Sample records for heparin cofactor ii

  1. [Heparin cofactor II, a thrombin inhibitor with a still not clarified physiologic role]. (United States)

    Rossi, E B; Duboscq, C L; Kordich, L C


    Heparin Cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of dermatan sulfate. Inhibition occurs by formation of a stable equimolar complex between HCII and thrombin. HCII association with thrombotic events has not always been observed, thus decreased HCII does not appear to be a strong risk factor for thromboembolic events. Reduced HCII levels have been detected in different clinical conditions, such as hepatic failure, disseminated intravascular coagulation, thalasemina, sickle cell anemia. Increased physiological levels have been found in pregnant women and oral contraception. In our laboratory, we measured HCII plasmatic levels in the normal Buenos Aires city population and in patients under different clinical conditions, such as sepsis, diabetis, burns, oral anticoagulation and in patients treated with heparin, hyperhomcysteinemia in whom septic and diabetic patients showed decreased values. HCII thrombin inhibition possibly takes place in extravascular sites where dermatan sulfate is present. HCII activity would be important in the regulation of wound healing, inflammation, or neuronal development.

  2. Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity

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    Sugahara Kazuyuki


    Full Text Available Abstract Background Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra and Stolidobranchia (Halocynthia pyriformis and Styela plicata. Despite the identical disaccharide backbone, consisting of [→4IdoA(2Sβ-1→3GalNAcβ-1→], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi and Phlebobranchia (Ciona intestinalis, aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. Results Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [→4IdoA(2-Sulfateβ-1→3GalNAcβ-1

  3. Correction: Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity

    Directory of Open Access Journals (Sweden)

    Sugahara Kazuyuki


    Full Text Available Abstract After the publication of the work entitled "Dermatan sulfate in tunicate phylogeny: Order-specific sulfation pattern and the effect of [→4IdoA(2-Sulfateβ-1→3GalNAc(4-Sulfateβ-1→] motifs in dermatan sulfate on heparin cofactor II activity", by Kozlowski et al., BMC Biochemistry 2011, 12:29, we found that the legends to Figures 2 to 5 contain serious mistakes that compromise the comprehension of the work. This correction article contains the correct text of the legends to Figures 2 to 5.

  4. Stoichiometry of Zn(II)-heparin-glycine complex, determined using data from elemental and thermal analysis (United States)

    Feofanova, M. A.; Skobin, M. I.; Kryukov, T. V.; Alekseev, V. G.; Ryasenskii, S. S.


    Ternary polymer Zn(II)-heparin-glycine complex with the composition {Na3[ZnHepGly]·H2O} n , where Hep4- is the monomer chain of a heparin polyanion and Gly- is the chain of a glycine anion, is isolated in a solid state from a water solution, and is characterized via elemental and thermal analysis.

  5. Controllable production of low molecular weight heparins by combinations of heparinase I/II/III. (United States)

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


    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.

  6. Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xanthinuria type II. (United States)

    Ichida, K; Matsumura, T; Sakuma, R; Hosoya, T; Nishino, T


    Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO. Copyright 2001 Academic Press.

  7. A peptide of heparin cofactor II inhibits endotoxin-mediated shock and invasive Pseudomonas aeruginosa infection

    DEFF Research Database (Denmark)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath


    Sepsis and septic shock remain important medical problems with high mortality rates. Today's treatment is based mainly on using antibiotics to target the bacteria, without addressing the systemic inflammatory response, which is a major contributor to mortality in sepsis. Therefore, novel treatment......-inflammatory responses by decreasing NF-κB/AP-1 activation in vitro. In mouse models of LPS-induced shock, KYE28 significantly enhanced survival by dampening the pro-inflammatory cytokine response. Finally, in an invasive Pseudomonas infection model, the peptide inhibited bacterial growth and reduced the pro...

  8. Successful management of acute thromboembolic disease complicated with heparin induced thrombocytopenia type II (HIT II: a case series

    Directory of Open Access Journals (Sweden)

    Trellopoulos George


    Full Text Available Abstract Heparin-induced thrombocytopenia type II (HIT II is a rare immune-mediated complication of heparin. The diagnosis of HIT is considered in patients exposed to heparin, presenting with thrombocytopenia and thrombosis. We present two cases with massive pulmonary embolism and HIT, successfully treated with the administration of fondaparinux, an alternative anticoagulant, combined with the insertion of an inferior vena cava filter for the prevention of new thromboembolic events. The two cases supplement the available data of the use of fondaparinux in patients with HIT and pulmonary embolism, before further large studies establish its efficacy and safety in this group of patients. Moreover, the management of these patients reveals the need for future evaluation of the combined therapy of alternative anticoagulant agents with the placement of vena cava filters.

  9. Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II). (United States)

    Yin, Ruichuan; Mo, Jiezhen; Dai, Jiayin; Wang, Hailin


    Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

  10. Randomized comparison of a novel anticoagulant, vasoflux, and heparin as adjunctive therapy to streptokinase for acute myocardial infarction: results of the VITAL study (Vasoflux International Trial for Acute Myocardial Infarction Lysis)

    NARCIS (Netherlands)

    Peters, R. J.; Spickler, W.; Théroux, P.; White, H.; Gibson, M.; Molhoek, P. G.; Anderson, H. V.; Weitz, J. I.; Hirsh, J.; Weaver, W. D.


    Vasoflux is a low-molecular-weight heparin derivative that inhibits factor IXa activation of factor X and catalyzes fibrin-bound thrombin inactivation by heparin cofactor II. We studied whether vasoflux improves the results of thrombolysis with streptokinase for acute myocardial infarction. We

  11. Deletion mutation in Drosophila ma-l homologous, putative molybdopterin cofactor sulfurase gene is associated with bovine xanthinuria type II. (United States)

    Watanabe, T; Ihara, N; Itoh, T; Fujita, T; Sugimoto, Y


    Defective xanthine dehydrogenase (XDH) activity in humans results in xanthinuria and xanthine calculus accumulation in kidneys. Bovine xanthinuria was demonstrated in a local herd and characterized as xanthinuria type II, similar to the Drosophila ma-l mutations, which lose activities of molybdoenzymes, XDH, and aldehyde oxidase, although sulfite oxidase activity is preserved. Linkage analysis located the disease locus at the centromeric region of bovine chromosome 24, where a ma-l homologous, putative molybdopterin cofactor sulfurase gene (MCSU) has been physically mapped. We found that a deletion mutation at tyrosine 257 in MCSU is tightly associated with bovine xanthinuria type II.

  12. Heparan sulfate, heparin, and heparinase activity detection on polyacrylamide gel electrophoresis using the fluorochrome tris(2,2'-bipyridine) ruthenium (II). (United States)

    Rozenberg, G I; Espada, J; de Cidre, L L; Eiján, A M; Calvo, J C; Bertolesi, G E


    The paper shows the ability of the fluorochrome tris(2,2'-bipyridine) ruthenium (II) (Rubipy) to detect heparan sulfate, heparin, and heparinase activity of M3 murine mammary adenocarcinoma cells as well as bacterial heparinases I, II, and III in native polyacrylamide gel electrophoresis (PAGE). The technique is based on the electrophoretic mobility of high molecular weight heparins and subsequent staining with Rubipy (50 micrograms/mL). The minimum content of heparin detected by fluorescence in a UV transilluminator was 25-50 ng. The number of Rubipy molecules bound to heparin, determined in relationship to the number of disaccharide units (DU), showed that two to six heparin disaccharide units are bound by each fluorochrome molecule. Scatchard plot analysis showed one Rubipy-binding site (Kd = (8.56 +/- 2.97) x 10(-5) M). Heparinase activity was determined by densitometric analysis of the fluorescence intensity of the heparin-containing band of the gel. While heparinase I (EC degraded heparin and, to a lower degree, partially N-desulfated N-acetylated heparin (N-des N-Ac), heparinase II (no EC number) could efficiently degrade heparan sulfate (HS) and partially N-des N-Ac heparin. Finally, heparinase III (EC degraded HS almost exclusively. Only heparin and N-des N-Ac heparin were substrates for M3 tumor cell heparinases. We describe a qualitative, sensitive and simple method to detect heparinase activity and determine its substrate specificity using Rubipy fluorescence with heparin and heparan sulfate in multiple biological samples tested in parallel.

  13. Platelet factor 4-positive thrombi adhering to the ventricles of a ventricular assist device in patients with heparin-induced thrombocytopenia type II. (United States)

    Beiras-Fernandez, A; Kanzler, I; Michel, S; Sadoni, S; Kilger, E; Beiras, A; Kur, F


    Thromboembolism is a major complication in patients with ventricular assist devices (VADs). Drug anticoagulation and the use of biocompatible surfaces, such as coating with heparin, aim to reduce thromboembolism in these patients. Administration of heparin can lead to heparin-induced thrombocytopenia (HIT) type II, mainly through heparin/platelet factor 4 (PF4) antibodies. We assessed the presence of PF4 antibodies in VAD thrombi of patients with heparin-coated VADs and HIT II. Thrombi (n = 6) were obtained from the replaced Excor ventricles of patients with HIT II after biventricular VAD implantation (Excor Adult; Berlin Heart, Germany). Excor ventricles were changed after clinical examination and suspicion of thrombi in the polyurethane valves. Expression of PF4- antibodies was assessed with the use of a polyclonal rabbit antibody (anti-PF4 antibody; Abcam, USA). Expression was assessed by 2 independent observers. Biopsies of all thrombi showed an extreme positive immunoreaction for PF4. No differences between the different thrombi and localization (left/right Excor ventricle) were observed. The thrombi were organized, without lamination of fibrin and cellular layers. Platelet surface expression of PF4 in the thrombi reflects HIT antigen presentation. The physical relationship between the PF4-positive thrombi and the heparin-coated surface suggests that onset of HIT II could be influenced by the immobilized heparin coating. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Investigation of Luminescence Characteristics of Osmium(II Complexes in the Presence of Heparin Polyanions

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    Yixi Xie


    Full Text Available The luminescence characteristics of six osmium carbonyl complexes with phenanthroline (phen or bipyridine (bpy and pyridine (py, 4-phenylpyridine (4-phpy, or triphenylphosphine (PPh3 complexes in the presence of polyanion heparin were studied in both ethanol and aqueous solutions. The influence of heparin on the luminescence of the complexes is heavily dependent on the type of ligands in the complexes and the solvent used. In the ethanol solutions, the heparin solution enhanced the luminescence of the five osmium complexes, with the strongest enhancement to the 4-phenylpyridine complexes; linear curves were obtained in the luminescence enhancement ratio (F/F0 versus the heparin concentration range of 1–40 μg/mL. In aqueous solutions, heparin quenching of the complexes was more significant; a linear quenching curve was obtained with [Os(phen2CO(PPh3](PF62 in the lower concentration range of 1–12 μg/mL. The interaction of these complexes with heparin in the solutions is discussed. The complexes are shown to be successful in the fast and sensitive detection of heparin in commercial injectable samples.

  15. Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15. (United States)

    Hyun, Y-J; Lee, K-S; Kim, D-H


    This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15. The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli, and rHepII was purified using two simple ion-exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC. The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l(-1) sodium phosphate buffer with 75 mmol l(-1) NaCl (pH 7.4) at 32 degrees C, and the activity is stable at 4 degrees C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure. This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.

  16. Identification of a new point mutation in the human molybdenum cofactor sulferase gene that is responsible for xanthinuria type II. (United States)

    Yamamoto, Tetsuya; Moriwaki, Yuji; Takahashi, Sumio; Tsutsumi, Zenta; Tuneyoshi, Ka; Matsui, Kiyoshi; Cheng, Jidong; Hada, Toshikazu


    A 43-year-old xanthinuric female was referred to our department because of hypouricemia. Routine laboratory data showed hypouricemia, a high level of plasma oxypurines, decreased urinary uric acid excretion, and increased urinary oxypurine excretion, with xanthine dehydrogenase activity in the duodenal mucosa below the limits of detection. In addition, allopurinol was not metabolized. From these findings, the patient was diagnosed with xanthinuria type II. To investigate the properties of xanthine dehydrogenase/xanthine oxidase (XDH/XO) deficiency, a cDNA sequence encoding XDH/XO, aldehyde oxidase (AO), and molybdenum cofactor sulferase (MCS), as well as immunoblotting analysis for XDH/XO protein, obtained from duodenal mucosa samples were performed. The XDH/XO cDNA and AO cDNA sequences of the xanthinuric patient were consistent with previously reported ones, whereas the MCS cDNA sequence revealed a point mutation of G to C in nucleotide 466, which changed codon 156 from GCC (Ala) to CCC (Pro). In addition, the MCS genomic DNA sequence including the site of the mutation revealed the same, suggesting that the xanthinuric patient was homozygous for this mutation. Such findings have not been previously reported for patients with xanthinuria type II.

  17. The MHC class II cofactor, HLA-DM, interacts with immunoglobulin in B cells (United States)

    Ayyangar, Sashi; Jiang, Wei; Rajasekaran, Narendiran; Spura, Armin; Hessell, Ann J.; Madec, Anne-Marie; Mellins, Elizabeth D.


    B cells internalize extracellular antigen into endosomes using the immunoglobulin (Ig) component of the B cell receptor. In endosomes, antigen-derived peptides are loaded onto MHC class II proteins (MHC-II). How these pathways intersect remains unclear. We find that HLA-DM (DM), a catalyst for MHC-II peptide loading, co-precipitates with Ig in lysates from human tonsillar B cells and B cell lines. The molecules in the Ig/DM complexes have mature glycans, and the complexes co-localize with endosomal markers in intact cells. A larger fraction of Ig precipitates with DM after BCR crosslinking, implying that complexes can form when DM meets endocytosed Ig. In vitro, in the endosomal pH range, soluble HLA-DM (sDM) directly binds the Ig Fab domain, and increases levels of free antigen released from immune complexes. Together, these results argue that DM and Ig intersect in the endocytic pathway of B cells with potential functional consequences. PMID:25098292

  18. Low-concentration heparin suppresses ionomycin-activated CAMK-II/EGF receptor- and ERK-mediated signaling in mesangial cells. (United States)

    Song, Lifang; Xiao, Weiqun; Templeton, Douglas M


    Heparin and endogenous heparinoids inhibit the proliferation of smooth muscle cells, including renal mesangial cells; multiple effects on signaling pathways are well established, including effects on PKC, Erk, and CaMK-II. Many studies have used heparin at concentrations of 100 microg/ml or higher, whereas endogenous concentrations of heparinoids are much lower. Here we report the effects of low-concentration (1 microg/ml) heparin on activation of several kinases and subsequent induction of the c-fos gene in mesangial cells in response to the calcium ionophore, ionomycin, in the absence of serum factors. Ionomycin rapidly increases the phosphorylation of CaMK-II (by 30 s), and subsequently of the EGF receptor (EGFR), c-Src, and Erk 1/2. Low-dose heparin suppresses the ionomycin-dependent phosphorylation of EGFR, c-Src, and Erk 1/2, but not of CaMK-II, whereas inhibition of activated CaMK-II reduces phosphorylation of EGFR, c-Src, and Erk. Our data support a mechanism whereby heparin acts at the cell surface to suppress downstream targets of CaMK-II, including EGFR, leading in turn to a decrease in Erk- (but not c-Src-) dependent induction of c-fos.

  19. Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor. (United States)

    Kleiner, O; Butenandt, J; Carell, T; Batschauer, A


    The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD-photolyases repair this DNA damage by a light-driven electron transfer. The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell 9, 199-207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.

  20. Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells.


    Miralem, T; Templeton, D M


    Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partially suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbol ester or platelet-derived growth factor, this response appears to result from the ability of heparin to suppress activation of the extracellular-signal-regulated kinase family of mitogen-activated protein kinases. H...

  1. Heparin Injection (United States)

    ... moisture (not in the bathroom). Do not freeze heparin. It is important to keep all medication out of sight and reach of children as many containers (such as weekly pill minders and those for eye drops, creams, patches, and inhalers) are not child-resistant ...

  2. Structural Snapshots of Heparin Depolymerization by Heparin Lyase I

    Energy Technology Data Exchange (ETDEWEB)

    Han, Young-Hyun; Garron, Marie-Line; Kim, Hye-Yeon; Kim, Wan-Seok; Zhang, Zhenqing; Ryu, Kyeong-Seok; Shaya, David; Xiao, Zhongping; Cheong, Chaejoon; Kim, Yeong Shik; Linhardt, Robert J.; Jeon, Young Ho; Cygler, Miroslaw; (SNU); (Korea BSI); (McGill); (UST-Korea); (Rensselaer)


    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 an ({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.

  3. Key cofactors of photosystem II cores from four organisms identified by 1.7-K absorption, CD and MCD. (United States)

    Arsköld, Sindra Peterson; Smith, Paul J; Shen, Jian-Ren; Pace, Ron J; Krausz, Elmars


    Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98-103]. Electrochromic shifts resulting from QA reduction by 1.7-K illumination were recorded, and the Qx and Qy absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Qx transition is approximately 3 nm (100 cm-1) to higher energy in cyanobacteria than in the plants. The predominant Qy shift appears in the range 683-686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Qy region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.

  4. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development. (United States)

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G; Graham, Mark J; Berliner, Judith A; Lusis, Aldons J; Cai, Lei; Temel, Ryan E; Rateri, Debra L; Lee, Sangderk


    The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

  5. Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event. (United States)

    Okafor, C Denise; Lanier, Kathryn A; Petrov, Anton S; Athavale, Shreyas S; Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean


    Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Heparin and heparin-induced thrombocytopenia

    African Journals Online (AJOL)


    Jun 15, 2007 ... pregnancy, renal failure and obesity.4 See Table I for a comparison of unfractionated heparin and LMWH. Fondaparinux (Arixtra) is a novel anticoagulant which takes the evolution of heparin even further. It is an indirect factor. Xa inhibitor which does not cross-react with HIT antibodies.5. Fondaparinux has ...

  7. Low molecular weight heparin (dalteparin) as adjuvant treatment to thrombolysis in acute myocardial infarction—a pilot study: Biochemical Markers in Acute Coronary Syndromes (BIOMACS II)

    National Research Council Canada - National Science Library

    Frostfeldt, Gunnar; Ahlberg, Greger; Gustafsson, Gunnar; Helmius, Gunnar; Lindahl, Bertil; Nygren, Anders; Siegbahn, Agneta; Swahn, Eva; Venge, Per; Wallentin, Lars


    ... (5–7) . The additional effect of aspirin as treatment of acute myocardial infarction is today a well-established standard therapy (8,9) . Heparin infusion after treatment with thrombolytic agents improves early patency in the infarct-related artery (10) . However, the influences of heparin on long-term mortality is less clear (11–13) ...

  8. Hypersensitivity reactions to heparins. (United States)

    Gonzalez-Delgado, Purificación; Fernandez, Javier


    This article provides an update on hypersensitivity reactions to heparins and novel oral anticoagulants, with special emphasis on diagnostic methods and management of patients. Although heparins are drugs widely used, hypersensitivity reactions are uncommon. Cutaneous delayed hypersensitivity reactions after subcutaneous administration affects up to 7.5% of patients. Heparin-induced thrombocytopenia is another unusual but severe condition in which early recognition is crucial. Immediate hypersensitivity reactions to heparins have been also reported, but with the novel oral anticoagulants are much more uncommon, although reports of exanthemas have been notified.Skin tests and subcutaneous provocation test are useful tools in the diagnosis of hypersensitivity reactions, except in heparin-induced thrombocytopenia in which biopsy of lesional skin and in-vitro tests are the modalities of choice to confirm the diagnosis.Management of hypersensitivity reactions includes finding an alternative depending on the type of reaction. Fondaparinux and novel oral anticoagulants may be safe alternatives. Delayed skin lesions after subcutaneous heparin are the most common type of hypersensitivity reactions, followed by life-threatening heparin-induced thrombocytopenia. Immediate reactions are uncommon. Allergologic studies may be useful to find an alternative option in patients with skin lesions in which heparin-induced thrombocytopenia has been previously excluded, as well as in heparin immediate reactions.

  9. Kinetic analysis of the clotting system in the presence of heparin and depolymerized heparin. (United States)

    Heuck, C C; Baumann, P


    The kinetics of the activation of the plasmatic clotting system in the presence of heparin and depolymerized heparin (Kabi 2165), respectively, was compared with the kinetics of activation in plasma with isolated factor deficiency. Measurements were made with a chromogenic substrate method using Tos-Gly-Pro-Arg-p-nitroanilide acetate. The extinction curves were analyzed to determine the characteristics of a curve that was fitted to the experimental data to sufficiently describe the slope of the curves by constants. In the activated extrinsic clotting system, the action of heparin and depolymerized heparin results in a distribution pattern for the two relevant constants. K(1), defining the time of the point of inflection of the curve, and K(2), relating to the slope of the curve at the point of inflection, which is identical with the pattern observed in plasma with factor II deficiency. This distribution pattern can be explained by an inhibitory reaction on factor IIa, which is accelerated by the anticoagulant. In contrast, the pattern of K(2)/K(1) for the activated intrinsic system is identical with the pattern for plasma with factor X deficiency. Qualitative differences in the action of heparin and depolymerized heparin are not evident. The investigation confirms that the molecular action of heparin and depolymerized heparin as accelerators of the plasmatic clotting system is qualitatively the same. However, their action in the extrinsic and intrinsic system has different effects. Furthermore, the study reveals that constant K(2) is a more sensitive indicator to measure low heparin and depolymerized heparin activities than K(1) or its equivalent, the clotting time.

  10. Identification and characterization of the first mutation (Arg776Cys) in the C-terminal domain of the Human Molybdenum Cofactor Sulfurase (HMCS) associated with type II classical xanthinuria. (United States)

    Peretz, Hava; Naamati, Meirav Shtauber; Levartovsky, David; Lagziel, Ayala; Shani, Esther; Horn, Ivona; Shalev, Hanna; Landau, Daniel


    Classical xanthinuria type II is an autosomal recessive disorder characterized by deficiency of xanthine dehydrogenase and aldehyde oxidase activities due to lack of a common sulfido-olybdenum cofactor (MoCo). Two mutations, both in the N-terminal domain of the Human Molybdenum Cofactor Sulfurase (HMCS), were reported in patients with type II xanthinuria. Whereas the N-terminal domain of HMCS was demonstrated to have cysteine desulfurase activity, the C-terminal domain hypothetically transfers the sulfur to the MoCo. We describe the first mutation in the C-terminal domain of HMCS identified in a Bedouin-Arab child presenting with urolithiasis and in an asymptomatic Jewish female. Patients were diagnosed with type II xanthinuria by homozygosity mapping and/or allopurinol loading test. The Bedouin-Arab child was homozygous for a c.2326C>T (p.Arg776Cys) mutation, while the female patient was compound heterozygous for this and a novel c.1034insA (p.Gln347fsStop379) mutation in the N-terminal domain of HMCS. Cosegregation of the homozygous mutant genotype with hypouricemia and hypouricosuria was demonstrated in the Bedouin family. Haplotype analysis indicated that p.Arg776Cys is a recurrent mutation. Arg776 together with six surrounding amino acid residues were found fully conserved and predicted to be buried in homologous eukaryotic MoCo sulfurases. Moreover, Arg776 is conserved in a diversity of eukaryotic and prokaryotic proteins that posses a domain homologous to the C-terminal domain of HMCS. Our findings suggest that Arg776 is essential for a core structure of the C-terminal domain of the HMCS and identification of a mutation at this site may contribute clarifying the mechanism of MoCo sulfuration.

