those criteria do not take into account the impact of potential structural modifications generated by the heparin manufacturing processes. In fact, starting from pig mucosa, heparin purification involves oxidizing reagents such as sodium peroxide, potassium permanganate and peracetic acid. In the present work, we demonstrate that potassium permanganate treated heparins show a small but characteristic extra signal at 2.10 ppm. Controlled heparinase I depolymerisation is used to target and excise the oligosaccharide responsible for this extra signal from the polysaccharide backbone. By using orthogonal chromatographic techniques, the fingerprint oligosaccharide was isolated and its structure elucidated. Without the identification of this structural moiety, such purified heparins may have been considered as non-compliant drug substance and not suitable for pharmaceutical use. Copyright © 2010 Elsevier B.V. All rights reserved.

Simultaneous estimation of vitamin K1 and heparin with low limit of detection using cascaded channels fiber optic surface plasmon resonance.

PubMed

Tabassum, Rana; Gupta, Banshi D

2016-12-15

We report an approach for the simultaneous estimation of vitamin K1 (VK1) and heparin via cascaded channel multianalyte sensing probe employing fiber optic surface plasmon resonance technique. Cladding from two well separated portions of the fiber is removed and are respectively coated with thin films of silver (channel-1) and copper (channel-2). The nanohybrid of multiwalled carbon nanotube in chitosan is fabricated over silver layer for the sensing of VK1 whereas core shell nanostructure of polybrene@ZnO is coated over copper layer for the sensing of heparin. Spectral interrogation method is used for the characterization of the sensor. Analyte selectivity of both the channels is performed by carrying out experiments using independent solutions of VK1 and heparin. Experiments performed on the solution of the mixture of VK1 and heparin show red shifts in both the channels on changing the concentration of both the analytes in the mixture. The operating range of both VK1 and heparin is from 0 to 10(-3)g/l. The limit of detection of the sensor is 2.66×10(-4)µg/l and 2.88×10(-4)µg/l for VK1 and heparin respectively which are lower than the reported ones. The additional advantages of the present sensor are low cost, possibility of online monitoring and remote sensing. Copyright © 2016 Elsevier B.V. All rights reserved.

Low molecular weight heparin and cancer survival: clinical trials and experimental mechanisms.

PubMed

Zhang, Ning; Lou, Weihua; Ji, Fang; Qiu, Lihua; Tsang, Benjamin K; Di, Wen

2016-08-01

The relationship between cancer and thrombosis is complex, as the hemostatic system is inextricably linked to the mechanisms of cancer growth and metastasis. The coagulation system thus appears to be a site for oncogenic events and necessary for the survival and spread of malignant cells. Although several meta-analyses on the effectiveness of unfractionated heparin and low molecular weight heparin (LMWH) in the treatment of venous thromboembolism (VTE) have suggested a lower mortality risk in cancer patients receiving LMWH, this contention has not received general acceptance. In fact, there exist no sufficiently powered studies to date supporting the routine use of LMWH to improve cancer survival. Meanwhile, the molecular mechanism underlying the anti-neoplastic effect of LMWH which is independent of its anti-coagulant function is largely unexplored and is a topic of active investigation. In this communication, we aimed to review comprehensively evidences from clinical trials, meta-analysis as well as experimental molecular research and to identify future research areas of importance so as to stimulate future research on the potential anti-tumor action of LMWH. Although benefit of LMWH on cancer patients' survival is controversial depending on the tumor type, cancer stage as well as LMWH type, it appears to be associated with a reduction in VTE and increased bleeding is minor and controllable; thus, randomized controlled trials targeting the survival benefit of certain specific LWMH are needed and justified, and more in-depth experimental researches are imperative to elucidate the anti-tumor effect of anticoagulants.

Cost Effectiveness of Antiplatelet and Antithrombotic Therapy in The Setting of Acute Coronary Syndrome: Current Perspective and Literature Review

PubMed Central

Fanari, Zaher; Weiss, Sandra; Weintraub, William S

2015-01-01

Acute Coronary Syndromes are associated with high rates of morbidity and mortality. The advances of antiplatelet and anticoagulation therapy over several years time have result in in improved in cardiac outcomes, but with increased health care costs. Multiple cost effectiveness studies have been performed to evaluate the use of available antiplatelet agents and anticoagulation in the setting of both ST Elevation myocardial infarction (STEMI) and Non–ST Elevation Acute Coronary Syndrome (NSTE-ACS). Early on the use of GPI prove to be economically attractive in the management of ACS, however the introduction of P2Y12 receptor antagonist limited their use to a bail out agents in complex interventions. Generic clopidogrel is probably still an economically attractive P2Y12 receptor antagonist choice especially in low risk ACS, while both ticagrelor and prasugrel present an economically attractive alternative option especially in high risk ACS and patients at risk for stent thrombosis. While enoxaparin presents an economically dominant alternative to heparin in NSTE-ACS, its role in STEMI in the contemporary era is unclear. During PCI, bivalirudin monotherapy was shown to be an economically dominant alternative to the combination of heparin and GPI in ACS. However, new studies may suggest that using heparin monotherapy may offer an attractive alternative. The comparative and cost effectiveness of different combinations of antiplatelet and antithrombotic therapy will be the focus of future expected clinical and economic assessments. PMID:26068886

High sensitivity and specificity of a new functional flow cytometry assay for clinically significant heparin-induced thrombocytopenia antibodies.

PubMed

Garritsen, H S; Probst-Kepper, M; Legath, N; Eberl, W; Samaniego, S; Woudenberg, J; Schuitemaker, J H N; Kroll, H; Gurney, D A; Moore, G W; Zehnder, J L

2014-04-01

Heparin-induced thrombocytopenia (HIT) is a life-threatening condition, in which the anticoagulant heparin, platelet factor 4 (PF4), and platelet-activating antibodies form complexes with prothrombotic properties. Laboratory tests to support clinical diagnosis are subdivided into functional, platelet activation assays, which lack standardization, or immunological assays, which have moderate specificity toward HIT. In this study, clinical performance of HITAlert, a novel in vitro diagnostic (IVD) registered platelet activation assay, was tested in a large cohort of HIT-suspected patients and compared with immunological assays. From 346 HIT-suspected patients (single center), clinical data including 4T pretest probability results, citrated platelet-poor plasmas, and sera were collected, allowing direct comparison of clinical observations with HITAlert results. HITAlert performance was compared with PF4 IgG ELISA (246 patients, three centers) and PF4 PaGIA (298 patients, single center). HITAlert showed high sensitivity (88.2%) and specificity (99.1%) when compared with clinical diagnosis. Agreement of HITAlert with PF4 ELISA- and PF4 PaGIA-positive patients is low (52.7 and 23.2%, respectively), while agreement with PF4 IgG ELISA- and PF4 PaGIA-negative patients is very high (98.1 and 99.1%, respectively). HITAlert performance is excellent when compared with clinical HIT diagnosis, making it a suitable assay for rapid testing of platelet activation due to anticoagulant therapy. © 2013 John Wiley & Sons Ltd.

Interaction of albumins and heparinoids investigated by affinity capillary electrophoresis and free flow electrophoresis.

PubMed

Mozafari, Mona; El Deeb, Sami; Krull, Friederike; Wildgruber, Robert; Weber, Gerhard; Reiter, Christian G; Wätzig, Hermann

2018-02-01

A fast and precise affinity capillary electrophoresis (ACE) method has been applied to investigate the interactions between two serum albumins (HSA and BSA) and heparinoids. Furthermore, different free flow electrophoresis methods were developed to separate the species which appears owing to interaction of albumins with pentosan polysulfate sodium (PPS) under different experimental conditions. For ACE experiments, the normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. ACE experiments were performed at two different temperatures (23 and 37°C). Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, the interactions can already be observed at low mg/L concentrations (3 mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The additional signals also appeared at higher concentrations at 37°C. Nevertheless, in most cases the binding data were similar at both temperatures. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The free flow electrophoresis methods have confirmed and completed the ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis and structural study of two new heparin-like hexasaccharides.

PubMed

Lucas, Ricardo; Angulo, Jesús; Nieto, Pedro M; Martín-Lomas, Manuel

2003-07-07

Two new heparin-like hexasaccharides, 5 and 6, have been synthesised using a convergent block strategy and their solution conformations have been determined by NMR spectroscopy and molecular modelling. Both hexasaccharides contain the basic structural motif of the regular region of heparin but with negative charge distributions which have been designed to get insight into the mechanism of fibroblast growth factors (FGFs) activation.

Sustained Thromboresistant Bioactivity with Reduced Intimal Hyperplasia of Heparin-Bonded Polytetrafluoroethylene Propaten Graft in a Chronic Canine Femoral Artery Bypass Model.

PubMed

Freeman, John; Chen, Aaron; Weinberg, Roy J; Okada, Tamuru; Chen, Changyi; Lin, Peter H

2018-05-01

Bypass graft thrombosis remains a significant mode of failure in prosthetic graft revascularization. The purpose of this investigation was to evaluate the long-term thromboresistant effect of heparin-bonded expanded polytetrafluoroethylene (ePTFE) graft using Carmeda BioActive Surface technology in a canine model. Bilateral femorofemoral artery bypass grafts with ePTFE grafts were performed in 25 adult grayhound dogs. In each animal, a heparin-bonded ePTFE graft (Propaten, WL Gore) was placed on one side, whereas a control nonheparin graft was placed on the contralateral side. The graft patency was assessed at 1, 6, 12, 18, and 24 months (n = 5 per group) following the bypass. Heparin bioactivity of the graft material was analyzed. The effect of intimal hyperplasia was also assessed. All bypass grafts were patent at 1 month. Significantly greater patency rates were noted in the Propaten group compared to the control group at 12, 18, and 24 months, which were 84%, 80%, and 80% vs. 55%, 35%, and 20%, respectively (P < 0.02). There was a significant reduction in the anastomotic neointimal area and neointimal cell proliferation in Propaten grafts compared with control grafts at all groups between 6 and 24 months (P < 0.05). Heparin bioactivity as measured by antithrombin binding assay was demonstrated in the Propaten graft between 1 and 24 months. Mean heparin activities on Propaten grafts ranged from 26.3 ± 6.4 pmol/cm 2 to 18.4 ± 8.7 pmol/cm 2 between 1 and 24 months, which were significantly greater than the control group (P < 0.001). Differences between mean heparin activities of explanted Propaten graft samples at the various time points were nonsignificant (P > 0.05). Heparin-bonded ePTFE graft provides a thromboresistant surface and reduced anastomotic intimal hyperplasia at 2 years. The stable heparin bioactivity of the Propaten graft confers an advantage in long-term graft patency. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Targeted use of heparin, heparinoids, or low-molecular-weight heparin to improve outcome after acute ischaemic stroke: an individual patient data meta-analysis of randomised controlled trials

PubMed Central

Whiteley, William N; Adams, Harold P; Bath, Philip MW; Berge, Eivind; Sandset, Per Morten; Dennis, Martin; Murray, Gordon D; Wong, Ka-Sing Lawrence; Sandercock, Peter AG

2013-01-01

Summary Background Many international guidelines on the prevention of venous thromboembolism recommend targeting heparin treatment at patients with stroke who have a high risk of venous thrombotic events or a low risk of haemorrhagic events. We sought to identify reliable methods to target anticoagulant treatment and so improve the chance of avoiding death or dependence after stroke. Methods We obtained individual patient data from the five largest randomised controlled trials in acute ischaemic stroke that compared heparins (unfractionated heparin, heparinoids, or low-molecular-weight heparin) with aspirin or placebo. We developed and evaluated statistical models for the prediction of thrombotic events (myocardial infarction, stroke, deep vein thrombosis, or pulmonary embolism) and haemorrhagic events (symptomatic intracranial or significant extracranial) in the first 14 days after stroke. We calculated the absolute risk difference for the outcome “dead or dependent” in patients grouped by quartiles of predicted risk of thrombotic and haemorrhagic events with random effect meta-analysis. Findings Patients with ischaemic stroke who were of advanced age, had increased neurological impairment, or had atrial fibrillation had a high risk of both thrombotic and haemorrhagic events after stroke. Additionally, patients with CT-visible evidence of recent cerebral ischaemia were at increased risk of thrombotic events. In evaluation datasets, the area under a receiver operating curve for prediction models for thrombotic events was 0·63 (95% CI 0·59–0·67) and for haemorrhagic events was 0·60 (0·55–0·64). We found no evidence that the net benefit from heparins increased with either increasing risk of thrombotic events or decreasing risk of haemorrhagic events. Interpretation There was no evidence that patients with ischaemic stroke who were at higher risk of thrombotic events or lower risk of haemorrhagic events benefited from heparins. We were therefore unable to define a targeted approach to select the patients who would benefit from treatment with early anticoagulant therapy. We recommend that guidelines for routine or selective use of heparin in stroke should be revised. Funding MRC. PMID:23642343

Heparin or 0.9% sodium chloride to maintain central venous catheter patency: a randomized trial.

PubMed

Schallom, Marilyn E; Prentice, Donna; Sona, Carrie; Micek, Scott T; Skrupky, Lee P

2012-06-01

To compare heparin (3 mL, 10 units/mL) and 0.9% sodium chloride (NaCl, 10 mL) flush solutions with respect to central venous catheter lumen patency. Single-center, randomized, open label trial. Medical intensive care unit and Surgical/Burn/Trauma intensive care unit at Barnes-Jewish Hospital, St. Louis, MO. Three hundred forty-one patients with multilumen central venous catheters. Patients with at least one lumen with a minimum of two flushes were included in the analysis. Patients were randomly assigned within 12 hrs of central venous catheter insertion to receive either heparin or 0.9% sodium chloride flush. The primary outcome was lumen nonpatency. Secondary outcomes included the rates of loss of blood return, inability to infuse or flush through the lumen (flush failure), heparin-induced thrombocytopenia, and catheter-related blood stream infection. Assessment for patency was performed every 8 hrs in lumens without continuous infusions for the duration of catheter placement or discharge from intensive care unit. Three hundred twenty-six central venous catheters were studied yielding 709 lumens for analysis. The nonpatency rate was 3.8% in the heparin group (n = 314) and 6.3% in the 0.9% sodium chloride group (n = 395) (relative risk 1.66, 95% confidence interval 0.86-3.22, p = .136). The Kaplan-Meier analysis for time to first patency loss was not significantly different (log rank = 0.093) between groups. The rates of loss of blood return and flush failure were similar between the heparin and 0.9% sodium chloride groups. Pressure-injectable central venous catheters had significantly greater rates of nonpatency (10.6% vs. 4.3%, p = .001) and loss of blood return (37.0% vs. 18.8%, p <.001) compared to nonpressure-injectable catheters. The frequencies of heparin-induced thrombocytopenia and catheter-related blood stream infection were similar between groups. 0.9% sodium chloride and heparin flushing solutions have similar rates of lumen nonpatency. Given potential safety concerns with the use of heparin, 0.9% sodium chloride may be the preferred flushing solution for short-term use central venous catheter maintenance.

Targeted use of heparin, heparinoids, or low-molecular-weight heparin to improve outcome after acute ischaemic stroke: an individual patient data meta-analysis of randomised controlled trials.

PubMed

Whiteley, William N; Adams, Harold P; Bath, Philip M W; Berge, Eivind; Sandset, Per Morten; Dennis, Martin; Murray, Gordon D; Wong, Ka-Sing Lawrence; Sandercock, Peter A G

2013-06-01

Many international guidelines on the prevention of venous thromboembolism recommend targeting heparin treatment at patients with stroke who have a high risk of venous thrombotic events or a low risk of haemorrhagic events. We sought to identify reliable methods to target anticoagulant treatment and so improve the chance of avoiding death or dependence after stroke. We obtained individual patient data from the five largest randomised controlled trials in acute ischaemic stroke that compared heparins (unfractionated heparin, heparinoids, or low-molecular-weight heparin) with aspirin or placebo. We developed and evaluated statistical models for the prediction of thrombotic events (myocardial infarction, stroke, deep vein thrombosis, or pulmonary embolism) and haemorrhagic events (symptomatic intracranial or significant extracranial) in the first 14 days after stroke. We calculated the absolute risk difference for the outcome "dead or dependent" in patients grouped by quartiles of predicted risk of thrombotic and haemorrhagic events with random effect meta-analysis. Patients with ischaemic stroke who were of advanced age, had increased neurological impairment, or had atrial fibrillation had a high risk of both thrombotic and haemorrhagic events after stroke. Additionally, patients with CT-visible evidence of recent cerebral ischaemia were at increased risk of thrombotic events. In evaluation datasets, the area under a receiver operating curve for prediction models for thrombotic events was 0·63 (95% CI 0·59-0·67) and for haemorrhagic events was 0·60 (0·55-0·64). We found no evidence that the net benefit from heparins increased with either increasing risk of thrombotic events or decreasing risk of haemorrhagic events. There was no evidence that patients with ischaemic stroke who were at higher risk of thrombotic events or lower risk of haemorrhagic events benefited from heparins. We were therefore unable to define a targeted approach to select the patients who would benefit from treatment with early anticoagulant therapy. We recommend that guidelines for routine or selective use of heparin in stroke should be revised. MRC. Copyright © 2013 Elsevier Ltd. All rights reserved.

Quantitation of heparosan with heparin lyase III and spectrophotometry.

PubMed

Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J

2014-02-15

Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

Structural Basis for Antagonism by Suramin of Heparin Binding to Vaccinia Complement Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ganesh, Vannakambadi K.; Muthuvel, Suresh Kumar; Smith, Scott A.

2010-07-19

Suramin is a competitive inhibitor of heparin binding to many proteins, including viral envelope proteins, protein tyrosine phosphatases, and fibroblast growth factors (FGFs). It has been clinically evaluated as a potential therapeutic in treatment of cancers caused by unregulated angiogenesis, triggered by FGFs. Although it has shown clinical promise in treatment of several cancers, suramin has many undesirable side effects. There is currently no experimental structure that reveals the molecular interactions responsible for suramin inhibition of heparin binding, which could be of potential use in structure-assisted design of improved analogues of suramin. We report the structure of suramin, in complexmore » with the heparin-binding site of vaccinia virus complement control protein (VCP), which interacts with heparin in a geometrically similar manner to many FGFs. The larger than anticipated flexibility of suramin manifested in this structure, and other details of VCP-suramin interactions, might provide useful structural information for interpreting interactions of suramin with many proteins.« less

[Heparin and antiheparin in childhood. 2. Clinical relevance of the phenomenon of heparin resistance].

PubMed

Domula, M; Weissbach, G

1982-01-01

Investigations of the content of platelet factor 4 in different thrombocyte lysates and platelet-rich plasma after induced release reaction were aimed at checking the efficiency of the own antiheparin measuring system. In this connection, the age dependent dynamics of platelet factor 4 could be first discovered. In platelet-poor plasma of healthy grown-up test persons there was an evidence of antiheparin titres which were five times higher as compared with those persons born maturely. All patients with disseminated intravascular coagulation processes of different aetiology, however, will have significantly increased values. As demonstrated in two children with hyperpyretic toxicosis, the liberated platelet factor 4 will only show a short plasma half decay period. From investigations made for refinding heparin in the plasma after in vitro addition the conclusion can be drawn that, in addition to platelet factor 4, even unspecific adhesions of heparin to certain plasma proteins may be responsible for increasing heparin resistance.

Preparation, Separation, and Conformational Analysis of Differentially Sulfated Heparin Octasaccharide Isomers using Ion Mobility Mass Spectrometry

PubMed Central

Seo, Youjin; Andaya, Armann; Leary, Julie A.

2012-01-01

Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated based on their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations. PMID:22283665

Chronic intravascular coagulation associated with chronic myelocytic leukemia. Use of heparin in connection with a surgical procedure.

PubMed

German, H J; Smith, J A; Lindenbaum, J

1976-10-01

A women with Philadelphia chromosome-positive chronic myelocytic leukemia lived nearly 12 years from the time of diagnosis. During most of this period she received no therapy, and marked cyclic oscillations in the white blood cell count were documented. The last two years of her illness were marked by a hemorrhagic disorder associated with hypofibrinogenemia, thrombocytopenia, increased plasma fibrinopeptide A concentration and markedly elevated serum levels of fibrin degradation products. The coagulation disorder was rapidly reversible on several occasions with heparin therapy. After treatment with heparin and platelet transfusions, the patient underwent successful resection of a large ovarian cyst with excellent hemostasis during the procedure. Postoperatively, the administration of heparin and platelets was discontinued and a large wound hematoma developed. After resumption of therapy with heparin and platelets, the remainder of her postoperative course was uneventful. The literature on the subject is reviewed and tentative guidelines are offered concerning the management of patients with intravascular coagulation who require diagnostic or therapeutic surgical procedures.

Hemocompatible ɛ-polylysine-heparin microparticles: A platform for detecting triglycerides in whole blood.

PubMed

Xu, Tingting; Chi, Bo; Chu, Meilin; Zhang, Qicheng; Zhan, Shuyue; Shi, Rongjia; Xu, Hong; Mao, Chun

2018-01-15

Triglycerides are clinically important marker for atherosclerosis, heart disease and hypertension. Here, a platform for detecting triglycerides in whole blood directly was developed based on hemocompatible ɛ-polylysine-heparin microparticles. The obtained products of ɛ-polylysine-heparin microparticles were characterized by fourier transform infrared (FT-IR) spectra, transmission electron microscopy (TEM) and ζ-potential. Moreover, the blood compatibility of ɛ-polylysine-heparin microparticles was characterized by in vitro coagulation tests, hemolysis assay and whole blood adhesion tests. Considering of uniform particle size, good dispersibility and moderate long-term anticoagulation capability of the microparticles, a Lipase-(ɛ-polylysine-heparin)-glassy carbon electrode (GCE) was constructed to detect triglycerides. The proposed biosensor had good electrocatalytic activity towards triglycerides, in which case the sensitivity was 0.40μAmg -1 dLcm -2 and the detection limit was 4.67mgdL -1 (S/N = 3). Meanwhile, the Lipase-(ɛ-polylysine-heparin)-GCE electrode had strong anti-interference ability as well as a long shelf-life. Moreover, for the detection of triglycerides in whole blood directly, the detection limit was as low as 5.18mgdL -1 . The new constructed platform is suitable for detecting triglycerides in whole blood directly, which provides new analytical systems for clinical illness diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Self-Assembled Multivalent (SAMul) Polyanion Binding-Impact of Hydrophobic Modifications in the Micellar Core on DNA and Heparin Binding at the Peripheral Cationic Ligands.

PubMed

Albanyan, Buthaina; Laurini, Erik; Posocco, Paola; Pricl, Sabrina; Smith, David K

2017-05-05

This paper reports a small family of cationic surfactants designed to bind polyanions such as DNA and heparin. Each molecule has the same hydrophilic cationic ligand and a hydrophobic aliphatic group with eighteen carbon atoms with one, two, or three alkene groups within the hydrophobic chain (C18-1, C18-2 and C18-3). Dynamic light scattering indicates that more alkenes lead to geometric distortion, giving rise to larger self-assembled multivalent (SAMul) nanostructures. Mallard Blue and Ethidium Bromide dye displacement assays demonstrate that heparin and DNA have markedly different binding preferences, with heparin binding most effectively to C18-1, and DNA to C18-3, even though the molecular structural differences of these SAMul systems are buried in the hydrophobic core. Multiscale modelling suggests that adaptive heparin maximises enthalpically favourable interactions with C18-1, while shape-persistent DNA forms a similar number of interactions with each ligand display, but with slightly less entropic cost for binding to C18-3-fundamental thermodynamic differences in SAMul binding of heparin or DNA. This study therefore provides unique insight into electrostatic molecular recognition between highly charged nanoscale surfaces in biologically relevant systems. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of currently marketed heparin products: key tests for quality assurance.

PubMed

Keire, David A; Ye, Hongping; Trehy, Michael L; Ye, Wei; Kolinski, Richard E; Westenberger, Benjamin J; Buhse, Lucinda F; Nasr, Moheb; Al-Hakim, Ali

2011-01-01

During the 2007-2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz (1)H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007-2008 crisis.

Binding affinities of NKG2D and CD94 to sialyl Lewis X-expressing N-glycans and heparin.

PubMed

Higai, Koji; Suzuki, Chiho; Imaizumi, Yuzo; Xin, Xin; Azuma, Yutaro; Matsumoto, Kojiro

2011-01-01

Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (K(d)) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. K(d) values obtained by linear reciprocal plots revealed good coincidence between the two methods. K(d) values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 µM for heparin-BSA >0.30 and 0.20 µM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 µM for HepTf >0.37 and 0.36 µM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.

Quantitative PCR and disaccharide profiling to characterize the animal origin of low-molecular-weight heparins.

PubMed

Houiste, Céline; Auguste, Cécile; Macrez, Céline; Dereux, Stéphanie; Derouet, Angélique; Anger, Pascal

2009-02-01

Low-molecular-weight heparins (LMWHs) are widely used in the management of thrombosis and acute coronary syndromes. They are obtained by the enzymatic or chemical depolymerization of porcine intestinal heparin. Enoxaparin sodium, a widely used LMWH, has a unique and reproducible oligosaccharide profile which is determined by the origin of the starting material and a tightly controlled manufacturing process. Although other enoxaparin-like LMWHs do exist, specific release criteria including the origin of the crude heparin utilized for their production, have not been established. A quantitative polymerase chain reaction method has been developed to ensure the purity of the porcine origin of crude heparin, with a DNA detection limit as low as 1 ppm for bovine, or 10 ppm for ovine contaminants. This method is routinely used as the release acceptance criterion during enoxaparin sodium manufacturing. Furthermore, when the process removes DNA, other analytical techniques can be used to assess any contamination. Disaccharide profiling after exhaustive depolymerization can determine the presence of at least 10% bovine or 20% ovine material; multivariate analysis is useful to perform the data analysis. Consistent with the availability of newer technology, these methods should be required as acceptance criteria for crude heparins used in the manufacture of LMWHs to ensure their safety, quality, and immunologic profile.