  11. Heparin-associated thrombocytopenia: an update. (United States)

    Fondu, P


    The use of heparin may be complicated by two types of thrombocytopenia (HAT): type I occurs early, is transient, and has no clinical relevance, while type II may lead to very severe manifestations (arterial or venous thromboses and more rarely bleedings), that are still underestimated by some clinicians. HAT-type II most frequently develops after use of therapeutic doses of unfractionated heparin (UH) but has also been described less frequently after use of very low doses of UH, of low molecular weight heparins (LMWH), and even of polysulfated glycosaminoglycosans devoid of anticoagulant action. The estimation of the incidence of HAT-type II and of related thromboses is a very difficult matter. Recent observations suggest that thromboses (notably venous) may be more frequent than previously estimated. HAT-type II pathophysiology includes the formation of immune complexes at the surface of platelets; the antigen has been shown to be most often platelet factor 4 bound to heparin while the antibody is recognized by platelet Fc gamma RII receptors. Thromboses result most probably from activation of both platelets (leading to the formation of microparticles) and endothelial cells. Several biological tests are presently available for diagnosing HAT-type II but none of them has been shown to be ideal. The prevention of HAT-type II requires history taking preference of LMWH to UH, early start of oral anticoagulation, and platelet monitoring from the fifth day of heparin therapy. The therapy of HAT-type II implies immediate discontinuation of heparin and avoidance of platelet transfusions, unless severe bleeding occurs. If further antithrombotic treatment is deemed necessary (probably in all cases), several options are possible but presently, the most recommended ones are Org 10172 or Ancrod; embolectomy or thrombolysis may also be required if a new thrombotic event has developed. A very difficult dilemma concerns patients previously sensitized to heparin and who present a

  12. Heparin pharmacovigilance in Brazil. (United States)

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


    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.

  13. Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor

    NARCIS (Netherlands)

    W. Klootwijk (Willem); T.J. Visser (Theo); G.G.J.M. Kuiper (George)


    textabstractHuman type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all

  14. Highly sensitive ratiometric detection of heparin and its oversulfated chondroitin sulfate contaminant by fluorescent peptidyl probe. (United States)

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


    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 (R2=0.99) in aqueous solutions, 2.98nM (R2=0.98) in 1% serum samples, and 3.43nM (R2=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.

  15. Heparin for assisted reproduction. (United States)

    Akhtar, Muhammad A; Sur, Shyamaly; Raine-Fenning, Nick; Jayaprakasan, Kannamannadiar; Thornton, Jim G; Quenby, Siobhan


    Heparin as an adjunct in assisted reproduction (peri-implantation heparin) is given at or after egg collection or at embryo transfer during assisted reproduction. Heparin has been advocated to improve embryo implantation and clinical outcomes.  It has been proposed that heparin enhances the intra-uterine environment by improving decidualisation with an associated activation of growth factors and a cytokine expression profile in the endometrium that is favourable to pregnancy. To investigate whether the administration of heparin around the time of implantation (peri-implantation heparin) improves clinical outcomes in subfertile women undergoing assisted reproduction. A comprehensive and exhaustive search strategy was developed in consultation with the Trials Search Co-ordinator of the Cochrane Menstrual Disorders and Subfertility Group (MDSG). The strategy was used in an attempt to identify all relevant studies regardless of language or publication status (published, unpublished, in press, and in progress). Relevant trials were identified from both electronic databases and other resources (last search 6 May 2013). All randomised controlled trials (RCTs) were included where peri-implantation heparin was given during assisted reproduction. Peri-implantation low molecular weight heparin (LMWH) during IVF/ICSI was given at or after egg collection or at embryo transfer in the included studies. Live birth rate was the primary outcome. Two review authors independently assessed the eligibility and quality of trials and extracted relevant data. The quality of the evidence was evaluated using GRADE methods. Three RCTs (involving 386 women) were included in the review.Peri-implantation LMWH administration during assisted reproduction was associated with a significant improvement in live birth rate compared with placebo or no LMWH (odds ratio (OR) 1.77, 95% confidence interval (CI) 1.07 to 2.90, three studies, 386 women, I(2) = 51%, very low quality evidence with high

  16. Low molecular weight heparins. (United States)

    Bergqvist, D


    Low molecular weight heparins (LMWHs) differ from unfractionated heparin (UFH) in a number of characteristics, which is probably due to differences in molecular weight distribution. From a clinical point of view the better subcutaneous bioavailability and longer biological half-life are important, making it sufficient to inject LMWHs once-daily only. For practical purposes it is also important that LMWHs be used without monitoring. They are effective as prophylaxis against postoperative venous thromboembolism after all types of surgery; in most studies, more effective than UFH. In most studies, this effect can be obtained safely and with less bleeding than with UFH. LMWHs compare favourably with UFH for starting treatment of deep vein thrombosis, as well as an anticoagulant during haemodialysis. Adverse effects such as thrombocytopenia and osteoporosis are more common with UFH than with LMWHs. Studies evaluating whether or not LMWHs can replace UFH in arterial diseases are still few with small sample sizes. Thus further systematic research is needed.

  17. Women with homozygous AT deficiency type II heparin-binding site (HBS) are at high risk of pregnancy loss and pregnancy complications. (United States)

    Kraft, Julia; Sunder-Plassmann, Raute; Mannhalter, Christine; Quehenberger, Peter; Tews, Gernot; Langer, Martin; Pabinger, Ingrid


    Data regarding outcome and therapy of pregnancies in patients with homozygous antithrombin (AT) deficiency are very rare. We conducted a retrospective, descriptive investigation with emphasis on the obstetric history of eight women with homozygous AT deficiency heparin-binding site (HBS), who had at least one pregnancy. The aim of the study was to get a better insight into the outcome and identify suitable management procedures of pregnancy in this rare disease. All patients suffered from homozygous AT deficiency caused by the mutation c.391C>T p.Leu131Phe in the AT gene (SERPINC1). The women reported in total 23 pregnancies; one pregnancy was excluded because of induced abortion. We found that only seven out of the 22 analyzed pregnancies ended with a live infant, all of them were born preterm. Among the 15 negative outcomes, seven were early pregnancy losses and eight were intrauterine fetal deaths. We found no clear association between treatment protocols and outcome. Eight pregnancies were not treated at all; all of them ended with pregnancy loss. We conclude that homozygous AT deficiency HBS, a form of severe thrombophilia, is associated with high risk of pregnancy loss and preterm delivery. Rigorous anticoagulation and/or replacement of AT during pregnancy may improve the outcome.

  18. Electrochemical behavior of Pb (II) on a heparin modified chitosan/graphene nanocomposite film coated glassy carbon electrode and its sensitive detection. (United States)

    T, Priya; N, Dhanalakshmi; N, Thinakaran


    In this study, we developed a novel composite material containing biological macromolecules like heparin and chitosan coated on reduced graphene oxide (rGO) for the modification of glassy carbon electrode (hep/CS-rGO/GC). It can be applied for the sensitive electrochemical detection of Pb2+ by square wave anodic stripping voltammetry (SWASV). The physicochemical analysis such as XRD, FTIR, FESEM and Raman spectroscopy techniques revealed that an effective functionalization occurred at the rGO surface. The consequence of deposition and stripping of metal ions by various electrochemical parameters such as supporting electrolytes, pH value, deposition potential, and deposition time were carefully studied and optimized. Under the optimized conditions, the linear calibration curve was calculated to be from 1.125 to 8.25μgL-1 for Pb2+ with the correlation coefficient (R2) 0.9988. The detection limit and sensitivity achieved for the modified electrode were 0.03μgL-1 and 1.34μA/nM respectively. Furthermore, the electrochemical investigation indicates that the hep/CS-rGO composite electrode exhibits high selectivity, strong adherence to the electrode surface, good stability and reproducibility towards the detection of Pb2+. Finally, hep/CS-rGO/GC electrode was assessed by the quantity of Pb2+ present in the practical samples, and the determined results were consistent with that of AAS. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Collection of heparinized plasma by plasmapheresis

    NARCIS (Netherlands)

    van der Meer, P. F.; Vrielink, H.; Pietersz, R. N.; Dekker, W. J.; Reesink, H. W.


    BACKGROUND AND OBJECTIVES: Heparinized plasma can be used for exchange transfusions in neonates and is usually collected by drawing whole blood using heparin as anticoagulant. The heparinized red blood cells and buffy coat cannot be used and are therefore discarded. To collect heparinized plasma

  20. Incorporation of heparin into biomaterials. (United States)

    Sakiyama-Elbert, Shelly E


    This review provides an overview of the incorporation of heparin into biomaterials with a focus on drug delivery and the use of heparin-based biomaterials for self-assembly of polymer networks. Heparin conjugation to biomaterials was originally explored to reduce the thrombogenicity of materials in contact with blood. Many of the conjugation strategies that were developed for these applications are still popular today for other applications. More recently heparin has been conjugated to biomaterials for drug delivery applications. Many of the delivery approaches have taken advantage of the ability of heparin to bind to a wide variety of growth factors, protecting them from degradation and potentiating interactions with cell surface receptors. More recently, the use of heparin as a base polymer for scaffold fabrication has also been explored, often utilizing non-covalent binding of heparin with peptides or proteins to promote self-assembly of hydrogel networks. This review will highlight recent advances in each of these areas. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  1. Cofactor engineering for advancing chemical biotechnology. (United States)

    Wang, Yipeng; San, Ka-Yiu; Bennett, George N


    Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Interactions between nattokinase and heparin/GAGs. (United States)

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


    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.

  3. Heparin release from thermosensitive hydrogels

    NARCIS (Netherlands)

    Gutowska, Anna; Bae, You Han; Feijen, Jan; Kim, Sung Wan


    Thermosensitive hydrogels (TSH) were synthesized and investigated as heparin releasing polymers for the prevention of surface induced thrombosis. TSH were synthesized with N-isopropyl acrylamide (NiPAAm) copolymerized with butyl methacrylate (BMA) (hydrophobic) or acrylic acid (AAc) (hydrophilic)

  4. Engineering redox balance through cofactor systems. (United States)

    Chen, Xiulai; Li, Shubo; Liu, Liming


    Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration. (United States)

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


    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.

  6. Quantitative compositional analysis of heparin using exhaustive heparinase digestion and strong anion exchange chromatography

    Directory of Open Access Journals (Sweden)

    Pierre Mourier


    Full Text Available Heparin is a linear sulfated polysaccharide widely used therapeutically as an anticoagulant. It is also the starting material for manufacturing low-molecular-weight heparins (LMWH. Quality control of heparin and LMWH is critical to ensure the safety and therapeutic activity of the final product. However due to their complex and heterogeneous structure, orthogonal analytical techniques are needed to characterize the building blocks of heparin. One of the state-of-the-art methods for heparin analysis is based on complete enzymatic digestion using a mixture of heparinases I, II, and III, followed by the separation of the resulting oligosaccharides by liquid chromatography. The European Pharmacopoeia strong anion-exchange chromatographic method, used to quantify 1,6-anhydro derivatives in enoxaparin, is here applied to the analysis of the heparin building blocks. Their quantification, namely the determination of their average w/w percentage in the heparin chain, is obtained after identification of all components including glycoserine derivatives and 3-O sulfated di- and tetrasaccharides. This work therefore provides a comprehensive overview of the building blocks of unfractionated heparin, including those chemically modified by the manufacturing process, either within the polysaccharide chain or at its reducing end.

  7. Heparin interaction with protein-adsorbed surfaces

    NARCIS (Netherlands)

    Winterton, Lynn C.; Andrade, Joseph D.; Feijen, Jan; Kim, Sung Wan


    Albumin and fibrinogen show no binding affinity to varied molecular weights of heparin at physiological pH. Human plasma fibronectin was shown to bind heparins in both the solution and adsorbed states. Fibronectin was shown to have six active binding sites for heparins which may be sterically

  8. Insights into hydrocarbon formation by nitrogenase cofactor homologs. (United States)

    Lee, Chi Chung; Hu, Yilin; Ribbe, Markus W


    The L-cluster is an all-iron homolog of nitrogenase cofactors. Driven by europium(II) diethylenetriaminepentaacetate [Eu(II)-DTPA], the isolated L-cluster is capable of ATP-independent reduction of CO and CN(-) to C1 to C4 and C1 to C6 hydrocarbons, respectively. Compared to its cofactor homologs, the L-cluster generates considerably more CH4 from the reduction of CO and CN(-), which could be explained by the presence of a "free" Fe atom that is "unmasked" by homocitrate as an additional site for methanation. Moreover, the elevated CH4 formation is accompanied by a decrease in the amount of longer hydrocarbons and/or the lengths of the hydrocarbon products, illustrating a competition between CH4 formation/release and C-C coupling/chain extension. These observations suggest the possibility of designing simpler synthetic clusters for hydrocarbon formation while establishing the L-cluster as a platform for mechanistic investigations of CO and CN(-) reduction without complications originating from the heterometal and homocitrate components. Nitrogenase is a metalloenzyme that is highly complex in structure and uniquely versatile in function. It catalyzes two reactions that parallel two important industrial processes: the reduction of nitrogen to ammonia, which parallels the Haber-Bosch process in ammonia production, and the reduction of carbon monoxide to hydrocarbons, which parallels the Fischer-Tropsch process in fuel production. Thus, the significance of nitrogenase can be appreciated from the perspective of the useful products it generates: (i) ammonia, the "fixed" nitrogen that is essential for the existence of the entire human population; and (ii) hydrocarbons, the "recycled" carbon fuel that could be used to directly address the worldwide energy shortage. This article provides initial insights into the catalytic characteristics of various nitrogenase cofactors in hydrocarbon formation. The reported assay system provides a useful tool for mechanistic

  9. Prenatal brain disruption in molybdenum cofactor deficiency. (United States)

    Carmi-Nawi, Nirit; Malinger, Gustavo; Mandel, Hanna; Ichida, Kimiyoshi; Lerman-Sagie, Tally; Lev, Dorit


    Molybdenum cofactor deficiency is a rare autosomal recessive disorder that may present during the neonatal period with intractable seizures and be mistaken for ischemic encephalopathy. We describe a patient whose prenatal sonography at 35 weeks' gestation revealed diffuse brain damage with multiple subcortical cavities, ventriculomegaly, dysgenesis of the corpus callosum, and a hypoplastic cerebellum with an enlarged cisterna magna. Magnetic resonance imaging (MRI) later revealed brain atrophy, and multicystic encephalomalacia with hypoplastic vermis and cerebellum. Neurological examination at 10 months showed microcephaly, profound mental retardation, and spasticity. Uric acid was low, and taurine and xanthine were increased in the urine. A sulfite test was positive. The diagnosis of molybdenum cofactor deficiency was made. Sulfite oxidase activity in fibroblasts was undetectable. The patient was found to be homozygous for the 251-418del in the MOCS1 gene. This is the first description of the prenatal development of severe brain disruption in molybdenum cofactor deficiency.

  10. Optimized Treatment of Heparinized Blood Fractions to Make Them Suitable for Analysis. (United States)

    Sánchez-Fito, María Teresa; Oltra, Elisa


    It has been known for decades that many cytokines, such as IL-2, IL-6, and IL-12, bind to heparin. Even though some enzyme-linked immunosorbent assays (ELISA) use antibody-recognizing epitopes not affected by this binding, ELISA manufacturers often warn that heparinized plasma or serum fractions containing more than 3 IU (international units)/mL of heparin should not be used in assays so as to prevent heparin interference in the reaction. In addition, enzyme-based nucleic acid amplifications from heparinized samples have been shown defective by several research groups. The aim of this study was to determine optimal degradation and/or removal of heparin from heparinized blood samples to best turn them into fractions for appropriate ELISA and RT-PCR analysis. A colorimetric reporter assay based on the metachromatic effect of the binding of heparin to toluidine blue was shown to be a low-cost effective method to discriminate assay compatible blood fractions with heparin levels below 3 IU/mL. Heparin removal from human blood fractions was best achieved by treatment with either Bacteroides Heparinase II or the less expensive Heparinase I at a final concentration of 0.1 U/μL and incubations at 30°C for a period between 30 min and 4 h, or by adsorption to Ecteola slurries at a concentration of 20 mg/mL for 20 min at room temperature (RT). The fact that both enzymatic and resin-based optimized treatments allowed for replication of the readings obtained with heparin-free equivalent fractions in both ELISA and RT-PCR assays indicates they should be appropriate for quantitative studies such as expression profiling at both the protein and nucleic acid level. The cost-effective protocols developed in this study could make heparinized, otherwise unusable, blood-derived collections suitable for analysis by ELISA and RT-PCR amplifications, among other analyses, enhancing the possibilities for studying valuable bio-banked heparinized human samples.

  11. Heparin-induced thrombocytopenia and thrombosis. (United States)

    Patel, Vipul P; Bong, Matthew; Di Cesare, Paul E


    Heparin-induced thrombocytopenia (HIT) and heparin induced thrombocytopenia with thrombosis (HITT) ar rare complications associated with use of unfractionate heparin (UFH) or low-molecular-weight heparin (LMWH) HIT is a benign clinical condition characterized by a mil drop in platelet count with no clinical significance. HIT is an immune-mediated reaction associated with a wide spread "hypercoagulable" state resulting in arterial an venous thrombosis. There is a higher incidence of HIT with UFH use than with LMWH use. Orthopedic surger patients are at higher risk for developing HITT than are patients who receive prophylactic heparin for cardiovascular surgery or medical reasons. Therapy for patients suspected of having HITT should begin with immedi ate discontinuation of heparin in any form followed by pharmacologic inhibition with thrombin (e.g., recombinant hirudin [lepirudin], argatroban, danaparoid sodium).

  12. Cofactors Influencing Prevalence and Intensity of Schistosoma ...

    African Journals Online (AJOL)

    An epidemiological study of sedentary Fulani settlements in Dumbi, Igabi Local Government Area of Kaduna State was undertaken to determine cofactors of Schistosoma haematobium prevalence and intensity of infection. Consenting individuals were recruited after sensitization from six settlements and administered a ...

  13. Low molecular weight heparin versus unfractionated heparin in the initial treatment of venous thromboembolism

    NARCIS (Netherlands)

    Hettiarachchi, R. J.; Prins, M. H.; Lensing, A. W.; Buller, H. R.


    In this review, we analyze data from randomized trials in which low molecular weight heparin was compared with unfractionated heparin, both to estimate the treatment effect of low molecular weight heparin in the initial treatment of venous thromboembolism and to evaluate the effect of the varied

  14. Replacing Electron Transport Cofactors with Hydrogenases

    KAUST Repository

    Laamarti, Rkia


    Enzymes have found applications in a broad range of industrial production processes. While high catalytic activity, selectivity and mild reaction conditions are attractive advantages of the biocatalysts, particularly costs arising from required cofactors pose a sever limitation. While cofactor-recycling systems are available, their use implies constraints for process set-up and conditions, which are a particular problem e.g. for solid-gas-phase reactions. Several oxidoreductases are able to directly exchange electrons with electrodes. Hence, the co-immobilization of both, an electron-utilizing and an electron-generating oxidoreductase on conductive nanoparticles should facilitate the direct electron flow from an enzymatic oxidation to a reduction reaction circumventing redox-cofactors requirements. In such a set-up, hydrogenases could generate and provide electrons directly form gaseous hydrogen. This thesis describes the co-immobilization of the oxygen tolerant hydrogenases from C. eutropha or C. metallidurans and cytochrome P450BM3 as test system. Conductive material in the form of carbon nanotubes (CNT) serves as a suitable support. A combination of the hydrogenase and the catalytic domain of P450BM3 immobilized on carbon nanotubes were tested for the oxidation of lauric acid in the presence of hydrogen instead of an electron-transport cofactor. The GC-MS analysis reveals the conversion of 4% of lauric acid (LA) into three products, which correspond to the hydroxylated lauric acid in three different positions with a total turnover (TON) of 34. The product distribution is similar to that obtained when using the wildtype P450BM3 with the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor. Such electronic coupling couldn’t be achieved for the conversion of other substrates such as propane and cyclohexane, probably due to the high uncoupling rate within the heme-domain of cytochrome P450BM3 when unnatural substrates are introduced.

  15. Applications of heparin and heparan sulfate microarrays. (United States)

    Yin, Jian; Seeberger, Peter H


    Carbohydrate microarrays have become crucial tools for revealing the biological interactions and functions of glycans, primarily because the microarray format enables the investigation of large numbers of carbohydrates at a time. Heparan sulfate (HS) and heparin are the most structurally complex glycosaminoglycans (GAGs). In this chapter, we describe the preparation of a small library of HS/heparin oligosaccharides, and the fabrication of HS/heparin microarrays that have been used to establish HS/heparin-binding profiles. Fibroblast growth factors (FGFs), natural cytotoxicity receptors (NCRs), and chemokines were screened to illuminate the very important biological functions of these glycans. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  16. Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients. (United States)

    Junqueira, Daniela R; Zorzela, Liliane M; Perini, Edson


    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

  17. Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xinyue, E-mail: [National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, 250100 (China); Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); St Ange, Kalib, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Wang, Xiaohua, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); School of Computer and Information, Hefei University of Technology, Hefei (China); Lin, Lei, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Zhang, Fuming, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); and others


    Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox{sup ®}, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox{sup ®}, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin. - Highlights: • Low molecular weight heparins prepared from different heparin parents were analyzed. • An integrated analytical

  18. Antiproliferative heparin (glycosaminoglycans) isolated from giant ...

    African Journals Online (AJOL)

    Heparin was isolated from two bivalve mollusks, Tridacna maxima (giant clam) and Perna viridis (green mussel). The isolated heparin was quantified in crude as well as purified samples and they were estimated as 2.72 and 2.2 g/kg (in crude) and 260 and 248 mg/g (in purified samples) in T. maxima and P. viridis, ...


    African Journals Online (AJOL)


    Dec 2, 2009 ... bleeding, allergic reactions, skin necrosis, osteoporosis ... This study found a significantly higher incidence of heparin induced antibody formation in the UFH group than in the. LMWH heparin group (20.7% vs 7.5% p<0.001). The patients on short term .... Tietge et al (30) presented a case report of a 76 year ...

  20. Complex coacervation of lysozyme and heparin

    DEFF Research Database (Denmark)

    van de Weert, Marco; Andersen, Mia Bendix; Frokjaer, Sven


    To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability.......To characterize complex coacervates/flocculates of lysozyme and heparin in terms of binding stoichiometry and to determine the effect of complexation on protein structure and stability....

  1. Sterilization of heparinized cuprophan hemodialysis membranes

    NARCIS (Netherlands)

    ten Hoopen, Hermina W.M.; Hinrichs, W.L.J.; Hinrichs, W.L.J.; Engbers, G.H.M.; Feijen, Jan


    The effects of sterilization of dry heparinized Cuprophan hemodialysis membranes by means of ethylene oxide (EtO) exposure, gamma irradiation, or steam on the anticoagulant activity and chemical characteristics of immobilized heparin and the permeability of the membrane were investigated.

  2. Allergic anaphylaxis due to subcutaneously injected heparin

    Directory of Open Access Journals (Sweden)

    Anders Diana


    Full Text Available Abstract Heparins are one of the most used class of anticoagulants in daily clinical practice. Despite their widespread application immune-mediated hypersensitivity reactions to heparins are rare. Among these, the delayed-type reactions to s.c. injected heparins are well-known usually presenting as circumscribed eczematous plaques at the injection sites. In contrast, potentially life-threatening systemic immediate-type anaphylactic reactions to heparins are extremely rare. Recently, some cases of non-allergic anaphylaxis could be attributed to undesirable heparin contaminants. A 43-year-old patient developed severe anaphylaxis symptoms within 5–10 minutes after s.c. injection of enoxaparin. Titrated skin prick testing with wheal and flare responses up to an enoxaparin dilution of 1:10.000 indicated a probable allergic mechanism of the enoxaparin-induced anaphylaxis. The basophil activation test as an additional in-vitro test method was negative. Furthermore, skin prick testing showed rather broad cross-reactivity among different heparin preparations tested. In the presented case, history, symptoms, and results of skin testing strongly suggested an IgE-mediated allergic hypersensitivity against different heparins. Therefore, as safe alternative anticoagulants the patient could receive beneath coumarins the hirudins or direct thrombin inhibitors. Because these compounds have a completely different molecular structure compared with the heparin-polysaccharides.

  3. Unfractionated heparin versus low molecular weight heparin for avoiding heparin-induced thrombocytopenia in postoperative patients. (United States)

    Junqueira, Daniela R G; Perini, Edson; Penholati, Raphael R M; Carvalho, Maria G


    Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction presenting as a prothrombotic disorder related to antibody-mediated platelet activation. It is a poorly understood 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 presenting a 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 adverse reactions such as HIT. We therefore sought to determine the relative impact of UFH and LMWH specifically on HIT in postoperative patients receiving thromboembolism prophylaxis. The objective of this review was to compare the incidence of HIT and HIT complicated by thrombosis in patients exposed to UFH versus LMWH in randomised controlled trials (RCTs) of postoperative heparin therapy. The Cochrane Peripheral Vascular Diseases Group searched their Specialised Register (March 2012) and CENTRAL (2012, Issue 2). In addition, the authors searched LILACS (March 2012) and additional trials were sought from reference lists of relevant publications. We were interested in comparing the incidence of HIT occurring during exposure to UFH or LMWH after any surgical intervention. Therefore, we studied RCTs in which participants were postoperative patients allocated to receive UFH or LMWH, in a blinded or unblinded fashion. Eligible studies were

  4. Cutaneous reactions to heparin therapy: when are they caused by heparin allergy?

    Directory of Open Access Journals (Sweden)

    Giuliana Zisa


    Full Text Available Introduction: Little is known about the incidence and causes of heparin-induced skin lesions. The most commonly reported causes are delayed-type hypersensitivity reactions. We describe 3 patients who were referred to our staff between March and October 2009 for suspected heparin allergies. All were scheduled to undergo major surgery (cardiovascular or orthopedic. Materials and methods: All 3 patients reported the development of itchy, erythematous rashes a few days after the subcutaneous administration of heparin (nadroparin calcium in cases 1 and 2, unspecified in case 3. Each of them underwent a diagnostic work-up for heparin allergy, which included prick and intradermal tests with commonly used heparins and patch testing with undiluted heparins and disinfectants. Results: Patch tests with disinfectants were negative in all 3 cases. In case 2, all allergological tests were negative. In cases 1 and 3, delayed positivity emerged for nadroparin calcium and at least one other heparin tested. Intravenous and/or subcutaneous provocation testing was done with an alternative heparin which produced negative results in skin tests (heparin sodium in case 1, pentasaccharide fondaparinux in case 3. In both cases the alternative drug was tolerated. After our evaluation, all 3 patients underwent surgery with no heparin-related complications. Discussion: The presenting clinical features in these 3 cases provided no information on which reactions were likely to be allergic: all 3 patients presented with similar local delayed reaction. The allergic reactions were identified only after cutaneous testing.

  5. [Heparin-induced thrombocytopenia: recent data]. (United States)

    Gruel, Y; Rollin, J; Leroux, D; Pouplard, C


    Despite less frequent, heparin-induced thrombocytopenia (HIT) remains a severe complication of treatment with heparin, and is important to diagnose and manage appropriately. HIT results from an atypical immune response to heparin, with the synthesis of IgG antibodies specific to heparin-modified platelet factor 4 (PF4) which activate platelets, leukocytes and the endothelium. This activation explains that low platelet count is associated with thrombotic events in 50% of patients. The diagnosis of HIT is sometimes evoked because of atypical manifestations (i.e. cutaneous necrosis, amnesia, hypotension or dyspnea following intravenous injection of heparin). Biological assays are always necessary to confirm HIT in case of clinical suspicion, and specific rapid tests are now available for detecting anti-PF4 antibodies. However, their specificity is poor and functional assays such as serotonin release assay or platelet aggregation test are often necessary. Argatroban that is a direct antithrombin drug can be used in patients with severe renal failure and will be preferred to danaparoid sodium in this situation. Fondaparinux is not licensed for treating confirmed HIT and can only be used in case of suspicion. The early detection of HIT is based on the monitoring of platelet count recommended in surgical patients receiving a low molecular weight heparin and in all patients treated with unfractionated heparin. Copyright © 2013. Published by Elsevier SAS.

  6. Antithrombin inactivation by neutrophil elastase requires heparin. (United States)

    Jordan, R E; Nelson, R M; Kilpatrick, J; Newgren, J O; Esmon, P C; Fournel, M A


    In certain thrombotic states, large declines in the levels of functional circulating antithrombin occur, which may reflect the highly active nature of the endothelial surface in suppressing excessive amounts of activated coagulation enzymes. Alternatively, we have recently observed an unexpected and paradoxical in vitro functioning of heparin that could result in the inactivation of antithrombin in pathologic conditions. Specifically, antithrombin was rendered nonfunctional as an inhibitor of clotting enzymes as a result of a limited, heparin-dependent cleavage by neutrophil elastase. This inactivation occurred only in the presence of the active anticoagulant heparin fraction, which suggested that the heparin-antithrombin complex was the substrate for elastase attack. Interestingly, neutrophil elastase was found to bind tightly to heparin and heparin-like materials. Neutrophil elastase has been previously linked to nonspecific proteinolysis occurring in inflammatory thrombotic reactions. This affinity of both antithrombin and elastase for heparin suggests a novel mechanism of potential specificity. An important component of this hypothesis is the localization of the elastase/antithrombin reaction away from the high circulating levels of elastase inhibitors. The proposed inactivation of antithrombin on the vascular surface would likely occur only in pathologic states associated with neutrophil sequestration and activation. Nevertheless, this mechanism could lead to a localized reversal of the nonthrombogenic nature of the endothelium and potentially lead to significant reductions of functional antithrombin in certain disease states.

  7. Revisiting the Response Mechanism of Polymeric Membrane Based Heparin Electrodes


    Bell, Andrea K.; Höfler, Lajos; Meyerhoff, Mark E.


    Potentiometric membrane electrodes that respond to heparin and other polyanions were introduced in the early 1990s. Herein, the mechanism of polymer membrane electrode type heparin sensors is revisited. The extraction/diffusion of heparin is studied via both potentiometric and impedance spectroscopic techniques using a pre-fractionated heparin preparation that contains polyanionic species > 10000 Daltons. The reversal in EMF response using this heparin preparation indicates diffusion of highe...

  8. Heparin-induced thrombocytopenia: towards standardization of platelet factor 4/heparin antigen tests. (United States)

    Greinacher, A; Ittermann, T; Bagemühl, J; Althaus, K; Fürll, B; Selleng, S; Lubenow, N; Schellong, S; Sheppard, J I; Warkentin, T E


    Laboratory confirmation of heparin-induced thrombocytopenia (HIT) is based on detection of heparin-dependent platelet-activating antibodies. Platelet factor 4 (PF4)/heparin enzyme-immunoassays (EIA) are a widely available surrogate for platelet-activating antibodies. Defining the optical density (OD) reactivity profiles of a PF4/heparin EIA in reference subject and patient populations and the correlation of the EIA results (expressed in OD units) with the prevalence of platelet-activating antibodies. Using quantile regression we determined the 97.5th percentile of PF4/heparin-immunoglobulin G (IgG) EIA reactivities in non-heparin-treated individuals [blood donors (n = 935)] and patients before heparin therapy (n = 1207). In patients with suspected HIT, we compared the correlation of EIA-IgG reactivities (Greifswald laboratory; n = 2821) and the heparin-induced platelet activation assay (HIPA) with the correlation of reactivities of another EIA-IgG (McMaster laboratory; n = 1956) with the serotonin-release assay (SRA). PF4/heparin-IgG EIA OD reactivities had a lower OD 97.5th percentile in blood donors compared with patient groups before heparin treatment (P 0.9) when normalized OD ranges (maximum OD divided by 10) were used instead of absolute OD values. Results of PF4/heparin-IgG EIA should not be reported as only positive or negative as there is no single acceptable cut-off value. Instead, reporting PF4/heparin-IgG EIA OD results in ranges allows for risk-stratified prediction for presence of platelet-activating antibodies. Use of normalized OD ranges permits a standardized approach for inter-laboratory comparisons. © 2010 International Society on Thrombosis and Haemostasis.

  9. How to give a heparin shot (United States)

    ... this page: // How to give a heparin shot To use the sharing ... nurse or other health professional will teach you how to prepare the medicine and give the shot. The ...

  10. Immune pathogenesis of heparin-induced thrombocytopenia. (United States)

    Khandelwal, Sanjay; Arepally, Gowthami M


    The immune response to heparin is one of the most common drug-induced allergies, and yet, atypical for a drug hypersensitivity reaction. Whereas most drug-induced allergies are rare, idiosyncratic and life-long, the allergic response to heparin is common, predictable in certain clinical settings and transient. Advances in the last decade with regards to structural characterisation of the PF4/heparin antigenic complex, contributions of innate immunity and development of animal models have provided insights into the distinctive features of the HIT immune response. Recent descriptions of the crystal structure of the PF4/heparin complex, alongside other biophysical studies, have clarified the structural requirements for immunogenicity and heparin-dependency of antibody formation. Studies of interactions of PF4 with bacterial cell walls as well as epidemiologic associations of anti-PF4/heparin antibody formation and infection suggest a role for immune priming and explain the rapid evolution of an isotype-switched immune response in sensitised patients. Murine models have greatly facilitated investigations of cellular basis of the HIT response and identified a major role for T-cells and marginal zone B-cells, but key findings have yet to be validated in human disease. This chapter will summarise recent investigations of the HIT immune response in the context of major pathways of immune activation and identify areas of uncertainty.

  11. Strategy for the sequence analysis of heparin. (United States)

    Liu, J; Desai, U R; Han, X J; Toida, T; Linhardt, R J


    The versatile biological activities of proteoglycans are mainly mediated by their glycosaminoglycan (GAG) components. Unlike proteins and nucleic acids, no satisfactory method for sequencing GAGs has been developed. This paper describes a strategy to sequence the GAG chains of heparin. Heparin, prepared from animal tissue, and processed by proteinases and endoglucuronidases, is 90% GAG heparin and 10% peptidoglycan heparin (containing small remnants of core protein). Raw porcine mucosal heparin was labelled on the amino termini of these core protein remnants with a hydrophobic, fluorescent tag [N-4-(6-dimethylamino-2-benzofuranyl) phenyl (NDBP)-isothiocyanate]. Enrichment of the NDBP-heparin using phenyl-Sepharose chromatography, followed by treatment with a mixture of heparin lyase I and III, resulted in a single NDBP-linkage region tetrasaccharide, which was characterized as deltaUAp(1-->3)-beta-D-Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp -(1-->O-Ser-NDBP (deltaUAp is 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid). Several NDBP-octasaccharides were isolated when NDBP-heparin was treated with only heparin lyase I. The structure of one of these NDBP-octasaccharides, deltaUAp2S(1-->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp(1-->3)-beta-D- Galp(1-->3)-beta-D-Galp(1-->4)-beta-Xylp-(1-->O-Ser NDBP (S is sulphate, Ac is acetate), was determined by 1H-NMR and enzymatic methods. Enriched NDBP-heparin was treated with lithium hydroxide to release heparin, and the GAG chain was then labelled at xylose with 7-amino-1,3-naphthalene disulphonic acid (AGA). The resulting AGA-Xyl-heparin was sequenced on gradient PAGE using heparin lyase I and heparin lyase III. A predominant sequence in heparin at the protein core attachment site was deduced to be -D-GlcNp2S6S(or 6OH)(1-->4)-alpha-L-IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S (or60H) (1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNp2S6S( or 6OH)(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha-D-GlcNpAc (1

  12. Early Heparin Administration Reduces Risk for Left Atrial Thrombus Formation during Atrial Fibrillation Ablation Procedures

    Directory of Open Access Journals (Sweden)

    Stefan Asbach


    Full Text Available Objective. Despite the use of anticoagulation during left atrial (LA ablation procedures, ischemic cerebrovascular accidents (CVAs are recognized as a serious complication. Heparin is usually given after safe transseptal access has been obtained, resulting in a short unprotected dwell time of catheters within the LA, which may account for CVAs. We investigated the frequency of CVAs and LA thrombus formation as detected by intracardiac ultrasound (ICE depending on the timing of heparin administration. Methods and Results. Sixty LA ablation procedures with the use of ICE were performed in 55 patients. Patients were grouped by heparin administration after (Group I, =13 and before (Group II, =47 transseptal access. Group I patients were younger (56.6±13.7 versus 65.9±9.9 years, =.01; other clinical and echocardiographic characteristics did not differ between groups. Early thrombus formation was observed in 2 (15.4% of group I patients as compared to 0% of group II patients (=.04. One CVA (2.1% occurred in one group II patient without prior thrombus detection, and none occurred in group I patients (=ns. Conclusion. Early administration of heparin reduces the risk of early intracardiac thrombus formation during LA ablation procedures. This did not result in reduced rate of CVAs.

  13. [Methods of heparin therapy in surgical patients with thrombohemorrhagic syndrome]. (United States)

    Gasanov, F D


    The results of comparative study of different heparin medications efficacy in patients with thrombohemorrhagic syndrome (THS) are presented. The study was conducted in 286 patients with THS as a result of peritonitis of various etiology (174 patients), massive hemorrhage, shock, microcirculation disorders (112 patients). Heparin therapy carried out in 249 patients (87.1%), 37 patients (12.9%) had no heparin therapy./ In "heparin" group 193 patients (77.5%) received low molecular weight heparin (LMWH), 56 patients (22.5%) took unfractionated heparin (UFH). LWMH demonstrated high efficiency with fewer hemorrhagic complications in comparison with UFH/.

  14. Extraction and purification of molybdenum cofactor from milk xanthine oxidase

    NARCIS (Netherlands)

    van Spanning, R J; Wansell-Bettenhaussen, C W; Oltmann, L F; Stouthamer, A.H.


    Molybdenum cofactor (mocofactor) is extracted efficiently, free of impurities and in high concentrations, by acid treatment of xanthine oxidase and subsequent incubation of the precipitate with phosphate buffer containing EDTA, molybdate and oxygen. It is suggested that cofactor is bound to the

  15. Metabolic impact of redox cofactor perturbations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Lages, Nuno; Oldiges, M.


    to induce widespread changes in metabolism. We present a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering. We enhanced NADH...

  16. Kinetics based reaction optimization of enzyme catalyzed reduction of formaldehyde to methanol with synchronous cofactor regeneration. (United States)

    Marpani, Fauziah; Sárossy, Zsuzsa; Pinelo, Manuel; Meyer, Anne S


    Enzymatic reduction of carbon dioxide (CO2 ) to methanol (CH3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced regeneration of the reducing equivalents during reaction is required. Herein, we report the optimization of the enzymatic conversion of formaldehyde (CHOH) to CH3 OH by alcohol dehydrogenase, the final step of the enzymatic redox reaction of CO2 to CH3 OH, with kinetically synchronous enzymatic cofactor regeneration using either glucose dehydrogenase (System I) or xylose dehydrogenase (System II). A mathematical model of the enzyme kinetics was employed to identify the best reaction set-up for attaining optimal cofactor recycling rate and enzyme utilization efficiency. Targeted process optimization experiments were conducted to verify the kinetically modeled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes to high concentrations of CHOH. System II was found to be superior to System I with a yield of 8 mM CH3 OH, a TTN of 160 and BPR of 24 μmol CH3 OH/U · h during 6 hr of reaction. The study demonstrates that an optimal reaction set-up could be designed from rational kinetics modeling to maximize the yield of CH3 OH, whilst simultaneously optimizing cofactor recycling and enzyme utilization efficiency. © 2017 Wiley Periodicals, Inc.

  17. CD/MCD/VTVH-MCD Studies of Escherichia coli Bacterioferritin Support a Binuclear Iron Cofactor Site. (United States)

    Kwak, Yeonju; Schwartz, Jennifer K; Huang, Victor W; Boice, Emily; Kurtz, Donald M; Solomon, Edward I


    Ferritins and bacterioferritins (Bfrs) utilize a binuclear non-heme iron binding site to catalyze oxidation of Fe(II), leading to formation of an iron mineral core within a protein shell. Unlike ferritins, in which the diiron site binds Fe(II) as a substrate, which then autoxidizes and migrates to the mineral core, the diiron site in Bfr has a 2-His/4-carboxylate ligand set that is commonly found in diiron cofactor enzymes. Bfrs could, therefore, utilize the diiron site as a cofactor rather than for substrate iron binding. In this study, we applied circular dichroism (CD), magnetic CD (MCD), and variable-temperature, variable-field MCD (VTVH-MCD) spectroscopies to define the geometric and electronic structures of the biferrous active site in Escherichia coli Bfr. For these studies, we used an engineered M52L variant, which is known to eliminate binding of a heme cofactor but to have very minor effects on either iron oxidation or mineral core formation. We also examined an H46A/D50A/M52L Bfr variant, which additionally disrupts a previously observed mononuclear non-heme iron binding site inside the protein shell. The spectral analyses define a binuclear and an additional mononuclear ferrous site. The biferrous site shows two different five-coordinate centers. After O2 oxidation and re-reduction, only the mononuclear ferrous signal is eliminated. The retention of the biferrous but not the mononuclear ferrous site upon O2 cycling supports a mechanism in which the binuclear site acts as a cofactor for the O2 reaction, while the mononuclear site binds the substrate Fe(II) that, after its oxidation to Fe(III), migrates to the mineral core.

  18. Recurrent left atrial and left ventricular thrombosis due to heparin-induced thrombocytopenia: case report and short review. (United States)

    Kuhl, Thomas; Wendt, Stefanie; Langebartels, Georg; Kröner, Axel; Wahlers, Thorsten


    The combination of heparin-induced thrombocytopenia (HIT) II, left ventricular failure with extracorporeal membrane oxygenation (ECMO) therapy, and recurrent left ventricular thrombosis is rare and predicts a poor outcome. In this case, HIT II occurred after an ischemic cardiogenic shock. We initiated ECMO and intra-aortic balloon pump therapy during coronary artery bypass grafting and ventricular thrombectomy. Despite continued therapeutic therapy with heparin and later argatroban, the patient developed solid and recurrent thrombotic masses in the left atrium and left ventricle. Georg Thieme Verlag KG Stuttgart · New York.

  19. Modulation of the homophilic interaction between the first and second Ig modules of neural cell adhesion molecule by heparin

    DEFF Research Database (Denmark)

    Kulahin, Nikolaj; Rudenko, Olga; Kiselyov, V.


    The second Ig module (IgII) of the neural cell adhesion molecule (NCAM) is known to bind to the first Ig module (IgI) of NCAM (so-called homophilic binding) and to interact with heparan sulfate and chondroitin sulfate glycoconjugates. We here show by NMR that the heparin and chondroitin sulfate......II. Accordingly, we show that treatment of cerebellar granule neurons (CGNs) with heparin inhibits NCAM-mediated outgrowth. In contrast, treatment with heparinase III or chondroitinase ABC abrogates NCAM-mediated neurite outgrowth in CGNs emphasizing the importance of the presence of heparan/chondroitin sulfates...

  20. Recurrent intradialytic heparin induced anaphylaxis: workup and management


    Santosa, Amelia; Tan, Seng Hoe; Cheng, Yew Kuang


    Heparin has been widely used for intradialytic anticoagulation since the 1940s. Heparin induced anaphylaxis can be life threatening, mandating early recognition and intervention. However, due to its relative rarity many physicians remain unaware. We report the case of a 70-year-old woman requiring dialysis, who developed recurrent anaphylaxis to intradialytic heparin. We describe a systematic approach to confirm the suspected heparin allergy, which must include an evaluation of predisposing f...