Immediate Type Hypersensitivity to Heparins: Two Case Reports and a Review of the Literature.

PubMed

Cesana, Philipp; Scherer, Kathrin; Bircher, Andreas J

2016-01-01

Immediate type hypersensitivity reactions due to heparins are rare, and the exact immunologic pathomechanism has not been identified so far. In our 2 case reports, we describe first a 50-year-old female who received dalteparin (Fragmin®) and developed signs of an immediate type hypersensitivity reaction. The personal history revealed a previous application of dalteparin (Fragmin®). Evaluation with a skin prick test showed positive results for dalteparin. The second case deals with a 73-year-old female with a suspected immediate type reaction after the administration of dalteparin (Fragmin®). A skin prick test was negative but intracutaneous tests showed a positive reaction to the causative agent. Both cases indicated cross-reactivity reactions for low-molecular-weight heparin (LMWH) but not for unfractioned heparin (UFH) or fondaparinux. In conclusion, our case reports including a review of published cases of immediate type hypersensitivity reactions after the application of heparins illustrate this rare complication. Mostly, the causative agent can be identified with a skin test, which is highly suggestive of an IgE-mediated reaction. Therapeutic alternatives for patients with sensitization to an LMWH are UFH and fondaparinux. Both agents have a small risk of cross-reactivity compared to heparins of the same substance class. © 2017 S. Karger AG, Basel.

Heparin-induced increase in serum levels of aminotranferases. A controlled clinical trial.

PubMed

Nielsen, H K; Husted, S E; Koopmann, H D; Fasting, H; Simonsen, O; Andersen, K; Husegaard, H C; Petersen, T K

1984-01-01

Sixty-four patients over the age of 40 years, undergoing elective surgery of at least one hour's duration, were randomized to treatment with either a thromboembolic deterrent ( TED ) stocking (Kendall Co.) or subcutaneous low-dose heparin 5 000 IU every 12 hours. Serum levels of alanine aminotransferase (S-ALAT), aspartate aminotransferase (S-ASAT), gamma-glutamyl transpeptidase (S-gamma-GT) and alkaline phosphatase (S-ALP) were measured. S-ALAT increased significantly on the 5th and 10th postoperative day, from 27 +/- 2 (x +/- SE) to 40 +/- 4 (p less than 0.01) and 55 +/- 7 U/l (p less than 0.001), respectively, in the heparin group and was significantly higher in the heparin than in the TED group both on the 5th (p less than 0.01) and 10th (p less than 0.05) postoperative day. S-ASAT and S-gamma-GT increased significantly during heparin treatment, but did not differ significantly from the values of the TED group. No change in S-ALP was registered in either group. It is concluded that prophylactic treatment with low-dose heparin induces a significant increase in S-aminotransferase levels, especially in S-ALAT. The phenomenon has profound differential diagnostic implications in conditions such as pulmonary embolism and acute myocardial infarction.

Effects of the repeated administration of adenosine and heparin on myocardial perfusion in patients with chronic stable angina pectoris.

PubMed

Barron, H V; Sciammarella, M G; Lenihan, K; Michaels, A D; Botvinick, E H

2000-01-01

The mechanism by which ischemia stimulates angiogenesis is unknown. Adenosine is released during myocardial ischemia and may be a mediator of this process. Experimental data suggest that heparin may enhance this effect. The purpose of this open-labeled, placebo-controlled trial was to determine whether repeated intravenous administration of adenosine and heparin could mimic physiologic angiogenesis and reduce the amount of exercise-induced myocardial ischemia in patients with coronary artery disease. Subjects with chronic stable angina refractory to conventional medical therapy and not suitable for revascularization received either adenosine (140 microg/kg/min for 6 minutes) and heparin (10,000 U bolus), (n = 14), or placebo, (n = 7) daily for 10 days. All patients underwent baseline and follow-up exercise testing with thallium-201 single-photon emission computed tomography myocardial perfusion imaging. A semiquantitative assessment of the extent and severity of the perfusion abnormalities was calculated by 2 blinded investigators. There was no significant change in exercise duration or in the peak heart rate systolic blood pressure product associated with adenosine and heparin compared with placebo treatment. There was, however, a 9% reduction in the extent (60.6 +/- 4.0 vs 54.9 +/- 4.1, p = 0.03) and a 14% improvement in severity (41.5 +/- 3.2 vs 35.7 +/- 2.9, p = 0.01) of the myocardial perfusion abnormalities seen in patients who received adenosine and heparin compared with placebo. Thus, in this pilot study, repeated administration of adenosine and heparin reduced the amount of exercise-induced ischemia in patients with chronic stable angina refractory to conventional treatment.

An economic evaluation of the costs and benefits of heparin rationalisation in a hospital pharmacy.

PubMed

Reeves, Penny; Cooke, Jonathan; Lloyd, Adam; Hutchings, Adam

2004-06-01

To estimate the costs and benefits for a UK hospital pharmacy of stocking a single low molecular weight heparin (LMWH), enoxaparin, compared to stocking unfractionated heparin (UFH) and stocking both UFH and multiple different LMWHs. A decision-tree model was developed which considered the use of heparins for five indications: prophylaxis against venous thromboembolism (VTE) in major orthopaedic surgery; VTE prophylaxis in major general surgery; VTE prophylaxis in acute medical inpatients; treatment of diagnosed VTE; and anticoagulation for patients with unstable angina and non-ST-elevation myocardial infarction (UA/NSTEMI). Previously published cost-effectiveness analyses for each indication were combined into a single model and updated to 2002 prices. The number of patients given heparin in each indication was estimated from the pharmacy records of a large UK teaching hospital. The model estimated the use of drugs, staff time, clinical events and resource use resulting from anti-coagulation. Costs were estimated from the perspective of the hospital and the UK National Health Service. Total annual cost was estimated to be pounds sterling 3.2 m (single LMWH), pounds sterling 4.4 m (UFH only) and pounds sterling 3.7 m (multiple heparins). The largest expected cost savings from using a single LMWH compared to UFH only resulted from reduced hospital stay for DVT treatment, reduced revascularisation in UA/NSTEMI and fewer VTE events in orthopaedic surgery. Expected cost savings from using a single LMWH compared to multiple heparins were more modest Sub-optimal choice of anticoagulants may result in substantial excess costs elsewhere in the hospital.

Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with low doses of heparins.

PubMed

Lider, O; Baharav, E; Mekori, Y A; Miller, T; Naparstek, Y; Vlodavsky, I; Cohen, I R

1989-03-01

The ability of activated T lymphocytes to penetrate the extracellular matrix and migrate to target tissues was found to be related to expression of a heparanase enzyme (Naparstek, Y., I. R. Cohen, Z. Fuks, and I. Vlodavsky. 1984. Nature (Lond.). 310:241-243; Savion, N., Z. Fuks, and I. Vlodavsky. 1984. J. Cell. Physiol. 118:169-176; Fridman, R., O. Lider, Y. Naparstek, Z. Fuks, I. Vlodavsky, and I. R. Cohen. 1987. J. Cell. Physiol. 130:85-92; Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We found previously that heparin molecules inhibited expression of T lymphocyte heparanase activity in vitro and in vivo, and administration of a low dose of heparin in mice inhibited lymphocyte traffic and delayed-type hypersensitivity reactions (Lider, O., J. Mekori, I. Vlodavsky, E. Baharav, Y. Naparstek, and I. R. Cohen, manuscript submitted for publication). We now report that treatment with commercial or chemically modified heparins at relatively low doses once daily (5 micrograms for mice and 20 micrograms for rats) led to inhibition of allograft rejection and the experimental autoimmune diseases adjuvant arthritis and experimental autoimmune encephalomyelitis. Higher doses of the heparins were less effective. The ability of chemically modified heparins to inhibit these immune reactions was associated with their ability to inhibit expression of T lymphocyte heparanase. There was no relationship to anticoagulant activity. Thus heparins devoid of anticoagulant activity can be effective in regulating immune reactions when used at appropriate doses.

Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla1

PubMed Central

Ardon, Florencia; Markello, Ross D.; Hu, Lian; Deutsch, Zarah I.; Tung, Chih-Kuan; Wu, Mingming; Suarez, Susan S.

2016-01-01

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium. PMID:27605344

Dynamics of Bovine Sperm Interaction with Epithelium Differ Between Oviductal Isthmus and Ampulla.

PubMed

Ardon, Florencia; Markello, Ross D; Hu, Lian; Deutsch, Zarah I; Tung, Chih-Kuan; Wu, Mingming; Suarez, Susan S

2016-10-01

In mammals, many sperm that reach the oviduct are held in a reservoir by binding to epithelium. To leave the reservoir, sperm detach from the epithelium; however, they may bind and detach again as they ascend into the ampulla toward oocytes. In order to elucidate the nature of binding interactions along the oviduct, we compared the effects of bursts of strong fluid flow (as would be caused by oviductal contractions), heparin, and hyperactivation on detachment of bovine sperm bound in vitro to epithelium on intact folds of isthmic and ampullar mucosa. Intact folds of oviductal mucosa were used to represent the strong attachments of epithelial cells to each other and to underlying connective tissue that exist in vivo. Effects of heparin on binding were tested because heparin binds to the Binder of SPerm (BSP) proteins that attach sperm to oviductal epithelium. Sperm bound by their heads to beating cilia on both isthmic and ampullar epithelia and could not be detached by strong bursts of fluid flow. Addition of heparin immediately detached sperm from isthmic epithelium but not ampullar epithelium. Addition of 4-aminopyridine immediately stimulated hyperactivation of sperm but did not detach them from isthmic or ampullar epithelium unless added with heparin. These observations indicate that the nature of binding of sperm to ampullar epithelium differs from that of binding to isthmic epithelium; specifically, sperm bound to isthmic epithelium can be detached by heparin alone, while sperm bound to ampullar epithelium requires both heparin and hyperactivation to detach from the epithelium. © 2016 by the Society for the Study of Reproduction, Inc.

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells.

PubMed

Wu, Chunxiao; Wang, Shu

2012-01-01

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.

Effect of heparin bonding on catheter-induced fibrin formation and platelet activation.

PubMed

Nichols, A B; Owen, J; Grossman, B A; Marcella, J J; Fleisher, L N; Lee, M M

1984-11-01

Pathologic and experimental evidence indicates that platelet activation and fibrin formation contribute to the pathogenesis of angina pectoris, coronary vasospasm and myocardial infarction. Detection of localized intravascular platelet activation and fibrin formation in vivo by selective blood sampling requires catheters that do not induce coagulation ex vivo. We studied the effect of heparin bonding of catheter surfaces on activation of the coagulation system by cardiovascular catheters. Woven Dacron, polyvinylchloride, and polyurethane catheters were tested and compared with identical catheters with heparin-bonded surfaces in 47 patients undergoing percutaneous cardiac catheterization. Platelet activation was measured by radioimmunoassay of plasma platelet factor 4 (PF4), beta-thromboglobulin (BTG), and thromboxane B2 (TXB2) in blood samples withdrawn through catheters, and fibrin formation was assessed by determination of fibrinopeptide A (FPA) levels. In blood samples collected through conventional catheters, FPA, PF4, BTG, and TXB2 levels were markedly elevated; blood sampling through heparin-bonded catheters had no significant effect on FPA, PF4, BTG, or TXB2 levels. Scanning electron microscopy disclosed extensive platelet aggregates and fibrin strands adherent to the surface of conventional catheters but not to heparin-bonded catheter surfaces. This study demonstrates that (1) collection of blood samples through cardiovascular catheters causes artifactual elevation of FPA, PF4, BTG, and TXB2 levels, and (2) heparin-bonded catheter surfaces effectively prevent catheter-induced platelet alpha-granule release and fibrin formation on catheter surfaces. Heparin-bonded catheters will facilitate investigation of the role of intravascular coagulation in coronary artery disease by eliminating catheter-induced fibrin formation and platelet activation.

Influence of porcine spermadhesins on the susceptibility of boar spermatozoa to high dilution.

PubMed

Centurion, Fernando; Vazquez, Juan M; Calvete, Juan J; Roca, Jordi; Sanz, Libia; Parrilla, Inmaculada; Garcia, Eva M; Martinez, Emilio A

2003-08-01

The effect of heparin-binding and non-heparin-binding spermadhesins on the viability, motility, and mitochondrial activity of boar spermatozoa at the high dilution (300,000 sperm/ml) to which sperm are exposed during the process of sex sorting by flow cytometry was investigated. Incubation of spermatozoa with heparin-binding spermadhesins caused a time- and dose-dependent decrease in the percentage of functional spermatozoa. The percentage of viable spermatozoa incubated at 38 degrees C with heparin-binding spermadhesins diluted in PBS (1 mg/ml) dropped from 75% (0.5 h) to 4% (5 h), whereas the percentage of viable spermatozoa incubated in PBS without proteins (control) decreased from 85% (0.5 h) to 19% (5 h). Addition of non-heparin-binding PSP-I/PSP-II spermadhesin to the PBS resulted in a concentration-dependent increment of the percentage of viable cells (65% after 5-h incubation), with maximum effect at 1.5 mg/ml. The heparin-binding spermadhesins totally suppressed sperm motility and mitochondrial activity after 5 h of incubation. The same parameters of sperm incubated in the presence of 1.5 mg/ml of PSP-I/PSP-II were 50% and 58%, respectively, and the percentages of control sperm displaying motility and mitochondrial activity were 21% and 26%, respectively. Moreover, the viability, motility, and mitochondrial activity all decreased on incubation of spermatozoa with mixtures of PSP-I/PSP-II and heparin-binding spermadhesins as the concentration of the latter increased. We conclude that PSP-I/PSP-II and the heparin-binding spermadhesins exert antagonistic effects on the functionality of highly diluted boar spermatozoa. The finding that PSP-I/PSP-II contributes to maintaining sperm with high viability, motility, and mitochondrial activity for at least 5 h at physiological temperature points to its potential use as an additive for sperm preservation, specifically of highly diluted, flow-sorted spermatozoa for sex preselection.

Overcoming Heparin-Associated RT-qPCR Inhibition and Normalization Issues for microRNA Quantification in Patients with Acute Myocardial Infarction.

PubMed

Coelho-Lima, Jose; Mohammed, Ashfaq; Cormack, Suzanne; Jones, Samuel; Das, Rajiv; Egred, Mohaned; Panahi, Pedram; Ali, Simi; Spyridopoulos, Ioakim

2018-06-11

 Cardiac-enriched micro ribonucleic acids (miRNAs) are released into the circulation following ST-elevation myocardial infarction (STEMI). Lack of standardized approaches for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) data normalization and presence of RT-qPCR inhibitors (e.g. heparin) in patient blood samples have prevented reproducible miRNA quantification in this cohort and subsequent translation of these biomarkers to clinical practice.  Using a RT-qPCR miRNA screening platform, we identified and validated an endogenous circulating miRNA as a normalization control. In addition, we assessed the effects of in vivo and in vitro anticoagulant drugs administration (heparin and bivalirudin) on three RT-qPCR normalization strategies (global miRNA mean, exogenous spike-in control [cel-miR-39] and endogenous miRNA control). Finally, we evaluated the effect of heparin and its in vitro inhibition with heparinase on the quantification of cardiac-enriched miRNAs in STEMI patients.  miR-425-5p was validated as an endogenous miRNA control. Heparin administration in vitro and in vivo inhibited all RT-qPCR normalization strategies. In contrast, bivalirudin had no effects on cel-miR-39 or miR-425-5p quantification. In vitro RNA sample treatment with 0.3 U of heparinase overcame heparin-induced over-estimation of cardiac-enriched miRNA levels and improved their correlation with high-sensitivity troponin T.  miRNA quantification in STEMI patients receiving heparin is jeopardized by its effect on all RT-qPCR normalization approaches. Use of samples from bivalirudin-treated patients or in vitro treatment of heparin-contaminated samples with heparinase are suitable alternatives for miRNA quantification in this cohort. Finally, we reinforce the evidence that cardiac-enriched miRNAs early after myocardial reperfusion reflect the severity of cardiac injury. Schattauer GmbH Stuttgart.

Deciphering the Role of Sulfonated Unit in Heparin-Mimicking Polymer to Promote Neural Differentiation of Embryonic Stem Cells.

PubMed

Lei, Jiehua; Yuan, Yuqi; Lyu, Zhonglin; Wang, Mengmeng; Liu, Qi; Wang, Hongwei; Yuan, Lin; Chen, Hong

2017-08-30

Glycosaminoglycans (GAGs), especially heparin and heparan sulfate (HS), hold great potential for inducing the neural differentiation of embryonic stem cells (ESCs) and have brought new hope for the treatment of neurological diseases. However, the disadvantages of natural heparin/HS, such as difficulty in isolating them with a sufficient amount, highly heterogeneous structure, and the risk of immune responses, have limited their further therapeutic applications. Thus, there is a great demand for stable, controllable, and well-defined synthetic alternatives of heparin/HS with more effective biological functions. In this study, based upon a previously proposed unit-recombination strategy, several heparin-mimicking polymers were synthesized by integrating glucosamine-like 2-methacrylamido glucopyranose monomers (MAG) with three sulfonated units in different structural forms, and their effects on cell proliferation, the pluripotency, and the differentiation of ESCs were carefully studied. The results showed that all the copolymers had good cytocompatibility and displayed much better bioactivity in promoting the neural differentiation of ESCs as compared to natural heparin; copolymers with different sulfonated units exhibited different levels of promoting ability; among them, copolymer with 3-sulfopropyl acrylate (SPA) as a sulfonated unit was the most potent in promoting the neural differentiation of ESCs; the promoting effect is dependent on the molecular weight and concentration of P(MAG-co-SPA), with the highest levels occurring at the intermediate molecular weight and concentration. These results clearly demonstrated that the sulfonated unit in the copolymers played an important role in determining the promoting effect on ESCs' neural differentiation; SPA was identified as the most potent sulfonated unit for copolymer with the strongest promoting ability. The possible reason for sulfonated unit structure as a vital factor influencing the ability of the copolymers may be attributed to the difference in electrostatic and steric hindrance effect. The synthetic heparin-mimicking polymers obtained here can offer an effective alternative to heparin/HS and have great therapeutic potential for nervous system diseases.

[Outpatient use of heparin: data from the Midi-Pyrenes Health Fund].

PubMed

Berchery, Delphine; Roussel, Henri; Bourrel, Robert; Sciortino, Vincent

2003-01-01

The risk of haemorrhagic complications associated with heparin therapy can be reduced by good clinical practice. The aim of this study was to describe outpatient heparin therapy by using the database of the National Health Fund. The study population consisted of affiliates of the salaried employees insured by the health fund branch of the Midi-Pyrénées region, and corresponded to 62% of the residents of that region. Analysis of treatments and biological monitoring was carried out on a 1-year period. During this period, 16,462 patients started a treatment with heparin, 92% for a single treatment. The mean age of the patients was 55 years (SD = 19.8) and the majority were women (53%). Nine percent of these patients were switched to oral anticoagulant therapy. Of the other patients, 52% received heparin for less than 10 days, 36% for between 10 days and 5 weeks, and 12% for more than 5 weeks; 33% of the last group where heparin was prescribed for more than 5 weeks corresponds to a prescription of more than 3 months. Seventy-three percent of the heparin treatment durations complied with the authorities' (l'Agence française de sécurité sanitaire des produits de santé [AFSSAPS]) recommendations. Biological monitoring comprised a platelet count, an APTT (activated partial thromboplastin time) or an anti-Xa check in 41.9%, 27.8% and 3.1% of treated patients, respectively. Creatininaemia was measured in 27% of patients aged > 75 years (a group at increased risk of adverse drug reactions). Even considering some of the differences noted between the medical prescriptions and the reimbursement data of the health fund, results from this study allowed an evaluation of medical practices and suggests that monitoring of patients receiving heparin treatments remains insufficient, thus decreasing the benefit/risk ratio of such therapies.

A single center retrospective cohort study comparing low-molecular-weight heparins to direct oral anticoagulants for the treatment of venous thromboembolism in patients with cancer - A real world experience.

PubMed

Phelps, Megan K; Wiczer, Tracy E; Erdeljac, H Paige; Van Deusen, Kelsey R; Porter, Kyle; Philips, Gary; Wang, Tzu-Fei

2018-01-01

Introduction Low-molecular-weight heparins are the standard treatment for cancer-associated thrombosis. Recently, direct oral anticoagulants are a new option for thrombosis treatment; however, data supporting the use of direct oral anticoagulants for cancer-associated thrombosis are limited. Objectives The primary objective of this study was to determine the rate of recurrent cancer-associated thrombosis and major bleeding within 6 months of starting either low-molecular-weight heparin or direct oral anticoagulant for treatment of cancer-associated thrombosis. Secondary objectives were to determine the rates of clinically relevant-non-major bleeding and all-cause mortality. Patients/methods This is a retrospective cohort study including adults with cancer-associated thrombosis treated with low-molecular-weight heparin or direct oral anticoagulant between 2010 and 2016 at the Ohio State University. Medical records were reviewed for 6 months after initiation of anticoagulation or until the occurrence of recurrent cancer-associated thrombosis, major bleeding, cessation of anticoagulation of interest, or death, whichever occurred first. Results Four hundred and eighty patients were included (290 low-molecular-weight heparin and 190 direct oral anticoagulant). Patients treated with direct oral anticoagulant were found to carry "lower risk" features including cancer with lower VTE risk and lower rate of metastatic disease. After adjustment for baseline differences, there was no significant difference in the rate of recurrent cancer-associated thrombosis (7.2% low-molecular-weight heparin vs 6.3% direct oral anticoagulant, p = 0.71) or major bleeding (7.6% low-molecular-weight heparin vs 2.6% direct oral anticoagulant, p = 0.08). Conclusions Our study demonstrates that in a select population of cancer patients with VTE, direct oral anticoagulant use can be as effective and safe compared to the standard therapy with low-molecular-weight heparin.

Anticoagulant Activity of a Unique Sulfated Pyranosic (1→3)-β-l-Arabinan through Direct Interaction with Thrombin*

PubMed Central

Fernández, Paula V.; Quintana, Irene; Cerezo, Alberto S.; Caramelo, Julio J.; Pol-Fachin, Laercio; Verli, Hugo; Estevez, José M.; Ciancia, Marina

2013-01-01

A highly sulfated 3-linked β-arabinan (Ab1) with arabinose in the pyranose form was obtained from green seaweed Codium vermilara (Bryopsidales). It comprised major amounts of units sulfated on C-2 and C-4 and constitutes the first polysaccharide of this type isolated in the pure form and fully characterized. Ab1 showed anticoagulant activity by global coagulation tests. Less sulfated arabinans obtained from the same seaweed have less or no activity. Ab1 exerts its activity through direct and indirect (antithrombin- and heparin cofactor II-mediated) inhibition of thrombin. Direct thrombin inhibition was studied in detail. By native PAGE, it was possible to detect formation of a complex between Ab1 and human thrombin (HT). Ab1 binding to HT was measured by fluorescence spectroscopy. CD spectra of the Ab1 complex suggested that ligand binding induced a small conformational change on HT. Ab1-thrombin interactions were studied by molecular dynamic simulations using the persulfated octasaccharide as model compound. Most carbohydrate-protein contacts would occur by interaction of sulfate groups with basic amino acid residues on the surface of the enzyme, more than 60% of them being performed by the exosite 2-composing residues. In these interactions, the sulfate groups on C-2 were shown to interact more intensely with the thrombin structure. In contrast, the disulfated oligosaccharide does not promote major conformational modifications at the catalytic site when complexed to exosite 1. These results show that this novel pyranosic sulfated arabinan Ab1 exerts its anticoagulant activity by a mechanism different from those found previously for other sulfated polysaccharides and glycosaminoglycans. PMID:23161548

Bivalirudin Versus Heparin Anticoagulation in Transcatheter Aortic Valve Replacement: The Randomized BRAVO-3 Trial.

PubMed

Dangas, George D; Lefèvre, Thierry; Kupatt, Christian; Tchetche, Didier; Schäfer, Ulrich; Dumonteil, Nicolas; Webb, John G; Colombo, Antonio; Windecker, Stephan; Ten Berg, Jurriën M; Hildick-Smith, David; Mehran, Roxana; Boekstegers, Peter; Linke, Axel; Tron, Christophe; Van Belle, Eric; Asgar, Anita W; Fach, Andreas; Jeger, Raban; Sardella, Gennaro; Hink, Hans Ulrich; Husser, Oliver; Grube, Eberhard; Deliargyris, Efthymios N; Lechthaler, Ilknur; Bernstein, Debra; Wijngaard, Peter; Anthopoulos, Prodromos; Hengstenberg, Christian

2015-12-29

Anticoagulation is required during transcatheter aortic valve replacement (TAVR) procedures. Although an optimal regimen has not been determined, heparin is mainly used. Direct thrombin inhibition with bivalirudin may be an effective alternative to heparin as the procedural anticoagulant agent in this setting. The goal of this study was to determine whether bivalirudin offers an alternative to heparin as the procedural anticoagulant agent in patients undergoing TAVR. A total of 802 patients with aortic stenosis were randomized to undergo transfemoral TAVR with bivalirudin versus unfractionated heparin during the procedure. The 2 primary endpoints were major bleeding within 48 h or before hospital discharge (whichever occurred first) and 30-day net adverse clinical events, defined as the combination of major adverse cardiovascular events (all-cause mortality, myocardial infarction, or stroke) and major bleeding. Anticoagulation with bivalirudin versus heparin did not meet superiority because it did not result in significantly lower rates of major bleeding at 48 h (6.9% vs. 9.0%; relative risk: 0.77; 95% confidence interval [CI]: 0.48 to 1.23; p = 0.27) or net adverse cardiovascular events at 30 days (14.4% vs. 16.1%; relative risk: 0.89; 95% CI: 0.64 to 1.24; risk difference: -1.72; 95% CI: -6.70 to 3.25; p = 0.50); regarding the latter, the prespecified noninferiority hypothesis was met (pnoninferiority < 0.01). Rates of major adverse cardiovascular events at 48 h were not significantly different (3.5% vs. 4.8%; relative risk: 0.73; 95% CI: 0.37 to 1.43; p = 0.35). At 48 h, the bivalirudin group had significantly fewer myocardial infarctions but more acute kidney injury events than the heparin group; at 30 days, these differences were no longer significant. In this randomized trial of TAVR procedural pharmacotherapy, bivalirudin did not reduce rates of major bleeding at 48 h or net adverse cardiovascular events within 30 days compared with heparin. Although superiority was not shown, the noninferiority hypothesis was met with respect to the latter factor. Given the lower cost, heparin should remain the standard of care, and bivalirudin can be an alternative anticoagulant option in patients unable to receive heparin in TAVR. (International, Multi-center, Open-label, Randomized Controlled Trial in Patients Undergoing TAVR to Determine the Treatment Effect [Both Safety and Efficacy] of Using Bivalirudin Instead of UFH [BRAVO-2/3]; NCT01651780). Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Perspective on the Use of Sulfated Polysaccharides from Marine Organisms as a Source of New Antithrombotic Drugs

PubMed Central

Mourão, Paulo A. S.