  1. The Interplay of Cofactor Interactions and Post-translational Modifications in the Regulation of the AAA+ ATPase p97 (United States)

    Hänzelmann, Petra; Schindelin, Hermann


    The hexameric type II AAA ATPase (ATPase associated with various activities) p97 (also referred to as VCP, Cdc48, and Ter94) is critically involved in a variety of cellular activities including pathways such as DNA replication and repair which both involve chromatin remodeling, and is a key player in various protein quality control pathways mediated by the ubiquitin proteasome system as well as autophagy. Correspondingly, p97 has been linked to various pathophysiological states including cancer, neurodegeneration, and premature aging. p97 encompasses an N-terminal domain, two highly conserved ATPase domains and an unstructured C-terminal tail. This enzyme hydrolyzes ATP and utilizes the resulting energy to extract or disassemble protein targets modified with ubiquitin from stable protein assemblies, chromatin and membranes. p97 participates in highly diverse cellular processes and hence its activity is tightly controlled. This is achieved by multiple regulatory cofactors, which either associate with the N-terminal domain or interact with the extreme C-terminus via distinct binding elements and target p97 to specific cellular pathways, sometimes requiring the simultaneous association with more than one cofactor. Most cofactors are recruited to p97 through conserved binding motifs/domains and assist in substrate recognition or processing by providing additional molecular properties. A tight control of p97 cofactor specificity and diversity as well as the assembly of higher-order p97-cofactor complexes is accomplished by various regulatory mechanisms, which include bipartite binding, binding site competition, changes in oligomeric assemblies, and nucleotide-induced conformational changes. Furthermore, post-translational modifications (PTMs) like acetylation, palmitoylation, phosphorylation, SUMOylation, and ubiquitylation of p97 have been reported which further modulate its diverse molecular activities. In this review, we will describe the molecular basis of p97

  2. Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

    Directory of Open Access Journals (Sweden)

    Wagner A S Judice

    Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

  3. Economic evaluation of low-molecularweight heparin in the ...

    African Journals Online (AJOL)

    Objective. To undertake an economic evaluation of the administration and monitoring costs of the two different forms of heparin in patients with unstable coronary artery disease (DCAD). Study design. Equivalent efficacy was found for lowmolecular- weight heparin (LMWH) and for unfractionated heparin (UFH) in the ...

  4. The suicide substrate reaction between plasminogen activator inhibitor 1 acid thrombin is regulated by the cofactors vitronectin and heparin

    NARCIS (Netherlands)

    van Meijer, M.; Smilde, A.; Tans, G.; Nesheim, M. E.; Pannekoek, H.; Horrevoets, A. J.


    The interaction of thrombin with plasminogen activator inhibitor 1 (PAI-1) is shown to result in the simultaneous formation of both cleaved PAI-1 and a sodium dodecyl sulfate-stable thrombin-PAI-1 complex. The kinetics of this reaction can be described by a ''suicide substrate'' mechanism that

  5. Antiproliferative heparin (glycosaminoglycans) isolated from giant ...

    African Journals Online (AJOL)



    May 18, 2009 ... source of these sulfated polysaccharides (Nader and. Dietrich, 1989) and it often corresponds up to 90% of the total GAG content of these organisms. Heparin and heap- rin-like substances have a wide range of important biolo- gical activities including inhibition of pulmonary artery smooth muscle cell ...

  6. Heparin-Induced Thrombocytopenia Antibody Test (United States)

    ... High-sensitivity C-reactive Protein (hs-CRP) Histamine Histone Antibody HIV Antibody and HIV Antigen (p24) HIV ... Sources Used in Previous Reviews (© 1995-2011). Unit Code 81904: Heparin-PF4 Antibody (HIT), Serum. Mayo Clinic ...

  7. Revisiting the Response Mechanism of Polymeric Membrane Based Heparin Electrodes. (United States)

    Bell, Andrea K; Höfler, Lajos; Meyerhoff, Mark E


    Potentiometric membrane electrodes that respond to heparin and other polyanions were introduced in the early 1990s. Herein, the mechanism of polymer membrane electrode type heparin sensors is revisited. The extraction/diffusion of heparin is studied via both potentiometric and impedance spectroscopic techniques using a pre-fractionated heparin preparation that contains polyanionic species > 10000 Daltons. The reversal in EMF response using this heparin preparation indicates diffusion of higher MW heparin fragments to the backside of the membrane. Diffusion coefficients are calculated using a novel formula derived from the phase boundary potential model and Fick's second law of diffusion. Impedance spectroscopy is also employed to show that high MW heparin species are extracted and diffuse across the PVC membranes.

  8. Enzyme cofactors: Double-edged sword for catalysis (United States)

    Ivanov, Ivaylo


    The metal cofactors responsible for the activity of CDK2 -- a representative member of the kinase superfamily of enzymes -- have now been shown to also have inhibitory effects during the catalytic cycle.

  9. Metabolic and Transcriptional Response to Cofactor Perturbations in Escherichia coli

    DEFF Research Database (Denmark)

    Holm, Anders Koefoed; Blank, L.M.; Oldiges, M.


    Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli...... of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli....

  10. Heparin-binding proteins of human seminal plasma: purification and characterization. (United States)

    Kumar, Vijay; Hassan, Md Imtaiyaz; Kashav, Tara; Singh, Tej P; Yadav, Savita


    Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity-chromatography on Heparin-Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield. (c) 2008 Wiley-Liss, Inc.

  11. Structural and binding studies of SAP-1 protein with heparin. (United States)

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


    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.

  12. What's in a covalent bond? On the role and formation of covalently bound flavin cofactors

    NARCIS (Netherlands)

    Heuts, Dominic P. H. M.; Scrutton, Nigel S.; McIntire, William S.; Fraaije, Marco W.

    Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to

  13. Cofactor requirement of HpyAV restriction endonuclease.

    Directory of Open Access Journals (Sweden)

    Siu-Hong Chan

    Full Text Available BACKGROUND: Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M systems in microorganisms. PRINCIPAL FINDINGS: We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. CONCLUSIONS/SIGNIFICANCE: Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  14. Cofactor requirement of HpyAV restriction endonuclease. (United States)

    Chan, Siu-Hong; Opitz, Lars; Higgins, Lauren; O'loane, Diana; Xu, Shuang-Yong


    Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg(++). The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

  15. Kinetics based reaction optimization of enzyme catalysed reduction of formaldehyde to methanol with synchronous cofactor regeneration

    DEFF Research Database (Denmark)

    Marpani, Fauziah Binti; Sárossy, Zsuzsa; Pinelo, Manuel


    Enzymatic reduction of carbon dioxide (CO2 ) to methanol (CH3 OH) can be accomplished using a designed set-up of three oxidoreductases utilizing reduced pyridine nucleotide (NADH) as cofactor for the reducing equivalents electron supply. For this enzyme system to function efficiently a balanced...... experiments were conducted to verify the kinetically modelled results. Repetitive reaction cycles were shown to enhance the yield of CH3 OH, increase the total turnover number (TTN) and the biocatalytic productivity rate (BPR) value for both system I and II whilst minimizing the exposure of the enzymes...

  16. Is nitric oxide and heparin treatment justified in inflammatory bowel disease? An experimental study. (United States)

    Dobosz, M; Mionskowska, L; Dobrowolski, S; Dymecki, D; Makarewicz, W; Hrabowska, M; Wajda, Z


    Microcirculatory disturbances of the colon may contribute to the pathogenesis of inflammatory bowel disease. The aim of the study was to investigate the alterations of rectal blood perfusion in experimental colitis with reference to nitric oxide and heparin treatment. The study was carried out on 36 rats, divided into six groups: group I, control; group II, control + NG-nitro-L-arginine (L-NNA); group III, colitis without treatment; group IV, colitis + L-arginine; group V, colitis + L-NNA; group VI, colitis + heparin treatment. Experimental colitis was induced by 4% acetic acid enema, and 48 h after the enema, besides the measurement of rectal capillary blood flow by means of laser Doppler flowmetry, the serum interleukin-6 (IL-6) level and histopathological alterations within the rectal mucosa were examined. Experimental colitis resulted in a drop in rectal wall perfusion. L-Arginine and heparin treatment improved the microcirculatory values. The highest IL-6 level and the most advanced histopathological alterations were observed in the rats treated with L-NNA. L-Arginine treatment had no influence on IL-6 concentration, however it aggravated the inflammatory changes within the rectal mucosa. Heparin administration reduced the IL-6 values and also had a positive impact on the microscopic alterations within the rectal wall. It is concluded that heparin treatment has a beneficial effect on the microcirculatory disturbances and inflammatory changes observed in experimental colitis. The inhibition of nitric oxide-synthase aggravated the course of experimental colitis. L-Arginine administration improves the rectal blood flow but aggravates the histopathological alterations within the rectal wall.

  17. Heparin-conjugated polyethylenimine for gene delivery. (United States)

    Jeon, Oju; Yang, Hee Seok; Lee, Tae-Jin; Kim, Byung-Soo


    A major problem when using cationic polymers for gene delivery is that transfection is strongly inhibited by the presence of serum. This shortcoming limits the application of cationic polymers for systematic gene delivery in vivo. Due to the shielding effect of heparin, heparin conjugation to cationic polymers may improve the in vivo gene transfection efficiency. In this study, the transfection efficiency of heparin-conjugated polyethylenimine (HCPEI) with a low molecular weight of 1800 Da was compared to the transfection efficiencies of polyethylenimine with a low molecular weight of 1800 Da (PEI1800), polyethylenimine with a high molecular weight of 25,000 Da (PEI25k), and Lipofectamine. The size of the HCPEI/plasmid DNA (pDNA) complex is approximately 250 nm. HCPEI has a proton-buffering effect and HCPEI/pDNA has higher blood compatibility and a lower cytotoxicity than PEI25k/pDNA and Lipofectamine/pDNA. For in vitro transfection of rabbit smooth muscle cells in serum-free medium, the transfection efficiency of HCPEI/pDNA was not significantly different from those of PEI25k/pDNA and Lipofectamine/pDNA. Importantly, in serum-containing medium, the transfection efficiency of HCPEI/pDNA was significantly higher than those of PEI25k/pDNA and Lipofectamine/pDNA. For vascular endothelial growth factor (VEGF) gene transfection to mouse ischemic limbs, HCPEI/pDNA exhibited significantly higher VEGF expression and more extensive neovascularization than PEI/pDNA and Lipofectamine/pDNA. Taken together, heparin conjugation to PEI improves the in vivo gene transfection efficiency of PEI.

  18. The Effect of Heparin on the Carbon Tetrachloride Induced Changes in Rat Testis

    Directory of Open Access Journals (Sweden)

    Elçin Hakan Terzi


    Full Text Available Objectives: To investigate the preventive effects of low molecular weight heparin on testis destruction created by low dose (0.25 ml/kg carbon tetrachloride (CCl4application.    Materials and Methods: Sixteen Spraque Dawley male rats were used. CCl4 induced testicular destruction was created. There were 4 rats in each group; Group I recieved 1 ml of intraperitoneal olive oil, Group recieved intraperitoneal 0,25ml/kg CCL4 in1 ml of olive oil, Group III recieved intraperitoneal 0,25ml/kg CCL4 in1 ml of olive oil plus subcutaneous 180 IU/kg low molecular weight heparin and Grup IV received only subcutaneous 180 IU/kg low molecular weight heparin. At the end of 4 weeks of study period, rats were sacrified and tissue samples were fixed in glutaraldehyde solution.Results: There was no statistically significant difference between the groups in terms of testis weight (p>0.05. Light microscopy showed vacuolization in the basal portion of the seminiferous tubules in Group II. Intercellular dehiscence between the spermatogenetic cells was significant in Group II compared to control group at the level of fine structure. Intercellular dehiscence was not significant in Group III compared to Group II. The seminiferous tubules structures in Group IV were found comparable with the control group. Low dose of CCl4 caused germ cell loss, inhibition of mitosis, and structural deterioration of the Sertoli cells in seminiferous tubules of rat testes. Conclusions: Low molecular weight heparin was found to be effective preventing CCl4 induced testis destruction.  

  19. A spectroscopic study of interaction of cationic dyes with heparin

    Directory of Open Access Journals (Sweden)

    R. Nandini


    Full Text Available The interaction of two cationic dyes namely, acridine orange and pinacyanol chloride with an anionic polyelectrolyte, heparin, has been investigated by spectrophotometric method.The polymer induced metachromasy in the dyes resulting in the shift of the absorption maxima of the dyes towards shorter wavelengths. The stability of the complexes formed between acridine orange and heparin was found to be lesser than that formed between pinacyanol chloride and heparin. This fact was further confirmed by reversal studies using alcohols, urea and surfactants. The interaction of acridine orange with heparin has also been investigated fluorimetrically.The interaction parameters revealed that binding between acridine orange and heparin arises due to electrostatic interaction while that between pinacyanol chloride and heparin is found to involve both electrostatic and hydrophobic forces. The effect of the structure of the dye in inducing metachromasy has also been discussed.

  20. Electrophoresis for the analysis of heparin purity and quality (United States)

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


    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

  1. Heparin Increases Food Intake through AgRP Neurons

    Directory of Open Access Journals (Sweden)

    Canjun Zhu


    Full Text Available 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 treatment increases food intake and body weight gain. By using electrophysiological, pharmacological, molecular biological, and chemogenetic approaches, we provide evidence that heparin increases food intake by stimulating AgRP neurons and increasing AgRP release. Our results support a model whereby heparin competes with insulin for insulin receptor binding on AgRP neurons, and by doing so it inhibits FoxO1 activity to promote AgRP release and feeding. Heparin may be a potential drug target for food intake regulation and body weight control.

  2. Heparin monitoring: From blood tube to microfluidic device


    Harris, L. F.; Killard, A.


    Heparin anticoagulant therapy has been pivotal in both the treatment and prophylaxis of thrombotic disease for many decades. It remains standard practice to monitor unfractionated heparin (UFH) therapy due to its unpredictable pharmacokinetics. The advent of low molecular weight heparins (LMWHs) reduced the need for continuous laboratory monitoring due to the improved dose-response relationships and pharmacokinetics of these drugs. However, special patient cohorts exist where monitoring becom...

  3. Dual chain synthetic heparin-binding growth factor analogs

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  4. Inhibitory Effect of Heparin on Gentamicin Concentrations in Blood (United States)

    Regamey, Claude; Schaberg, Dennis; Kirby, William M. M.


    In monitoring gentamicin concentrations in the blood of patients with renal insufficiency, the assayed antibiotic concentration was found to be lower when the sample was drawn as heparinized plasma rather than as serum. This lowering of gentamicin concentrations by heparin was studied further by adding increasing doses of heparin and various amounts of gentamicin to human serum. With a range of 2 to 100 units of heparin per ml, gentamicin concentrations in the serum were lowered by 9 to 14%; with higher heparin concentrations, an even greater and increasing inhibition was noticed, reaching 56% for 1,000 units/ml. This inhibitory effect of heparin on gentamicin was reversible by dilution, indicating that it was not due to degradation or to formation of an inactive chemical complex. Underestimation by the laboratory of gentamicin concentrations in blood is likely to be greatest with capillary tubes, with which the concentration of heparin is especially high. With clinical heparinization, the amount of active heparin in the blood does not exceed 10 units/ml and is for the most part under 3 units/ml; thus, therapeutically significant inhibition of the antibiotic is unlikely in patients receiving anticoagulation. PMID:4670696

  5. Dual chain synthetic heparin-binding growth factor analogs (United States)

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


    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.

  6. Does ′heparin-induced thrombocytopenia′ hit our minds?

    Directory of Open Access Journals (Sweden)

    Arun R Thangavel


    Full Text Available Unfractionated heparin is a widely used drug to prevent deep vein thrombosis and pulmonary emboli in patients at risk. With the advent of newer anticoagulants having lesser side effects, its use has diminished but not out of service. Here, we report a case of deep venous thrombosis, in a patient on prophylactic dose of heparin, which was later found to be a manifestation of heparin-induced thrombocytopenia (HIT. Thrombosis in the presence of heparin prophylaxis should be considered as HIT rather than a failure of anticoagulation.

  7. Heparin in the treatment of burns: a review. (United States)

    Saliba, M J


    Burns are difficult to treat, wounds with complex local and systemic pathology and high mortality, that often heal slowly with scars and contractures. Glycosaminoglycans (GAGs) have been used in parenteral and topical application studies. These studies have uncovered anticoagulative, antiinflammatory and neoangiogenic properties, which may stimulate tissue repair and reepithelializing effects. The endogenous GAGs utilized in treating burns are heparin, dermatan sulfate, heparan sulfate, keratin sulfate, chondroitin-4- and chondroitin-6-sulfate, and hyaluronic acid. Heparin, the most sulfated and acidic GAG, has been used parenterally, topically, by inhalation, in pellet, and in bioengineered membranes. Heparin relieved pain, inhibited clotting and inflammation, restored blood flow, and enhanced healing. Heparin effects that improved and reduced burn care were time, dose, pH, site, source and duration related in studies. Potential adverse effects with heparin use are bleeding, thrombocytopenia and allergy. Heparin preserved lung and improved function. Heparin preserved intestinal integrity and reduced bacterial translocation. Collagen restoration was enhanced. The healed skin was smooth. Heparin reduced needs for pain medicine, topical antibiotics, resuscitation fluids, blood, water baths, debridement, surgery and grafts. Cost of treatments were reduced. Although not as yet fully substantiated, topical heparin therapy of burns may be a useful addition to the range of available treatments for burn wounds.

  8. Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

    DEFF Research Database (Denmark)

    Henriksen, Jens; Roepstorff, Peter; Ringborg, Lene H.


    Heparin and heparin-derived components are widely applied anticoagulant drugs used for amongst other applications medical treatment of deep vein thrombosis and pulmonary embolism. Depolymerisation of native heparin results in complex mixtures of sulfated linear oligosaccharides that are usually...

  9. Emerging roles for tubulin folding cofactors at the centrosome (United States)

    Fanarraga, Mónica López; Carranza, Gerardo; Castaño, Raquel; Jiménez, Victoria; Villegas, Juan Carlos


    Despite its fundamental role in centrosome biology, procentriole formation, both in the canonical and in the de novo replication pathways, remains poorly understood, and the molecular components that are involved in human cells are not well established. We found that one of the tubulin cofactors, TBCD, is localized at centrosomes and the midbody, and is required for spindle organization, cell abscission, centriole formation and ciliogenesis. Our studies have established a molecular link between the centriole and the midbody, demonstrating that this cofactor is also necessary for microtubule retraction during cell abscission. TBCD is the first centriolar protein identified that plays a role in the assembly of both “centriolar rosettes” during early ciliogenesis, and at the procentriole budding site by S/G2, a discovery that directly implicates tubulin cofactors in the cell division, cell migration and cell signaling research fields. PMID:20798813

  10. Molybdenum cofactor deficiency mimics cerebral palsy: differentiating factors for diagnosis. (United States)

    Kikuchi, Kenjiro; Hamano, Shin-ichiro; Mochizuki, Hiroshi; Ichida, Kimiyoshi; Ida, Hiroyuki


    We describe an infant with molybdenum cofactor deficiency, initially diagnosed as cerebral palsy. Clinical features of molybdenum cofactor deficiency, e.g., neonatal seizures, hypertonus/hypotonus, and feeding and respiratory difficulties, resemble those of neonatal hypoxic-ischemic encephalopathy. Our patient, a 2-year-old boy, presented with spastic quadriplegia and mental retardation. He manifested intractable neonatal seizures and diffuse cerebral atrophy. When admitted with bronchitis at age 18 months, his uric acid levels in blood and urine were undetectable. A urinary sulfite test revealed positive results. Further tests revealed elevated urinary levels of xanthine, hypoxanthine, and S-sulfocystein. Sequencing of the MOCS2A gene revealed heterozygosity for c.[265T>C] + [266A>G], diagnosed as molybdenum cofactor deficiency type B. Neonatal seizures, progressive cerebral atrophy, and low serum levels of uric acid may provide diagnostic clues in patients with cerebral palsy of undetermined cause. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. [Xanthine oxidase deficiency (hereditary xanthinuria), molybdenum cofactor deficiency]. (United States)

    Sumi, S; Wada, Y


    Hereditary xanthinuria is a rare autosomal recessive disorder, with xanthine oxidase deficiency. Patients often display renal symptoms because they excrete a large amounts of xanthine in urine. An high-fluid-intake, alow-purine-food, and alkalinization of urine are effective in the patients. Molybdenum cofactor is essential for xanthine oxidase, sulfite oxidase and aldehyde oxidase. Patients with molybdenum cofactor deficiency display severe neurological symptoms, such as severe convulsions. The patients increase urinary excretions of xanthine and sulfite. Treatments are ineffective for neurological symptoms.

  12. The oxidation product of molybdenum cofactor from milk xanthine oxidase

    NARCIS (Netherlands)

    van Spanning, R J; Wansell-Bettenhaussen, C W; Oltmann, L F; Stouthamer, A.H.

    In extracts of acid treated molybdenum cofactor containing xanthine oxidase, fluorescence is maximally developed upon a three hours incubation. Analysis by means of reversed phase HPLC revealed the presence of several fluorescent compounds, the main one being a blue fluorescent compound with an

  13. Structure-Based Redesign of Cofactor Binding in Putrescine Oxidase

    NARCIS (Netherlands)

    Kopacz, Malgorzata M.; Rovida, Stefano; van Duijn, Esther; Fraaije, Marco W.; Mattevi, Andrea


    Putrescine oxidase (PuO) from Rhodococcus erythropolis is a soluble homodimeric Flavoprotein, which oxidizes small aliphatic diamines. In this study, we report the crystal structures and cofactor binding properties of wild-type and mutant enzymes. From a structural viewpoint, PuO closely resembles

  14. Insight into cofactor recognition in arylamine N-acetyltransferase enzymes

    DEFF Research Database (Denmark)

    Xu, Ximing; Li de la Sierra-Gallay, Inés; Kubiak, Xavier Jean Philippe


    for Bacillus anthracis NAT1 and Homo sapiens NAT2. Therefore, in contrast to previous data, this study shows that different orthologous NATs can bind their cofactors in a similar way, suggesting that the mode of binding CoA in this family of enzymes is less diverse than previously thought. Moreover...

  15. [Therapeutic indications of low molecular weight heparins]. (United States)

    Samama, M M; Michaut-Paterno, F


    The depolymerisation of the various chains of unfractionated heparin (UFH) by chemical or enzymatic reactions provides so-called low molecular weight heparin (LMWH), with an average molecular weight of approximately 5000 daltons. The specific biological and pharmacokinetic properties of LMWH with greater inhibition of factor Xa than of thrombin activity, less interaction with platelets, better bioavailability and a longer half life of anti-Xa activity, suggest possible new therapeutic applications. The hypothesis of reducing the risk of haemorrhage related to the antithrombin activity and the incidence of heparin-induced thrombocytopenia whilst preserving effective antithrombotic action has stimulated clinical and biological research. Clinical trials of prophylaxis of venous thrombo-embolism have been undertaken mainly in surgical patients. The results have shown identical if not better efficacy of LMWH compared to UFH in general surgical and above all orthopedic patients in whom subcutaneous heparin is only effective with a strict protocol which is difficult to adhere to in routine practice (adaptation of dosage to activated partial thromboplastin time). The risk of bleeding was not significantly lower using LMWH at the specified dosage, which in the latter indication, is twice that used in general surgery. There are many indications of prophylaxis of thromboembolism in the medical specialties but, paradoxically, LMWH has not been widely studied because of the difficulties in performing the therapeutic trials. Except in rare cases (extreme body weights, renal failure, haemorrhagic disease, thrombotic or haemorrhagic complications) the evaluation of amidolytic anti-Xa activity does not seem to be necessary. More recently, LMWH has been studied in a small number of trials for the treatment of deep venous thrombosis (DVT). The therapeutic efficacy is identical if not better than that of UFH without increasing the risk of bleeding. Biological monitoring seems to be

  16. Heparin Leakage in Central Venous Catheters by Hemodynamic Transport (United States)

    Barbour, Michael; McGah, Patrick; Gow, Kenneth; Aliseda, Alberto


    Central venous catheters (CVCs), placed in the superior vena cava for hemodialysis, are routinely filled with heparin, an anticoagulant, while not in use to maintain patency and prevent thrombus formation at the catheter tip. However, the heparin-lock procedure places the patient at risk for systemic bleeding incidences, as heparin is known to leak into the blood stream. We propose that the driving mechanism behind heparin leakage is advective-diffusive transport due to the pulsatile blood flow surrounding the catheter tip. This novel hypothesis is based on Planar Laser Induced Fluorescence (PLIF) measurements of heparin transport from a CVC placed inside an in vitro pulsatile flow loop and validated with CFD simulations. The results show an initial, fast (catheter lumen, where concentration is still high, that is insufficient at replenishing the lost heparin at the tip. These results, which estimate leakage rates consistent with published in vivo data, predict that the concentration of heparin at the catheter tip is effectively zero for the majority of the interdialytic phase, rendering the heparin lock ineffective.

  17. Anticoagulant effect of low molecular weight heparin on central ...

    African Journals Online (AJOL)

    Purpose: To analyse the effect of low molecular weight heparin on venous catheters in haemodialysis patients. Methods: This study included 140 eligible patients who were randomly and evenly divided into two groups, viz, a study group that received low molecular weight heparin and a control group that received ...

  18. 99m Tc-labeled heparin test in orthopaedic surgery

    Energy Technology Data Exchange (ETDEWEB)

    Bouvier, J.F.; Lafon, J.C.; Colin, M.; Chatelut, J.; Beaubatie, F. (Hopital Universitaire Dupuytren, Limoges (France))


    99m Tc-labeled heparin test was performed for early detection of phlebitis or pulmonary embolism after orthopaedic prothesis. Heparinic treatment and surgery per se were demonstrated to have no effect on the results. If this test demonstrates a statistical difference for pathologic patients, it is of greater value to consider ratio between rates before and after intervention.

  19. Citrate anticoagulation for CRRT in children: comparison with heparin. (United States)

    Fernández, Sara Nicole; Santiago, Maria José; López-Herce, Jesús; García, Miriam; Del Castillo, Jimena; Alcaraz, Andrés José; Bellón, Jose María


    Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P = 0.028). 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I) of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  20. Citrate Anticoagulation for CRRT in Children: Comparison with Heparin

    Directory of Open Access Journals (Sweden)

    Sara Nicole Fernández


    Full Text Available Regional anticoagulation with citrate is an alternative to heparin in continuous renal replacement therapies, which may prolong circuit lifetime and decrease hemorrhagic complications. A retrospective comparative cohort study based on a prospective observational registry was conducted including critically ill children undergoing CRRT. Efficacy, measured as circuit survival, and secondary effects of heparin and citrate were compared. 12 patients on CRRT with citrate anticoagulation and 24 patients with heparin anticoagulation were analyzed. Median citrate dose was 2.6 mmol/L. Median calcium dose was 0.16 mEq/kg/h. Median heparin dose was 15 UI/kg/h. Median circuit survival was 48 hours with citrate and 31 hours with heparin (P=0.028. 66.6% of patients treated with citrate developed mild metabolic alkalosis, which was directly related to citrate dose. There were no cases of citrate intoxication: median total calcium/ionic calcium index (CaT/I of 2.16 and a maximum CaT/I of 2.33, without metabolic acidosis. In the citrate group, 45.5% of patients developed hypochloremia and 27.3% hypomagnesemia. In the heparin group, 27.8% developed hypophosphatemia. Three patients were moved from heparin to citrate to control postoperatory bleeding. In conclusion citrate is a safe and effective anticoagulation method for CRRT in children and it achieves longer circuit survival than heparin.

  1. Increased accuracy in heparin and protamine administration decreases bleeding

    DEFF Research Database (Denmark)

    Runge, Marx; Møller, Christian H; Steinbrüchel, Daniel A


    Three to 5 percent of the patients undergoing cardiac surgery are reoperated because of bleeding. When a surgical cause can be excluded, heparin/protamine mismatch may be considered. Insufficient reversal of heparin and overdosing of protamine may cause postoperative bleeding. The purpose of the ...

  2. Heparin-containing block copolymers; Part I: Surface characterization

    NARCIS (Netherlands)

    Vulić, I.; Pijpers, A.P.; Okano, T.; Kim, S.W.; Feijen, Jan


    Newly synthesized heparin-containing block copolymers, consisting of a hydrophobic block of polystyrene (PS), a hydrophilic spacer-block of poly(ethylene oxide) (PEO) and covalently bound heparin (Hep) as bioactive block, were coated on aluminium, glass, polydimethylsiloxane (PDMS), PS or Biomer

  3. ORIGINAL ARTICLES Endogenous heparin levels in the controlled ...

    African Journals Online (AJOL)

    the hypothesis that asthmatic patients have lower levels of circulating endogenous heparin than healthy ... of stimuli in guinea pig and rat models of allergic asthma, as well as in asthmatic patients.3 .... 2008; 585: 375-384. 4. Ceyhan B, Celikel T. Effect of inhaled heparin on methacholine-induced bronchial hyperreactivity.

  4. Release of macromolecules from albumin-heparin microspheres

    NARCIS (Netherlands)

    Kwon, Glen S.; Bae, You Han; Cremers, H.F.M.; Cremers, Harry; Feijen, Jan; Kim, Sung Wan


    Hydrophilic microspheres based on albumin-heparin conjugates have been prepared as a macromolecular delivery system. The soluble albumin-heparin conjugate was synthesized and crosslinked in a water-in-oil emulsion with glutaraldehyde to form microspheres in the same manner as for albumin microsphere

  5. Anticoagulant effect of low molecular weight heparin on central ...

    African Journals Online (AJOL)

    Thus, low molecular weight heparin seems to be more suitable as an anticoagulant solution in patients with venous catheters. Keywords: Low molecular weight heparin, Haemodialysis, Central venous catheter, Vascular access. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), ...

  6. Voltammetric determination of heparin based on its interaction with

    African Journals Online (AJOL)


    For example, Jiao et al. investigated the interaction between some cationic dyes such as azure A, methylene blue and azure B with heparin by absorption spectrophotometry [13]. Sun et al. applied brilliant cresyl blue and neutral red for spectrophotometric determination of heparin [14,. 15]. Liu et al. established a resonance ...

  7. A systematic review of heparin to treat burn injury. (United States)

    Oremus, Mark; Hanson, Mark D; Whitlock, Richard; Young, Edward; Archer, Carolyn; Dal Cin, Arianna; Gupta, Alok; Raina, Parminder


    This systematic review was conducted to assess the evidence for using heparin to treat burn injury. The following databases were searched for relevant studies: MEDLINE, EMBASE, CINAHL, The Cochrane Central Database of Controlled Trials, Web of Science, and BIOSIS. Additional searches involved the reference lists of included studies, the "grey " literature (eg, government reports), and consultations with experts to obtain unpublished manuscripts. Included studies were summarized descriptively and in tabular form, and assessed for methodological quality. A metaanalysis was conducted to obtain a summary estimate for the association between heparin use and postburn mortality. Nine studies were abstracted and included in the review. Five studies contained adult and pediatric patients, one contained adults only, and three contained pediatric patients only. Burn etiologies included flame, scald, thermal, or smoke inhalation. Heparin administration was done topically, subcutaneously, intravenously, or via aerosol. Heparin was reported to have a beneficial impact on mortality, graft and wound healing, and pain control. For mortality, the overall estimate (relative risk) of heparin's effect was 0.32 (95% confidence interval = 0.18-0.57). Heparin's reported benefits may be severely biased because the abstracted studies were beset by poor methodological quality (eg, inadequate definitions of treatment and outcome, no control of confounding). Given poor study quality, there is no strong evidence to indicate that heparin can improve clinical outcomes in the treatment of burn injury. Further research is needed to assess the clinical utility of using heparin in the treatment of burn injury.

  8. Prophylaxis of postoperative thromboembolism with low molecular weight heparins

    DEFF Research Database (Denmark)

    Jørgensen, L N; Wille-Jørgensen, P; Hauch, O


    to placebo or dextran and at least as efficient as unfractionated heparin in the prevention of deep vein thrombosis (DVT). Compared with unfractionated heparin, one of the LMWH preparations significantly reduced the total incidence of DVT. The rate of non-fatal pulmonary embolism was 0.49 per cent...

  9. [Delayed-type hypersensitivity to heparin: diagnosis and therapeutic management]. (United States)

    Nosbaum, A; Pralong, P; Rozieres, A; Dargaud, Y; Nicolas, J-F; Bérard, F


    Heparin is widely used as an anticoagulant and is indicated in the prevention and treatment of thromboembolic disorders. Heparin-induced delayed-type hypersensitivity presents as eczematous lesions, either at the injection site or generally, and affects 7.5% of patients on heparin. This poses diagnostic and therapeutic issues, since an alternative anticoagulant treatment is essential and the risk of cross-reactivity may be as high as 80%, depending on the type of heparin used. If delayed-type hypersensitivity is suspected, heparin-induced thrombocytopenia must first be ruled out, and heparin should be stopped. Fondaparinux is currently the first-line alternative, with a risk of cross-reactivity estimated at only 10%. The switch from a low-molecular-weight heparin (LMWH) to another LMWH is no longer recommended. The use of unfractionated heparin, danaparoid or hirudin may be warranted in the event of recurrence with fondaparinux, and an immuno-allergological work-up is needed to specify the exact profile of cross-allergies. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  10. Three different chromogenic methods do not give equivalent anti-Xa levels for patients on therapeutic low molecular weight heparin (dalteparin) or unfractionated heparin. (United States)

    Kovacs, M J; Keeney, M; MacKinnon, K; Boyle, E


    In this study we compare three chromogenic methods (IL-Heparin, Stachrom Heparin and Heparin Sigma) on two different instruments (ACL300+ and AMAX CS190) for patients on dalteparin (n = 41), a low molecular weight heparin or unfractionated heparin (n = 50). For dalteparin the mean anti-Xa levels for IL-Heparin, Stachrom Heparin and Heparin Sigma were 0.27, 0.30 and 0.21 U/ml, respectively, while for heparin they were 0.52, 0.55 and 0.41 U/ml, respectively. To test for instrument specific effects, IL-Heparin and Stachrom Heparin were repeated on both instruments on 42 patients receiving unfractionated heparin. For IL-Heparin the mean anti-Xa levels on the AMAX CS190 and ACL300+ were 0.51 and 0.59 U/ml, respectively, while for Stachrom Heparin they were 0.55 and 0.67 anti-Xa U/ml. We conclude that different chromogenic anti-Xa methods do not give equivalent anti-Xa levels for the same samples. Moreover, the differences are clinically significant. This is not explained entirely by instrumentation effects. Recommended therapeutic ranges may need to be method and instrument specific.

  11. Hairy transcriptional repression targets and cofactor recruitment in Drosophila.

    Directory of Open Access Journals (Sweden)

    Daniella Bianchi-Frias


    Full Text Available Members of the widely conserved Hairy/Enhancer of split family of basic Helix-Loop-Helix repressors are essential for proper Drosophila and vertebrate development and are misregulated in many cancers. While a major step forward in understanding the molecular mechanism(s surrounding Hairy-mediated repression was made with the identification of Groucho, Drosophila C-terminal binding protein (dCtBP, and Drosophila silent information regulator 2 (dSir2 as Hairy transcriptional cofactors, the identity of Hairy target genes and the rules governing cofactor recruitment are relatively unknown. We have used the chromatin profiling method DamID to perform a global and systematic search for direct transcriptional targets for Drosophila Hairy and the genomic recruitment sites for three of its cofactors: Groucho, dCtBP, and dSir2. Each of the proteins was tethered to Escherichia coli DNA adenine methyltransferase, permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells, as well as 155 loci recruiting Groucho, 107 loci recruiting dSir2, and wide genomic binding of dCtBP to 496 loci. We also adapted DamID profiling such that we could use tightly gated collections of embryos (2-6 h and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box-containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets in both the Kc cell and embryo DamID experiments corresponds to Hairy binding sites in vivo on polytene chromosomes. Similarly, the distributions of loci recruiting each of Hairy's cofactors are detected as cofactor binding sites in vivo on polytene chromosomes. We have identified 59 putative transcriptional targets of Hairy. In addition to finding putative targets for

  12. Cefotaxime-heparin lock prophylaxis against hemodialysis catheter-related sepsis among Staphylococcus aureus nasal carriers

    Directory of Open Access Journals (Sweden)

    Anil K Saxena


    Full Text Available Staphylococcus aureus nasal carriers undergoing hemodialysis (HD through tunneled cuffed catheters (TCCs form a high-risk group for the development of catheter-related bloodstream infections (CRBSI and ensuing morbidity. The efficacy of antibiotic-locks on the outcomes of TCCs among S. aureus nasal carriers has not been studied earlier. Persistent nasal carriage was defined by two or more positive cultures for methicillin-susceptible (MSSA or methicillin-resistant (MRSA S. aureus of five standardized nasal swabs taken from all the participants dialyzed at a large out-patient HD center affiliated to a tertiary care hospital. Of 218 participants, 82 S. aureus nasal carriers dialyzed through TCCs (n = 88 were identified through April 2005 to March 2006 and randomized to two groups. Group I comprised of 39 nasal carriers who had TCCs (n = 41 "locked" with cefotaxime/heparin while group II included 43 patients with TCCs (n = 47 filled with standard heparin. The CRBSI incidence and TCC survival at 365 days were statistically compared between the two groups. A significantly lower CRBSI incidence (1.47 vs. 3.44/1000 catheter-days, P <0.001 and higher infection-free TCC survival rates at 365 days (80.5 vs. 40.4%, P <0.0001 were observed in the cefotaxime group compared with the stan-dard heparin group. However, no significant difference in MRSA-associated CRBSI incidence was observed between the two groups. Cefotaxime-heparin "locks" effectively reduced CRBSI-incidence associated with gram-positive cocci, including MSSA, among S. aureus nasal carriers. There remains a compelling requirement for antibiotic-locks effective against MRSA.

  13. Budd-Chiari syndrome and heparin-induced thrombocytopenia in polycythemia vera: Successful treatment with repeated TIPS and interferon alpha

    Directory of Open Access Journals (Sweden)

    Akoum Riad


    Full Text Available Polycythemia vera (PV is a common cause of Budd-Chiari syndrome (BCS and portal vein thrombosis (PVT. The postpartum period is a precipitating cofactor. An additional heparin-induced thrombocytopenia/thrombosis (HIT/T leads to a life-threatening condition in which transjugular intrahepatic portosystemic shunting (TIPS seems to be the only life-saving procedure. We describe the case of a subacute BCS and PVT in the late postpartum period. The diagnosis was established using CT scan, MRI, and Doppler ultrasonography of abdominal vessels and the laboratory findings were compatible with PV. After a successful creation of TIPS, a HIT/T worsened the hemorrhagic and thrombotic picture. TIPS procedure was successfully repeated and heparin was replaced with Fondaparinux and then vitamin K antagonist. The treatment with interferon alpha-2A, started after the normalization of liver functions, resulted in a complete remission within 6 months. The JAK2 V617F mutation clone remained undetectable after 2 years′ follow-up.

  14. Dimethylsulfoxide with heparin in the treatment of smoke inhalation injury. (United States)

    Brown, M; Desai, M; Traber, L D; Herndon, D N; Traber, D L


    Recent studies suggest that lung lesions occurring in inhalation injury result from actions of oxygen-free radicals released from polymorphonuclear leukocytes marginating in the pulmonary microcirculation and tracheobronchial region. Peroxide and hydroxyl ions have been implicated as mediators in the increased microvascular permeability and pulmonary edema noted after inhalation injury. In this study we evaluated the use of an O2-free radical scavenger, dimethylsulfoxide (DMSO), and heparin in the treatment of smoke inhalation. Ewes (N = 26) that had been surgically prepared five days earlier were insufflated with smoke from burning cotton. There were four groups: controls (n = 7), DMSO (n = 6), heparin (n = 6), and DMSO plus heparin (n = 7). All animals were given ventilatory support to maintain their PO2 above 60 mmHg and their PCO2 below 45 mmHg. There was a significant difference in survival rates between groups. By 72 hours all seven of the control group were dead. All animals in the DMSO plus heparin group survived, four of the DMSO group died, and two of the heparin group died. Lung lymph flow was not as high in the DMSO plus heparin group as in the heparin-only group. DMSO was proved effective in reducing the lung injury associated with smoke inhalation.

  15. An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase. (United States)

    Cotruvo, Joseph A; Stubbe, Joanne


    Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: alpha2 (NrdE), where nucleotide reduction occurs, and beta2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)(6)-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with Mn(II) and incubated with fully reduced NrdI (NrdI(hq)) and O(2). Active RNR was rapidly produced with 0.25 +/- 0.03 tyrosyl radical (Y*) per beta2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is Mn(III)(2)-Y*, which we propose is generated by Mn(II)(2)-NrdF reacting with two equivalents of HO(2)(-), produced by reduction of O(2) by NrdF-bound NrdI(hq). In the absence of NrdI(hq), with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with Fe(II) and incubated with O(2) in the presence or absence of NrdI(hq) gave 0.2 and 0.7 Y*/beta2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdI(hq) hinders Fe(III)(2)-Y* cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the Mn(III)(2)-Y* cofactor, not the diferric-Y* one, is the active metallocofactor in vivo.

  16. Intratracheal heparin improves plastic bronchitis due to sulfur mustard analog. (United States)

    Houin, Paul R; Veress, Livia A; Rancourt, Raymond C; Hendry-Hofer, Tara B; Loader, Joan E; Rioux, Jacqueline S; Garlick, Rhonda B; White, Carl W


    Inhalation of sulfur mustard (SM) and SM analog, 2-chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology-induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation. Sprague-Dawley rats were exposed to 7.5% CEES via nose-only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non-treated, phosphate-buffered saline (PBS)-treated, and heparin-treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation. Heparin, given intratracheally, improved survival (100%) when compared to non-treated (75%) and PBS-treated (90%) controls. Heparin-treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin-treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin-treated rats compared to PBS-treated control rats. Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation. © 2014 Wiley Periodicals, Inc.

  17. A Heparin Purification Process Removes Spiked Transmissible Spongiform Encephalopathy Agent. (United States)

    Bett, Cyrus; Grgac, Ksenija; Long, Dianna; Karfunkle, Michael; Keire, David A; Asher, David M; Gregori, Luisa


    In 2000, bovine heparin was withdrawn from the US market for fear of contamination with bovine spongiform encephalopathy (BSE) agent, the cause of variant Creutzfeldt-Jakob disease in humans. Thus, US heparin is currently sourced only from pig intestines. Availability of alternative sources of crude heparin, a life-saving drug, would benefit public health. Bovine heparin is an obvious option, but BSE clearance by the bovine heparin manufacturing process should be evaluated. To this end, using hamster 263K scrapie as a surrogate for BSE agent, we applied a four-step bench-scale heparin purification protocol resembling a typical heparin manufacturing process to investigate removal of the spiked scrapie agent. We removed aliquots from each step and analyzed them for residual abnormal prion protein (PrPTSE) using a sensitive in vitro method, real-time quaking-induced conversion (RT-QuIC) assay, and for infectivity using animal bioassays. The purification process reduced infectivity by 3.6 log10 and removed PrPTSE, measured as seeding activity, by 3.4 log10. NaOH treatment was the most effective removal step tested. We also investigated NaOH at different concentrations and pH: the results showed that as much as 5.2 log10 of PrPTSE seeding activity was removed at pH 12.5. Thus, changes to the concentration, treatment time, and temperature of alkaline extraction might further improve removal. Our results, using a basic heparin manufacturing process, inform efforts to reintroduce safe bovine heparin in the USA.

  18. Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

    Directory of Open Access Journals (Sweden)

    Teresa Anna eGiancaspero


    Full Text Available The primary role of the water-soluble vitamin B2 (riboflavin in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD chaperone.The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.- and dimethylglycine dehydrogenase (Me2GlyDH, EC, respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

  19. Capillary electrophoresis for characterization of low molecular weight heparins. (United States)

    Ramasamy, I; Kennedy, J; Tan, K


    In many instances, low molecular weight heparins (LMWH) have replaced unfractionated heparins for prevention and treatment of venous thromboembolism. Each LMWH is a specific compound with its own physicochemical and pharmacological properties. These properties are critical in determining the therapeutic efficacy of the product. In recent times, capillary electrophoresis (CE) has emerged as a means of analytical separation of biological molecules. There are few reports, however, detailing the separation of whole heparins by capillary electrophoresis. This paper reports the use of CE to characterize LMWH without prior oligosaccharide release, labeling, or derivatization. The paper also focuses on the advantages of CE separation for the monitoring of product consistency.

  20. Prophylaxis of postoperative thromboembolism with low molecular weight heparins

    DEFF Research Database (Denmark)

    Jørgensen, L N; Wille-Jørgensen, P; Hauch, O


    to placebo or dextran and at least as efficient as unfractionated heparin in the prevention of deep vein thrombosis (DVT). Compared with unfractionated heparin, one of the LMWH preparations significantly reduced the total incidence of DVT. The rate of non-fatal pulmonary embolism was 0.49 per cent...... in patients receiving LMWH and 1.22 per cent in controls. Seven orthopaedic patients (0.15 per cent) died from pulmonary embolism, none of whom received LMWH. In general surgery, the LMWHs were at least as efficient as unfractionated heparin, with a trend towards a lower risk of pulmonary embolism...