2015-01-01

Thromboembolic diseases are increasing worldwide and always require anticoagulant therapy. We still need safer and more secure antithrombotic drugs than those presently available. Sulfated polysaccharides from marine organisms may constitute a new source for the development of such drugs. Investigation of these compounds usually attempts to reproduce the therapeutic effects of heparin. However, we may need to follow different routes, focusing particularly in the following aspects: (1) defining precisely the specific structures required for interaction of these sulfated polysaccharides with proteins of the coagulation system; (2) looking for alternative mechanisms of action, distinct from those of heparin; (3) identifying side effects (mostly pro-coagulant action and hypotension rather than bleeding) and preparing derivatives that retain the desired antithrombotic action but are devoid of side effects; (4) considering that sulfated polysaccharides with low anticoagulant action on in vitro assays may display potent effects on animal models of experimental thrombosis; and finally (5) investigating the antithrombotic effect of these sulfated polysaccharides after oral administration or preparing derivatives that may achieve this effect. If these aspects are successfully addressed, sulfated polysaccharides from marine organisms may conquer the frontier of antithrombotic therapy and open new avenues for treatment or prevention of thromboembolic diseases. PMID:25955754

Heparin-induced thrombocytopenia and heart operation: management with tedelparin.

PubMed

Altés, A; Martino, R; Gari, M; Cámara, M L; Garín, R; Casas, J I; Fontcuberta, J

1995-02-01

Anticoagulation for cardiopulmonary bypass in the infrequent clinical setting of thrombocytopenia associated with the use of unfractionated heparin is a very serious problem. We describe a case in which a low-molecular-weight heparin (tedelparin) was selected for this purpose based on a platelet aggregation test, permitting adequate anticoagulation during cardiopulmonary bypass for valve replacement. This case report might help establish an adequate anticoagulation protocol when faced with a patient suffering from this condition.

Extracorporeal Perfusion without Exogenous Anticoagulation: Its Protective Role in Endotoxin Shock.

DTIC Science & Technology

1982-02-19

lethal effects of endotoxin . Group E: Heparinized dogs given endotoxin ; no perfusion. This group served to evaluate the effects of heparin on...recovery from endotoxin shock. Group F: Heparinized dogs perfused 90 minutes; then given endotoxin . This group served to assay the effects of exogenous...stable model and is preperfusion neces- sary to provide protection against the lethal effects of endotoxin ? The first series of experiments (Group B) were

Successful Implantation of a Left Ventricular Assist Device in a Patient with Heparin-Induced Thrombocytopenia and Thrombosis

PubMed Central

Garland, Cassandra; Somogyi, David

2014-01-01

Abstract: We report the case of a 27-year-old woman with signs of heparin-induced thrombocytopenia and thrombosis (HITT) and left heart failure presenting for urgent implantation of a left ventricular assist device (LVAD). HITT can occur in 4.2–6.1% of patients with LVADs. If the patient remains hemodynamically stable, implantation can be delayed for several months until the heparin/PF-4 antibodies decline allowing the use of heparin on cardiopulmonary bypass, However, in most cases related to cardiogenic shock, surgery cannot be delayed. We present the case of a patient who underwent implantation of a HeartMate II LVAD and discuss management strategy using bivalirudin during cardiopulmonary bypass. PMID:25208434

Effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro.

PubMed

Lee, J S; Kim, J M; Hong, E K; Kim, S-O; Yoo, Y-J; Cha, J-H

2009-02-01

A growing amount of attention has been placed on periodontal regeneration and wound healing for periodontal therapy. This study was conducted in an effort to determine the effects of heparin-binding epidermal growth factor-like growth factor on cell repopulation and signal transduction in periodontal ligament cells after scratch wounding in vitro. Human periodontal ligament cells were acquired from explant tissue of human healthy periodontal ligament. After the wounding of periodontal ligament cells, the change in expression of heparin-binding epidermal growth factor-like growth factor and epidermal growth factor receptors 1-4 mRNA was assessed. The effects of heparin-binding epidermal growth factor-like growth factor on periodontal ligament cell proliferation and repopulation were assessed in vitro via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and by photographing the injuries, respectively. Extracellular signal-regulated kinase (Erk)1/2, p38 and Akt phosphorylation was characterized via western blotting. Scratch wounding resulted in a significant up-regulation of heparin-binding epidermal growth factor-like growth factor mRNA expression, whereas wounding had no effect on the expression levels of epidermal growth factor receptors 1-4. Interestingly, no expression of epidermal growth factor receptors 2 and 4 was detectable prior to or after wounding. Heparin-binding epidermal growth factor-like growth factor treatment promoted the proliferation and repopulation of periodontal ligament cells. The scratch wounding also stimulated the phosphorylation of Erk1/2 and p38, but not of Akt, in periodontal ligament cells, and heparin-binding epidermal growth factor-like growth factor treatment applied after wounding amplified and extended the activations of Erk1/2 and p38, but not of Akt. Furthermore, Erk1/2 inhibition blocked the process of cell repopulation induced by heparin-binding epidermal growth factor-like growth factor, whereas the inhibition of p38 delayed the process. These results indicate that heparin-binding epidermal growth factor-like growth factor may constitute a critical factor in the wound healing of human periodontal ligament cells by a mechanism that requires the activation of Erk1/2 via specific interaction with epidermal growth factor receptor 1.

Comparison of two doses of heparin on outcome in off-pump coronary artery bypass surgery patients: A prospective randomized control study.

PubMed

Chakravarthy, Murali; Prabhakumar, Dattatreya; Thimmannagowda, Patil; Krishnamoorthy, Jayaprakash; George, Antony; Jawali, Vivek

2017-01-01

While off pump coronary artery bypass surgery is practiced with an intention to reduce the morbidity associated with cardiopulmonary bypass, the resultant 'hypercoagulability' needs to be addressed. Complications such as cavitary thrombus possibly due to the hyper coagulability after off pump coronary artery bypass surgery have been described. Many clinicians use higher doses of heparin - up to 5 mg/kg in order to thwart this fear. Overall, there appears to be no consensus on the dose of heparin in off pump coronary artery bypass surgeries. The aim of the study was understand the differences in outcome of such as transfusion requirement, myocardial ischemia, and morbidity when two different doses were used for systemic heparinization. Elective patients scheduled for off pump coronary artery bypass surgery were included. Ongoing anti platelet medication was not an exclusion criteria, however, anti platelet medications were ceased about a week prior to surgery when possible. Thoracic epidural anesthesia was administered as an adjunct in patients who qualified for it. By computer generated randomization chart, patients were chosen to receive either 2 or 3 mg/kg of intravenous unfractioned heparin to achieve systemic heparinization with activated clotting time targeted at >240 secs. Intraoperative blood loss, postoperative blood loss, myocardial ischemic episodes, requirement of intraaortic balloon counter pulsation and transfusion requirement were analyzed. Sixty two patients participated in the study. There was one conversion to cardiopulmonary bypass. The groups had comparable ACT at baseline (138.8 vs. 146.64 seconds, P = 0.12); 3 mg/kg group had significantly higher values after heparin, as expected. But after reversal with protamine, ACT and need for additional protamine was similar among the groups. Intraoperative (685.56 ± 241.42 ml vs. 675.15 ± 251.86 ml, P = 0.82) and postoperative blood loss (1906.29 ± 611.87 ml vs 1793.65 ± 663.54 ml , p value 0.49) were similar among the groups [Table 4]. The incidence of ECG changes of ischemia, arrhythmias, conversion to CPB, or need for intra-aortic balloon counter pulsation were not different. Use of either 2 or 3 mg/kg heparin for systemic heparinization in patients undergoing OPCAB did not affect the outcome.

Demonstration of specific binding of heparin to Plasmodium falciparum-infected vs. non-infected red blood cells by single-molecule force spectroscopy

NASA Astrophysics Data System (ADS)

Valle-Delgado, Juan José; Urbán, Patricia; Fernàndez-Busquets, Xavier

2013-04-01

Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level.Glycosaminoglycans (GAGs) play an important role in the sequestration of Plasmodium falciparum-infected red blood cells (pRBCs) in the microvascular endothelium of different tissues, as well as in the formation of small clusters (rosettes) between infected and non-infected red blood cells (RBCs). Both sequestration and rosetting have been recognized as characteristic events in severe malaria. Here we have used heparin and pRBCs infected by the 3D7 strain of P. falciparum as a model to study GAG-pRBC interactions. Fluorescence microscopy and fluorescence-assisted cell sorting assays have shown that exogenously added heparin has binding specificity for pRBCs (preferentially for those infected with late forms of the parasite) vs. RBCs. Heparin-pRBC adhesion has been probed by single-molecule force spectroscopy, obtaining an average binding force ranging between 28 and 46 pN depending on the loading rate. No significant binding of heparin to non-infected RBCs has been observed in control experiments. This work represents the first approach to quantitatively evaluate GAG-pRBC molecular interactions at the individual molecule level. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32821f

Anticoagulation of pregnant women with mechanical heart valves: a systematic review of the literature.

PubMed

Chan, W S; Anand, S; Ginsberg, J S

2000-01-24

The management of women with prosthetic heart valves during pregnancy poses a particular challenge as there are no available controlled clinical trials to provide guidelines for effective antithrombotic therapy. Oral anticoagulants such as warfarin sodium cause fetal embryopathy; subcutaneous administration of heparin sodium has been reported to be ineffective in preventing thromboembolic complications. To identify the risks of maternal and fetal complications in women with mechanical heart valves treated with different anticoagulation regimens during pregnancy. We performed a systematic review of the literature to determine pooled estimates of maternal and fetal risks associated with the 3 commonly used approaches: (1) oral anticoagulants (OA) throughout pregnancy, (2) replacing OA with heparin in the first trimester (from 6-12 weeks' gestation), and (3) heparin use throughout pregnancy. Fetal outcomes included spontaneous abortions and fetopathic effects, and maternal outcomes were major bleeding, thromboembolic complications, and death. The use of OA throughout pregnancy is associated with warfarin embryopathy in 6.4% (95% confidence interval [CI], 4.6%-8.9%) of livebirths. The substitution of heparin at or prior to 6 weeks, and continued until 12 weeks, eliminated this risk. Overall risks for fetal wastage (spontaneous abortion, stillbirths, and neonatal deaths) were similar in women treated with OA throughout, compared with women treated with heparin in the first trimester. Maternal mortality was 2.9% (95% CI, 1.9%-4.2%). Maj or bleeding events occurred in 2.5% (95% CI, 1.7%-3.5%) of all pregnancies, most at the time of delivery. The regimen associated with the lowest risk of valve thrombosis (3.9%; 95% CI, 2.9-5.9%) was the use of OA throughout; using heparin only between 6 and 12 weeks' gestation was associated with an increased risk of valve thrombosis (9.2%; 95% CI, 5.9%-13.9%). Thromboembolic prophylaxis of women with mechanical heart valves during pregnancy is best achieved with OA; however, this increases the risk of fetal embryopathy. Substituting OA with heparin between 6 and 12 weeks reduces the risk of fetopathic effects, but with an increased risk of thromboembolic complications. The use of low-dose heparin is definitely inadequate; the use of adjusted-dose heparin warrants aggressive monitoring and appropriate dose adjustment. Large prospective trials to determine the best regimen for these women are needed.

Heparin molecularly imprinted polymer thin flm on gold electrode by plasma-induced graft polymerization for label-free biosensor.

PubMed

Orihara, Kouhei; Hikichi, Atsushi; Arita, Tomohiko; Muguruma, Hitoshi; Yoshimi, Yasuo

2018-03-20

Heparin, a highly sulfated glycosaminoglycan, is an important biomaterial having biological and therapeutic functionalities such as anticoagulation, regeneration, and protein stabilization. This study addresses a label-free quartz crystal microbalance (QCM) biosensor for heparin detection based on a macromolecularly imprinted polymer (MIP) as an artificial recognition element. We demonstrate the novel strategy for MIP in the form of thin film on a gold (Au) electrode with the plasma-induced graft polymerization (PIP) technique. The procedure of PIP is as follows: (i) Hexamethyldisiloxane plasma-polymerized thin film (PPF) as a pre-coating scaffold of active species for PIP (post-polymerization) is deposited on an Au electrode. (ii) The PPF/Au electrode is soaked in an water solution containing heparin (template), (2-(methacryloxy)-ethyl)trimethylammonium chloride acrylamide (functional monomer), acrylamide, and N,N-methylenebisacrylamide (crosslinker). Double bonds of monomer and crosslinker attacked by residually active species in pre-coating PPF cause radical chain reaction. Consequently, a growing polymer network of 20 nm thickness of PIP-MIP thin film is formed and grafted on the PPF/Au surface. (iii) The PIP-MIP/PPF/Au is washed by sodium chloride solution so as to remove the template. Non-imprinted polymer (NIP) is carried out like the same procedure without a template. The AFM, XPS, and QCM measurements show that the PIP process facilitates macromolecularly surface imprinting of template heparin where the template is easily removed and is rapidly rebound to PIP-MIP without a diffusional barrier. The heparin-PIP-MIP specifically binds to heparin compared with heparin analog chondroitin sulfate C (selective factor: 4.0) and a detectable range of heparin in the presence of CS (0.1 wt%) was 0.001-0.1 wt%. The PIP-NIP does not show selectivity between them. The evaluated binding kinetics are association (k a  = 350 ± 100 M -1  s -1 ), dissociation (k d  = (5.0 ± 2.0) × 10 -4  s -1 ), and binding (K D  = 1.3 ± 0.6 μM) constants, demonstrating that the PIP-MIP as a synthetic antibody can be applied to analytical chemistry. Copyright © 2018 Elsevier B.V. All rights reserved.

Heparin Microparticle Effects on Presentation and Bioactivity of Bone Morphogenetic Protein-2

PubMed Central

Hettiaratchi, Marian H.; Miller, Tobias; Temenoff, Johnna S.; Guldberg, Robert E.; McDevitt, Todd C.

2014-01-01

Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration. PMID:24881028

Bivalirudin versus heparin in primary PCI: clinical outcomes and cost analysis

PubMed Central

Deharo, Pierre; Johnson, Thomas W; Rahbi, Hazim; Kandan, Raveen; Bowles, Ruth; Mozid, Abdul; Dorman, Stephen; Strange, Julian W; Baumbach, Andreas

2018-01-01

Background The evidence for benefits of bivalirudin over heparin has recently been challenged. We aimed to analyse the safety and cost-effectiveness following reintroduction of heparin instead of bivalirudin as the standard anticoagulation for primary percutaneous coronary intervention (PPCI) in a high-volume centre. Methods and results This analysis was an open-label, prospective registry including all patients admitted to our centre for PPCI from April 2014 to April 2016. Heparin was reintroduced as standard anticoagulant in April 2015. During the 2 years, 1291 patients underwent a PPCI, 662 in the Bivalirudin protocol period (Cohort B) and 629 in the Heparin protocol period (Cohort H). Baseline and procedural characteristics were not significantly different, except for a higher use of thromboaspiration and femoral access in the earlier Cohort B. Glycoprotein 2b3a (Gp2b3a) antagonists were used in 24% of the patients in Cohort B versus 28% in Cohort H (P<0.01). We did not observe any differences in death at 180 days (11.03% in Cohort B vs 11.29% in Cohort H)(HR 95% CI 0.98 (0.72 to 1.33), P=0.88). The incidence of any bleeding complications at 30 days did not differ between the two periods (21.9% vs 21.9%, P=0.99). The cost related to the anticoagulants amounted to £246 236 in Cohort B versus £4483 in Cohort H (£324 406 vs £102 347 when adding Gp2b3a antagonists). Conclusion We did not find clinically relevant changes in patient outcomes, including bleeding complications with reintroduction of heparin in our PPCI protocol. However, the use of heparin was associated with a major reduction in treatment costs. PMID:29765614

Real-time measurement of free thrombin: evaluation of the usability of a new thrombin assay for coagulation monitoring during extracorporeal circulation.

PubMed

Krajewski, Stefanie; Krauss, Sabrina; Kurz, Julia; Neumann, Bernd; Schlensak, Christian; Wendel, Hans P

2014-03-01

In patients undergoing cardiac surgery with heart-lung machine support, adequate anticoagulation to mitigate blood clotting caused by the artificial surfaces of the extracorporeal circulation (ECC) system is essential. These patients routinely receive heparin, whose effectiveness is monitored by measurements of the activated clotting time (ACT). However, ACT values only poorly correlate with the actual hemostatic status. The aim of our study was to evaluate the detection of free thrombin in heparinized human blood as a monitor of anticoagulation during ECC. Human whole blood was anticoagulated with different concentrations of heparin (0.75, 1, 2 or 3 IU/ml) and circulated in the Chandler-loop model for up to 240 min at 37 °C. Next to ACT, ECC-mediated changes in free active thrombin, prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin-III (TAT) levels were measured before and during circulation. Platelet activation and cell count parameters were further investigated. Our study shows that detection of ECC-mediated changes in free thrombin is possible in blood anticoagulated with 0.75 or 1 IU/ml heparin, whereas no thrombin was detectable at higher heparin concentrations. Thrombin generation during 240 min of ECC is comparable to F 1+2 and TAT plasma levels during ECC. Thrombin is the key enzyme in the coagulation cascade and hence represents a promising marker for monitoring the coagulation status of patients. Although detection of free thrombin was not feasible at high heparin concentrations, the employed test represents an additional test to current laboratory methods investigating blood coagulation at low heparin concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma.

PubMed

de Barros, Cintia M; Andrade, Leonardo R; Allodi, Silvana; Viskov, Christian; Mourier, Pierre A; Cavalcante, Moisés C M; Straus, Anita H; Takahashi, Helio K; Pomin, Vitor H; Carvalho, Vinicius F; Martins, Marco A; Pavão, Mauro S G

2007-01-19

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

Gold Medal Forum Winner. Unfractionated heparin three times a day versus enoxaparin in the prevention of deep vein thrombosis in trauma patients.

PubMed

Arnold, Joshua D; Dart, Benjamin W; Barker, Donald E; Maxwell, Robert A; Burkholder, Hans C; Mejia, Vicente A; Smith, Philip W; Longley, Joy M

2010-06-01

Venous thromboembolic disease is a significant source of morbidity and mortality in hospitalized trauma patients. Multiple drugs and dosing regimens have been suggested for pharmacoprophylaxis. In this study, we compared efficacy, complications, and cost of unfractionated heparin administered subcutaneously three times a day with standard-dosed enoxaparin for prophylaxis of deep venous thrombosis (DVT) in adult trauma patients over 1 year. Patients admitted for greater than 72 hours who received pharmacoprophylaxis as part of a comprehensive DVT protocol were included. A change was made in the protocol from enoxaparin (30 mg twice a day or 40 mg per day) to heparin (5000 U three times a day) at midyear. Surveillance lower extremity venous ultrasound was performed according to established institutional guidelines. Data, including demographics, associated injuries, complications, and cost, were collected and analyzed. Four hundred seventy-six patients met inclusion criteria. Two hundred thirty-seven (49.8%) patients received enoxaparin and 239 (50.2%) received heparin. Proximal lower extremity DVTs were detected in 16 (6.75%) patients in the enoxaparin group and 17 (7.11%) in the heparin group (P = 0.999). Risk factors for DVT in these patients included spinal cord injury (P = 0.001) and closed head injury (P = 0.031). There was no difference between the incidence of pulmonary emboli and bleeding. There was an estimated yearly pharmacy cost savings of $135,606. In trauma patients, subcutaneous heparin dosed three times a day may be as effective as standard-dosed enoxaparin for prophylaxis of venous thromboembolism without increased complications. Heparin three times a day for venous thromboembolism prophylaxis was associated with significant pharmaceutical cost savings.

Comparison of telavancin and vancomycin lock solutions in eradication of biofilm-producing staphylococci and enterococci from central venous catheters.

PubMed

Luther, Megan K; Mermel, Leonard A; LaPlante, Kerry L

2016-03-01

Results of a study of the activity of antibiotic lock solutions of vancomycin and telavancin against biofilm-forming strains of Staphylococcus epidermidis, Enterococcus faecalis, and Staphylococcus aureus are reported. An established in vitro central venous catheter model was used to evaluate lock solutions containing vancomycin (5 mg/mL) or telavancin (5 mg/mL), with and without preservative-containing heparin sodium (with 0.45% benzyl alcohol) 2500 units/mL, heparin, and 0.9% sodium chloride solution. Lock solutions were introduced after 24-hour bacterial growth in catheters incubated at 35 °C. After 72 hours of exposure to the lock solutions, catheters were drained, flushed, and cut into segments for quantification of colony-forming units. Against S. epidermidis, vancomycin and telavancin (with or without heparin) had similar activity. Against E. faecalis, vancomycin alone was more active than telavancin alone (p < 0.01). Against S. aureus, vancomycin plus heparin had activity similar to that of vancomycin alone; both lock agents had greater activity than telavancin (p < 0.02). The addition of heparin was associated with reduced activity of the vancomycin lock solution against S. epidermidis and E. faecalis (p < 0.01). Telavancin activity was not significantly changed with the addition of heparin. In a central venous catheter model, vancomycin and telavancin activity was similar in reducing biofilm-producing S. epidermidis. However, vancomycin was more active than telavancin against E. faecalis and S. aureus. None of the tested agents eradicated biofilm-forming strains. The addition of preservative-containing heparin sodium 2500 units/mL to vancomycin was associated with reduced activity against S. epidermidis and E. faecalis. Copyright © 2016 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Heparin-Based Coacervate of FGF2 Improves Dermal Regeneration by Asserting a Synergistic Role with Cell Proliferation and Endogenous Facilitated VEGF for Cutaneous Wound Healing.

PubMed

Wu, Jiang; Ye, Jingjing; Zhu, Jingjing; Xiao, Zecong; He, Chaochao; Shi, Hongxue; Wang, Yadong; Lin, Cai; Zhang, Hongyu; Zhao, Yingzheng; Fu, Xiaobing; Chen, Hong; Li, Xiaokun; Li, Lin; Zheng, Jie; Xiao, Jian

2016-06-13

Effective wound healing requires complicated, coordinated interactions and responses at protein, cellular, and tissue levels involving growth factor expression, cell proliferation, wound closure, granulation tissue formation, and vascularization. In this study, we develop a heparin-based coacervate consisting of poly(ethylene argininylaspartate digylceride) (PEAD) as a storage matrix, heparin as a bridge, and fibroblast growth factor-2 (FGF2) as a cargo (namely heparin-FGF2@PEAD) for wound healing. First, in vitro characterization demonstrates the loading efficiency and control release of FGF2 from the heparin-FGF2@PEAD coacervate. The following in vivo studies examine the wound healing efficiency of the heparin-FGF2@PEAD coacervate upon delivering FGF2 to full-thickness excisional skin wounds in vivo, in comparison with the other three control groups with saline, heparin@PEAD as vehicle, and free FGF2. Collective in vivo data show that controlled release of FGF2 to the wounds by the coacervate significantly accelerates the wound healing by promoting cell proliferation, stimulating the secretion of vascular endothelial growth factor (VEGF) for re-epithelization, collagen deposition, and granulation tissue formation, and enhancing the expression of platelet endothelial cell adhesion molecule (CD31) and alpha-smooth muscle actin (α-SMA) for blood vessel maturation. In parallel, no obvious wound healing effect is found for the control, vehicle, and free FGF2 groups, indicating the important role of the coavervate in the wound healing process. This work designs a suitable delivery system that can protect and release FGF2 in a sustained and controlled manner, which provides a promising therapeutic potential for topical treatment of wounds.

A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

PubMed

Fernández, Silvina; Córdoba, Mariana

2017-04-01

Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC. Copyright © 2017 Elsevier B.V. All rights reserved.

Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance.

PubMed

Rajabi, Mohsen; Struble, Evi; Zhou, Zhaohua; Karnaukhova, Elena

2012-01-01

Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states. SCOPE OF RESEARCH: We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout. Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation. This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies. Published by Elsevier B.V.

Cost-effectiveness analysis of low-molecular-weight heparin versus aspirin thromboprophylaxis in patients newly diagnosed with multiple myeloma.

PubMed

Chalayer, Emilie; Bourmaud, Aurélie; Tinquaut, Fabien; Chauvin, Franck; Tardy, Bernard

2016-09-01

The aim of this study was to assess the cost-effectiveness of low molecular weight heparin versus aspirin as primary thromboprophylaxis throughout chemotherapy for newly diagnosed multiple myeloma patients treated with protocols including thalidomide from the perspective of French health care providers. We used a modeling approach combining data from the only randomized trial evaluating the efficacy of the two treatments and secondary sources for costs, and utility values. We performed a decision-tree analysis and our base case was a hypothetical cohort of 10,000 patients. A bootstrap resampling technique was used. The incremental cost-effectiveness ratio was calculated using estimated quality-adjusted life years as the efficacy outcome. Incremental costs and effectiveness were estimated for each strategy and the incremental cost-effectiveness ratio was calculated. One-way sensitivity analyses were performed. The number of quality-adjusted life years was estimated to be 0.300 with aspirin and 0.299 with heparin. The estimated gain with aspirin was therefore approximately one day. Over 6months, the mean total cost was € 1518 (SD=601) per patient in the heparin arm and € 273 (SD=1019) in the aspirin arm. This resulted in an incremental cost of € 1245 per patient treated with heparin. The incremental cost-effectiveness ratio for the aspirin versus heparin strategy was calculated to be - 687,398 € (95% CI, -13,457,369 to -225,385). Aspirin rather than heparin thromboprophylaxis, during the first six months of chemotherapy for myeloma, is associated with significant cost savings per patient and also with an unexpected slight increase in quality of life. Copyright © 2016 Elsevier Ltd. All rights reserved.

Synthesis and characterization of Poloxamer 188-grafted heparin copolymer.

PubMed

Tian, Ji-Lai; Zhao, Ying-Zheng; Jin, Zhuo; Lu, Cui-Tao; Tang, Qin-Qin; Xiang, Qi; Sun, Chang-Zheng; Zhang, Lu; Xu, Yan-Yan; Gao, Hui-Sheng; Zhou, Zhi-Cai; Li, Xiao-Kun; Zhang, Ying

2010-07-01

Poloxamer 188 is a safe biocompatible polymer that can be used in protein drug delivery system. In this study, a new heparin-poloxamer 188 conjugate (HP) was synthesized and its physicochemical properties were investigated. HP structure was confirmed by Fourier transform infrared spectroscopy (FTIR) and Hydrogen-1 nuclear magnetic resonance spectroscopy ((1)H-NMR). Content of the conjugated heparin was analyzed using Toluidine Blue. The critical micelle concentration (CMC) of the copolymer was determined by a fluorescence probe technique. The effect of HP on the gelation of poloxamer 188 was characterized by the rheological properties of the HP-poloxamer hydrogels. Solubility and viscosity of HP were also evaluated compared with poloxamer 188. From the results, the solubility of the conjugated heparin was increased compared with free heparin. The content of heparin in HP copolymer was 62.9%. The CMC of HP and poloxamer 188 were 0.483 and 0.743 mg/mL, respectively. The gelation temperature of 0.4 g/mL HP was 43.5 degrees C, whereas that of the same concentration of poloxamer 188 was 37.3 degrees C. With HP content in poloxamer 188 solution increasing, a V-shape change of gelation temperature was observed. Considering the importance of poloxamer 188 in functional material, HP may prove to be a facile temperature-sensitive material for protein drug-targeted therapy.

Biologically Derived Soft Conducting Hydrogels Using Heparin-Doped Polymer Networks

PubMed Central

2015-01-01

The emergence of flexible and stretchable electronic components expands the range of applications of electronic devices. Flexible devices are ideally suited for electronic biointerfaces because of mechanically permissive structures that conform to curvilinear structures found in native tissue. Most electronic materials used in these applications exhibit elastic moduli on the order of 0.1–1 MPa. However, many electronically excitable tissues exhibit elasticities in the range of 1–10 kPa, several orders of magnitude smaller than existing components used in flexible devices. This work describes the use of biologically derived heparins as scaffold materials for fabricating networks with hybrid electronic/ionic conductivity and ultracompliant mechanical properties. Photo-cross-linkable heparin–methacrylate hydrogels serve as templates to control the microstructure and doping of in situ polymerized polyaniline structures. Macroscopic heparin-doped polyaniline hydrogel dual networks exhibit impedances as low as Z = 4.17 Ω at 1 kHz and storage moduli of G′ = 900 ± 100 Pa. The conductivity of heparin/polyaniline networks depends on the oxidation state and microstructure of secondary polyaniline networks. Furthermore, heparin/polyaniline networks support the attachment, proliferation, and differentiation of murine myoblasts without any surface treatments. Taken together, these results suggest that heparin/polyaniline hydrogel networks exhibit suitable physical properties as an electronically active biointerface material that can match the mechanical properties of soft tissues composed of excitable cells. PMID:24738911

Intra-articular TSG-6 delivery from heparin-based microparticles reduces cartilage damage in a rat model of osteoarthritis.

PubMed

Tellier, Liane E; Treviño, Elda A; Brimeyer, Alexandra L; Reece, David S; Willett, Nick J; Guldberg, Robert E; Temenoff, Johnna S

2018-05-01

As a potential treatment for osteoarthritis (OA), we have developed injectable and hydrolytically degradable heparin-based biomaterials with tunable sulfation for the intra-articular delivery of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a protein known to inhibit plasmin which may degrade extracellular matrix within OA joints. We first assessed the effect of heparin sulfation on TSG-6 anti-plasmin activity and found that while fully sulfated (Hep) and heparin desulfated at only the N position (Hep-N) significantly enhanced TSG-6 bioactivity in vitro, fully desulfated heparin (Hep-) had no effect, indicating that heparin sulfation plays a significant role in modulating TSG-6 bioactivity. Next, TSG-6 loaded, degradable 10 wt% Hep-N microparticles (MPs) were delivered via intra-articular injection into the knee at 1, 7, and 15 days following medial meniscal transection (MMT) injury in a rat model. After 21 days, cartilage thickness, volume, and attenuation were significantly increased with soluble TSG-6, indicating degenerative changes. In contrast, no significant differences were observed with TSG-6 loaded MP treatment, demonstrating that TSG-6 loaded MPs reduced cartilage damage following MMT injury. Ultimately, our results indicate that Hep-N can enhance TSG-6 anti-plasmin activity and that Hep-N-based biomaterials may be an effective method for TSG-6 delivery to treat OA.

Heparin sodium compliance to USP monograph: structural elucidation of an atypical 2.18 ppm NMR signal.

PubMed

Mourier, Pierre A J; Guichard, Olivier Y; Herman, Fréderic; Viskov, Christian

2012-01-01

The ¹H nuclear magnetic resonance (NMR) acceptance criteria in the new heparin US Pharmacopeia (USP) monograph do not take into account potential structural modifications responsible for any extra signals observed in ¹H NMR spectra, some purified heparins may be non-compliant under the proposed new USP guidelines and incorrectly classified as unsuitable for pharmaceutical use. Heparins from the "ES" source, containing an extra signal at 2.18 ppm, were depolymerized under controlled conditions using heparinases I, II, and III. The oligosaccharides responsible for the 2.18 ppm signal were enriched using orthogonal chromatographic techniques. After multiple purification steps, we obtained an oligosaccharide mixture containing a highly enriched octasaccharide bearing the structural modification responsible for the extra signal. Following heparinase I depolymerization, a pure tetrasaccharide containing the fingerprint structural modification was isolated for full structural determination. Using 1D and 2D ¹H NMR spectroscopy, the structural moiety responsible for the extra signal at 2.18 ppm was identified as an acetyl group on the heparin backbone, most likely resulting from a very minor manufacturing process side reaction that esterifies the uronic acid at position 3. Such analytical peculiarity has always been present in this heparin source and it was used safety over the years. Copyright © 2012 Elsevier B.V. All rights reserved.

Liquid chromatography-diode array detection-mass spectrometry for compositional analysis of low molecular weight heparins.

PubMed

Wang, Zhangjie; Li, Daoyuan; Sun, Xiaojun; Bai, Xue; Jin, Lan; Chi, Lianli

2014-04-15

Low molecular weight heparins (LMWHs) are important artificial preparations from heparin polysaccharide and are widely used as anticoagulant drugs. To analyze the structure and composition of LMWHs, identification and quantitation of their natural and modified building blocks are indispensable. We have established a novel reversed-phase high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry approach for compositional analysis of LMWHs. After being exhaustively digested and labeled with 2-aminoacridone, the structural motifs constructing LMWHs, including 17 components from dalteparin and 15 components from enoxaparin, were well separated, identified, and quantified. Besides the eight natural heparin disaccharides, many characteristic structures from dalteparin and enoxaparin, such as modified structures from the reducing end and nonreducing end, 3-O-sulfated tetrasaccharides, and trisaccharides, have been unambiguously identified based on their retention time and mass spectra. Compared with the traditional heparin compositional analysis methods, the approach described here is not only robust but also comprehensive because it is capable of identifying and quantifying nearly all components from lyase digests of LMWHs. Copyright © 2014 Elsevier Inc. All rights reserved.

Impact of autoclave sterilization on the activity and structure of formulated heparin.

PubMed

Beaudet, Julie M; Weyers, Amanda; Solakyildirim, Kemal; Yang, Bo; Takieddin, Majde; Mousa, Shaker; Zhang, Fuming; Linhardt, Robert J

2011-08-01

The stability of a formulated heparin was examined during its sterilization by autoclaving. A new method to follow loss in heparin binding to the serine protease inhibitor, antithrombin III, and the serine protease, thrombin, was developed using a surface plasmon resonance competitive binding assay. This loss in binding affinity correlated well with loss in antifactor IIa (thrombin) activity as well as antifactor Xa activity as measured using conventional amidolytic assays. Autoclaving also resulted in a modest breakdown of the heparin backbone as confirmed by a slight reduction in number-averaged and weight-averaged molecular weight and an increase in polydispersity. Although no clear changes were observed by nuclear magnetic resonance spectroscopy, disaccharide composition analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry suggested that loss of selected sulfo groups had taken place. It is this sulfo group loss that probably accounts for a decrease in the binding of autoclaved heparin to antithrombin III and thrombin as well as the observed decrease in its amidolytic activity. Copyright © 2011 Wiley-Liss, Inc.

Top-down and bottom-up analysis of commercial enoxaparins.

PubMed

Liu, Xinyue; St Ange, Kalib; Lin, Lei; Zhang, Fuming; Chi, Lianli; Linhardt, Robert J

2017-01-13

A strategy for the comprehensive analysis of low molecular weight (LMW) heparins is described that relies on using an integrated top-down and bottom-up approach. Liquid chromatography-mass spectrometry, an essential component of this approach, is rapid, robust, and amenable to automated processing and interpretation. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues comprising a low molecular weight heparin. Using our integrated approach four different low molecular weight heparins prepared from porcine heparin through chemical β-eliminative cleavage were comprehensively analyzed. Lovenox™ and Clexane™, the innovator versions of enoxaparin marketed in the US and Europe, respectively, and two generic enoxaparins, from Sandoz and Teva, were analyzed. The results which were supported by analysis of variation (ANOVA), while showing remarkable similarities between different versions of the product and good lot-to-lot consistency of each product, also detects subtle differences that may result from differences in their manufacturing processes or differences in the source (or parent) porcine heparin from which each product is prepared. Copyright © 2016 Elsevier B.V. All rights reserved.

A prospective randomized trial comparing the clinical effectiveness and biocompatibility of heparin-coated circuits and PMEA-coated circuits in pediatric cardiopulmonary bypass.

PubMed

Itoh, Hideshi; Ichiba, Shingo; Ujike, Yoshihito; Douguchi, Takuma; Kasahara, Shingo; Arai, Sadahiko; Sano, Shunji

2016-04-01

We compared the clinical effectiveness and biocompatibility of poly-2-methoxyethyl acrylate (PMEA)-coated and heparin-coated cardiopulmonary bypass (CPB) circuits in a prospective pediatric trial. Infants randomly received heparin-coated (n=7) or PMEA-coated (n=7) circuits in elective pediatric cardiac surgery with CPB for ventricular septum defects. Clinical and hematologic variables, respiratory indices and hemodynamic changes were analyzed perioperatively. Demographic and clinical variables were similar in both groups. Leukocyte counts were significantly lower 5 minutes after CPB in the PMEA group than the heparin group. Hemodynamic data showed that PMEA caused hypotension within 5 minutes of CPB. The respiratory index was significantly higher immediately after CPB and 1 hour after transfer to the intensive care unit (ICU) in the PMEA group, as were levels of C-reactive protein 24 hours after transfer to the ICU. Our study shows that PMEA-coated circuits, unlike heparin-coated circuits, cause transient leukopenia during pediatric CPB and, perhaps, systemic inflammatory respiratory syndrome after pediatric CPB. © The Author(s) 2015.

Chemoenzymatic Synthesis of Heparin Oligosaccharides with both Anti-factor Xa and Anti-factor IIa Activities*

PubMed Central

Xu, Yongmei; Pempe, Elizabeth H.; Liu, Jian

2012-01-01

Heparan sulfate (HS) and heparin are highly sulfated polysaccharides. Heparin is a commonly used anticoagulant drug that inhibits the activities of factors Xa and IIa (also known as thrombin) to prevent blood clot formation. Here, we report the synthesis of a series of size-defined oligosaccharides to probe the minimum size requirement for an oligosaccharide with anti-IIa activity. The synthesis was completed by a chemoenzymatic approach involving glycosyltransferases, HS sulfotransferases, and C5-epimerase. We demonstrate the ability to synthesize highly purified N-sulfo-oligosaccharides having up to 21 saccharide residues. The results from anti-Xa and anti-IIa activity measurements revealed that an oligosaccharide longer than 19 saccharide residues is necessary to display anti-IIa activity. The oligosaccharides also exhibit low binding toward platelet factor 4, raising the possibility of preparing a synthetic heparin with a reduced effect of heparin-induced thrombocytopenia. The results from this study demonstrate the ability to synthesize large HS oligosaccharides and provide a unique tool to probe the structure and function relationships of HS that require the use of large HS fragments. PMID:22773834

Biological and structural analyses of bovine heparin fractions of intermediate and high molecular weight.

PubMed

Nogueira, Alexsandro V; Drehmer, Daiana L; Iacomini, Marcello; Sassaki, Guilherme L; Cipriani, Thales R

2017-02-10

Low molecular weight heparin, which is generally obtained by chemical and enzymatic depolymerization of unfractionated heparin, has high bioavailability and can be subcutaneously injected. The aim of the present investigation was to fractionate bovine heparin using a physical method (ultrafiltration through a 10kDa cut-off membrane), avoiding structural modifications that can be caused by chemical or enzymatic treatments. Two fractions with different molecular weights were obtained: the first had an intermediate molecular weight (B-IMWH; Mn=9587Da) and the other had a high molecular weight (B-HMWH; 22,396Da). B-IMWH and B-HMWH have anticoagulant activity of 103 and 154IU/mg respectively, which could be inhibited by protamine. Both fractions inhibited α-thrombin and factor Xa in vitro and showed antithrombotic effect in vivo. Moreover, ex vivo aPTT assay demonstrated that B-IMWH is absorbed by subcutaneous route. The results showed that ultrafiltration can be used to obtain two bovine heparin fractions, which differ on their molecular weights, structural components, anticoagulant potency, and administration routes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Matrix metalloproteinase and heparin-stimulated serine proteinase activities in post-prostate massage urine of men with prostate cancer.

PubMed

Muñoz, David; Serrano, Maria K; Hernandez, Maria E; Haller, Ross; Swanson, Tamara; Slaton, Joel W; Sinha, Akhouri A; Wilson, Michael J

2017-12-01

Proteinases secreted by the prostate gland have a reproductive function in cleaving proteins in the ejaculate and in the female reproductive tract, but some may have a fundamental role in disease and pathological processes including cancer. The purpose of this study was to determine if there were differences in proteinase activities in urine samples collected following prostate massage of men positive (CaP) or negative (no evidence of malignancy, NEM) for biopsy determined prostate cancer. Matrix metalloproteinase (MMP) and serine proteinase activities were detected using protein substrate zymography. There were no differences in activities of MMP-2, proMMP-9, and MMP-9/NGAL (neutrophil gelatinase associated lipocalin) complex (gelatin substrate) in men with detected prostate cancer, although the latter two were somewhat diminished. A caseinolytic activity of about 75kDa inhibited by calcium did not differ between the NEM and CaP groups. Heparin stimulated calcium sensitive gelatinolytic activities of approximately 22, 42, and 60kDa, but did not affect activities of MMP-2, MMP-9, or the 75kDa caseinolytic activity. The 22, 42, and 60kDa activities appear to be serine proteinases since they were inhibited by benzamidine. There was a significant decrease in the 22kDa heparin-stimulated serine proteinase activity in urines of men with cancer. Proteinase expression and activities, perhaps in combination with other potential markers, may prove useful in urine for detection and evaluation of prostate cancer. Copyright © 2017. Published by Elsevier Inc.

Vitamin K antagonists or low-molecular-weight heparin for the long term treatment of symptomatic venous thromboembolism.

PubMed

van der Heijden, J F; Hutten, B A; Büller, H R; Prins, M H

2002-01-01

People with venous thromboembolism are generally treated for five days with intravenous unfractionated heparin or subcutaneous low-molecular-weight heparin followed by three months of vitamin K antagonists treatment. Treatment with vitamin K antagonists requires regular laboratory measurements and some patients have contraindications for treatment. To evaluate the efficacy and safety of long-term treatment of venous thromboembolism with low-molecular-weight heparins compared to vitamin K antagonists. Searches of MEDLINE, EMBASE and ISI Web of Science, the Specialised Trials Register of the Cochrane Peripheral Vascular Disease Group and the Cochrane Controlled Trials Register were made and relevant journals were hand-searched. Additional trials were sought through communication with colleagues and pharmaceutical companies. Two reviewers evaluated studies independently for methodological quality. Two reviewers extracted data independently. Primary analysis concerned all trial participants during the period of randomized treatment. Separate analyses were performed for category I and category II studies; i.e. studies using similar treatments initially in both study arms, and those that did not; and the different periods of follow-up. All seven studies fulfilling our criteria combined, a statistically non-significant reduction in the risk of recurrent venous thromboembolism favoring low-molecular-weight heparin treatment (OR 0.70; 95% CI [0.42, 1.16]) was found. Analysis of pooled data for category I studies showed a non-significant reduction in the risk of recurrent venous thromboembolism favoring low-molecular-weight heparin treatment (OR 0.75; 95% CI [0.40, 1.39]). Omitting a potentially-confounded study, a statistically non-significant reduction in the risk of recurrent venous thromboembolism favoring vitamin K antagonist treatment remained (OR 1.95; 95% CI [0.74, 5.19]). All studies combined, the difference in bleeding significantly favored treatment with low-molecular-weight heparin (OR 0.38; 95% CI [0.15, 0.94]), however, considering only category I studies a non-significant trend favoring low-molecular-weight heparin remained (OR 0.80; 95% CI [0.21, 3.00]). No difference was observed in mortality (OR 1.13; 95% CI [0.47, 2.69]). Low-molecular-weight heparins are possibly as effective as vitamin K antagonists in preventing symptomatic venous thromboembolism after an episode of symptomatic deep venous thrombosis, but are much more expensive. Treatment with low-molecular-weight heparin is significantly safer than treatment with vitamin K antagonists and is possibly a safe alternative in some patients; especially those in geographically inaccessible places, reluctant to visit the thrombosis service regularly, or with contraindications to vitamin K antagonists. However, treatment with vitamin K antagonists remains the treatment of choice for the majority of patients.

The structure of myostatin:follistatin 288: insights into receptor utilization and heparin binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cash, Jennifer N.; Rejon, Carlis A.; McPherron, Alexandra C.

2009-09-29

Myostatin is a member of the transforming growth factor-{beta} (TGF-{beta}) family and a strong negative regulator of muscle growth. Here, we present the crystal structure of myostatin in complex with the antagonist follistatin 288 (Fst288). We find that the prehelix region of myostatin very closely resembles that of TGF-{beta} class members and that this region alone can be swapped into activin A to confer signalling through the non-canonical type I receptor Alk5. Furthermore, the N-terminal domain of Fst288 undergoes conformational rearrangements to bind myostatin and likely acts as a site of specificity for the antagonist. In addition, a unique continuousmore » electropositive surface is created when myostatin binds Fst288, which significantly increases the affinity for heparin. This translates into stronger interactions with the cell surface and enhanced myostatin degradation in the presence of either Fst288 or Fst315. Overall, we have identified several characteristics unique to myostatin that will be paramount to the rational design of myostatin inhibitors that could be used in the treatment of muscle-wasting disorders.« less

Heparin/heparan sulfate analysis by covalently modified reverse polarity capillary zone electrophoresis-mass spectrometry.

PubMed

Sanderson, Patience; Stickney, Morgan; Leach, Franklin E; Xia, Qiangwei; Yu, Yanlei; Zhang, Fuming; Linhardt, Robert J; Amster, I Jonathan

2018-04-13

Reverse polarity capillary zone electrophoresis coupled to negative ion mode mass spectrometry (CZE-MS) is shown to be an effective and sensitive tool for the analysis of glycosaminoglycan mixtures. Covalent modification of the inner wall of the separation capillary with neutral or cationic reagents produces a stable and durable surface that provides reproducible separations. By combining CZE-MS with a cation-coated capillary and a sheath flow interface, a rapid and reliable method has been developed for the analysis of sulfated oligosaccharides from dp4 to dp12. Several different mixtures have been separated and detected by mass spectrometry. The mixtures were selected to test the capability of this approach to resolve subtle differences in structure, such as sulfation position and epimeric variation of the uronic acid. The system was applied to a complex mixture of heparin/heparan sulfate oligosaccharides varying in chain length from dp3 to dp12 and more than 80 molecular compositions were identified by accurate mass measurement. Copyright © 2018 Elsevier B.V. All rights reserved.

Collagen/heparin sulfate scaffolds fabricated by a 3D bioprinter improved mechanical properties and neurological function after spinal cord injury in rats.

PubMed

Chen, Chong; Zhao, Ming-Liang; Zhang, Ren-Kun; Lu, Gang; Zhao, Chang-Yu; Fu, Feng; Sun, Hong-Tao; Zhang, Sai; Tu, Yue; Li, Xiao-Hong

2017-05-01

Effective treatments promoting axonal regeneration and functional recovery for spinal cord injury (SCI) are still in the early stages of development. Most approaches have been focused on providing supportive substrates for guiding neurons and overcoming the physical and chemical barriers to healing that arise after SCI. Although collagen has become a promising natural substrate with good compatibility, its low mechanical properties restrict its potential applications. The mechanical properties mainly rely on the composition and pore structure of scaffolds. For the composition of a scaffold, we used heparin sulfate to react with collagen by crosslinking. For the structure, we adopted a three-dimensional (3D) printing technology to fabricate a scaffold with a uniform pore distributions. We observed that the internal structure of the scaffold printed with a 3D bioprinter was regular and porous. We also found that both the compression modulus and strengths of the scaffold were significantly enhanced by the collagen/heparin sulfate composition compared to a collagen scaffold. Meanwhile, the collagen/heparin sulfate scaffold presented good biocompatibility when it was co-cultured with neural stem cells in vitro. We also demonstrated that heparin sulfate modification significantly improved bFGF immobilization and absorption to the collagen by examining the release kinetics of bFGF from scaffolds. Two months after implantating the scaffold into transection lesions in T10 of the spinal cord in rats, the collagen/heparin sulfate group demonstrated significant recovery of locomotor function and according to electrophysiological examinations. Parallel to functional recovery, collagen/heparin sulfate treatment further ameliorated the pathological process and markedly increased the number of neurofilament (NF) positive cells compared to collagen treatment alone. These data suggested that a collagen/heparin sulfate scaffold fabricated by a 3D bioprinter could enhance the mechanical properties of collagen and provide continuous guidance channels for axons, which would improve the neurological function after SCI. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1324-1332, 2017. © 2017 Wiley Periodicals, Inc.

Influence of spacer length on heparin coupling efficiency and fibrinogen adsorption of modified titanium surfaces

PubMed Central

Tebbe, David; Thull, Roger; Gbureck, Uwe

2007-01-01

Background Chemical bonding of the drug onto surfaces by means of spacer molecules is accompanied with a reduction of the biological activity of the drug due to a constricted mobility since normally only short spacer molecule like aminopropyltrimethoxysilane (APMS) are used for drug coupling. This work aimed to study covalent attachment of heparin to titanium(oxide) surfaces by varying the length of the silane coupling agent, which should affect the biological potency of the drug due to a higher mobility with longer spacer chains. Methods Covalent attachment of heparin to titanium metal and TiO2 powder was carried out using the coupling agents 3-(Trimethoxysilyl)-propylamine (APMS), N- [3-(Trimethoxysilyl)propyl]ethylenediamine (Diamino-APMS) and N1- [3-(Trimethoxy-silyl)-propyl]diethylenetriamine (Triamino-APMS). The amount of bound coupling agent and heparin was quantified photometrically by the ninhydrin reaction and the tolidine-blue test. The biological potency of heparin was determined photometrically by the chromogenic substrate Chromozym TH and fibrinogen adsorption to the modified surfaces was researched using the QCM-D (Quartz Crystal Microbalance with Dissipation Monitoring) technique. Results Zeta-potential measurements confirmed the successful coupling reaction; the potential of the unmodified anatase surface (approx. -26 mV) shifted into the positive range (> + 40 mV) after silanisation. Binding of heparin results in a strongly negatively charged surface with zeta-potentials of approx. -39 mV. The retaining biological activity of heparin was highest for the spacer molecule Triamino-APMS. QCM-D measurements showed a lower viscosity for adsorbed fibrinogen films on heparinised surfaces by means of Triamino-APMS. Conclusion The remaining activity of heparin was found to be highest for the covalent attachment with Triamino-APMS as coupling agent due to the long chain of this spacer molecule and therefore the highest mobility of the drug. Furthermore, the adsorption of fibrinogen on the differently heparinised surfaces in real time demonstrated that with longer spacer chains the ΔD/Δf ratios became higher, which is also associated with better biocompatible properties of the substrates in contact with a biosystem. PMID:17640335

Is Taurolidine-citrate an effective and cost-effective hemodialysis catheter lock solution? A systematic review and cost- effectiveness analysis.