  1. Proteinuria-lowering effect of heparin therapy in diabetic nephropathy without affecting the renin-angiotensin-aldosterone system. (United States)

    Benck, Urs; Haeckel, Sarah; Clorius, John H; van der Woude, Fokko J


    Angiotensin-converting enzyme inhibitors and angiotensin II (AngII) type 1 receptor blockers lower proteinuria and preserve renal function in diabetic nephropathy (DN). The antiproteinuric effects are greater than their blood pressure reduction, involving the sieving properties of the glomerular filter. In DN, glomerular staining for heparan sulfate proteoglycans is decreased. AngII inhibits heparan sulfate synthesis. Also, heparins modulate AngII signaling in glomerular cells, inhibiting aldosterone synthesis and lowering proteinuria in DN. Is the antiproteinuric effect of heparins due to its interference with the renin-angiotensin-aldosterone system? Ten volunteers each with DN and glomerulonephritis and control subjects were examined before and after low-dosage enoxaparin. Renal hemodynamics were determined with (99m)Tc-DTPA and (131)I-hippurate clearance. Glomerular filtration rate (GFR), effective renal plasma flow, mean arterial pressure, and heart rate were measured at baseline and during AngII infusion before and after enoxaparin while on normal salt and salt restriction. Enoxaparin did not lower aldosterone levels. GFR remained stable in all groups. AngII caused a significant decrease in effective renal plasma flow, whereas mean arterial pressure and heart rate increased significantly. Enoxaparin did not influence the AngII-induced changes of renal hemodynamics during normal salt intake or salt restriction. All groups showed identical responses to AngII before and after enoxaparin. In patients with diabetes, enoxaparin caused a significant decrease in proteinuria. It is concluded that the antiproteinuric effect of heparins in DN cannot be explained via interaction with the renin-angiotensin-aldosterone system. The absence of hemodynamic changes combined with reduced proteinuria point to intrinsic alterations in the glomerular filter. The effects were seen only in DN, not in glomerulonephritis.

  2. Engineering of routes to heparin and related polysaccharides. (United States)

    Bhaskar, Ujjwal; Sterner, Eric; Hickey, Anne Marie; Onishi, Akihiro; Zhang, Fuming; Dordick, Jonathan S; Linhardt, Robert J


    Anticoagulant heparin has been shown to possess important biological functions that vary according to its fine structure. Variability within heparin's structure occurs owing to its biosynthesis and animal tissue-based recovery and adds another dimension to its complex polymeric structure. The structural variations in chain length and sulfation patterns mediate its interaction with many heparin-binding proteins, thereby eliciting complex biological responses. The advent of novel chemical and enzymatic approaches for polysaccharide synthesis coupled with high throughput combinatorial approaches for drug discovery have facilitated an increased effort to understand heparin's structure-activity relationships. An improved understanding would offer potential for new therapeutic development through the engineering of polysaccharides. Such a bioengineering approach requires the amalgamation of several different disciplines, including carbohydrate synthesis, applied enzymology, metabolic engineering, and process biochemistry.

  3. Heparin removal by ecteola-cellulose pre-treatment enables the use of plasma samples for accurate measurement of anti-Yellow fever virus neutralizing antibodies. (United States)

    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


    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.

  4. The effect of heparin and EDTA on the NBT test. (United States)

    Rothwell, D J; Doumas, B T


    Heparin precipitates nitroblue tetrazolium (NBT) in the absence of protein (plasma). This NBT-heparin precipitate, when presented to neutrophils, results in NBT scores higher than those obtained with NBT in solution. It is postulated that the high NBT scores obtained with increasing heparin concentrations are due to increased NBT precipitation and hence increased phagocytosis. It is proposed that the variability of NBT scores obtained when heparin is used as the anticoagulant is related to the amount of precipitate formed and to inhibition of phagocytosis by excess heparin. EDTA, in the presence of plasma and NBT, forms a precipitate consisting of protein and little, if any, NBT. In the presence of EDTA, NBT scores are lower than those obtained with heparin. EDTA, at concentrations used to prevent coagulation, inhibits phagocytosis and this could explain the low NBT scores. These observations provide a mechanism for entry of NBT dye into neutrophils and may help explain the inconsistent results found in both normal and disease states.

  5. Heparin conjugated quantum dots for in vitro imaging applications. (United States)

    Maguire, Ciaran Manus; Mahfoud, Omar Kazem; Rakovich, Tatsiana; Gerard, Valerie Anne; Prina-Mello, Adriele; Gun'ko, Yurii; Volkov, Yuri


    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.

  6. 78 FR 38058 - Guidance for Industry on Heparin for Drug and Medical Device Use: Monitoring Crude Heparin for... (United States)


    ... written comments to the Division of Dockets Management, Food and Drug Administration, 5630 Fishers Lane... pharmaceutical ingredient (API) manufacturers, pharmaceutical and medical device manufacturers of finished... suppliers and heparin API suppliers to ensure conformance to appropriate quality standards. Manufacturers...

  7. Rates of clinically apparent heparin-induced thrombocytopenia for unfractionated heparin vs. low molecular weight heparin in non-surgical patients are low and similar

    Directory of Open Access Journals (Sweden)

    Gerber Jonathan


    Full Text Available Abstract With the growing use of low-molecular-weight heparins (LMWH for the treatment and prevention of venous thromboembolism (VTE, it is important to provide an evidence-based comparison with unfractionated heparin (UFH concerning rates of heparin-induced thrombocytopenia (HIT. Such comparisons are essential in clinical decision-making and cost-modeling. In this paper we review data regarding non-surgical (medical patients. We conclude that the lack of uniform evaluation and standardized testing for HIT in the current literature precludes making a reliable estimate of the relative risk of HIT in UFH vs. LMWH in either the treatment or prevention of VTE in non-surgical patients. However, current data suggest that the risk of thrombocytopenia and HIT is low and similar for non-surgical patients who receive either LMWH or UFH.

  8. Heparinization of a biomimetic bone matrix: integration of heparin during matrix synthesis versus adsorptive post surface modification. (United States)

    König, Ulla; Lode, Anja; Welzel, Petra B; Ueda, Yuichiro; Knaack, Sven; Henß, Anja; Hauswald, Anke; Gelinsky, Michael


    This study intended to evaluate a contemporary concept of scaffolding in bone tissue engineering in order to mimic functions of the extracellular matrix. The investigated approach considered the effect of the glycosaminoglycan heparin on structural and biological properties of a synthetic biomimetic bone graft material consisting of mineralized collagen. Two strategies for heparin functionalization were explored in order to receive a three-component bone substitute material. Heparin was either incorporated during matrix synthesis by mixing with collagen prior to simultaneous fibril reassembly and mineralization (in situ) or added to the matrix after fabrication (a posteriori). Both methods resulted in an incorporation of comparable amounts of heparin, though its distribution in the matrix varied as indicated by TOF-SIMS analyses, and a similar modulation of their protein binding properties. Differential scanning calorimetry revealed that the thermal stability and thereby the degree of crosslinking of the heparinized matrices was increased. However, in contrast to the a posteriori modification, the in situ integration of heparin led to considerable changes of morphology and composition of the matrix: a more open network of collagen fibers yielding a more porous surface and a reduced mineral content were observed. Cell culture experiments with human mesenchymal stem cells (hMSC) revealed a strong influence of the mode of heparin functionalization on cellular processes, as demonstrated for proliferation and osteogenic differentiation of hMSC. Our results indicate that not only heparin per se but also the way of its incorporation into a collagenous matrix determines the cell response. In conclusion, the a posteriori modification was beneficial to support adhesion, proliferation and differentiation of hMSC.

  9. Domain analysis of the tubulin cofactor system: a model for tubulin folding and dimerization

    Directory of Open Access Journals (Sweden)

    Jaroszewski Lukasz


    Full Text Available Abstract Background The correct folding and dimerization of tubulins, before their addition to the microtubular structure, needs a group of conserved proteins called cofactors A to E. The biochemical analysis of cofactors gave an insight to their general functions, however not much is known about the domain structure and detailed, molecular function of these proteins. Results Combining modelling and fold prediction tools, we present 3D models of all cofactors, including several previously unannotated domains of cofactors B-E. Apart from the new HEAT and Armadillo domains in cofactor D and an unusual spectrin-like domain in cofactor C, we have identified a new subfamily of ubiquitin-like domains in cofactors B and E. Together, these observations provide a reliable, molecular level model of cofactor complex. Conclusion Distant homology searches allowed the identification of unknown regions of cofactors as self-reliant domains and allow us to present a detailed hypothesis of how a cofactor complex performs its function.

  10. Alzheimer's Disease: Another Target for Heparin Therapy

    Directory of Open Access Journals (Sweden)

    Luigi Bergamaschini


    Full Text Available Alzheimer's disease (AD is the leading cause of dementia and cognitive decline in the elderly. Brain tissue changes indicate that the two main proteins involved in AD are amyloid-β(A-β, which is associated with the formation of senile amyloid plaques, and tau, which is associated with the formation of neurofibrillary tangles. Although a central role for A-β in the pathogenesis of AD is indisputable, considerable evidence indicates that A-β production is not the sole culprit in AD pathology. AD is also accompanied by an inflammatory response that contributes to irreversible changes in neuronal viability and brain function, and accumulating evidence supports the pivotal role of complement and contact systems in its pathogenesis and progression. The complexity of AD pathology provides numerous potential targets for therapeutic interventions. Compounds that interact directly with A-β protein or interfere with its production and/or aggregation can reduce the inflammatory and neurotoxic effects of A-β, and heparin, a glycosaminoglycan mixture currently used in the prophylaxis and treatment of thrombosis, might be a candidate, as recent research has been extended to consider its nonanticoagulant properties, including its modulation of various proteases and anti-inflammatory activity.

  11. Profiling Heparin-Chemokine Interactions Using Synthetic Tools (United States)

    de Paz, Jose L.; Moseman, E. Ashley; Noti, Christian; Polito, Laura; von Andrian, Ulrich H.; Seeberger, Peter H.


    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

  12. Stability of sildenafil in combination with heparin and dopamine. (United States)

    Luu, Yao; Thigpen, Jim; Brown, Stacy D


    The stability of sildenafil in combination with heparin and dopamine was evaluated. A stability-indicating high-performance liquid chromatography method with ultraviolet detection was developed for sildenafil citrate and validated. The method was applied to the investigation of sildenafil alone, sildenafil with heparin, sildenafil with dopamine, and sildenafil with heparin and with dopamine, all in 5% dextrose injection at room temperature and under refrigeration for 30 days. Samples of 100 μL were pulled from each storage bottle on each sampling day, diluted in mobile phase, and assayed in duplicate. Samples were tested on days 0, 1, 2, 3, 4, 5, 7, 9, 12, 14, 21, and 30. Each preparation was visually inspected for precipitation and color change. The percent recovery in each study sample was determined by comparing the peak area of sildenafil in the sample with the peak area of sildenafil from a freshly prepared 100-μg/mL standard in mobile phase. The sildenafil alone, sildenafil with heparin, and sildenafil with dopamine remained within 90-110% of the expected sildenafil potency for at least 30 days at both temperatures. The preparation of sildenafil with both heparin and dopamine fell below 90% potency after 3 days at room temperature and 21 days in the refrigerator. Sildenafil prepared in 5% dextrose injection alone, with heparin, and with dopamine retained over 90% potency after 30 days of storage at room temperature and under refrigeration. Sildenafil prepared with both heparin and dopamine had a potency of <90% after 3 days of storage at room temperature and 21 days of storage under refrigeration. Copyright © 2017 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

  13. Sulphur shuttling across a chaperone during molybdenum cofactor maturation. (United States)

    Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, Marina I; Toci, René; Mendel, Ralf R; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne


    Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

  14. Exercise–induced Anaphylaxis: the Role of Cofactors (United States)

    Zogaj, Dukagjin; Ibranji, Alkerta; Hoxha, Mehmet


    Introduction: Anaphylaxis is a dramatic clinical emergency. It is a very severe, life-threatening generalized or systemic hypersensitivity reaction. Based on immunologic mechanism the anaphylaxis is divided in IgE, IgG, complement, or immune complexes-mediated vs non allergic anaphylaxis. There are a lot of etiologic factors of anaphylaxis, but the three principal immunologic triggers are drugs, insect stings, and foods. Regarding the clinical severity there are several proposed grading systems. The diagnosis of anaphylaxis is mainly clinical. Discussion: The anaphylaxis markers measured in clinical laboratories are total tryptase and histamine. There are some conditions that modulate the onset of anaphylaxis, acting as co- or augmentation factors, which significantly lower the allergen dose necessary for triggering anaphylaxis. The well-documented cofactors of anaphylaxis are physical exercise, alcohol consumption, some foods, co-administration of nonsteroidal anti-inflammatory drugs (NSAID), and concomitant infectious diseases. Development of anaphylaxis depends on the sensitization pattern, the proportion of the involved immunoglobulin classes, characteristics of the allergen, the proportion of the involved immunoglobulin classes, the avidity and affinity of immunoglobulins to bind an allergen, the route of allergen application, and, last but not least, the presence of cofactors of anaphylaxis. Conclusion: Anaphylaxis remains a continuous challenge for the diagnosis and treatment. The adequate management of anaphylaxis requires rapid diagnosis, implementation of primary and secondary prevention measures, and immediate administration of subcutaneous epinephrine. PMID:25685088

  15. Unfractionated heparin and low-molecular-weight heparin in acute coronary syndrome without ST elevation: a meta-analysis. (United States)

    Eikelboom, J W; Anand, S S; Malmberg, K; Weitz, J I; Ginsberg, J S; Yusuf, S


    In acute coronary syndrome without ST elevation, the role of unfractionated and low-molecular-weight heparin in aspirin-treated patients remains unclear, and there is conflicting evidence regarding the efficacy and safety of low-molecular-weight heparin (LMWH) relative to unfractionated heparin. We did a systematic overview of the randomised trials to assess the effect of unfractionated heparin and LMWH on death, myocardial infarction, and major bleeding. Randomised trials comparing unfractionated heparin or LMWH with placebo or untreated control, or comparing unfractionated heparin with LMWH, for the short-term and long-term management of patients with acute coronary syndrome without ST elevation, were identified by electronic and manual searches and through contact with experts and industry representatives. Odds ratios for death, myocardial infarction, and major bleeding were calculated for each trial, and results for the individual trials were combined by a modification of the Mantel-Haenszel method. 12 trials, involving a total of 17157 patients, were included. The summary odds ratio (OR) for myocardial infarction or death during short-term (up to 7 days) unfractionated heparin or LMWH compared with placebo or untreated control was 0.53 (95% CI 0.38-0.73; p=0.0001) or 29 events prevented per 1000 patients treated; during short-term LMWH compared with unfractionated heparin was 0.88 (0.69-1.12; p=0.34); and during long-term LMWH (up to 3 months) compared with placebo or untreated control was 0.98 (0.81-1.17; p=0.80). Long-term LMWH was associated with a significantly increased risk of major bleeding (OR 2.26, [95% CI 1.63-3.14], paspirin-treated patients with acute coronary syndrome without ST elevation, short-term unfractionated heparin or LMWH halves the risk of myocardial infarction or death. There is no convincing difference in efficacy or safety between LMWH and unfractionated heparin. Long-term LMWH has not been proven to confer benefit additional to

  16. Manual control of catalytic reactions: Reactions by an apoenzyme gel and a cofactor gel (United States)

    Kobayashi, Yuichiro; Takashima, Yoshinori; Hashidzume, Akihito; Yamaguchi, Hiroyasu; Harada, Akira


    Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme–cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes. PMID:26537172

  17. Global substitution of hemeproteins with noncanonical amino acids in Escherichia coli with intact cofactor maturation machinery. (United States)

    Völler, Jan-Stefan; Thi To, Tuyet Mai; Biava, Hernan; Koksch, Beate; Budisa, Nediljko


    Global substitution of canonical amino acids (cAAs) with noncanonical (ncAAs) counterparts in proteins whose function is dependent on post-translational events such as cofactor binding is still a methodically challenging and difficult task as ncAA insertion generally interferes with the cofactor biosynthesis machinery. Here, we report a technology for the expression of fully substituted and functionally active cofactor-containing hemeproteins. The maturation process which yields an intact cofactor is timely separated from cAA→ncAA substitutions. This is achieved by an optimised expression and fermentation procedure which includes pre-induction of the heme cofactor biosynthesis followed by an incorporation experiment at multiple positions in the protein sequence. This simple strategy can be potentially applied for engineering of other cofactor-containing enzymes. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Cofactory: Sequence-based prediction of cofactor specificity of Rossmann folds

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Blom, Nikolaj; Feist, Adam


    cofactor specificity using only primary amino acid sequence information. The algorithm identifies potential cofactor binding Rossinann folds and predicts the specificity for the cofactors FAD(H2), NAD(H), and NADP(H) The Rossmann fold sequence search is carried out using hidden Markov models whereas......Obtaining optimal cofactor balance to drive production is a challenge metabolically engineered microbial strains. To facilitate identification of heterologous enzymes with desirable altered cofactor requirements from native content, we have developed Cofactory, a method for prediction of enzyme...... artificial neural networks are used for specificity prediction. Training was carried out using experimental data from protein cofactor structure complexes. The overall performance was benchmarked against an independent evaluation set obtaining Matthews correlation coefficients of 0.94, 0.79, and 0.65 for FAD...

  19. The use of heparin in preparing samples for blood-gas analysis. (United States)

    Higgins, Chris


    Heparin is the only anticoagulant used to prepare samples for blood-gas analysis. There are two ways in which heparin can interfere with results. The first is high heparin concentration in blood, and the second is heparin dilution of blood if liquid rather than dried (lyophilized) heparin is used. Traditional blood-gas analytes (pH, pCO2, and pO2) are less affected than electrolytes (particularly ionized calcium), also measured on modern blood-gas analyzers. The sample requirements as far as heparin is concerned are thus less exacting if only pH, pCO2, and pO2 are to be measured. For these analytes, it is still essential that the heparin (either sodium or lithium) concentration is less than 200 IU/mL blood and that the blood is not diluted more than 5%. The inclusion of electrolytes in the test repertoire excludes the use of sodium heparin in favor of lithium heparin. The inclusion of ionized calcium in the test repertoire demands that the heparin should be lyophilized, and the concentration should not exceed 10 IU/mL blood, unless a specialized heparin that eliminates the effect of calcium binding by heparin is used. Whatever the heparin formulation, it is essential for accurate results that the correct volume of blood is sampled to achieve a correct heparin concentration (and dilution, if liquid heparin is used), and that blood and anticoagulant are well mixed immediately after sampling. One of the most common practical problems associated with blood-gas analysis is inadequate anticoagulation and the formation of small blood clots that can block the sample pathway of blood-gas analyzers and invalidate results. Inadequate mixing of specimen with heparin is usually the problem. Clearly, the lower the heparin concentration the greater is the risk that poor mixing technique will give rise to inadequate anticoagulation and the associated problems.

  20. Heparin-induced thrombocytopenia among patients of a comprehensive cancer center

    Directory of Open Access Journals (Sweden)

    Weixin Wu


    Full Text Available Most clinical studies of heparin-induced thrombocytopenia have not included cancer patients who have high risk of thromboembolism, frequent exposure to heparin, and many potential causes of thrombocytopenia other than heparin-induced thrombocytopenia. To estimate the incidence and prevalence of heparin-induced thrombocytopenia in cancer patients, we identified cases based on diagnostic codes, anti-heparin antibody testing, and clinical characteristics (4T score at a comprehensive cancer center between 1 October 2008 and 31 December 2011. We estimated that the prevalence of heparin-induced thrombocytopenia to be 0.02% among all cancer patients and 0.24% among cancer patients exposed to heparin. The annual incidence of heparin-induced thrombocytopenia was 0.57 cases per 1000 cancer patients exposed to heparin. Of the 40 cancer patients with the International Classification of Diseases (Ninth Revision; ICD-9 code for heparin-induced thrombocytopenia, positive anti-heparin antibody, and 4T score ≥4, 5 (12.5% died of related thromboembolic or hemorrhagic complications. In a multivariate logistic regression model, male gender was a significant (p = 0.035 factor, and non-hematological malignancy was a significant (p = 0.017 factor associated with anti-heparin antibody positivity. Future studies may further examine the risk factors associated with heparin-induced thrombocytopenia in larger cohorts.