PubMed

Kavosi, Zahra; Sarikhani Khorrami, Maryam; Keshavarz, Khosro; Jafari, Abdosaleh; Hashemi Meshkini, Amir; Safaei, Hamid Reza; Nikfar, Shekoufeh

2016-01-01

Prevention of catheter-related infection is of prime importance,. However, because of the risks caused by the leakage of circulating antibiotics and development of resistance to antibiotics, they are replaced by lock solutions. The aim of this study was to evaluate the efficacy and cost- effectiveness of taurolidine-citrate as a hemodialysis catheter lock solution compared to other common alternatives in Iran. To evaluate the efficacy of taurolidine-citrate, a systematic review was conducted by searching electronic databases. The outcomes of interest for cost-effectiveness analysis were as follows: "Catheter-related bacteremia episodes"; "catheter-related bacteremia-free survival"; "catheter thrombosis rate" for efficacy evaluation and "reduction of catheter-related infection". For evidence synthesis, a meta-analysis was conducted on the extracted efficacy data. To evaluate the cost of treatments, direct medical costs were included, and the incremental cost-effectiveness ratio was calculated for each comparison. The payers' (patients and insurance companies) perspectives were used for cost analysis. After carrying out the systematic process, three articles were included in the analysis. Considering 95% confidence interval, the relative difference was -0.16 (-0.25 to -0.07) for catheterrelated bacteremia episode, indicating that the rate of catheter-related infections in hemodialysis patients who used taurolidine-citrate was 16% less than in those hemodialysis patients who received heparin. Considering 95% confidence interval, the relative difference was 0.13 (-0.06 0.32) for catheter thrombosis, showing that the rate of catheter-related thrombosis in hemodialysis patients who used taurolidine-citrate was 13% more than in hemodialysis patients who received heparin. The results of this analysis indicated that taurolidine-citrate, compared to heparin, was more effective in preventing catheter-related infection; therefore, it could be considered as a superior strategy. Nevertheless, compared to heparin-gentamicin combination, taurolidine-citrate is an inferior strategy because of its higher cost and lower infection prevention. Compared to heparin, taurolidine-citrate is a superior option, but it is an inferior strategy compared to heparin-gentamicin combination. The clinical evidences on taurolidine-citrate, heparin and gentamicin/heparin are not sufficient for making confident decisions.

Heparin-binding peptide amphiphile supramolecular architectures as platforms for angiogenesis and drug delivery

NASA Astrophysics Data System (ADS)

Chow, Lesleyann W.

A fascinating phenomenon in nature is the self-assembly of molecules into a functional, hierarchical structure. In the past decade, the Stupp Laboratory has developed several classes of self-assembling biomaterials, one of which is the synthetic peptide amphiphile (PA). Self-assembling PAs are attractive and versatile biomolecules that can be customized for specific applications in regenerative medicine. In particular, a heparin-binding peptide amphiphile (HBPA) containing a specific heparin-binding peptide sequence was used here to induce angiogenesis and serve as a delivery vehicle for growth factors and small hydrophobic molecules. Throughout this dissertation, the HBPA/heparin system is used in different architectures for a variety of regenerative medicine applications. In one aspect of this work, hybrid scaffolds made from HBPA/heparin gelled on a poly(L-lactic acid) (PLLA) fiber mesh were used to promote angiogenesis to facilitate pancreatic islet transplantation for the treatment of type 1 diabetes. Delivery of growth factors with HBPA/PLLA scafflolds increased vessel density in vivo and correlated with improved transplant outcomes in a streptozotocin-induced diabetic mouse model. Soluble HBPA nanofiber architectures were also useful for islet transplantation applications. These nanofibers were used at concentrations below gelation to deliver growth factors into the dense islet cell aggregate, promoting cell survival and angiogenesis in vitro. The nanostructures infiltrated the islets and promoted the retention of heparin and growth factors within the islet. Another interesting growth factor release system discussed here is the HBPA membrane structure. HBPA was found to self-assemble with hyaluronic acid, a large biopolymer found in the body, into macroscopic, hierarchically-ordered membranes. Heparin was incorporated into these membranes and affected the membrane's mechanical properties and growth factor release. Human mesenchymal stem cells were also shown to attach and maintain viability on these membranes. Finally, HBPA nanofibers were used to control the release of small hydrophobic molecules. HBPA nanofiber gels released nitric oxide (NO) to inhibit neointimal hyperplasia, a major cause for vascular graft or stent failure. HBPA/heparin gels were shown to prolong the release of NO generated from NO donors, significantly reducing neointimal hyperplasia in injured carotid arteries in vivo.

Ultrasound has synergistic effects in vitro with tirofiban and heparin for thrombus dissolution.

PubMed

Birnbaum, Y; Atar, S; Luo, H; Nagai, T; Siegel, R J

1999-12-15

Previous studies have shown synergism between ultrasound and thrombolytic agents or microbubbles on blood clot dissolution. It has not been investigated whether heparin or glycoprotein IIb/IIIa blockers enhance clot lysis by ultrasound. We compared the blood clot dissolution effect of saline, heparin, tissue plasminogen activator (tPA), tirofiban, and an echocardiographic contrast media (Optison) without and with ultrasound application. Human blood clots from four donors, 2 to 4 hours old, were cut into 200- to 400-mg sections, weighed, and immersed for 2 minutes in 1 L of normal saline 0.9% solution containing either heparin 1000 U, tirofiban 150 microg, tPA 20 mg, Optison 0.5 mL, or normal saline alone. Clots were randomized to 2 minutes ultrasound application or immersion alone without ultrasound. Ultrasound was applied with a 19.5 KHz catheter. After treatment, the clots were weighed, and the absolute and percent difference in weight was calculated. Immersion in heparin, tirofiban, and tPA without ultrasound did not augment clot disruption relative to normal saline alone. Immersion in Optison (p = 0.07) tended to result in less lysis than saline alone. Ultrasound enhanced clot dissolution compared to immersion alone with: saline (48.1+/-15.3% vs. 26.0+/-13.8%, p<0.0000002); heparin (60.8+/-17.5% vs. 30.8+/-15.1%, p = 0.000001); tirofiban (61.8+/-13.6% vs. 30.1+/-12.2%, p<0.0000001); tPA (53.1+/-15.3% vs. 30.2+/-11.5%, p<0.000002); and Optison (47.8+/-16.0% vs. 18.4+/-11.5%, p<0.0000001). The combination of tirofiban with ultrasound, as well as heparin with ultrasound, was associated with a significant augmentation of clot dissolution compared with the saline plus ultrasound group (p = 0.002, 0.013, respectively). Ultrasound with tPA or with Optison had no significant augmentation of clot dissolution over the ultrasound + saline effect. This in vitro study of catheter-delivered high-intensity low-frequency ultrasound demonstrates that: (1) tirofiban and heparin, as well as perfluorocarbon microbubbles, augment clot dissolution by ultrasound; (2) augmentation of clot dissolution is evident even after only brief exposure of ultrasound and the drug studied.

EFFECT OF THE TREATMENT WITH P-32 ON BLOOD COAGULATION FACTORS IN POLYCYTHEMIA VERA (in Italian)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Austoni, M.; Masetto, I.; Butto, M.

1961-01-01

Blood coagulation factors were investigated in 14 patients with polycythemia vera, before and after P/sup 32/ treatment. In connection with the improvement of clinical signs of thrombosis or hemorrhage and with the red cell count decrease, significant changes were observed, especially in the platelet sector. A general normalization of the platelet function occurred with a decrease from 420000 before to 186200/cmm after the therapy. This was evident with the Heparin tolerance test in vitro: the mean values, which were 6'39"/3 heparin Units/ml, 8'57"/7 heparin U/ml, and 12'31"/10 heparin U/ml before treatment, passed to 7'36", 11'10", and 16'08", in connection withmore » the improvement of the plasmatic thrombogenic diathesis. (P.C.H.)« less

Metabolic aspects and viability of heparin/CPDA-1-stored red cell concentrate as a by-product of a high-yield factor VIII production method.

PubMed

de Jonge, J; Smit Sibinga, C T; Das, P C

1983-01-01

As a by-product of a new high-yield method of production of freeze-dried factor VIII, red cell concentrate (RCC) containing a small amount of heparin besides CPDA-1 was studied. Compared to CPDA-1 stored RCC no difference was found in hematology parameters and 2,3-DPG levels during 28 days storage. Although still in the normal range for transfusion, ATP levels were significantly lower compared to CPDA-1-stored RCC after 30 days shelf life. A survival study with 51Cr-labelled red cells showed good recovery and normal red cell half-life. Rapid transfusion of heparin/CPDA-1 RCC in 6 volunteers did not have any effect on aPTT. Heparin could not be detected in posttransfusion plasma samples.

Hydrophilic interaction chromatography-multiple reaction monitoring mass spectrometry method for basic building block analysis of low molecular weight heparins prepared through nitrous acid depolymerization.

PubMed

Sun, Xiaojun; Guo, Zhimou; Yu, Mengqi; Lin, Chao; Sheng, Anran; Wang, Zhiyu; Linhardt, Robert J; Chi, Lianli

2017-01-06

Low molecular weight heparins (LMWHs) are important anticoagulant drugs that are prepared through depolymerization of unfractionated heparin. Based on the types of processing reactions and the structures of the products, LMWHs can be divided into different classifications. Enoxaparin is prepared by benzyl esterification and alkaline depolymerization, while dalteparin and nadroparin are prepared through nitrous acid depolymerization followed by borohydride reduction. Compositional analysis of their basic building blocks is an effective way to provide structural information on heparin and LMWHs. However, most current compositional analysis methods have been limited to heparin and enoxaparin. A sensitive and comprehensive approach is needed for detailed investigation of the structure of LMWHs prepared through nitrous acid depolymerization, especially their characteristic saturated non-reducing end (NRE) and 2,5-anhydro-d-mannitol reducing end (RE). A maltose modified hydrophilic interaction column offers improved separation of complicated mixtures of acidic disaccharides and oligosaccharides. A total of 36 basic building blocks were unambiguously identified by high-resolution tandem mass spectrometry (MS). Multiple reaction monitoring (MRM) MS/MS quantification was developed and validated in the analysis of dalteparin and nadroparin samples. Each group of building blocks revealed different aspects of the properties of LMWHs, such as functional motifs required for anticoagulant activity, the structure of heparin starting materials, cleavage sites in the depolymerization reaction, and undesired structural modifications resulting from side reactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and in vivo evaluation of an oral delivery system for low molecular weight heparin based on thiolated polycarbophil.

PubMed

Kast, Constantia E; Guggi, Davide; Langoth, Nina; Bernkop-Schnürch, Andreas

2003-06-01

It was the purpose of this study to develop a new oral drug delivery system for low molecular weight heparin (LMWH) providing an improved bioavailability and a prolonged therapeutic effect. The permeation enhancing polycarbophil-cysteine conjugate (PCP-Cys) used in this study displayed 111.4 +/- 6.4 microM thiol groups per gram polymer. Permeation studies on freshly excised intestinal mucosa were performed in Ussing chambers demonstrating a 2-fold improved uptake of heparin as a result of the addition of 0.5% (w/v) PCP-Cys and the permeation mediator glutathione (GSH). Tablets containing PCP-Cys, GSH, and 279 IU of LMWH showed a sustained drug release over 4 h. To guarantee the swelling of the polymeric carrier matrix in the small intestine tablets were enteric coated. They were orally given to rats. For tablets being based on the thiomer/GSH system an absolute bioavailability of 19.9 +/- 9.3% (means +/- SD; n = 5) vs. intravenous injection could be achieved. whereas tablets comprising unmodified PCP did not lead to a significant (p < 0.01) heparin concentration in plasma. The permeation enhancing effect and subsequently a therapeutic heparin level was maintained for 24 h after a single dose. Because of the strong and prolonged lasting permeation enhancing effect of the thiomer/GSH system, the oral bioavailability of LMWH could be significantly improved. This new delivery system represents therefore a promising tool for the oral administration of heparin.

Think of HIT.

PubMed

Warkentin, Theodore E

2006-01-01

Heparin-induced thrombocytopenia, or HIT, can present in many ways, ranging from common-isolated thrombocytopenia, venous thromboembolism, acute limb ischemia-to less common but specific presentations-necrotizing skin lesions at heparin injection sites, post-bolus acute systemic reactions, and adrenal hemorrhagic necrosis (secondary to adrenal vein thrombosis). Many patients with HIT have mild or moderate thrombocytopenia: the median platelet count nadir is 60 x 10(9)/L, and ranges from 15 to 150 x 10(9)/L in 90% of patients, most of whom evince a 50% or greater fall in the platelet count. HIT that begins after stopping heparin ("delayed-onset HIT") is increasingly recognized. Factors influencing risk of HIT include type of heparin (unfractionated heparin > low-molecular-weight heparin), type of patient (surgical > medical), and gender (female > male). Since timely diagnosis and treatment of HIT may reduce the risk of adverse outcomes, this review focuses on those clinical circumstances that should prompt the clinician to "think of HIT." Coumarin anticoagulants such as warfarin are ineffective in acute HIT and can even be deleterious by predisposing to micro-thrombosis via protein C depletion (venous limb gangrene and skin necrosis syndromes). Thus, it is important to avoid or postpone coumarin while managing HIT hypercoagulability, focusing on agents that inhibit thrombin directly (lepirudin, argatroban) or that inhibit its generation (danaparoid, fondaparinux). Post-marketing experience suggests that standard dosing of lepirudin is too high; current recommendations are to avoid the initial lepirudin bolus and to begin with lower infusion rates, even in patients without overt renal dysfunction.

Lack of difference between continuous versus intermittent heparin infusion on maintenance of intra-arterial catheter in postoperative pediatric surgery: a randomized controlled study

PubMed Central

Witkowski, Maria Carolina; de Moraes, Maria Antonieta P.; Firpo, Cora Maria F.

2013-01-01

OBJECTIVE: To compare two systems of arterial catheters maintenance in postoperative pediatric surgery using intermittent or continuous infusion of heparin solution and to analyze adverse events related to the site of catheter insertion and the volume of infused heparin solution. METHODS: Randomized control trial with 140 patients selected for continuous infusion group (CIG) and intermittent infusion group (IIG). The variables analyzed were: type of heart disease, permanence time and size of the catheter, insertion site, technique used, volume of heparin solution and adverse events. The descriptive variables were analyzed by Student's t-test and the categorical variables, by chi-square test, being significant p<0.05. RESULTS: The median age was 11 (0-22) months, and 77 (55%) were females. No significant differences between studied variables were found, except for the volume used in CIG (12.0±1.2mL/24 hours) when compared to IIG (5.3±3.5mL/24 hours) with p<0.0003. CONCLUSIONS: The continuous infusion system and the intermittent infusion of heparin solution can be used for intra-arterial catheters maintenance in postoperative pediatric surgery, regardless of patient's clinical and demographic characteristics. Adverse events up to the third postoperative day occurred similarly in both groups. However, the intermittent infusion system usage in underweight children should be considered, due to the lower volume of infused heparin solution [ClinicalTrials.gov Identifier: NCT01097031]. PMID:24473958

Heparin for prolonging peripheral intravenous catheter use in neonates: a randomized controlled trial.

PubMed

Upadhyay, A; Verma, K K; Lal, P; Chawla, D; Sreenivas, V

2015-04-01

To determine the efficacy of heparinized saline administered as intermittent flush on functional duration of the peripheral intravenous catheter (PIVC) in neonates. Randomized, double-blind and placebo-controlled trial. Neonatal intensive care unit of a teaching hospital. Term and preterm neonates born at >32 weeks of gestation who required PIVC only for intermittent administration of antibiotics. Eligible neonates were randomized to receive 1 ml of either heparinized saline (10 U ml(-1)) (n=60) or normal saline (n=60) every 12 h before and after intravenous antibiotics. Functional duration of first peripheral intravenous catheter. A total of 120 neonates were randomized to two groups of 60 neonates each. The mean (s.d.) of age of babies in case and control group was 5.7 (2.5) days and 4.6 (3.1) days, respectively. The average weight of babies in both the groups was 2.1 kg. Mean functional duration of first catheter was more in heparinized saline group, mean (s.d.) of 71.68 h  (27.3) as compared with 57.7 h (23.6) in normal saline group (P<0.005). The mean (95% confidence interval) difference in functional duration in the two groups was 13.9 h (4.7-23.15). Mean duration of patency for any catheter was also significantly more in heparinized saline group than control group. Heparinized saline flush increases the functional duration of peripheral intravenous catheter.

Efficacy of Minocycline and EDTA Lock Solution in Preventing Catheter-Related Bacteremia, Septic Phlebitis, and Endocarditis in Rabbits

PubMed Central

Raad, Issam; Hachem, Ray; Tcholakian, Robert K.; Sherertz, Robert

2002-01-01

To determine the efficacy of antibiotic catheter lock solution in preventing catheter-related infections, silicone catheters were tunneled and inserted into the jugular veins of 18 rabbits. The catheters were challenged with an intraluminal injection of 105 CFU of slime-producing Staphylococcus epidermidis in 0.1 ml of water. The catheters were maintained on heparin (100 IU/ml) flush for the first 3 days. On day 3, quantitative blood samples for culture were obtained from the catheters and ear veins, which documented catheter-related bacteremia, and the rabbits were randomized to have their catheters flushed as follows: five animals were continued on heparin (100 IU/ml), five animals received vancomycin (3 mg/ml) with heparin (100 IU/ml), and eight animals received 3 mg of minocycline per ml with 30 mg of EDTA per ml (M-EDTA). All animals were killed at day 7. Blood, catheters, jugular veins, and heart valves were cultured quantitatively. Animals maintained on heparin developed catheter-related colonization, bacteremia, septic phlebitis, and endocarditis. Vancomycin-heparin partially prevented catheter colonization, bacteremia, and phlebitis (P = 0.2). M-EDTA completely prevented catheter colonization, catheter-related bacteremia, and phlebitis in all of the animals (P < 0.01). Tricuspid endocarditis was equally prevented by vancomycin-heparin and M-EDTA (P ≤ 0.06). In conclusion, the M-EDTA catheter flush solution was highly efficacious in preventing catheter-related colonization, bacteremia, septic phlebitis, and endocarditis in rabbits. PMID:11796338

High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin.

PubMed

Zhang, J; Suflita, M; Fiaschetti, C M; Li, G; Li, L; Zhang, F; Dordick, J S; Linhardt, R J

2015-01-01

One of six heparin biosynthetic enzymes, cloned and expressed in Escherichia coli as a soluble fusion protein, requires large-scale preparation for use in the chemoenzymatic synthesis of heparin, an important anticoagulant drug. The 6-O-sulfotransferase isoform-3 (6-OST-3) can be conveniently prepared at mg/L levels in the laboratory by culturing E. coli on Luria-Bertani medium in shake flasks and inducing with isopropyl β-D-1-thiogalactopyranoside at an optical density of 0·6-0·8. The production of larger amounts of 6-OST-3 required fed-batch cultivation of E. coli in a stirred tank fermenter on medium containing an inexpensive carbon source, such as glucose or glycerol. The cultivation of E. coli on various carbon sources under different feeding schedules and induction strategies was examined. Conditions were established giving yields (5-20 mg g-cell-dry weight(-1)) of active 6-OST-3 with excellent productivity (2-5 mg l(-1) h(-1)). The production of 6-OST-3 in a fed-batch fermentation on an inexpensive carbon source has been demonstrated. The ability to scale-up the production of heparin biosynthetic enzymes, such as 6-OST-3, is critical for scaling-up the chemoenzymatic synthesis of heparin. The success of this project may someday lead to a commercially viable bioengineered heparin to replace the animal-sourced anticoagulant product currently on the market. © 2014 The Society for Applied Microbiology.

Collaborative study for the calibration of replacement batches for the heparin low-molecular-mass for assay biological reference preparation.

PubMed

Terao, E; Daas, A

2016-01-01

The European Pharmacopoeia (Ph. Eur.) prescribes the control of the activity of low molecular mass heparins by assays for anti-Xa and anti-IIa activities (monograph 0828), using a reference standard calibrated in International Units (IU). An international collaborative study coded BSP133 was launched in the framework of the Biological Standardisation Programme (BSP) run under the aegis of the Council of Europe and the European Commission to calibrate replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay Biological Reference Preparation (BRP) batch 8. Thirteen official medicines control and manufacturers laboratories from European and non-European countries took part in this study to calibrate two freeze-dried candidate batches against the 3rd International Standard (IS) for heparin, low molecular weight (11/176; 3rd IS). The Heparin low-molecular-mass for assay BRP (batch 8) was also included in the test panel to check the continuity between subsequent BRP batches. Taking into account the stability data, the results of this collaborative study and on the basis of the central statistical analysis performed at the European Directorate for the Quality of Medicines & HealthCare (EDQM), the 2 candidate batches were officially adopted by the Commission of the European Pharmacopoeia as Heparin low-molecular-mass for assay BRP batches 9 and 10 with assigned anti-Xa activities of 102 and 100 IU/vial and anti-IIa activities of 34 and 33 IU/vial respectively.

Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

PubMed

Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

2017-07-01

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic core-shell heparin-PEG microparticles presented here overcome this limitation by sequestering proteins through a PEG-based shell onto a protein-protective heparin core, temporarily isolating bound proteins from the cellular microenvironment, and re-delivering proteins only after degradation of the PEG-based shell. Thus, these core-shell microparticles have potential to be a novel tool to harness and isolate proteins produced in the cellular environment and then control when proteins are re-introduced for the most effective tissue regeneration and repair. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Persistent staphylococcal bacteremia in an intravenous drug abuser.

PubMed

Barg, N L; Supena, R B; Fekety, R

1986-02-01

A patient with methicillin-resistant Staphylococcus aureus bacteremia received vancomycin (MIC = 0.8 microgram/ml, MBC = 15 micrograms/ml) and heparin simultaneously through the same intravenous line to treat a septic deep venous thrombosis. Bacteremia persisted for 7 days. Bacteremia terminated when the simultaneous infusion of heparin and vancomycin through the same line was stopped. This suggested that an interaction between vancomycin and heparin may have occurred, which resulted in a reduction in vancomycin activity. To test for such an interaction, mixtures of heparin and vancomycin in various concentrations were made and tested for antimicrobial activity against the organisms in the patient. A precipitate formed at the concentrations achieved in the intravenous lines, and when the vancomycin concentrations were measured by bioassay, a 50 to 60% reduction in activity was noted. In contrast, when these solutions were prepared and mixed at microgram concentrations, a precipitate was no longer observed, and antimicrobial activity was not reduced. Heparin appeared to interact unfavorably with vancomycin at the concentrations in the intravenous lines when these drugs were administered simultaneously to patients. This may be the cause of poor therapeutic responses to vancomycin in some patients, especially those infected with tolerant organisms.

Fondaparinux for intra and perioperative anticoagulation in patients with heparin-induced thrombocytopenia candidates for peripheral vascular surgery: Report of 4 cases.

PubMed

Illuminati, Giulio; Calio', Francesco G; Pizzardi, Giulia; Amatucci, Chiara; Masci, Federica; Palumbo, Piergaspare

2016-01-01

Intra and perioperative anticoagulation in patients with heparin induced thrombocytopenia (HIT), candidates for peripheral vascular surgery remains a challenge, as the best alternative to heparin has not yet been established. We evaluated the off-label use of fondaparinux in four patients with HIT, undergoing peripheral vascular surgery procedures. Four patients of whom 3 men of a mean age of 66 years, with proven heparin induced thrombocytopenia (HIT) underwent two axillo-femoral bypasses, one femoro-popliteal bypass and one resection of a splenic artery aneurysm under fondaparinux. No intra or perioperative bleeding or thrombosis of new onset was observed. In the absence of a valid alternative to heparin for intra and perioperative anticoagulation in HIT, several other anticoagulants can be used in an off-label setting. However, no general consensus exist on which should be the one of choice. In this small series fondaparinux appeared to be both safe and effective. These preliminary results seem to justify the off-label use of fondaparinux for intra and perioperative anticoagulation in patients with HIT, candidates for peripheral vascular surgery interventions. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

0.9% sodium chloride injection with and without heparin for maintaining peripheral indwelling intermittent-infusion devices in infants.

PubMed

Nelson, T J; Graves, S M

1998-03-15

The use of 0.9% sodium chloride injection with and without heparin sodium for maintaining peripheral indwelling intermittent-infusion devices (PIIIDs) in infants was studied. In this double-blind study, children up to one year of age who had a 24-gauge PIIID through which a continuous i.v. infusion was no longer running were randomly assigned to have their PIIID capped with 0.9% sodium chloride injection with or without heparin sodium 10 units/mL. PIIIDs were capped every eight hours if no medications were administered; otherwise, they were capped after each dose of an i.v. drug. The heparin group had 26 patients with 28 evaluable PIIIDs, and the 0.9% sodium chloride injection group had 32 patients with 46 evaluable PIIIDs. The two groups did not differ significantly in variables assessing the duration of PIIID use, reasons for removal of PIIIDs, mean number of cappings, irritant potential of administered drugs, or severity of medication-related irritation. There was no significant difference between 0.9% sodium chloride injection with and without heparin sodium 10 units/mL in maintaining 24-gauge PIIIDs in children younger than one year.

Sulfated polysaccharide from the leaves of Artemisia Princeps activates heparin cofactor II independently of the Lys173 and Arg189 residues of heparin cofactor II.

PubMed

Hayashi, T; Hayakawa, Y; Hayashi, T; Sasaki, H; Sakuragawa, N

1997-07-01

A sulfated polysaccharide (AFE-HCD) purified from the leaves of Artemisia princeps Pamp selectively accelerated the rate of thrombin inhibition by heparin cofactor II (HCII). By using plasma derived HCII and bacterial expressed recombinant HCII molecules, the interaction between each HCII molecule and AFE-HCD was analyzed. AFE-HCD accelerated thrombin inhibition by plasma derived HCII or bacterial expressed wild type HCII to the same extent (IC50: 0.056 micrograms/ml for plasma derived HCII and 0.066 micrograms/ml for recombinant HCII under the experimental condition). The recombinant HCII (rHCII) molecule with Lys173-->Leu or Arg189-->His substitution, which is defective in interactions with heparin and dermatan sulfate, respectively, is activated by AFE-HCD to inhibit thrombin in a manner similar to wild type rHCII. These results suggested that activation of HCII was independent of its Lys173 or Arg189 residue. Although AFE-HCD is a selective activator of HCII like dermatan sulfate, the amino acid residue required for the activation of HCII was distinct form that of dermatan sulfate as well as heparin.

Clinical Efficacy of Low Molecular Heparin on Unexplained Recurrent Spontaneous Abortion.

PubMed

Xu, Guang-Li; Hu, Xiao-Fang; Han, Yong-Mei; Wei, Ai-Wu

2018-06-01

To study the clinical effect of low molecular heparin on unexplained recurrent spontaneous abortion (URSA). A total of 120 URSA patients were collected in our hospital from October 2015 to September 2017. They were divided into two groups: control group (n = 60) and observation group (n = 60). The patients in the control group were administered with progesterone and human chorionic gonadotropin, and the observation group with low molecular heparin. Pregnancy outcomes, incidence of complications in pregnancy and adverse drug reactions were compared in the two groups. The pregnancy success rate of patients in the observation group (90.00%) is higher than that in the control group (68.33%) (p < 0.05). The incidence of complications in pregnancy in the observation group (90.00%) is lower than those in the control group (68.33%) (p < 0.05). The incidence of adverse drug reactions between the patients in the observation group (20.00%) and those in the control group (23.33%) showed no significant difference (p > 0.05). Low molecular heparin treatment can improve pregnancy success rate and reduce the incidence of complications in the URSA patients. Low molecular heparin is characterized by safety and reliability and has potential for application in clinic.

Low-molecular-weight heparin for thromboprophylaxis.

PubMed

Camporese, Giuseppe; Bernardi, Enrico

2009-09-01

Venous thromboembolism represents a potentially threatening complication in surgical and medical patients. Thromboprophylaxis showed a significant reduction of venous thromboembolic events, and low-molecular-weight heparins have been considered the standardized prophylactic regimen for a long time. The purpose of this review is to provide updated evidence on the use of low-molecular-weight heparins for prevention of venous thromboembolism after the publication of the latest American College of Chest Physicians Evidence-Based Clinical Practice Guidelines on antithrombotic and thrombolytic therapy. Low-molecular-weight heparins, used as comparator or investigational drug, have been investigated in several studies not included in the analysis of the latest American College of Chest Physicians Guidelines on Antithrombotic and Thrombolytic Therapy. Data gathered from studies published from December 2007 up to May 2009 dealing with surgical and medical patients have been collected and discussed. Low-molecular-weight heparins are expanding their application, but progressively they will be replaced by other new antithrombotics for the prophylaxis of venous thromboembolism. Surgical patients undergo a more concerted approach to thromboprophylaxis than medical patients. Future research should aim at improving prophylaxis in the latter setting in order to significantly reduce the rate of venous thromboembolic events.

[Effect of early intervention with heparin on function of coagulopathy, liver and kidney in rats with exertional heatstroke under the ambient air of high temperature and low humidity].

PubMed

Yu, Yang; Wei, Yuying; Zhang, Xiangrong; Li, Xinyu

2018-03-01

To explore the effects of early intervention with heparin on function of coagulopathy, liver and kidney as well as the prognosis in rats with exertional heatstroke (EHS) under the ambient air of high temperature and low humidity. 108 healthy SPF male Sprague-Dawley (SD) rats were randomly divided into normal temperature control group, EHS + normal saline (NS) group and EHS + heparin group. Of which 54 rats were collected for survival analysis (18 rats in each group), the weight change and 8-hour survival rate were observed, and Kaplan-Meier survival curves were drawn. Other 54 rats were collected for intervention experiment, the rats in each group were subdivided into 0, 1, 2 hours subgroups according to the time points of intervention with heparin after model reproduction, with 6 rats in each subgroup. The rats were placed in an artificial experiment cabin with northwest special environment, and the temperature and the relative humidity were (25.0±1.0) centigrade and (35±5)%, respectively, in normal temperature control group, and the rats were not treated in the cabin. The rats in EHS + NS group and EHS + heparin group kept running in the cabin which temperature and relative humidity were set at (43.0±0.5) centigrade and (35±5)% until the anus temperature of rats reached 43.0 centigrade, and then the rats were placed in room temperature. The rats were injected with 1 mL/kg NS or 250 U/kg heparin sodium injection through their caudal veins at 0, 1, and 2 hours, respectively, and then the blood was collected after 1.5 hours to determine the biochemical parameters including coagulation, liver and kidney as well as platelet count (PLT). (1) The weight loss of EHS + NS group and EHS + heparin group was more significant than that of normal temperature control group (g: 8.28±1.41, 8.39±1.38 vs. 2.06±1.06, both P < 0.05), but there was no significant difference between EHS + NS group and EHS + heparin group. (2) As the time went on after modeling, serum creatinine (SCr), blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), activated partial thromboplastin time (APTT), and D-dimer of EHS rats showed a tendency of increasing, but fibrinogen (FBG), antithrombin III (AT III) and PLT were decreased gradually, which were obviously abnormal as compared with those at corresponding time point of the normal temperature control group. Heparin intervention 0 hour after modeling could improve the function of liver and kidney, FBG, D-dimer, AT III and PLT, but APTT was prolonged further. The SCr, BUN, ALT, AST and CK in EHS 2 hours + heparin group were still better than EHS + NS group [SCr (μmol/L): 93.33±7.69 vs. 110.50±13.56, BUN (mmol/L): 20.55±1.35 vs. 24.88±2.41, ALT (U/L): 322.17±36.36 vs. 492.33±64.19, AST (U/L): 1 252.33±240.86 vs. 2 725.67±461.17, CK (U/L): 1 4628.67±2 784.68 vs. 2 6843.00±2 637.16, all P < 0.01], APTT was significantly prolonged (s: 51.83±6.11 vs. 33.83±4.31, P < 0.01), and AT III and PLT were significantly increased [AT III: (78.03±9.15)% vs. (64.28±12.55)%, PLT (×10 9 /L): 457.67±32.33 vs. 415.83±26.45, both P < 0.05], however, there was no obvious influence on FBG or D-dimer. (3) The rats in normal temperature control group were all survived within 8 hours, and all dead in EHS + NS group. The survival rate of rats given heparin intervention at 0, 1, 2 hours after successfully modeling was 50.0%, 33.3% and 0%, respectively. Kaplan-Meier survival curve analysis showed that 8-hour cumulative survival rate in EHS 0 hour, 1 hour + heparin groups was higher than that in EHS 0 hour, 1 hour + NS groups (χ 1 2 = 7.930, P 1 = 0.005; χ 2 2 = 4.408, P 2 = 0.036), however, there was no significant difference between EHS 2 hours + heparin group and EHS 2 hours + NS group (χ 2 = 2.660, P = 0.103). Early heparin intervention can improve the coagulation function and organ function of EHS rats, while improving the survival rate of rats, indicating the earlier intervention of heparin, the better prognosis of rats is.

Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients.

PubMed

Junqueira, Daniela R; Zorzela, Liliane M; Perini, Edson

2017-04-21

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a paradoxical immune reaction resulting in thrombin generation in vivo, which leads to a hypercoagulable state and the potential to initiate venous or arterial thrombosis. A number of factors are thought to influence the incidence of HIT including the type and preparation of heparin (unfractionated heparin (UFH) or low molecular weight heparin (LMWH)) and the heparin-exposed patient population, with the postoperative patient population at higher risk.Although LMWH has largely replaced UFH as a front-line therapy, there is evidence supporting a lack of superiority of LMWH compared with UFH regarding prevention of deep vein thrombosis and pulmonary embolism following surgery, and similar frequencies of bleeding have been described with LMWH and UFH. The decision as to which of these two preparations of heparin to use may thus be influenced by harmful effects such as HIT. We therefore sought to determine the relative impact of UFH and LMWH on HIT in postoperative patients receiving thromboembolism prophylaxis. This is an update of a review first published in 2012. The objective of this review was to compare the incidence of heparin-induced thrombocytopenia (HIT) and HIT complicated by venous thromboembolism in postoperative patients exposed to unfractionated heparin (UFH) versus low molecular weight heparin (LMWH). For this update, the Cochrane Vascular Information Specialist searched the Specialised Register (May 2016), CENTRAL (2016, Issue 4) and trials registries. The authors searched Lilacs (June 2016) and additional trials were sought from reference lists of relevant publications. We included randomised controlled trials (RCTs) in which participants were postoperative patients allocated to receive prophylaxis with UFH or LMWH, in a blinded or unblinded fashion. Studies were excluded if they did not use the accepted definition of HIT. This was defined as a relative reduction in the platelet count of 50% or greater from the postoperative peak (even if the platelet count at its lowest remained greater than 150 x 10 9 /L) occurring within five to 14 days after the surgery, with or without a thrombotic event occurring in this timeframe. Additionally, we required circulating antibodies associated with the syndrome to have been investigated through laboratory assays. Two review authors independently extracted data and assessed the risk of bias. Disagreements were resolved by consensus with participation of a third author. In this update, we included three trials involving 1398 postoperative participants. Participants were submitted to general surgical procedures, minor and major, and the minimum mean age was 49 years. Pooled analysis showed a significant reduction in the risk of HIT with LMWH compared with UFH (risk ratio (RR) 0.23, 95% confidence interval (CI) 0.07 to 0.73); low-quality evidence. The number needed to treat for an additional beneficial outcome (NNTB) was 59. The risk of HIT was consistently reduced comparing participants undergoing major surgical procedures exposed to LMWH or UFH (RR 0.22, 95% CI 0.06 to 0.75); low-quality evidence. The occurrence of HIT complicated by venous thromboembolism was significantly lower in participants receiving LMWH compared with UFH (RR 0.22, 95% CI 0.06 to 0.84); low-quality evidence. The NNTB was 75. Arterial thrombosis occurred in only one participant who received UFH. There were no amputations or deaths documented. Although limited evidence is available, it appears that HIT induced by both types of heparins is common in people undergoing major surgical procedures (incidence greater than 1% and less than 10%). This updated review demonstrated low-quality evidence of a lower incidence of HIT, and HIT complicated by venous thromboembolism, in postoperative patients undergoing thromboprophylaxis with LMWH compared with UFH. Similarily, the risk of HIT in people undergoing major surgical procedures was lower when treated with LMWH compared to UFH (low-quality evidence). The quality of the evidence was downgraded due to concerns about the risk of bias in the included studies and imprecision of the study results. These findings may support current clinical use of LMWH over UFH as front-line heparin therapy. However, our conclusions are limited and there was an unexpected paucity of RCTs including HIT as an outcome. To address the scarcity of clinically-relevant information on HIT, HIT must be included as a core harmful outcome in future RCTs of heparin.

In vivo bioactivity of rhBMP-2 delivered with novel polyelectrolyte complexation shells assembled on an alginate microbead core template.

PubMed

Abbah, Sunny-Akogwu; Liu, Jing; Lam, Raymond W M; Goh, James C H; Wong, Hee-Kit

2012-09-10

Electrostatic interactions between polycations and polyanions are being explored to fabricate polyelectrolyte complexes (PEC) that could entrap and regulate the release of a wide range of biomolecules. Here, we report the in vivo application of PEC shells fabricated from three different polycations: poly-l-ornithine (PLO), poly-l-arginine (PLA) and DEAE-dextran (DEAE-D) to condense heparin on the surface of alginate microbeads and further control the delivery of recombinant human bone morphogenetic protein 2 (rhBMP-2) in spinal fusion application. We observed large differences in the behavior of PEC shells fabricated from the cationic polyamino acids (PLO and PLA) when compared to the cationic polysaccharide, DEAE-D. Whereas DEAE-D-based PEC shells eroded and released rhBMP-2 over 2 days in vitro, PLO- and PLA-based shells retained at least 60% of loaded rhBMP-2 after 3 weeks of incubation in phosphate-buffered saline. In vivo implantation in a rat model of posterolateral spinal fusion revealed robust bone formation in the PLO- and PLA-based PEC shell groups. This resulted in a significantly enhanced mechanical stability of the fused segments. However, bone induction and biomechanical stability of spine segments implanted with DEAE-D-based carriers were significantly inferior to both PLO- and PLA-based PEC shell groups (p<0.01). From these results, we conclude that PEC shells incorporating native heparin could be used for growth factor delivery in functional bone tissue engineering application and that PLA- and PLO-based complexes could represent superior options to DEAE-D for loading and in vivo delivery of bioactive BMP-2 in this approach. Copyright © 2012 Elsevier B.V. All rights reserved.

The guanidinium group as a key part of water-soluble polymer carriers for siRNA complexation and protection against degradation.

PubMed

Tabujew, Ilja; Freidel, Christoph; Krieg, Bettina; Helm, Mark; Koynov, Kaloian; Müllen, Klaus; Peneva, Kalina

2014-07-01

Here, the preparation of a novel block copolymer consisting of a statistical copolymer N-(2-hydroxypropyl) methacrylamide-s-N-(3-aminopropyl) methacrylamide and a short terminal 3-guanidinopropyl methacrylamide block is reported. This polymer structure forms neutral but water-soluble nanosized complexes with siRNA. The siRNA block copolymer complexes are first analyzed using agarose gel electrophoresis and their size is determined with fluorescence correlation spectroscopy. The protective properties of the polymer against RNA degradation are investigated by treating the siRNA block copolymer complexes with RNase V1. Heparin competition assays confirm the efficient release of the cargo in vitro. In addition, the utilization of microscale thermophoresis is demonstrated for the determination of the binding strength between a fluorescently labeled polyanion and a polymer molecule. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Spurious hypocalcemia in hemodialysis patients after heparinization. In-vitro formation of calcium soaps.

PubMed

Godolphin, W; Cameron, E C; Frohlich, J; Price, J D

1979-02-01

Patients on long-term hemodialysis via arteriovenous fistula received heparin when the fistula needle was inserted, before a sample of blood was obtained for chemical analysis. The resultant release of lipoprotein lipase activity in vivo and continued lipolytic activity in vitro sometimes produced sufficient free fatty acid to precipitate calcium soaps. The consequent spurious hypocalcemia was most frequently observed when the patients had chylomicronemia. This cause of apparent hypocalcemia was eliminated either by immediate analyses of the blood samples or by obtaining samples before systemic heparinization.

Ethanol combined with heparin as a locking solution for the prevention of catheter related blood stream infections in hemodialysis patients: A prospective randomized study.

PubMed

Sofroniadou, Sofia; Revela, Ioanna; Kouloubinis, Alexandros; Makriniotou, Ioanna; Zerbala, Sinodi; Smirloglou, Despina; Kalocheretis, Petros; Drouzas, Apostolos; Samonis, George; Iatrou, Christos

2017-10-01

Ethanol lock solution has been mainly administered in paediatric and home parenteral nutrition patients in order to prevent catheter related blood stream infections (CRBSI). Its utility in hemodialysis (HD) patients with non-tunneled-uncuffed catheter (NTC) has been poorly explored. We conducted a prospective randomized study in chronic HD patients requiring a newly inserted NTC-while awaiting for the maturation of an already established arteriovenous fistula (AVF) or arteriovenous graft (AVG) or tunneled-cuffed catheter insertion. Patients were randomized in two groups: Group A, where the lock solution was ethanol 70% + unfractionated heparin 2000 U/mL and group B, that received only unfractionated heparin 2000 U/mL. Primary end point was CRBSIs whereas exit site infections, thrombotic and bleeding episodes were the secondary end points. One hundred three HD patients were enrolled in the study (group A, n = 52; group B, n = 51). The median number of catheter days was 32 for group A (range: 23-39) and 34 (range: 27-40) for group B with no statistically significant difference between the two groups. Group A (ethanol + heparin) demonstrated 4/52 episodes (7.69%) of CRBSI whereas Group B (heparin) 11/51 episodes (21.57%) (P = 0.04). CRBSI rates per 1000 catheter days were 2.53/1000 catheter days for group A and 6.7/1000 catheter days for group B (P = 0.04). Mean cumulative infection-free catheter survival in the ethanol group did not differ significantly compared to the heparin group (log-rank test = 2.99, P = 0.08). Thrombotic episodes did not differ between the two groups. Locking of NTCs in HD patients with ethanol 70% + unfractionated heparin reduces CRBSI rates without increasing the thrombotic episodes. © 2017 International Society for Hemodialysis.

Bivalirudin therapy is associated with improved clinical and economic outcomes in ST-elevation myocardial infarction patients undergoing percutaneous coronary intervention: results from an observational database.

PubMed

Pinto, Duane S; Ogbonnaya, Augustina; Sherman, Steven A; Tung, Patricia; Normand, Sharon-Lise T

2012-01-01

Randomized trials show improved outcomes among acute coronary syndrome patients treated with bivalirudin. The objective of this analysis was to compare clinical and economic outcomes in ST-elevation myocardial infarction (STEMI) patients encountered in routine clinical practice undergoing primary percutaneous coronary intervention (PPCI), treated with bivalirudin or heparin+GP IIb/IIIa receptor inhibitor (heparin+GPI). STEMI admissions from January 1, 2004 through March 31, 2008 among patients receiving PPCI and bivalirudin or heparin+GPI in the Premier hospital database were identified. The probability of receiving bivalirudin was estimated using individual and hospital variables; using propensity scores, each bivalirudin patient was matched to 3 heparin+GPI treated patients. The primary outcome was in-hospital death. Rates of bleeding, transfusion, length of stay, and in-hospital cost were secondary outcomes. There were 59,917 STEMI PPCIs receiving bivalirudin (n=6735) or heparin+GPI (n=53,182). Seventy-nine percent of bivalirudin patients matched, resulting in 21,316 STEMI PPCIs for analysis. Compared with heparin+GPI patients, bivalirudin patients had fewer deaths (3.2% versus 4.0%; P=0.011) and less inpatient bleeding (clinically apparent bleeding [6.9% versus 10.5%, P<0.0001], clinically apparent bleeding with transfusion [1.6% versus 3.0%, P<0.0001], and transfusion [5.9% versus 7.6%, P<0.0001]). Patients receiving bivalirudin had shorter average length of stay (mean 4.3 versus 4.5 days; P<0.0001), with lower in-hospital cost (mean $18,640 versus $19,967 [median $14,462 versus $16,003], P<0.0001). This large "real-world" retrospective analysis demonstrates that bivalirudin therapy compared with heparin+GPI is associated with a lower rate of inpatient death, inpatient bleeding, and decreased overall in-hospital cost in STEMI patients undergoing PPCI.

Angiogenic Factor Profiles in Pregnant Women With a History of Early-Onset Severe Preeclampsia Receiving Low-Molecular-Weight Heparin Prophylaxis.

PubMed

Lecarpentier, Edouard; Gris, Jean Christophe; Cochery-Nouvellon, Eva; Mercier, Erick; Touboul, Cyril; Thadhani, Ravi; Karumanchi, S Ananth; Haddad, Bassam

2018-01-01

To evaluate whether daily low-molecular-weight (LMW) heparin prophylaxis during pregnancy alters profile of circulating angiogenic factors that have been linked with the pathogenesis of preeclampsia and fetal growth restriction. This is a planned ancillary study of the Heparin-Preeclampsia trial, a randomized trial in pregnant women with a history of severe early-onset preeclampsia (less than 34 weeks of gestation). In the parent study, all women were treated with aspirin and then randomized to receive LMW heparin or aspirin alone. In this study, we measured serum levels of circulating angiogenic factors (soluble fms-like tyrosine kinase-1, placental growth factor, and soluble endoglin by immunoassay) at the following gestational windows: 10-13 6/7 weeks, 14-17 6/7 weeks, 18-21 6/7 weeks, 22-25 6/7 weeks, 26-29 6/7 weeks, 30-33 6/7 weeks, and 34-37 6/7 weeks. Samples were available from 185 patients: LMW heparin+aspirin (n=92) and aspirin alone (n=93). The two groups had comparable baseline characteristics and had similar adverse composite outcomes (35/92 [38.0%] compared with 36/93 [38.7%]; P=.92). There were no significant differences in serum levels of soluble fms-like tyrosine kinase-1, placental growth factor, and soluble endoglin in the participants who received LMW heparin and aspirin compared with those who received aspirin alone regardless of gestational age period. Finally, women who developed an adverse composite outcome at less than 34 weeks of gestation demonstrated significant alterations in serum angiogenic profile as early as 10-13 6/7 weeks that was most dramatic 6-8 weeks preceding delivery. Prophylactic LMW heparin therapy when beginning from before 14 weeks of gestation with aspirin during pregnancy is not associated with an improved angiogenic profile. This may provide a molecular explanation for the lack of clinical benefit noted in recent trials. ClinicalTrials.gov, NCT00986765.

Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering.

PubMed

Zhang, Kuihua; Huang, Dianwu; Yan, Zhiyong; Wang, Chunyang

2017-07-01

Biomimicing topological structure of natural nerve tissue to direct axon growth and controlling sustained release of moderate neurotrophic factors are extremely propitious to the functional recovery of damaged nervous systems. In this study, the heparin/collagen encapsulating nerve growth factor (NGF) multilayers were coated onto the aligned poly-L-lactide (PLLA) nanofibrous scaffolds via a layer-by-layer (LbL) self-assembly technique to combine biomolecular signals, and physical guidance cues for peripheral nerve regeneration. Scanning electronic microscopy (SEM) revealed that the surface of aligned PLLA nanofibrous scaffolds coated with heparin/collagen multilayers became rougher and appeared some net-like filaments and protuberances in comparison with PLLA nanofibrous scaffolds. The heparin/collagen multilayers did not destroy the alignment of nanofibers. X-ray photoelectron spectroscopy and water contact angles displayed that heparin and collagen were successfully coated onto the aligned PLLA nanofibrous scaffolds and improved its hydrophilicity. Three-dimensional (3 D) confocal microscopy images further demonstrated that collagen, heparin, and NGF were not only coated onto the surface of aligned PLLA nanofibrous scaffolds but also permeated into the inner of scaffolds. Moreover, NGF presented a sustained release for 2 weeks from aligned nanofibrous scaffolds coated with 5.5 bilayers or above and remained good bioactivity. The heparin/collagen encapsulating NGF multilayers coated aligned nanofibrous scaffolds, in particular 5.5 bilayers or above, was more beneficial to Schwann cells (SCs) proliferation and PC12 cells differentiation as well as the SC cytoskeleton and neurite growth along the direction of nanofibrous alignment compared to the aligned PLLA nanofibrous scaffolds. This novel scaffolds combining sustained release of bioactive NGF and aligned nanofibrous topography presented an excellent potential in peripheral nerve regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1900-1910, 2017. © 2017 Wiley Periodicals, Inc.

Heparin-Binding Protein Measurement Improves the Prediction of Severe Infection With Organ Dysfunction in the Emergency Department

PubMed Central

Arnold, Ryan; Boyd, John H.; Zindovic, Marko; Zindovic, Igor; Lange, Anna; Paulsson, Magnus; Nyberg, Patrik; Russell, James A.; Pritchard, David; Christensson, Bertil; Åkesson, Per

2015-01-01

Objectives: Early identification of patients with infection and at risk of developing severe disease with organ dysfunction remains a difficult challenge. We aimed to evaluate and validate the heparin-binding protein, a neutrophil-derived mediator of vascular leakage, as a prognostic biomarker for risk of progression to severe sepsis with circulatory failure in a multicenter setting. Design: A prospective international multicenter cohort study. Setting: Seven different emergency departments in Sweden, Canada, and the United States. Patients: Adult patients with a suspected infection and at least one of three clinical systemic inflammatory response syndrome criteria (excluding leukocyte count). Intervention: None. Measurements and Main Results: Plasma levels of heparin-binding protein, procalcitonin, C-reactive protein, lactate, and leukocyte count were determined at admission and 12–24 hours after admission in 759 emergency department patients with suspected infection. Patients were defined depending on the presence of infection and organ dysfunction. Plasma samples from 104 emergency department patients with suspected sepsis collected at an independent center were used to validate the results. Of the 674 patients diagnosed with an infection, 487 did not have organ dysfunction at enrollment. Of these 487 patients, 141 (29%) developed organ dysfunction within the 72-hour study period; 78.0% of the latter patients had an elevated plasma heparin-binding protein level (> 30 ng/mL) prior to development of organ dysfunction (median, 10.5 hr). Compared with other biomarkers, heparin-binding protein was the best predictor of progression to organ dysfunction (area under the receiver operating characteristic curve = 0.80). The performance of heparin-binding protein was confirmed in the validation cohort. Conclusion: In patients presenting at the emergency department, heparin-binding protein is an early indicator of infection-related organ dysfunction and a strong predictor of disease progression to severe sepsis within 72 hours. PMID:26468696

Molecular dynamics simulations on networks of heparin and collagen.