  1. The uses of heparin to treat burn injury. (United States)

    Oremus, Mark; Hanson, Mark; Whitlock, Richard; Young, Ed; Gupta, Alok; Dal Cin, Arianna; Archer, Carolyn; Raina, Parminder


    To assess the evidence for using heparin in the treatment of burn injury or the complications of burn injury in adults and children. The following databases were searched: MEDLINE (1966-current), EMBASE (1980-current), Cumulative Index to Nursing & Allied Health (CINAHL) (1982-current), The Cochrane Central Database of Controlled Trials (1995-current), Web of Science (1976-current), and BIOSIS (1976-current). Additional data sources included the U.S. and European Patent Offices, technical experts, the partner organization, and reference lists. Studies identified from the data sources went through two levels of title and abstract screening. Passing studies advanced to full text screening. Studies that met the full text screening criteria were abstracted. Criteria for abstraction included publication in any language, human patients of any age, and burns of any type, grade, or total body surface area. All formulations of heparin, and all application methods (e.g., topical, subcutaneous), were eligible for inclusion in the report. Abstracted studies required a comparison group. Outcomes of interest included mortality, pain, length of stay in hospital, thrombosis and emboli, psychiatric adjustment, and adverse effects (e.g., bleeding). Nineteen articles from 18 unique studies were abstracted and included in this report. In these articles, there were multiple uses of heparin to treat burns (e.g., wound healing, inhalation injury, sepsis, pain). However, the overall quality of the articles was weak. Examples of weakness included unclear or inappropriate treatment allocation, no blinding, no control of confounding, poorly defined burn characteristics (e.g., thickness), unclear duration of treatment, incomplete description of heparin treatment, and use of inadequately described or invalid outcome measures. Overall, the evidence from these weak articles was insufficient to determine whether the effectiveness of heparin to treat burn injury was different from the

  2. Mechanical prophylaxis is a heparin-independent risk for anti–platelet factor 4/heparin antibody formation after orthopedic surgery (United States)

    Bito, Seiji; Migita, Kiyoshi; Nakamura, Mashio; Shinohara, Kazuhito; Sato, Tomotaro; Tonai, Takeharu; Shimizu, Motoyuki; Shibata, Yasuhiro; Kishi, Kazuhiko; Kubota, Chikara; Nakahara, Shinnosuke; Mori, Toshihito; Ikeda, Kazuo; Ota, Shusuke; Minamizaki, Takeshi; Yamada, Shigeru; Shiota, Naofumi; Kamei, Masataka; Motokawa, Satoru


    Platelet-activating antibodies, which recognize platelet factor 4 (PF4)/heparin complexes, induce spontaneous heparin-induced thrombocytopenia (HIT) syndrome or fondaparinux-associated HIT without exposure to unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). This condition mostly occurs after major orthopedic surgery, implying that surgery itself could trigger this immune response, although the mechanism is unclear. To investigate how surgery may do so, we performed a multicenter, prospective study of 2069 patients who underwent total knee arthroplasty (TKA) or hip arthroplasty. Approximately half of the patients received postoperative thromboprophylaxis with UFH, LMWH, or fondaparinux. The other half received only mechanical thromboprophylaxis, including dynamic (intermittent plantar or pneumatic compression device), static (graduated compression stockings [GCSs]), or both. We measured anti-PF4/heparin immunoglobulins G, A, and M before and 10 days after surgery using an immunoassay. Multivariate analysis revealed that dynamic mechanical thromboprophylaxis (DMT) was an independent risk factor for seroconversion (odds ratio [OR], 2.01; 95% confidence interval [CI], 1.34-3.02; P = .001), which was confirmed with propensity-score matching (OR, 1.99; 95% CI, 1.17-3.37; P = .018). For TKA, the seroconversion rates in patients treated with DMT but no anticoagulation and in patients treated with UFH or LMWH without DMT were similar, but significantly higher than in patients treated with only GCSs. The proportion of patients with ≥1.4 optical density units appeared to be higher among those treated with any anticoagulant plus DMT than among those not treated with DMT. Our study suggests that DMT increases risk of an anti-PF4/heparin immune response, even without heparin exposure. This trial was registered to as #UMIN000001366. PMID:26659923

  3. Therapeutic monitoring of unfractionated heparin - trials and tribulations. (United States)

    Baluwala, Israfil; Favaloro, Emmanuel J; Pasalic, Leonardo


    Heparin is one of the oldest biological medicines with an established role in prevention and treatment of arterial and venous thromboembolism. Published therapeutic ranges for unfractionated heparin (UFH) mostly precede the large increase in the number of activated partial thromboplastin time (APTT) reagent/instrument combinations that now show wide variability. Areas covered: This paper explores the use of UFH, the development of heparin therapeutic ranges (HTRs), and the strengths and limitations of the methods used to monitor heparin's anticoagulant effect. Expert commentary: Despite longstanding use of UFH for management of thromboembolic conditions, the optimal test for monitoring UFH remains undetermined. Although used extensively for monitoring UFH, routine APTT-derived HTRs are based on limited science that may have little relevance to current laboratory practice. Anti-FXa levels may provide better and more reliable HTRs; however, even these levels show considerable inter-laboratory variation, and there are insufficient clinical studies proving improved clinical efficacy. Alternative tests for monitoring UFH reported over time have not been proven effective nor feasible, secondary to technical or cost issues, or lack of general adoption. Thus, despite limited evidence of clinical utility, an uncomfortable marriage of convenience represented by heparin laboratory monitoring is unlikely to be terminated in the immediate future.

  4. Relationship of serum S1P and HC-II levels with vasoactive substances and cytokines in patients with cerebral vascular restenosis after stent implantation

    Directory of Open Access Journals (Sweden)

    Yong Liu


    Full Text Available Objective: To study the relationship of serum sphingosine 1-phosphate (S1P and heparin cofactor II (HCII levels with vasoactive substances and cytokines in patients with cerebral vascular restenosis after stent implantation. Methods: 52 patients who received cerebrovascular stent implantation and developed restenosis in our hospital between May 2012 and December 2015 were collected as observation group, and 40 healthy patients with cerebrovascular stent implantation who had re-examination in our hospital during the same period were selected as control group. ELISA method was used to detect serum S1P and HC-II levels as well as vasoactive substance and inflammatory factor contents. Spearman correlation analysis was used to evaluate the relationship of serum S1P and HC-II levels with vasoactive substances and inflammatory factors. Results: Serum S1P and HC-II levels of observation group were lower than those of control group (P<0.05; serum vasoactive substances endothelin (ET, angiotensin II (AngII and thromboxane B2 (TXB2 contents of observation group were higher than those of control group while nitric oxide (NO content was lower than that of control group (P<0.05; serum inflammatory factors hypersensitive C-reactive protein (hs-CRP, interleukin-1 (IL-1, IL-6, IL-8 and IL-11 contents of observation group were higher than those of control group (P<0.05. Serum S1P and HC-II levels in patients with cerebral vascular restenosis after stent implantation were directly correlated with vasoactive substance and inflammatory factor contents. Conclusion: Serum S1P and HC-II levels decrease in patients with cerebral vascular restenosis after stent implantation, and it is an important cause of cerebral vascular dysfunction and systemic inflammatory response.

  5. 78 FR 36786 - Linking Marketplace Heparin Product Attributes and Manufacturing Processes to Bioactivity and... (United States)


    ... antibodies to complexes formed between platelet factor 4 (PF4) and heparin which can occur in patients who undergo major trauma (e.g. broken bones and cardiovascular surgery) and receive heparin. The condition...

  6. Heparin effect on plasma fibrinogen in the thrombophilic syndrome. (United States)

    Ruggiero, H A; Castellanos, H; Caprissi, L F; de Caprissi, E S; Ruggiero, L H


    The effects of heparin were studied in a group of 42 patients with preinfarction angina (PA) and acute myocardial infarction (AMI) whose plasma fibrinogen was increased. Plasma fibrinogen was measured by the turbidimetric method in timol turbidimetric units. Statistically significant results proved that heparin reduces the plasma fibrinogen progressively over a treatment period of 6 weeks. During the first three weeks a dose of 1 cc (50 mg or 5000 IU) was given by intravenous injection at 6-h intervals, this was followed by a dose of 2 cc (100 mg or 10,000 IU) given by subcutaneous injection at 12-h intervals for a further three weeks. Hyperfibrinogenemia is perhaps one of the most important factors in the thrombophilic syndrome, and at the same time it is one of the fundamental physiopathological alterations observed in AMI and PA. Because heparin reduces hyperfibrinogenemia it has a beneficial effect in these diseases.

  7. Quantitation of heparosan with heparin lyase III and spectrophotometry. (United States)

    Huang, Haichan; Zhao, Yingying; Lv, Shencong; Zhong, Weihong; Zhang, Fuming; Linhardt, Robert J


    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.

  8. Heparin oligosaccharides as potential therapeutic agents in senile dementia. (United States)

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


    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.

  9. Neutral polyethylene oxide with a cofactor recommended for particle flocculation

    Directory of Open Access Journals (Sweden)

    M. R Abdallah/Qasaimeh


    Full Text Available Conventional and neutral high molecular weight polyethylene oxide (PEO adsorbs on some colloids and fines, flocculating them into flocs. Addition of a cofactor (CF makes PEO adsorb on all types of colloids and fines, flocculating them into larger flocs. Homoflocculation of fines with PEO alone and with CF added prior to PEO were investigated in this work at low and high effective shear rates. CF role was investigated: it enhanced flocculation amplitude and rate by several magnitudes relative to PEO used alone, and was ascribed to the CF action to stiffen and extend PEO coils. Considering CF-PEO abilities in homoflocculation and in heteroflocculation as recorded in the literature, combination of homo - and heteroflocculation can now be applied to processes. Formed flocs and individual particles will simultaneously deposit onto fibers and, when filtered, particles will be retained in the fiber cake. This technique can be applied in industry processes and water treatment.

  10. Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins. (United States)

    Bjarnadóttir, Stefanía Guðrún; Flengsrud, Ragnar


    Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Changes in heparin dose response slope during cardiac surgery: possible result in inaccuracy in predicting heparin bolus dose requirement to achieve target ACT. (United States)

    Ichikawa, Junko; Mori, Tetsu; Kodaka, Mitsuharu; Nishiyama, Keiko; Ozaki, Makoto; Komori, Makiko


    The substantial interpatient variability in heparin requirement has led to the use of a heparin dose response (HDR) technique. The accuracy of Hepcon-based heparin administration in achieving a target activated clotting time (ACT) using an HDR slope remains controversial. We prospectively studied 86 adult patients scheduled for cardiac surgery requiring cardiopulmonary bypass. The total dose of calculated heparin required for patient and pump priming was administered simultaneously to achieve a target ACT of 450 s for HDR on the Hepcon HMS system. Blood samples were obtained after the induction of anesthesia, at 3 min after heparin administration and after the initiation of CPB to measure kaolin ACT, HDR slope, whole-blood heparin concentration based on the HDR slope and anti-Xa heparin concentration, antithrombin and complete blood count. The target ACT of 450 s was not achieved in 68.6% of patients. Compared with patients who achieved the target ACT, those who failed to achieve their target ACT had a significantly higher platelet count at baseline. Correlation between the HDR slope and heparin sensitivity was poor. Projected heparin concentration and anti-Xa heparin concentration are not interchangeable based on the Bland-Altman analysis. It can be hypothesized that the wide discrepancy in HDR slope versus heparin sensitivity may be explained by an inaccurate prediction of the plasma heparin level and/or the change in HDR of individual patients, depending on in vivo factors such as extravascular sequestration of heparin, decreased intrinsic antithrombin activity level and platelet count and/or activity.

  12. A comparison of seven methods to analyze heparin in biomaterials: quantification, location, and anticoagulant activity

    NARCIS (Netherlands)

    Lammers, G.; Westerlo, E.M.A. van de; Versteeg, E.M.M.; Kuppevelt, A.H.M.S.M. van; Daamen, W.F.


    Glycosaminoglycans, like heparin, are frequently incorporated in biomaterials because of their capacity to bind and store growth factors and because of their hydrating properties. Heparin is also often used in biomaterials for its anticoagulant activity. Analysis of biomaterial-bound heparin is

  13. Use of a heparin nomogram for treatment of patients with venous thromboembolism in a community hospital

    NARCIS (Netherlands)

    de Groot, M. R.; Büller, H. R.; ten Cate, J. W.; van Marwijk Kooy, M.


    The application of a heparin dosing nomogram in the treatment of patients with venous thromboembolism resulted in improvement of heparin therapy in clinical research settings. In 1992 a heparin nomogram was introduced in our hospital, which is a community hospital where anticoagulant therapy is

  14. We Use Heparin as the Anticoagulant for CRRT. (United States)

    Karakala, Nithin; Tolwani, Ashita


    Continuous Renal Replacement Therapy (CRRT) usually requires anticoagulation to prevent clotting of the extracorporeal circuit. Interruptions due to filter clotting significantly reduce total therapy time and CRRT efficacy. Although heparin has traditionally been the most common anticoagulant used for CRRT, increasing evidence suggests that heparin is less effective than regional citrate in prolonging circuit life and considerably increases patient bleeding risk. Advantages of regional citrate anticoagulation (RCA) include less bleeding, increased circuit life, and less blood transfusion requirement. RCA should be the anticoagulant of choice for CRRT. © 2016 Wiley Periodicals, Inc.

  15. Prophylaxis of postoperative thromboembolism with low molecular weight heparins

    DEFF Research Database (Denmark)

    Jørgensen, L N; Wille-Jørgensen, P; Hauch, O


    To evaluate the thromboprophylactic use of low molecular weight heparins (LMWHs), publications from 27 orthopaedic trials and 35 studies of patients undergoing general or gynaecological surgery were scrutinized and subjected to a partial meta-analysis. In orthopaedic surgery, LMWHs were superior...... in patients receiving LMWH and 1.22 per cent in controls. Seven orthopaedic patients (0.15 per cent) died from pulmonary embolism, none of whom received LMWH. In general surgery, the LMWHs were at least as efficient as unfractionated heparin, with a trend towards a lower risk of pulmonary embolism...

  16. The non-enzymatic reduction of azo dyes by flavin and nicotinamide cofactors under varying conditions. (United States)

    Morrison, Jessica M; John, Gilbert H


    Azo dyes are ubiquitous in products and often become environmental pollutants due to their anthropogenic nature. Azoreductases are enzymes which are present within many bacteria and are capable of breaking down the azo dyes via reduction of the azo bond. Often, though, carcinogenic aromatic amines are formed as metabolites and are of concern to humans. Azoreductases function via an oxidation-reduction reaction and require cofactors (a nicotinamide cofactor and sometimes a flavin cofactor) to perform their function. Non-enzymatic reduction of azo dyes in the absence of an azoreductase enzyme has been suggested in previous studies, but has never been studied in detail in terms of varying cofactor combinations, different oxygen states or pHs, nor has the enzymatic reduction been compared to azoreduction in terms of dye reduction or metabolites produced, which was the aim of this study. Reduction of azo dyes by different cofactor combinations was found to occur under both aerobic and anaerobic conditions and under physiologically-relevant pHs to produce the same metabolites as an azoreductase. Our results show that, in some cases, the non-enzymatic reduction by the cofactors was found to be equal to that seen with the azoreductase, suggesting that all dye reduction in these cases is due to the cofactors themselves. This study details the importance of the use of a cofactor-only control when studying azoreductase enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Application of NAD(P)H oxidase for cofactor regeneration in dehydrogenase catalyzed oxidations

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; Woodley, John


    alcohol dehydrogenases. However, their effective use requires an effective regeneration of the oxidized nicotinamide cofactor (NAD(P)+), which is critical for the economic feasibility of the process. NAD(P)H oxidase is an enzyme class of particular interest for this cofactor regeneration since it enables...

  18. Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome

    NARCIS (Netherlands)

    Houwman, J.A.; Westphal, A.H.; Berkel, van W.J.H.; Mierlo, van C.P.M.


    Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent

  19. Nuclear Receptor Cofactors in PPARγ-Mediated Adipogenesis and Adipocyte Energy Metabolism

    Directory of Open Access Journals (Sweden)

    Emily Powell


    Full Text Available Transcriptional cofactors are integral to the proper function and regulation of nuclear receptors. Members of the peroxisome proliferator-activated receptor (PPAR family of nuclear receptors are involved in the regulation of lipid and carbohydrate metabolism. They modulate gene transcription in response to a wide variety of ligands, a process that is mediated by transcriptional coactivators and corepressors. The mechanisms by which these cofactors mediate transcriptional regulation of nuclear receptor function are still being elucidated. The rapidly increasing array of cofactors has brought into focus the need for a clear understanding of how these cofactors interact in ligand- and cell-specific manners. This review highlights the differential effects of the assorted cofactors regulating the transcriptional action of PPARγ and summarizes the recent advances in understanding the physiological functions of corepressors and coactivators.

  20. The emerging role of low-molecular-weight heparin in cardiovascular medicine. (United States)

    Hirsh, J; Bates, S M


    Although unfractionated heparin is widely used in the treatment of acute coronary syndromes, it has several pharmacokinetic, biophysical, and biological limitations. The practical advantages and success of low-molecular-weight heparin administered subcutaneously without laboratory monitoring for the treatment of venous thromboembolism have prompted a number of randomized studies investigating the efficacy and safety of these agents in patients with acute coronary syndromes. This article will review the limitations of unfractionated heparin and the mechanisms by which low-molecular-weight heparin overcomes these limitations, as well as the results of recent trials involving low-molecular-weight heparin in the management of patients with acute coronary syndromes.

  1. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)


    capacitation in the female reproductive tract is aided by. HBPs secreted by the male accessory sex glands (Miller et al. 1990). Seminal fluid HBPs are supposed to attach themselves to the sperm surface, especially lipids containing the phosphoryl-choline group, thus allowing heparin-like. GAGs in the female reproductive ...

  2. Proteomic analysis of heparin-binding proteins from human seminal ...

    Indian Academy of Sciences (India)

    Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser ...

  3. Heparin and Aspirin in Pregnant Sudanese women with Recurrent ...

    African Journals Online (AJOL)

    This was a prospective clinical trial conducted at Khartoum Fertility Center, during the period June 2002 to February 2005 to investigate the efficacy of unfractionated heparin and low-dose aspirin as prophylaxis against pregnancy loss in 58 pregnant Sudanese women with recurrent (>3) miscarriages associated with ...

  4. Clinical effects of low-molecular-weight heparin combined with ...

    African Journals Online (AJOL)

    Purpose: To explore the clinical effects of low-molecular-weight heparin (LMWH) combined with ulinastatin (UTI) in children with acute pancreatitis. Methods: In total, 560 patients with severe acute pancreatitis treated at Binzhou People's Hospital,. Shandong, China, from April 2012 to June 2014 were enrolled in this study.

  5. Low-molecular-weight heparins allow selected outpatient treatment ...

    African Journals Online (AJOL)

    DVT) at present consists of an initial continuous intravenous infusion of unfractionated heparin, administered for a minimum of 5 - 7 days.' Oral anticoagulation is started at the same time, while the patient is still in hospital, and is continued for at ...

  6. Heparin removal in three intraoperative blood savers in cardiac surgery. (United States)

    Rougé, P; Fourquet, D; Depoix-Joseph, J P; Nguyen, F; Barthélémy, R


    The aim of the study was to compare the residual heparin in the composition of autologous blood retransfusion units harvested during cardiac surgery under extra-corporeal circulation with three different intraoperative autologous blood savers. In this institutionally approved study, thirty patients undergoing CABG were randomly assigned to three groups according to the intraoperative blood saver used during the procedure: {HAEMONETICS Cell Saver IV (n=10)--DIDECO/SHILEY STAT (n=11)--BRAT 250 (n=9)}. Anaesthesia and conduct of bypass were identical for all patients. The initial heparin dose was 300IU-kg -1 and was supplemented to maintain an activated coagulation time over 480s. The harvested blood was processed according to the procedure defined by each equipment manufacturer. The biological study was performed on the first blood sediments sampled before administering protamine to the patient. Blood cell count, residual heparinemia assessed by its anti-Xa activity using an amidolytic method {STACHROM HEPARIN--DIAGNOSTICA STAGO}, and weight of the blood sediment proteins were determined. Demographic data did not differ between groups. Despite a slight but significant difference between groups, the three devices provided virtual elimination of heparin. The total protein content was significantly higher in the BRAT 250 group. There was a highly significant positive correlation between the anti-Xa activity and total protein content. Haematologic data were within clinically acceptable ranges.

  7. Low molecular weight heparins in acute ischaemic syndromes. (United States)

    White, H D; Ellis, C J; French, J K


    Thrombus formation and ongoing generation play a major role in the pathogenesis of unstable angina and myocardial infarction (MI). Unfractionated heparin is widely used as an adjunctive therapy in the management of acute ischaemic syndromes, but it has a number of limitations including inter-patient variability, need for monitoring, and the fact that its use may be associated with thrombocytopenia. Low molecular weight heparins have pharmacological and pharmacokinetic properties that may result in better clinical outcomes and safety. They are easy to administer and do not require monitoring. Low molecular weight heparins have been shown to reduce mortality and the incidence of MI and recurrent ischaemia compared with placebo in patients with unstable angina and non-Q-wave MI, and have a more predictable anticoagulant effect than standard unfractionated heparin. They also can be used long term in the outpatient setting, in the form of self-administered subcutaneous injections. The clinical relevance of these new developments is still being defined in ongoing clinical trials and cost-effectiveness analyses.

  8. Heparin bridge therapy and post-polypectomy bleeding. (United States)

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


    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 bridge therapy were significant risk factors for PPB (P bridge therapy might be responsible for the increased PPB in patients taking anticoagulants.

  9. Preventive role of low-molecular-weight heparin in unexplained ...

    African Journals Online (AJOL)

    Preventive role of low-molecular-weight heparin in unexplained recurrent pregnancy loss. E S Khan,1 MBBS, FCPS ... with thromboprophylaxis as preventive therapy has been proposed. This open-access article is distributed under ... placental abruption or preeclampsia. A relationship with early pregnancy loss is less clear ...

  10. Light harvesting in photosystem II

    NARCIS (Netherlands)

    van Amerongen, H.; Croce, R.


    Water oxidation in photosynthesis takes place in photosystem II (PSII). This photosystem is built around a reaction center (RC) where sunlight-induced charge separation occurs. This RC consists of various polypeptides that bind only a few chromophores or pigments, next to several other cofactors. It

  11. Falsely elevated INR results due to the sensitivity of a thromboplastin reagent to heparin. (United States)

    Leech, B F; Carter, C J


    The aim of this study was to determine the effect of heparin therapy on the international normalized ratio (INR). In vitro heparin sensitivity curves were created using normal and warfarinized plasma samples from patients. The INR results were measured with each of two reagents. In addition, a comparison of INR values with these two reagents was performed on plasma from patients receiving therapeutic heparin and warfarin. The INR values were measured before and after heparin removal with a heparin adsorbent system. While one of the reagents was found to be sensitive to even low levels of therapeutic heparin, the other thromboplastin was resistant up to at least 0.9 U/mL. The INR values determined for patients with one of the reagents were found to be erroneously elevated by an average of 16%. The error ranged from 2% to 55% depending on the in vivo heparin concentration. With the other reagent, the INR values were not substantially affected by heparin. Previous studies have described the effect or lack thereof of heparin on the prothrombin time. The present study demonstrates that the degree to which INR results are prolonged by heparin therapy depends on the reagent formulation. As the therapeutic index for monitoring warfarin is relatively narrow (2.0-3.0), an INR value that is falsely elevated due to reagent sensitivity may precipitate premature cessation of heparin therapy and place certain patients at risk for recurrent thrombosis in the short term.

  12. Crystallographic Analysis of Calcium-dependent Heparin Binding to Annexin A2

    Energy Technology Data Exchange (ETDEWEB)

    Shao,C.; Zhang, F.; Kemp, M.; Lindhardt, R.; Waisman, D.; Head, J.; Seaton, B.


    Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

  13. Bovine and porcine heparins: different drugs with similar effects on human haemodialysis (United States)


    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

  14. Treatment and Prevention of Heparin-Induced Thrombocytopenia (United States)

    Dans, Antonio L.; Moores, Lisa K.; Bona, Robert; Davidson, Bruce L.; Schulman, Sam; Crowther, Mark


    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

  15. 77 FR 7584 - Draft Guidance for Industry on Heparin for Drug and Medical Device Use; Monitoring Crude Heparin... (United States)


    ... Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852. See the... Quality.'' This draft guidance provides recommendations that will help API manufacturers, pharmaceutical... contaminant OSCS in heparin API manufactured in China. FDA is also concerned about the potential for...