PubMed

Kulke, Martin; Geist, Norman; Friedrichs, Wenke; Langel, Walter

2017-06-01

Synthetic scaffolds containing collagen (Type I) are of increasing interest for bone tissue engineering, especially for highly porous biomaterials in combination with glycosaminoglycans. In experiments the integration of heparin during the fibrillogenesis resulted in different types of collagen fibrils, but models for this aggregation on a molecular scale were only tentative. We conducted molecular dynamic simulations investigating the binding of heparin to collagen and the influence of the telopeptides during collagen aggregation. This aims at explaining experimental findings on a molecular level. Novel structures for N- and C-telopeptides were developed with the TIGER2 replica exchange algorithm and dihedral principle component analysis. We present an extended statistical analysis of the mainly electrostatic interaction between heparin and collagen and identify several binding sites. Finally, we propose a molecular mechanism for the influence of glycosaminoglycans on the morphology of collagen fibrils. Proteins 2017; 85:1119-1130. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Venous thrombosis after abdominal surgery. A comparison between subcutaneous heparin and antithrombotic stockings, or both

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rasmussen, A.; Hansen, P.T.; Lindholt, J.

1988-01-01

In an open controlled study, 248 consecutive patients (age more than 40 yrs) admitted for major abdominal surgery were randomized to one of three prophylactic antithrombotic treatments. Eighty-five patients received subcutaneous heparin, 74 patients had graduated compression stockings to the knee (TED stockings), and 89 patients had both subcutaneous heparin and stockings. Treatment began on the evening before operation and continued to complete mobilization, or for not less than five days postoperatively. On the fourth or fifth postoperative day, the patients underwent a /sup 99m/Tc-plasmin test of the lower limbs as a test for deep vein thrombosis. There were 29.7%more » positive tests in the stocking group, 29.4% in the group with heparin prophylaxis, and 25.8% in the combined group. Differences between treatments were not statistically significant.« less

Effects of oversulfated and fucosylated chondroitin sulfates on coagulation. Challenges for the study of anticoagulant polysaccharides.

PubMed

Fonseca, Roberto J C; Oliveira, Stephan-Nicollas M C G; Pomin, Vitor H; Mecawi, André S; Araujo, Iracema G; Mourão, Paulo A S

2010-05-01

We report the effects of a chemically oversulfated chondroitin sulfate and a naturally fucosylated chondroitin sulfate on the coagulation system. The former has been recently identified as a contaminant of heparin preparations and the latter has been proposed as an alternative anticoagulant. The mechanism of action of these polymers on coagulation is complex and target different components of the coagulation system. They have serpin-independent anticoagulant activity, which preponderates in plasma. They also have serpin-dependent anticoagulant activity but differ significantly in the target coagulation protease and preferential serpin. Their anticoagulant effects differ even more markedly when tested as inhibitors of coagulation proteases using plasma as a source of serpins. It is possible that the difference is due to the high availability of fucosylated chondroitin sulfate whereas oversulfated chondroitin sulfate has strong unspecific binding to plasma protein and low availability for the binding to serpins. When tested using a venous thrombosis experimental model, oversulfated chondroitin sulfate is less potent as an antithrombotic agent than fucosylated chondroitin sulfate. These highly sulfated chondroitin sulfates activate factor XII in in vitro assays, based on kallikrein release. However, only fucosylated chondroitin sulfate induces hypotension when intravenously injected into rats. In conclusion, the complexity of the regulatory mechanisms involved in the action of highly sulfated polysaccharides in coagulation requires their analysis by a combination of in vitro and in vivo assays. Our results are relevant due to the urgent need for new anticoagulant drugs or alternative sources of heparin.

Coagulation activation by MC28 fibrosarcoma cells facilitates lung tumor formation.

PubMed

Amirkhosravi, M; Francis, J L

1995-01-01

Tumor cells interact with the hemostatic system in various ways and may thus influence malignant growth and spread. MC28 fibrosarcoma cells possess a potent procoagulant activity (PCA) and form lung tumors following intravenous injection. The aim of this work was to study the relationship between PCA, intravascular coagulation and lung seeding in the MC28 model. MC28 cells were injected into control, warfarinized and heparinized hooded Lister rats. Coagulation changes were monitored by thromboelastography (TEG) and Sonoclot analysis (SA), lung fibrin formation by light and electron microscopy, tumor seeding by macroscopic counting and tumor cell and platelet deposition in the lungs by radiolabelling. PCA was measured by chromogenic assay. MC28 PCA was characterized as a tissue factor-factor VIIa complex that probably arose during cell culture or disaggregation of solid tumors. Injection of tumor cells caused marked coagulopathy and was rapidly (within 30 min) followed by fibrin deposition in the lungs and accumulation of radiolabelled platelets. Heparin and warfarin significantly reduced lung seeding (p < 0.001) and reduced retention of radiolabelled tumor cells in the pulmonary circulation (p < 0.01). Inhibition of cellular PCA by prior treatment with concanavalin A markedly reduced intravascular coagulation and lung seeding. We conclude that MC28 cells cause intravascular coagulation as a direct result of their procoagulant activity. The data suggest that tumor cells form complexes with platelets and fibrin which are retained in the lungs long enough for extravasation and seeding to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of replacement batches for heparin low-molecular-mass for calibration CRS, and the International Standard Low Molecular Weight Heparin for Calibration.

PubMed

Mulloy, B; Heath, A; Behr-Gross, M-E

2007-12-01

An international collaborative study involving fourteen laboratories has taken place, organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) with National Institute for Biological Standards & Control (NIBSC) (in its capacity as a World Health Organisation (WHO) Laboratory for Biological Standardisation) to provide supporting data for the establishment of replacement batches of Heparin Low-Molecular-Mass (LMM) for Calibration Chemical Reference Substance (CRS), and of the International Reference Reagent (IRR) Low Molecular Weight Heparin for Molecular Weight Calibration. A batch of low-molecular-mass heparin was donated to the organisers and candidate preparations of freeze-dried heparin were produced at NIBSC and EDQM. The establishment study was organised in two phases: a prequalification (phase 1, performed in 3 laboratories in 2005) followed by an international collaborative study (phase 2). In phase 2, started in March 2006, molecular mass parameters were determined for seven different LMM heparin samples using the current CRS batch and two batches of candidate replacement material with a defined number average relative molecular mass (Mn) of 3,700, determined in phase 1. The values calculated using the candidates as standard were systematically different from values calculated using the current batch with its assigned number-average molecular mass (Mna) of 3,700. Using raw data supplied by participants, molecular mass parameters were recalculated using the candidates as standard with values for Mna of 3,800 and 3,900. Values for these parameters agreed more closely with those calculated using the current batch supporting the fact that the candidates, though similar to batch 1 in view of the production processes used, differ slightly in terms of molecular mass distribution. Therefore establishment of the candidates was recommended with an assigned Mna value of 3,800 that is both consistent with phase 1 results and guarantees continuity with the current CRS batch. In phase 2, participants also determined molecular weight parameters for the seven different LMM heparin samples using both the 1st IRR (90/686) and its Broad Standard Table and the candidate World Health Organization (WHO) 2nd International Standard (05/112) (2nd IS) using a Broad Standard Table established in phase 1. Mean molecular weights calculated using 2nd IS were slightly higher than with 1st IRR, and participants in the study indicated that this systematic difference precluded establishment of 2nd IS with the table supplied. A replacement Broad Standard Table has been devised on the basis of the central recalculations of raw data supplied by participants; this table gives improved agreement between values derived using the 1st IRR and the candidate 2nd IS. On the basis of this study a recommendation was made for the establishment of 2nd IS and its proposed Broad Standard Table as a replacement for the 1st International Reference Reagent Low Molecular Weight Heparin for Molecular Weight Calibration. Unlike the 1st IRR however, the candidate material 2nd IS is not suitable for use with the method of Nielsen. The candidate materials were established as heparin low-molecular-mass for calibration batches 2 and 3 by the Ph. Eur. Commission in March 2007 and as 2nd IS low-molecular-weight heparin for molecular weight calibration (05/112) by the Expert Committee on Biological Standardization in November 2007.

Heparin-benzyl alcohol enhancement of biofilms formation and antifungal susceptibility of vaginal Candida species isolated from pregnant and nonpregnant Saudi women

PubMed Central

Al-akeel, Raid A; El-kersh, Talat A; Al-Sheikh, Yazeed A; Al-Ahmadey, Ziab Zakey

2013-01-01

Biofilm formation by Candida species is a major contribute to their pathogenic potential.The aim of this study was to determine in vitro effects of EDTA, cycloheximide, and heparin-benzyl alcohol preservative on C. albicans (126) and non-albicans (31)vaginal yeast isolates biofilm formations and their susceptibility against three antifungal Etest strips. Results of the crystal violet-assay, indicated that biofilms formation were most commonly observed [100%] for C. kefyr, C. utilis, C. famata, and Rhodotorula mucilaginosa, followed by C. glabrata [70%], C. tropicalis [50%], C. albicans [29%], Saccharomyces cerevisiae [0.0%]. EDTA (0.3mg/ml) significantly inhibited biofilm formation in both C. albicans and non-albicans isolates (P=0.0001) presumably due to chelation of necessary metal cations for the process-completion. In contrast, heparin (-benzyl alcohol preservative) stimulated biofilm formation in all tested isolates, but not at significant level (P=0.567). Conversely, cycloheximide significantly (P=0.0001) inhibited biofilm formation in all C. albicans strains(126) and its effect was even 3 fold more pronounced than EDTA inhibition, probably due to its attenuation of proteins (enzymes) and/or complex molecules necessary for biofilm formation. Results also showed that all nonalbicans yeasts isolates were susceptible to 5-flucytosine (MIC50, 0.016 µg/ml; MIC90, 0.064 µg/ml), but 14% of C. albicans isolates were resistant (MIC50, 0.064 µg/ml; MIC90 >32 µg/ml). The MIC50 value of amphotricin B for all C. albicans and non-albicans isolates was at a narrow range of 0.023 µg /ml, and the MIC90 values were 0.047 µg/ml and 0.064 µg/ml respectively, thereby confirming its efficacy as a first line empiric- treatment of Candida spp infections. PMID:23750080

Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.

PubMed

Maun, Henry R; Liu, Peter S; Franke, Yvonne; Eigenbrot, Charles; Forrest, William F; Schwartz, Lawrence B; Lazarus, Robert A

2018-04-16

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.

The antiviral potency of Fagus sylvatica 4OMe-glucuronoxylan sulfates.

PubMed

Pujol, C A; Damonte, E B; Turjan, J; Yanbo, K Z; Capek, P

2016-06-01

Herpes simplex virus belongs to Herpesviridae family and causes infection of humans from ancient times. 4OMe-glucuronoxylans as the renewable biopolymers can be promising glycomaterials for various applications in pharmacy. Control enzymatic degradation of the native 4OMe-glucuronoxylan (GX1) followed by targeted sulfation procedure afforded a range of 4OMe-glucuronoxylan sulfates differed in the degree of sulfation (10-16%) and molecular mass (21,000-5000g/mol; GXS1>GXS2>GXS3>GXS4). Antiviral activity tests on GXS1-4 against herpes simplex virus (HSV) types 1 and 2 revealed the positive effect of all compounds against strains of herpes virus. Of them, the compounds GXS1 and GXS4 were shown to be the most active for both HSV serotypes. The antiviral activity of GXS1 and GXS4 was similar to those of heparin or dextran sulfate, used as reference compounds. It was found that GXS1 and GXS4 were active as well against Polio and dengue viruses, however, on a smaller scale. The mode of antiviral action of 4OMe-glucuronoxylan sulfates is due to inhibition of the virus binding to the cell receptors. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessment of Heparin Anticoagulation Measured Using i-STAT and Hemochron Activated Clotting Time.

PubMed

Maslow, Andrew; Chambers, Alison; Cheves, Tracey; Sweeney, Joseph

2018-01-31

Adequate anticoagulation, measured using activated clotting time (ACT), is important during vascular and cardiac surgeries. Unfractionated heparin is the most common anticoagulant used. The purpose of this analysis was to compare the i-STAT ACT (iACT) to the Hemochron ACT (hACT), both of which were then compared to anti-factor Xa (anti-Xa) assay, a representation of heparin level and activity. Prospective study. Tertiary care cardiovascular center. Eleven consecutive elective adult cardiac surgical patients. Prior to cardiopulmonary bypass, ACTs were measured using i-STAT and Hemochron technologies and compared to each other and to anti-Xa assay prior to and during a cumulative administration of heparin. Data were compared using bias analyses. Heparin (300 U/kg) was administered in quarterly doses. Coagulation labs were collected prior to and 3 minutes after each quarterly dose of heparin. The baseline ACTs for i-STAT and Hemochron were 147 and 142 seconds, respectively. A significant association was found between iACT and hACT (p = 0.002). The iACT measurements underestimated hACT at ACT levels >180 seconds or anti-Xa levels >0.75 U/mL. No significant difference was found between ACT data at anti-Xa levels <0.5 U/mL. There was a good association between the iACT and hACT; however, the 2 tests are not equivalent. Overall, the iACT underestimated the hACT. Agreement between the ACT technologies was good at lower ACTs and anti-Xa levels, but declined with an anti-Xa >0.75 U/mL. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of the Becton-Dickinson rapid serum tube: does it provide a suitable alternative to lithium heparin plasma tubes?

PubMed

Dimeski, Goce; Masci, Paul P; Trabi, Manuela; Lavin, Martin F; de Jersey, John

2010-05-01

Obtaining a suitable specimen for analysis in a timely manner is pivotal in clinical chemistry service provision. Serum is recognized as the preferred specimen for most assays, but because of time constraints for completion of clotting and an increasing number of patients on anti-coagulant therapy, latent clotting or no clotting is an outcome which can lead to errors and delay in delivery of critical results. Although lithium heparin plasma has unique problems, it has become an alternative in hospital-based laboratories. The Becton-Dickinson (BD) rapid serum tube (RST) was evaluated in a hospital environment using a total of 53 participants, both healthy and anticoagulated, for 31 analytes against BD PST II and BD SST II tubes measured with Beckman DxC800 and DxI800 analyzers. Most results from the RST tube were comparable with those from the SST II tube. Potassium results were closer to the PST II plasma concentrations. Incomplete and latent clotting was encountered in the RST specimens from participants (cardiac and dialysis) who had received a total of >7000 units of heparin [activated partial thromboplastin time (APTT) >150 s], warfarin/heparin combination, and specimens from cardiac surgery patients who had received a total of >25,000 units of heparin (APTT >200 s) at the time of collection of specimens. The RST tube provides a suitable alternative to lithium heparin plasma tubes for most patients in a hospital environment. However, latent clotting continued to occur in specimens collected from participants who had received high concentrations of anticoagulants.

Heparin-Induced Thrombocytopenia in Contemporary Cardiac Surgical Practice and Experience With a Protocol for Early Identification.

PubMed

Sun, Xiumei; Hill, Peter C; Taylor-PaneK, Sharon L; Corso, Paul J; Lindsay, Joseph

2016-01-15

This analysis was designed to (1) examine the impact of heparin-induced thrombocytopenia (HIT) on contemporary cardiac surgical practice and (2) describe the results of a protocol designed for early identification of the presence of the immune mechanisms involved. Consecutive patients who underwent cardiac surgery were screened postoperatively for thrombocytopenia. Patients with thrombocytopenia were tested for antiplatelet factor 4 (PF4)/heparin antibodies by ELISA and clinical evidence of thrombosis sought. Demographics, co-morbidities, operative details, and outcomes were abstracted from the departmental registry. Of 14,415 consecutive patients undergoing cardiac surgery, 1,849 patients (13%) had thrombocytopenia. Of them, 277 patients (15%) had PF4/heparin antibodies and 76 patients (4%) had both antibodies and clinical thrombosis. Antibodies were more frequent: (1) in women (p = 0.01), (2) in patients with an increased body mass index (p <0.01), and (3) in patients with clinical heart failure before surgery (p <0.01). Thirty-day mortality was greatest among the 76 patients with the triad of thrombocytopenia, antibodies, and clinical thrombosis (30%). Of the 1,849 patients with thrombocytopenia, the presence of PF4/heparin antibodies was an independent predictor of 30-day mortality (odds ratio 2.09, 95% CI 1.46 to 2.49; p <0.001). HIT remains an infrequent but very serious complication of heparin therapy in contemporary cardiac surgical practice. The possibility that the presence of HIT antibodies in patients with thrombocytopenia independently increases operative mortality deserves further study. Copyright © 2016 Elsevier Inc. All rights reserved.

Treatment and Prevention of Heparin-Induced Thrombocytopenia

PubMed Central

Dans, Antonio L.; Moores, Lisa K.; Bona, Robert; Davidson, Bruce L.; Schulman, Sam; Crowther, Mark

2012-01-01

Background: Heparin-induced thrombocytopenia (HIT) is an antibody-mediated adverse drug reaction that can lead to devastating thromboembolic complications, including pulmonary embolism, ischemic limb necrosis necessitating limb amputation, acute myocardial infarction, and stroke. Methods: The methods of this guideline follow the Methodology for the Development of Antithrombotic Therapy and Prevention of Thrombosis Guidelines: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines in this supplement. Results: Among the key recommendations for this article are the following: For patients receiving heparin in whom clinicians consider the risk of HIT to be > 1%, we suggest that platelet count monitoring be performed every 2 or 3 days from day 4 to day 14 (or until heparin is stopped, whichever occurs first) (Grade 2C). For patients receiving heparin in whom clinicians consider the risk of HIT to be < 1%, we suggest that platelet counts not be monitored (Grade 2C). In patients with HIT with thrombosis (HITT) or isolated HIT who have normal renal function, we suggest the use of argatroban or lepirudin or danaparoid over other nonheparin anticoagulants (Grade 2C). In patients with HITT and renal insufficiency, we suggest the use of argatroban over other nonheparin anticoagulants (Grade 2C). In patients with acute HIT or subacute HIT who require urgent cardiac surgery, we suggest the use of bivalirudin over other nonheparin anticoagulants or heparin plus antiplatelet agents (Grade 2C). Conclusions: Further studies evaluating the role of fondaparinux and the new oral anticoagulants in the treatment of HIT are needed. PMID:22315270

Collaborative study for the establishment of replacement batches of heparin low- molecular-mass for assay biological reference preparations.

PubMed

Terao, E; Daas, A; Rautmann, G; Buchheit, K-H

2010-10-01

A collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM) in the context of the Biological Standardisation Programme (BSP), under the aegis of the Council of Europe and the European Commission, to establish replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay European Pharmacopoeia Biological Reference Preparation (BRP). The replacement batches of BRP are intended to be used in the assays for anti-Xa and anti-IIa activities, as described in the European Pharmacopoeia (Ph. Eur.) monograph Heparins, low-molecular-mass (0828). Three freeze-dried candidate batches were calibrated against the current International Standard (IS) for Heparin, lowmolecular- weight (2nd IS, 01/608). For the purpose of the continuity check between subsequent BRP batches, the current Heparin low-molecular-mass for assay BRP (batch 5) was also included in the test panel. Thirteen official medicines control and manufacturers laboratories from European and non-European countries contributed data. A central statistical analysis of the datasets was performed at the EDQM. On the basis of the results, the 3 candidate materials were assigned a potency of 104 IU/vial for the anti-Xa activity and 31 IU/vial for the anti-IIa activity. Taken into account the preliminary stability data and the results of this collaborative study, the 3 batches of candidate BRP were adopted in June 2010 by the Commission of the Ph. Eur. as Heparin low-molecular-mass for assay BRP batches 6, 7 and 8.

Novel method for the determination of average molecular weight of natural polymers based on 2D DOSY NMR and chemometrics: Example of heparin.

PubMed

Monakhova, Yulia B; Diehl, Bernd W K; Do, Tung X; Schulze, Margit; Witzleben, Steffen

2018-02-05

Apart from the characterization of impurities, the full characterization of heparin and low molecular weight heparin (LMWH) also requires the determination of average molecular weight, which is closely related to the pharmaceutical properties of anticoagulant drugs. To determine average molecular weight of these animal-derived polymer products, partial least squares regression (PLS) was utilized for modelling of diffused-ordered spectroscopy NMR data (DOSY) of a representative set of heparin (n=32) and LMWH (n=30) samples. The same sets of samples were measured by gel permeation chromatography (GPC) to obtain reference data. The application of PLS to the data led to calibration models with root mean square error of prediction of 498Da and 179Da for heparin and LMWH, respectively. The average coefficients of variation (CVs) did not exceed 2.1% excluding sample preparation (by successive measuring one solution, n=5) and 2.5% including sample preparation (by preparing and analyzing separate samples, n=5). An advantage of the method is that the sample after standard 1D NMR characterization can be used for the molecular weight determination without further manipulation. The accuracy of multivariate models is better than the previous results for other matrices employing internal standards. Therefore, DOSY experiment is recommended to be employed for the calculation of molecular weight of heparin products as a complementary measurement to standard 1D NMR quality control. The method can be easily transferred to other matrices as well. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhaled nebulised unfractionated heparin improves lung function in moderate to very severe COPD: A pilot study.

PubMed

Shute, Janis K; Calzetta, Luigino; Cardaci, Vittorio; di Toro, Stefania; Page, Clive P; Cazzola, Mario

2018-02-01

COPD is an inflammatory airway disease characterised by progressive airflow limitation and air trapping, leading to lung hyperinflation and exercise limitation. Acute worsening of symptoms, including dyspnea, cough and sputum production, occurs during exacerbations which are associated with significantly reduced health related quality of life, and increased morbidity and mortality. Chronic bronchial mucus production and productive cough are risk factors for exacerbations. Medicines targeting bronchoconstriction and airway inflammation are the current mainstays of COPD therapy. However, there is growing concern with an increased risk of pneumonia in patients with COPD receiving regular inhaled corticosteroids and there is therefore a need to find safer alternative treatments. Previous studies have indicated that inhalation of unfractionated heparin (UFH) treats local inflammation, mucus hypersecretion and lung injury, without systemic anticoagulation, and is safe. Therefore, our primary objective was to demonstrate that inhaled UFH significantly improves lung function (FEV 1 ) over 21 days of treatment in patients with COPD receiving pulmonary rehabilitation and that UFH provides a novel, safe and effective way of treating this complex disease. Forty patients with moderate to very severe COPD admitted to the IRCCS San Raffaele Pisana Hospital for 21 days pulmonary rehabilitation were randomised to receive nebulised inhaled UFH (75,000 or 150,000 IU BID) or placebo for 21 days. All patients also received nebulised salbutamol (1 mg) and beclomethasone dipropionate (400 μg) BID over the same period. Lung function was measured at day 0, 7, 14 and 21 of treatment and at a follow-up visit 7 days post-treatment. Exercise capacity (6MWT) and dyspnoea (Borg score) were measured before and after treatment. In pre-clinical studies, the ability of basic proteins found in COPD sputum to neutralise the anticoagulant activity of heparin was determined using the AMAX heparin assay kit. At both doses, UFH significantly increased FVC following 7 days of treatment and 150,000 IU BID significantly increased FEV1 (+249 ± 69 ml compared with placebo) at this time, an effect maintained to the 28 day follow-up. Clinically significant improvement in exercise capacity and dyspnoea were seen after 21 days of treatment with both doses of UFH. There were no serious adverse events or effects on systemic coagulation. Pre-clinical studies demonstrated that the basic proteins lactoferrin, platelet factor-4 (PF-4), IL-8 and polyarginine, as a model of the eosinophil cationic protein (ECP), found in COPD sputum neutralise the anticoagulant activity of heparin. Inhaled nebulised UFH is safe and provides additional clinical benefit for patients with moderate to very severe COPD through effects that are independent of its anticoagulant activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heparin as a pharmacologic intervention to induce positive scintiscan in occult gastrointestinal bleeding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chaudhuri, T.K.; Brantly, M.

1984-04-01

The value of using heparin as a pharmacologic intervention to induce a positive scintiscan was studied in a patient with chronic occult gastrointestinal bleeding. When all standard diagnostic tests (upper and lower gastrointestinal series, upper and lower endoscopy, and conventional noninterventional Tc-99m RBC imaging) fail to detect and localize gastrointestinal bleeding in a patient who has definite clinical evidence (guaiac positive stool and dropping hemoglobin, hematocrit) of chronic occult gastrointestinal oozing, heparin may be used (with proper precaution) as a last resort to aid in the scintigraphic detection and localization of chronic occult gastrointestinal bleeding.

Cardiopulmonary bypass with bivalirudin in type II heparin-induced thrombocytopenia.

PubMed

Clayton, Stephanie B; Acsell, Jeffrey R; Crumbley, Arthur J; Uber, Walter E

2004-12-01

Cardiopulmonary bypass in patients with type II heparin induced-thrombocytopenia poses significant challenges. Inadequate pharmacokinetic profiles, monitoring, reversibility, and availability often limit alternative anticoagulation strategies. Bivalirudin, a semisynthetic direct thrombin inhibitor, was recently approved for use in patients undergoing percutaneous coronary interventions. Its unique properties, including a relatively short half-life, an anticoagulation effect that closely correlates with activated clotting time, and an alternate metabolic pathway for elimination, make bivalirudin an attractive agent for cardiopulmonary bypass in patients with type II heparin induced-thrombocytopenia. We report our experience using bivalirudin in 2 patients undergoing coronary artery bypass grafting.

Effects of Chitosan Derivative N-[(2-Hydroxy-3-Trimethylammonium)Propyl]Chloride on Anticoagulant Activity of Guinea Pig Plasma.

PubMed

Drozd, N N; Shagdarova, B Ts; Il'ina, A V; Varlamov, V P

2017-07-01

Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 70 aIIa U/kg non-fractionated heparin shortened plasma clotting time (shown by partial activated thromboplastin time, thrombin time, and prothrombin time). Intravenous injection of protamine sulfate or quarternized chitosan derivative to guinea pigs after injection of 1 mg/kg (100 aXa U/kg) low-molecular-weight heparin (clexane) led to shortening of plasma clotting time in the ReaClot Heparin test and to prolongation of plasma amidolytic activity in the factor Xa chromogenic substrate test.

Cooperative control of blood compatibility and re-endothelialization by immobilized heparin and substrate topography.

PubMed

Ding, Yonghui; Yang, Meng; Yang, Zhilu; Luo, Rifang; Lu, Xiong; Huang, Nan; Huang, Pingbo; Leng, Yang

2015-03-01

A wide variety of environmental cues provided by the extracellular matrix, including biophysical and biochemical cues, are responsible for vascular cell behavior and function. In particular, substrate topography and surface chemistry have been shown to regulate blood and vascular compatibility individually. The combined impact of chemical and topographic cues on blood and vascular compatibility, and the interplay between these two types of cues, are subjects that are currently being explored. In the present study, a facile polydopamine-mediated approach is introduced for immobilization of heparin on topographically patterned substrates, and the combined effects of these cues on blood compatibility and re-endothelialization are systematically investigated. The results show that immobilized heparin and substrate topography cooperatively modulate anti-coagulation activity, endothelial cell (EC) attachment, proliferation, focal adhesion formation and endothelial marker expression. Meanwhile, the substrate topography is the primary determinant of cell alignment and elongation, driving in vivo-like endothelial organization. Importantly, combining immobilized heparin with substrate topography empowers substantially greater competitive ability of ECs over smooth muscle cells than each cue individually. Moreover, a model is proposed to elucidate the cooperative interplay between immobilized heparin and substrate topography in regulating cell behavior. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Biopolymer-modified graphite oxide nanocomposite films based on benzalkonium chloride-heparin intercalated in graphite oxide

NASA Astrophysics Data System (ADS)

Meng, Na; Zhang, Shuang-Quan; Zhou, Ning-Lin; Shen, Jian

2010-05-01

Heparin is a potent anticoagulant agent that interacts strongly with antithrombin III to prevent the formation of fibrin clots. In the present work, poly(dimethylsiloxane)(PDMS)/graphite oxide-benzalkonium chloride-heparin (PDMS/modified graphite oxide) nanocomposite films were obtained by the solution intercalation technique as a possible drug delivery system. The heparin-benzalkonium chloride (BAC-HEP) was intercalated into graphite oxide (GO) layers to form GO-BAC-HEP (modified graphite oxide). Nanocomposite films were characterized by XRD, SEM, TEM, ATR-FTIR and TGA. The modified graphite oxide was observed to be homogeneously dispersed throughout the PDMS matrix. The effect of modified graphite oxide on the mechanical properties of the nanocomposite film was investigated. When the modified graphite oxide content was lower than 0.2 wt%, the nanocomposites showed excellent mechanical properties. Furthermore, nanocomposite films become delivery systems that release heparin slowly to make the nanocomposite films blood compatible. The in vitro studies included hemocompatibility testing for effects on platelet adhesion, platelet activation, plasma recalcification profiles, and hemolysis. Results from these studies showed that the anticoagulation properties of PDMS/GO-BCA-HEP nanocomposite films were greatly superior to those for no treated PDMS. Cell culture assay indicated that PDMS/GO-BCA-HEP nanocomposite films showed enhanced cell adhesion.

Colorimetric assay of heparin in plasma based on the inhibition of oxidase-like activity of citrate-capped platinum nanoparticles.

PubMed

You, Jyun-Guo; Liu, Yao-Wen; Lu, Chi-Yu; Tseng, Wei-Lung; Yu, Cheng-Ju

2017-06-15

We report citrate-capped platinum nanoparticles (Pt NPs) as oxidase mimetics for effectively catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), dopamine, and methylene blue in the presence of O 2 . To confirm oxidase-like activity of citrate-capped Pt NPs, their activity toward oxygen reduction reaction was studied using cyclic voltammetry and rotating ring-disk electrode method. The results obtained showed that Pt NP NPs can catalyze the oxidation of organic substrates to the colored product and the reduction of oxygen to water through a four-electron exchange process. Because the aggregation of Pt NPs can inhibit their oxidase-like activity and protamine can recognize heparin, we prepared the protamine-modified Pt NPs through direct adsorption on the surface of citrate-capped Pt NPs. The electrostatic attraction between heparin and protamine-stabilized Pt NPs induced nanoparticle aggregation, inhibiting their catalytic activity. Therefore, the lowest detectable heparin concentrations through UV-vis absorption and by the naked eye were estimated to be 0.3 and 60nM, respectively. Moreover, the proposed system enabled the determination of the therapeutic heparin concentration in a single drop of blood. Copyright © 2016 Elsevier B.V. All rights reserved.

Heparin-binding growth factor isolated from human prostatic extracts.

PubMed

Mydlo, J H; Bulbul, M A; Richon, V M; Heston, W D; Fair, W R

1988-01-01

Prostatic tissue extracts from patients with benign prostatic hyperplasia (BPH) and prostatic carcinoma were fractionated using heparin-Sepharose chromatography. The mitogenic activity of eluted fractions on quiescent subconfluent Swiss Albino 3T3 fibroblasts was tested employing a tritiated-thymidine-incorporation assay. Two peaks of activity were consistently noted--one in the void volume and a second fraction which eluted with 1.3-1.6 M NaCl and contained the majority of the mitogenic activity. Both non-heparin- and heparin-binding fractions increased tritiated incorporation into a mouse osteoblast cell line (MC3T3), while only the heparin-binding fractions stimulated a human umbilical vein endothelial cell line (HUV). No increased uptake of thymidine was seen using a human prostatic carcinoma cell line (PC-3). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of lyophilized active fractions showed a persistent band at 17,500 daltons. The purified protein demonstrated angiogenic properties using the chick embryo chorioallantoic membrane (CAM) assay. Western blot analysis using antibodies specific to basic fibroblast growth factor (bFGF) or acidic FGF (aFGF) demonstrated that the former, but not the latter, bound to prostatic growth factor (PrGF), and inhibited its mitogenic activity as well. It appears that PrGF shares homology with basic fibroblast growth factors.

Development of the Integrated Info Tech System

DTIC Science & Technology

2007-01-01

withdrawn from the study:  Treatment with immunosuppressive  Cytotoxic  Corticosteroids  Anticoagulant agents such as Heparin, Plavix or Coumadin...such as Heparin, Plavix or Coumadin o Needing radiation or chemotherapy o Those who develop infection, Osteomyelitis, or dermatitis o At the

Multicenter, dose-ranging study of efegatran sulfate versus heparin with thrombolysis for acute myocardial infarction: The Promotion of Reperfusion in Myocardial Infarction Evolution (PRIME) trial.

PubMed

2002-01-01

Adjunctive therapies that increase the incidence of normal reperfusion after thrombolysis for acute myocardial infarction (MI) could enhance clinical outcomes. Direct thrombin inhibitors may offer an advantage over standard adjunctive therapies. We randomized 336 patients with acute MI at 33 sites to receive 1 of 5 doses of efegatran sulfate, a direct thrombin inhibitor, or heparin for 72 to 96 hours, both with accelerated alteplase and aspirin. The primary end point was the incidence of thrombolytic failure (death, reinfarction, or TIMI grade 0-2 flow in the infarct artery from 90 minutes to discharge or 30 days, whichever occurred earlier). Significantly more patients randomized to efegatran had evidence of heart failure at admission. The lowest-dose efegatran arm was terminated at 15 patients because of unacceptably increased thrombolytic failure. The primary end point occurred in 53.0% of patients treated with heparin, in 53.8% of patients treated with efegatran overall (P =.90), and in 55.4% of patients given intermediate-dose efegatran (P =.74). These findings were unaffected after adjustment was done for baseline differences. Most bleeding was minor; major bleeding and the use of blood transfusions did not differ significantly by treatment. Three patients in the high-dose efegatran group had intracranial hemorrhage, as did 1 patient in the heparin group. Continuous ST monitoring showed a shorter time to recovery for the efegatran group (median 107 minutes) compared with the heparin group (154 minutes; P =.025). Efegatran sulfate appeared to offer no clear advantage over heparin as an adjunct to thrombolysis for acute myocardial infarction, although there may be a modest improvement in time to reperfusion.

The influence of dialyzer geometry on blood coagulation and biocompatibility.

PubMed

Lins, L E; Boberg, U; Jacobson, S H; Kjellstrand, C; Ljungberg, B; Skröder, R

1993-11-01

The influence of dialyzer geometry on blood coagulation, heparin requirement and complement activation was studied in fourteen chronic hemodialysis patients. Each patient was dialyzed with two different cuprophan dialyzers, hollow fiber GF 120M and parallel plate Lundia IC5N. Both dialyzers had a wall thickness of 11 microns, surface area of 1.2 m2 and both were sterilized with ethylene oxide. Heparin doses were individually titrated. The mean heparin dose was 6089 +/- 988 U. Platelet count decreased from 218 x 10(9)/l to 193 x 10(9)/l and from 235 x 10(9)/l to 197 x 10(9)/l respectively (hollow fiber/plate dialyzer, ns). The number of leucocytes decreased at 15 min after start of dialysis by 56% and 61% (hollow fiber/plate dialyzer, ns). The heparin requirement, measured as prolongation of whole blood activated coagulation time after identical doses of heparin, were the same in hollow fiber and plate dialysis sessions. The arterial fibrinopeptide A concentrations increased during dialysis from 5.4 to 7.1 nmol/l and 8.5 to 9.6 nmol/l respectively (hollow fiber/plate dialyzer, ns). The residual blood volume in the hollow fiber dialyzers was 1.3 +/- 1.1 ml and in the plate dialyzers 1.5 +/- 0.9 ml (ns). C3a activation, indicated by a marked arterio-venous difference, was observed at 15 min after start of dialysis with hollow fiber as well as plate dialyzers. The arterio-venous difference was less pronounced at the end of dialysis. There were no differences in C3a activation between hollow fiber and plate dialyzers at any timepoint. It is concluded that dialyzer geometry does not significantly influence platelet count, blood coagulation, heparin requirement or complement activation.

Efficacy of intravascular catheter lock solutions containing preservatives in the prevention of microbial colonization

PubMed Central

Shenep, L.E.; Shenep, M.A.; Cheatham, W.; Hoffman, J.M.; Hale, A.; Williams, B.F.; Perkins, R.; Hewitt, C.B.; Hayden, R.T.; Shenep, J. L.

2016-01-01

SUMMARY There is little published evidence regarding whether heparin lock solutions containing preservatives prevent catheter-related infections. However, adverse effects from preservative-containing flushes have been documented in neonates, leading many hospitals to avoid their use altogether. Infection control records from 1982 to 2008 at St. Jude Children’s Research Hospital (SJCRH) were reviewed regarding the incidence of CRIs and the use of preservative-containing intravenous locks. In addition, the antimicrobial activities of heparin lock solution containing the preservatives parabens (0.165%) or benzyl alcohol (0.9%), and 70% ethanol were examined against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Candida albicans, and compared with preservative-free saline with and without heparin. Growth was assessed after exposure to test solutions for 0, 2, 4 and 24 h at 35°C. The activities of preservatives were assessed against both planktonic (free-floating) and sessile (biofilm-embedded) micro-organisms using the MBEC Assay. Infection control records revealed two periods of increased catheter-related infections, corresponding with two intervals when preservative-free heparin was used at SJCRH. Heparin solution containing preservatives demonstrated significant antimicrobial activity against both planktonic and sessile forms of all six microbial species. Ethanol demonstrated the greatest antimicrobial activity, especially following short incubation periods. Heparin lock solutions containing the preservatives parabens or benzyl alcohol, and 70% ethanol demonstrated significant antimicrobial activity against both planktonic and sessile micro-organisms commonly responsible for CRIs. These findings, together with the authors’ historical infection control experience, support the use of preservatives in intravenous lock solutions to reduce catheter related infections in patients beyond the neonatal period. PMID:21945067

Efficacy of intravascular catheter lock solutions containing preservatives in the prevention of microbial colonization.

PubMed

Shenep, L E; Shenep, M A; Cheatham, W; Hoffman, J M; Hale, A; Williams, B F; Perkins, R; Hewitt, C B; Hayden, R T; Shenep, J L

2011-12-01

There is little published evidence regarding whether heparin lock solutions containing preservatives prevent catheter-related infections. However, adverse effects from preservative-containing flushes have been documented in neonates, leading many hospitals to avoid their use altogether. Infection control records from 1982 to 2008 at St. Jude Children's Research Hospital (SJCRH) were reviewed regarding the incidence of catheter-related infections and the use of preservative-containing intravenous locks. In addition, the antimicrobial activities of heparin lock solution containing the preservatives parabens (0.165%) or benzyl alcohol (0.9%), and 70% ethanol were examined against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa and Candida albicans, and compared with preservative-free saline with and without heparin. Growth was assessed after exposure to test solutions for 0, 2, 4 and 24h at 35 °C. The activities of preservatives were assessed against both planktonic (free-floating) and sessile (biofilm-embedded) micro-organisms using the MBEC Assay. Infection control records revealed two periods of increased catheter-related infections, corresponding with two intervals when preservative-free heparin was used at SJCRH. Heparin solution containing preservatives demonstrated significant antimicrobial activity against both planktonic and sessile forms of all six microbial species. Ethanol demonstrated the greatest antimicrobial activity, especially following short incubation periods. Heparin lock solutions containing the preservatives parabens or benzyl alcohol, and 70% ethanol demonstrated significant antimicrobial activity against both planktonic and sessile micro-organisms commonly responsible for catheter-related infections. These findings, together with the authors' historical infection control experience, support the use of preservatives in intravenous lock solutions to reduce catheter related infections in patients beyond the neonatal period. Copyright © 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Comparison of heparinized saline and 0.9% sodium chloride for maintaining peripheral intravenous catheter patency in dogs.

PubMed

Ueda, Yu; Odunayo, Adesola; Mann, F A

2013-01-01

To determine whether heparinized saline would be more effective in maintaining the patency of peripheral IV catheters in dogs compared to 0.9% sodium chloride. Prospective blinded randomized study. University Veterinary Teaching Hospital. Thirty healthy purpose bred dogs, intended for use in the junior surgery laboratory, were utilized. The dogs were randomized into 1 of 3 groups, 2 treatment groups and a control group. An 18-Ga cephalic catheter was placed in the cephalic vein of each dog. Each dog in the treatment group had their catheter flushed with either 10 IU/mL heparinized saline or 0.9% sodium chloride every 6 hours for 42 hours. The dogs in the control group did not have their catheters flushed until the end of the study period. Immediately prior to flushing catheters, each catheter was evaluated for patency by aspiration of blood and the catheter site was evaluated for phlebitis. All dogs in the heparinized saline and 0.9% sodium chloride group had catheters that flushed easily at each evaluation point. More dogs in the saline group had catheters from which blood could not be aspirated, but there was no significant difference between these groups. All dogs in the control group had catheters that flushed easily at the end of the assigned 6 hour interval except in 1 dog. Phlebitis was not detected in any dog. Flushes of 0.9% sodium chloride were found to be as effective as 10 IU/mL heparinized saline flushes in maintaining patency of 18-Ga peripheral venous catheters in dogs for up to 42 hours. For peripheral catheters placed with the intention of performing serial blood draws, heparinized flushes may be warranted. © Veterinary Emergency and Critical Care Society 2013.

Nonthrombogenic Hydrogel Coatings with Carbene-Cross-Linking Bioadhesives.

PubMed

Nanda, Himansu Sekhar; Shah, Ankur Harish; Wicaksono, Gautama; Pokholenko, Oleksandr; Gao, Feng; Djordjevic, Ivan; Steele, Terry W J

2018-05-14

Bioadhesives are a current unmet clinical need for mending of blood contacting soft tissues without inducing thrombosis. Recent development of carbene precursor bioadhesives with the advantages of on-demand curing, tuneable modulus, and wet adhesion have been synthesized by grafting diazirine onto poly (amidoamine) (PAMAM-G5) dendrimers. Herein, the structure activity relationships of platelet adhesion and activation is evaluated for the first time on the cured PAMAM-g-diazirine bioadhesives. Three strategies were employed to prevent healthy human donor platelets from adhering and activating on light-cured bioadhesive surfaces: (1) Attenuation of cationic surface charge, (2) antifouling composites by incorporating heparin and alginate in uncured formulation, and (3) heparin wash of cured bioadhesive surface. Topographical imaging of cured and ethanol dehydrated bioadhesive surfaces was used to quantify the adhered and activated platelets with scanning electron microscopy, whose resolution allowed identification of round senescent, short dendritic, and long dendritic platelets. Cured surfaces of PAMAM-g-diazirine (15%) had 10300 ± 500 adhered platelets mm -2 with 99.7% activation into short/long dendritic cells. Reduction of primary amines by higher degree of diazirine grafting or capping of free amines by acetylation reduces platelet adherence (2400 ± 200 vs 3000 ± 300, respectively). Physical incorporation of heparin and alginate in the formulations reduced the activated platelet; 1300 ± 300 and 300 ± 50, activated platelets mm -2 , in comparison with additive free adhesive formulation. Similarly, heparin rinse of the surface of additive free bioadhesive reduced the activated platelet to platelets of heparin composites at 600 ± 100 platelets mm -2 . PAMAM-g-diazirine (15%) bioadhesive retained the photocured mechanical properties and lap shear adhesion despite the addition of heparin and alginate additives.

Development of decellularized aortic valvular conduit coated by heparin-SDF-1α multilayer.

PubMed

Zhou, Jingxin; Ye, Xiaofeng; Wang, Zhe; Liu, Jun; Zhang, Busheng; Qiu, Jiapei; Sun, Yanjun; Li, Haiqing; Zhao, Qiang

2015-02-01

Decellularization can reduce the immune response to aortic valve allograft tissue, but the thrombogenicity and in vivo remolding of these grafts remain controversial. The aim of the present study was to modify the surface of decellularized valvular conduits with heparin-stromal cell-derived factor-1α (SDF-1α) polyelectrolyte multilayer film and to test the thrombogenicity and biocompatibility in vitro and recellularization in vivo. The donor aortic valvular conduits were decellularized with a combination of different detergents and were coated with heparin and SDF-1α alternately to form a polyelectrolyte multilayer. Platelet adhesion and lactate dehydrogenase assay were used to evaluate the antiplatelet property. The adhesion, growth, and migration of bone marrow stem cells (BMSCs) to the scaffolds were assessed. For in vivo studies, the grafts were anastomosed to the infrarenal aorta, without or with heparin and SDF-1α multilayer. Functional assessment was performed by Doppler echography and micro-computed tomography at 2-week and 4-week time points after implantation. Explanted grafts were examined histologically and by immunohistochemistry. In vitro studies demonstrated that the heparin-SDF-1α multilayer film improved hemocompatibility with respect to a substantial reduction of platelet adhesion. BMSCs also achieved better adhesion, proliferation, and migration on the modified graft. For in vivo studies, the grafts in both groups remained patent after 4 weeks, but it was demonstrated that the modified decellularized grafts had better self-endothelialization and recruitment of endothelial progenitor cells. These results indicate that heparin-SDF-1α multilayer film can be used to cover the decellularized aortic valvular graft to decrease platelet adhesion while precipitating regeneration of the decellularized aortic valve allograft in vivo. Copyright © 2015 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability.

PubMed

Quinsey, Noelene S; Fitton, Hazel L; Coughlin, Paul; Whisstock, James C; Dafforn, Timothy R; Carrell, Robin W; Bottomley, Stephen P; Pike, Robert N

2003-09-02

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.

Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.

The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonarymore » hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension.« less

Heparin in acute ischemic stroke revisited.

PubMed

Chamorro, A

2008-10-01

The evidence gathered in clinical trials of low molecular weight heparins (LMWHs) or with unfractionated heparin (UH) given subcutaneously at low or medium doses to patients with acute stroke cannot be extrapolated to the insufficiently tested effects of intravenous, weight-adjusted UH. Recent small studies have provided encouraging results but are potentially confounded and deserve confirmation in larger randomized controlled trials. In accordance with the current understanding of the biology of acute ischemic stroke and the pharmacology of UH, the new randomized controlled trials on heparin should give appropriate credit to the importance of a short therapeutic window, adequate dose adjustment of the drug, intravenous administration, and close monitoring of biological effects. UH is an orphan drug and only an academic driven trial would be able to face such an enterprise. Meanwhile, recommendations against the value of "early" anticoagulation with full dose of weight adjusted UH in the setting of acute ischemic stroke are not based on direct evidence but on extrapolations.

[Meta-analysis on randomized trials comparing the results of low-molecular weight heparins to those of fractioned heparins in the prevention of deep venous thrombosis].

PubMed

Daures, J P; Schved, J F; Momas, I; Gril, J C; Azoulay, P; Gremy, F

1989-01-01

The main reason for using low molecular weight heparin (LMWH) is its capacity to keep the antithrombotic effect of standard non-fractioned heparin (NFH) while reducing its hemorrhagic power. Various studies comparing LMWH to NFH gave contradictory results, so we carried out a meta-analysis of randomized trials comparing these two treatments in prevention of deep-vein thrombosis (DVT) (and in prevention of deep-vein thrombosis or pulmonary embolism). From a selection of ten trials we could not show any significant difference in the DVT rates, as well as for the whole of surgical indications as for the subgroups of abdominal and orthopedic surgery. The weighted global difference between the DVT rates is so low that, in spite of the use of meta-analysis, the power is clearly insufficient. The hemorrhagic risk is not statistically reduced as well for the whole of surgical indications as for the subgroups.

Inhibition of heparin precipitation, bacterial growth, and fungal growth with a combined isopropanol-ethanol locking solution for vascular access devices.

PubMed

Restrepo, Daniel; Laconi, Nicholas S; Alcantar, Norma A; West, Leigh A; Buttice, Audrey L; Patel, Saumil; Kayton, Mark L

2015-03-01

Clinical reports of ethanol-lock use for the prevention of catheter-related bloodstream infections have been marked by the occurrence of serious catheter occlusions, particularly among children with mediports. We hypothesized that precipitate forms when ethanol mixes with heparin at the concentrations relevant for vascular access devices, but that the use of a combination of two alcohols, ethanol and isopropanol, would diminish heparin-related precipitation, while retaining anti-bacterial and anti-fungal effects. Heparin (0-100units/mL) was incubated in ethanol-water solutions (30%-70% vol/vol) or in an aqueous solution containing equal parts (35% and 35% vol/vol) of isopropanol and ethanol. Precipitation at temperatures from 4 to 40°C was measured in nephelometric turbidity units using a benchtop turbidimeter. Growth of Escherichia coli, Staphylococcus aureus, and Candida albicans colonies were measured following exposure to solutions of ethanol or isopropanol-ethanol. Groupwise comparisons were performed using analysis of variance with Bonferroni-corrected, post-hoc T-testing. Seventy percent ethanol and heparin exhibit dose-dependent precipitation that is pronounced and significant at the concentrations typically used in mediports (p<0.05). Precipitate is significantly reduced by use of a combined 35% isopropanol-35% ethanol solution rather than 70% ethanol (p<0.05), while maintaining the solution's anti-bacterial and anti-fungal properties. On the other hand, although ethanol solutions under 70% form less precipitate with heparin, such concentrations are also less effective at bacterial colony inhibition than solutions of either 70% ethanol or 35% isopropanol-35% ethanol (p<0.05). A combined 35% isopropanol-35% ethanol locking solution inhibits bacterial and fungal growth similarly to 70% ethanol, but results in less precipitate than 70% ethanol when exposed to heparin. Further study of a combined isopropanol-ethanol locking solution for the prevention of catheter-related bloodstream infections should focus on the determination as to whether such a locking solution may reduce the rate of precipitation-related catheter occlusion, and whether it may be administered with low systemic toxicity. Copyright © 2015 Elsevier Inc. All rights reserved.

Bivalirudin for Pediatric Procedural Anticoagulation: A Narrative Review.

PubMed

Zaleski, Katherine L; DiNardo, James A; Nasr, Viviane G

2018-02-14

Bivalirudin (Angiomax; The Medicines Company, Parsippany, NJ), a direct thrombin inhibitor, has found increasing utilization as a heparin alternative in the pediatric population, most commonly for the treatment of thrombosis secondary to heparin-induced thrombocytopenia. Due to the relative rarity of heparin-induced thrombocytopenia as well as the lack of Food and Drug Administration-approved indications in this age group, much of what is known regarding the pharmacokinetics and pharmacodynamics of bivalirudin in this population has been extrapolated from adult data. This narrative review will present recommendations regarding the use of bivalirudin for procedural anticoagulation in the pediatric population based on the published literature.

A caution regarding the use of low-molecular weight heparin in pediatric otogenic lateral sinus thrombosis.

PubMed

Shah, Udayan K; Jubelirer, Tracey F; Fish, Jonathan D; Elden, Lisa M

2007-02-01

Lateral sinus thrombosis (LST), a rare complication of otitis media, is managed by antibiotics, surgery and anticoagulation. Traditionally, post-operative anticoagulation has been achieved by intravenous unfractionated heparin followed by oral warfarin. Fractionated, or low-molecular weight heparin derivatives (LMWH) have been introduced recently. There has been minimal literature to date regarding their use for the management of LST. We present use of the LMWH enoxaparin (Lovenox) for otogenic LST in two children, both of whom experienced hemorrhagic complications. On this basis and in the context of a literature review, we urge caution when using LMWH for pediatric otogenic LST.

21 CFR 864.5680 - Automated heparin analyzer.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated heparin analyzer. 864.5680 Section 864.5680 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology Devices § 864...

21 CFR 864.5680 - Automated heparin analyzer.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated heparin analyzer. 864.5680 Section 864.5680 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology Devices § 864...