  16. Heparin and Heparin-Derivatives in Post-Subarachnoid Hemorrhage Brain Injury: A Multimodal Therapy for a Multimodal Disease

    Directory of Open Access Journals (Sweden)

    Erik G. Hayman


    Full Text Available Pharmacologic efforts to improve outcomes following aneurysmal subarachnoid hemorrhage (aSAH remain disappointing, likely owing to the complex nature of post-hemorrhage brain injury. Previous work suggests that heparin, due to the multimodal nature of its actions, reduces the incidence of clinical vasospasm and delayed cerebral ischemia that accompany the disease. This narrative review examines how heparin may mitigate the non-vasospastic pathological aspects of aSAH, particularly those related to neuroinflammation. Following a brief review of early brain injury in aSAH and heparin’s general pharmacology, we discuss potential mechanistic roles of heparin therapy in treating post-aSAH inflammatory injury. These roles include reducing ischemia-reperfusion injury, preventing leukocyte extravasation, modulating phagocyte activation, countering oxidative stress, and correcting blood-brain barrier dysfunction. Following a discussion of evidence to support these mechanistic roles, we provide a brief discussion of potential complications of heparin usage in aSAH. Our review suggests that heparin’s use in aSAH is not only safe, but effectively addresses a number of pathologies initiated by aSAH.

  17. X-ray absorption spectroscopy on the calcium cofactor to the manganese cluster in photosynthetic oxygen evolution

    Energy Technology Data Exchange (ETDEWEB)

    Cinco, Roehl M. [Univ. of California, Berkeley, CA (United States)


    Along with Mn, calcium and chloride ions are necessary cofactors for oxygen evolution in Photosystem II (PS II). To further test and verify whether Ca is close to the Mn cluster, the authors substituted strontium for Ca and probed from the Sr point of view for any nearby Mn. The extended X-ray absorption fine structure (EXAFS) of Sr-reactivated PS II indicates major differences between the intact and NH2OH-treated samples. In intact samples, the Fourier transform of the Sr EXAFS shows a Fourier peak that is missing in inactive samples. This peak II is best simulated by two Mn neighbors at a distance of 3.5 Angstrom, confirming the proximity of Ca (Sr) cofactor to the Mn cluster. In addition, polarized Sr EXAFS on oriented Sr-reactivated samples shows this peak II is dichroic: large magnitude at 10 degrees (angle between the PS II membrane normal and the x-ray electric field vector) and small at 80 degrees. Analysis of the dichroism yields the relative angle between the Sr-Mn vector and membrane normal (23 degrees ± 4 degrees), and the isotropic coordination number for these layered samples. X-ray absorption spectroscopy has also been employed to assess the degree of similarity between the manganese cluster in PS II and a family of synthetic manganese complexes containing the distorted cubane [Mn4O3X] core (X = benzoate, acetate, methoxide, hydroxide, azide, fluoride, chloride or bromide). In addition, Mn4O3Cl complexes containing three or six terminal Cl ligands at three of the Mn were included in this study. The EXAFS method detects the small changes in the core structures as X is varied in this series, and serves to exclude these distorted cubanes of C3v symmetry as a topological model for the Mn catalytic cluster. The sulfur K-edge x-ray absorption near-edge structure (XANES) spectra for the amino acids cysteine, methionine, their corresponding oxidized forms cystine and methionine sulfoxide, and

  18. Probing the impact of GFP tagging on Robo1-heparin interaction. (United States)

    Zhang, Fuming; Moniz, Heather A; Walcott, Benjamin; Moremen, Kelley W; Wang, Lianchun; Linhardt, Robert J


    Green fluorescent proteins (GFPs) and their derivatives are widely used as markers to visualize cells, protein localizations in in vitro and in vivo studies. The use of GFP fusion protein for visualization is generally thought to have negligible effects on cellular function. However, a number of reports suggest that the use of GFP may impact the biological activity of these proteins. Heparin is a glycosaminoglycan (GAG) that interacts with a number of proteins mediating diverse patho-physiological processes. In the heparin-based interactome studies, heparin-binding proteins are often prepared as GFP fusion proteins. In this report, we use surface plasmon resonance (SPR) spectroscopy to study the impact of the GFP tagging on the binding interaction between heparin and a heparin-binding protein, the Roundabout homolog 1 (Robo1). SPR reveals that heparin binds with higher affinity to Robo1 than GFP-tagged Robo1 and through a different kinetic mechanism. A conformational change is observed in the heparin-Robo1 interaction, but not in the heparin-Robo1-GFP interaction. Furthermore the GFP-tagged Robo1 requires a shorter (hexasaccharide) than the tag-free Robo1 (octadecasaccharide). These data demonstrate that GFP tagging can reduce the binding affinity of Robo1 to heparin and hinder heparin binding-induced Robo1 conformation change.

  19. Low Anticoagulant Heparin Blocks Thrombin-Induced Endothelial Permeability in a PAR-Dependent Manner (United States)

    Gonzales, Joyce N.; Kim, Kyung-mi; Zemskova, Marina A.; Rafikov, Ruslan; Heeke, Brenten; Varn, Matthew N.; Black, Stephen; Kennedy, Thomas P.; Verin, Alexander D.; Zemskov, Evgeny A.


    Acute lung injury and acute respiratory distress syndrome are accompanied by thrombin activation and fibrin deposition that enhances lung inflammation, activates endothelial cells and disrupts lung paracellular permeability. Heparin possesses anti-inflammatory properties but its clinical use is limited by hemorrhage and heparin induced thrombocytopenia. We studied the effects of heparin and low anticoagulant 2-O, 3-O desulfated heparin (ODSH) on thrombin-induced increases in paracellular permeability of cultured human pulmonary endothelial cells (EC). Pretreatment with heparin or ODSH blocked thrombin-induced decrease in the EC transendothelial electrical resistance (TER), attenuated thrombin-stimulated paracellular gap formation and actin cytoskeletal rearrangement. Our data demonstrated that heparin and ODSH had inhibitory effects on thrombin-induced RhoA activation and intracellular calcium elevation. Thrombin-stimulated phosphorylation of the cytoskeletal regulatory proteins, myosin light chain and ezrin/radixin/moesin, were also reduced. In these effects, low anticoagulant ODSH was more potent than heparin. Heparin or ODSH alone produced decreases in the EC TER that were abolished by siRNA-mediated depletion of the thrombin receptor, PAR-1. We also demonstrated that, in contrast to heparin, ODSH did not possess thrombin-binding activity. Results suggest that heparin and low anticoagulant ODSH, can interfere with thrombin-activated signaling. PMID:24469066

  20. Liposomal Heparin-Spraygel in Comparison with Subcutaneous Low Molecular Weight Heparin in Patients with Superficial Venous Thrombosis. A Randomized, Controlled, Open Multicentre Study


    Katzenschlager R; Hirschl M; Minar E; Ugurluoglu A


    Liposomales Heparin-Spraygel im Vergleich zu subkutanem niedermolekularem Heparin bei Patienten mit oberflächlicher Venenthrombose. Eine randomisierte, kontrollierte, offene Multicenter-Studie. Ziel: Die oberflächliche Venenthrombose (superficial vein thrombosis, SVT), die durch Kompressionsbehandlung sowie lokal angewandte oder systemische nichtsteroidale Antiphlogistika hinreichend behandelt werden kann, wird generell als relativ harmlos eingestuft. Allerdings kann eine SVT auch tiefer ge...

  1. HIV-1 evades innate immune recognition through specific cofactor recruitment (United States)

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.


    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

  2. In vitro effects of heparin and tissue factor pathway inhibitor on factor VII assays. possible implications for measurements in vivo after heparin therapy

    DEFF Research Database (Denmark)

    Bladbjerg, E-M; Larsen, L F; Ostergaard, P


    The coagulant activity of blood coagulation factor VII (FVII:C) can be lowered by changes in lifestyle and by therapeutic intervention, e.g. heparin infusion. The question is, however, whether FVII:C determined ex vivo is a valid measure of the FVII activity in vivo. We measured plasma FVII......:C, activated FVII (FVIIa), FVII protein (FVII:Ag), tissue factor pathway inhibitor (TFPI), triglycerides, and free fatty acids (FFA) before and 15 min after infusion of a bolus of unfractionated heparin (50 IU/kg body weight) in 12 healthy subjects. Additionally, we conducted in vitro experiments...... activity by means of FVII clotting assays. These assays should therefore not be used to measure the coagulation status of patients in heparin therapy, unless extraordinary precautions are taken to eliminate TFPI and heparin effects ex vivo. The observed effect of heparin on FVII:Ag should be investigated...

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

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


    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 1H 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.

  4. Comparison of the therapeutic effects of sildenafil citrate, heparin and neuropeptides in a rat model of acetic acid-induced gastric ulcer. (United States)

    Kalayci, Mehmet; Kocdor, Mehmet Ali; Kuloglu, Tuncay; Sahin, İbrahim; Sarac, Mehmet; Aksoy, Aziz; Yardim, Meltem; Dalkilic, Semih; Gursu, Onur; Aydin, Suna; Akkoc, Ramazan Fazil; Ugras, Meltem; Artas, Gokhan; Ozercan, İbrahim Hanifi; Ugur, Kader; Aydin, Suleyman


    The purpose of our investigative work has been to determine whether there can be therapeutic roles in the administration of sildenafil citrate, heparin and several neuropeptides on an animal model where gastric ulcers were induced with acetic acid, and to compare their efficacy. The animals were divided into 13 groups, with 4 animals in each. Gastric ulcers was induced in the animals of 12 groups with one untreated group being left as the control (Group I - control; given normal saline (NS)). The other groups were: Group II (ulcer+NS); Group III (5mg/kg sildenafil citrate, low dose); Group IV (10mg/kg sildenafil citrate, high dose); Group V (0.6mg/kg heparin, low dose); Group VI (6mg/kg heparin, high dose); Group VII (20nmol/kg des-acyl ghrelin); Group VIII (40nmol/kg des-acyl ghrelin); Group IX (4nmol/kg acyl ghrelin); Group X (8nmol/kg acly ghrelin); Group XI (20pmol/kg Nesfatin-1); Group XII (15nmol/kg Obestatin) and Group XIII (5nmol/kg Neuropeptide Y). Gastric neuropeptide expression was measured using an immunohistochemical method, and the amount in circulation was detected using ELISA. To compare with no treatment, the controls and other treatment groups, we recorded loss of the surface epithelium of the stomach, erosion, bleeding and inflammatory cell infiltration in the upper halves of the gastric glands. The muscularis and the layers beneath it were, however, apparently normal. The gastric mucosa healed with little or no inflammation when sildenafil citrate, low dose heparin, ghrelin, NUCB2/Nesfatin-1, obestatin, Neuropeptide Y were administered. Overall the data indicate that low dose heparin, and especially sildenafil citrate and neuropeptides, can be used clinically as an alternative approach in the treatment of the gastric ulcer. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Different effects of low weight molecular heparin and unfractioned heparin on lipid profile and coagulation at haemodialysis patients. (United States)

    Resić, Halima; Kukavica, Nihad; Sahović, Vahidin; Masnić, Fahrudin


    Each haemodialysis treatment requires the application of anticoagulation medicines, which will prevent coagulation in extracorporal blood circulation. In this study we try to determine the quality of admitted anticoagulant and his effect on lipid profile on hemodialysis patients after twelve months. We were applying standard heparin and low weight molecular heparin (LWMH). During our study we was analyzed effect of anticoagulant therapy on lipid profile of hemodialysis patients. In that parameters was included triglycerides, cholesterol, lipoprotein fractions, complete blood count, Hgb, HCT; All of these parameters was analyzed in correlation with duration of hemodialysis treatment, sex and age of the patients. Our research was carried out as a prospective study, for the period of 12 months. In the study were included 60 patients (34M/26F), who were on chronic hemodialysis program. All patients were divided into two groups. The first group of patients was included 27 patients (15M/12F) who were treated with standard heparin. The second group was included 33 patients (19M/14F) treated with LWMH (enoxaparin). The average length of hemodialysis was 4.15 +/- 0.52 years. Each patient had a protocol in which is marked parameters such as flushing dialysator, creating fibrin-ring in vein and arterial dropper and the time it takes to stop the bleeding. In the results the average age amounted to 58.54 +/- 2.24 years. The average value of cholesterol in the blood was 5.38 +/- 2.26. Values of HDL-cholesterol in patients treated with LWMH were significantly lower (Pprofile. After the first six months of study in male patients treated with standard heparin in relation to the female part of the observed patients was significantly better anticoagulation effect in the first half of the study (1.85 +/- 0.05 compared to 2.09 +/- 0.10) (Pprofiles. Patients treated with standard heparin had a statistically significant reduction in the rate of blood clots than patients who received

  6. Heparin and Carboxymethylchitosan Metal Nanoparticles: An Evaluation of Their Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Adriana Bava


    Full Text Available In the search for noninvasive diagnostic techniques and new therapies, “nanosystems”, which are capable of binding and targeting bioactive molecules, are becoming increasingly important. In this context, biocompatible coatings are gaining interest, not only for their biological effects but also because they are considered capable to mask nanoparticle toxicity. In this work, we have compared the toxicity of nanoparticles coated with heparin and carboxymethylchitosan in the SKOV-3 cell line. Our results indicate that heparin and carboxymethylchitosan coatings do not guarantee the decrease of nanoparticle intrinsic toxicity which is often envisaged. Nonetheless, these coatings provide the opportunity for further functionalization with a variety of biomolecules for their use in theranostics.

  7. Low molecular weight heparin (CY-216) versus unfractionated heparin in chronic hemodialysis. (United States)

    Grau, E; Sigüenza, F; Maduell, F; Linares, M; Olaso, M A; Martinez, R; Caridad, A


    In 14 patients undergoing chronic hemodialysis, we investigated the safety and efficacy of the low molecular fragment (CY-216) in comparison to unfractionated heparin (UFH) in the prevention of clotting in the extracorporeal circuit (ECC). In this study, 168 hemodialysis sessions were undertaken with UFH in 2 bolus doses (5,437 +/- 1,477 SD IU) and 231 with CY-216 in a single bolus dose [initial dose 150 anti-Xa U Institut Choay (IC)/kg]. There were no clots in the bubble trap in any UFH sessions, and 14.8% had coagulated fibers in the dialyzer. Clotting in the bubble trap was observed in 2 CY-216 sessions (0.8%) and coagulated fibers in 22.6% of the sessions. At the end of the study, the mean dose of CY-216 was 250 anti-Xa UIC/kg but a dose of 350 anti-Xa UIC/kg was needed in the 2 patients treated by recombinant human erythropoietin. Anti-Xa levels at the end of the runs were higher (0.47 +/- 0.1 U/ml) in the CY-216 group than in the UFH group (0.28 +/- 0.1 U/ml). There was a correlation between anti-Xa levels and efficacy in the CY-216 group. An anti-Xa activity above 0.4 U/ml was needed in order to minimize thrombus formation. Antithrombin III-protease complexes (ATM) and D dimer fibrin derivatives (D dimer) were used as thrombotic markers but they were of little value for the detection of fibrin formation in the ECC. Our findings suggest that CY-216 administered as a single bolus dose seems to be of similar effectiveness to UFH.

  8. Multifunctional silk-heparin biomaterials for vascular tissue engineering applications


    Seib, F. Philipp; Herklotz, Manuela; Burke, Kelly A; Maitz, Manfred F.; Werner, Carsten; Kaplan, David L.


    Over the past 30 years, silk has been proposed for numerous biomedical applications that go beyond its traditional use as a suture material. Silk sutures are well tolerated in humans, but the use of silk for vascular engineering applications still requires extensive biocompatibility testing. Some studies have indicated a need to modify silk to yield a hemocompatible surface. This study examined the potential of low molecular weight heparin as a material for refining silk properties by acting ...

  9. Influence of low molecular weight heparin on cancer patients’ survival

    Directory of Open Access Journals (Sweden)

    V. V. Ptushkin


    Full Text Available There is an evidence of interaction between the hemostasis system and tumor progression factors. It is known that in addition to the fibrin formation and platelets activation, thrombin can influence many cells function interacting with protease-activating receptors including tumor cells. These receptors are involved in the malignant cell phenotype formation (adhesion, proliferation, proteolysis. Thrombin can also affect angiogenesis by stimulating endothelial cells penetration through basal membrane and its migration with new vessels formation. Furthermore, it can cause the release of main neoangiogenesis promoter – vascular endothelial growth factor. All of the above and many other linkages of coagulation and tumor create a theoretical background of possible affecting tumor by regulation of the coagulation activity. Thepromise of this approach is controversial, but there is some clinical and experimental evidence of their effectiveness. The most used group ofdrugs for this purpose was heparins. Several retrospective studies have shown a benefit of low molecular weight heparins (LMWH over unfractionated heparin in cancer patient survival. The appearance of a new heparins group – ultra LMWH are of interest from this point ofview and their possible use in cancer patients. To date bemiparin and semuloparin are used in clinic. Both (bemiparin about 3600 kDa,semuloparin 3000 kDa have substancially reduced molecular weight as compared with the smallest of LMWH – enoxaparin (4600 kDa.Use of bemiparin in patients with small cell lung cancer receiving chemotherapy resulted in increased of 2-year survival rate compared to the control group (68.6 % vs. 29.4 %, p = 0.0042.

  10. Influence of low molecular weight heparin on cancer patients’ survival

    Directory of Open Access Journals (Sweden)

    V. V. Ptushkin


    Full Text Available There is an evidence of interaction between the hemostasis system and tumor progression factors. It is known that in addition to the fibrin formation and platelets activation, thrombin can influence many cells function interacting with protease-activating receptors including tumor cells. These receptors are involved in the malignant cell phenotype formation (adhesion, proliferation, proteolysis. Thrombin can also affect angiogenesis by stimulating endothelial cells penetration through basal membrane and its migration with new vessels formation. Furthermore, it can cause the release of main neoangiogenesis promoter – vascular endothelial growth factor. All of the above and many other linkages of coagulation and tumor create a theoretical background of possible affecting tumor by regulation of the coagulation activity. Thepromise of this approach is controversial, but there is some clinical and experimental evidence of their effectiveness. The most used group ofdrugs for this purpose was heparins. Several retrospective studies have shown a benefit of low molecular weight heparins (LMWH over unfractionated heparin in cancer patient survival. The appearance of a new heparins group – ultra LMWH are of interest from this point ofview and their possible use in cancer patients. To date bemiparin and semuloparin are used in clinic. Both (bemiparin about 3600 kDa,semuloparin 3000 kDa have substancially reduced molecular weight as compared with the smallest of LMWH – enoxaparin (4600 kDa.Use of bemiparin in patients with small cell lung cancer receiving chemotherapy resulted in increased of 2-year survival rate compared to the control group (68.6 % vs. 29.4 %, p = 0.0042.

  11. The efficiency of mapping of quantitative trait loci using cofactor analysis in half-sib design

    Directory of Open Access Journals (Sweden)

    Lund Mogens


    Full Text Available Abstract This simulation study was designed to study the power and type I error rate in QTL mapping using cofactor analysis in half-sib designs. A number of scenarios were simulated with different power to identify QTL by varying family size, heritability, QTL effect and map density, and three threshold levels for cofactor were considered. Generally cofactor analysis did not increase the power of QTL mapping in a half-sib design, but increased the type I error rate. The exception was with small family size where the number of correctly identified QTL increased by 13% when heritability was high and 21% when heritability was low. However, in the same scenarios the number of false positives increased by 49% and 45% respectively. With a liberal threshold level of 10% for cofactor combined with a low heritability, the number of correctly identified QTL increased by 14% but there was a 41% increase in the number of false positives. Also, the power of QTL mapping did not increase with cofactor analysis in scenarios with unequal QTL effect, sparse marker density and large QTL effect (25% of the genetic variance, but the type I error rate tended to increase. A priori, cofactor analysis was expected to have higher power than individual chromosome analysis especially in experiments with lower power to detect QTL. Our study shows that cofactor analysis increased the number of false positives in all scenarios with low heritability and the increase was up to 50% in low power experiments and with lower thresholds for cofactors.

  12. Molecular simulation to investigate the cofactor specificity for pichia stipitis Xylose reductase. (United States)

    Xia, Xiao-Le; Cong, Shan; Weng, Xiao-Rong; Chen, Jin-Hua; Wang, Jing-Fang; Chou, Kuo-Chen


    Xylose is one of the most abundant carbohydrates in nature, and widely used to produce bioethanol via fermentation in industry. Xylulose can produce two key enzymes: xylose reductase and xylitol dehydrogenase. Owing to the disparate cofactor specificities of xylose reductase and xylitol dehydrogenase, intracellular redox imbalance is detected during the xylose fermentation, resulting in low ethanol yields. To overcome this barrier, a common strategy is applied to artificially modify the cofactor specificity of xylose reductase. In this study, we utilized molecular simulation approaches to construct a 3D (three-dimensional) structural model for the NADP-dependent Pichia stipitis xylose reductase (PsXR). Based on the 3D model, the favourable binding modes for both cofactors NAD and NADP were obtained using the flexible docking procedure and molecular dynamics simulation. Structural analysis of the favourable binding modes showed that the cofactor binding site of PsXR was composed of 3 major components: a hydrophilic pocket, a hydrophobic pocket as well as a linker channel between the aforementioned two pockets. The hydrophilic pocket could recognize the nicotinamide moiety of the cofactors by hydrogen bonding networks, while the hydrophobic pocket functioned to position the adenine moiety of the cofactors by hydrophobic and Π-Π stacking interactions. The linker channel contained some key residues for ligand-binding; their mutation could have impact to the specificity of PsXR. Finally, it was found that any of the two single mutations, K21A and K270N, might reverse the cofactor specificity of PsXR from major NADP- to NADdependent, which was further confirmed by the additional experiments. Our findings may provide useful insights into the cofactor specificity of PsXR, stimulating new strategies for better designing xylose reductase and improving ethanol production in industry.

  13. Role of heparin in the antithrombotic treatment of valvulopathies. (United States)

    Hanania, Guy


    Heparin is indicated to replace warfarin in patients with valve disease requiring antithrombotic treatment. Its use in thus necessary for short periods during which warfarin is contraindicated, but the thromboembolic risk persists. These circumstances, which are common in patients with mechanical prostheses, include: hemorrhagic risk or event complicating an existing thromboembolic risk (heart or extracardiac surgery, severe hemorrhage, end of pregnancy); when an unstable situation develops and imposes the rapid diminution or interruption of anticoagulants (stroke, infectious endocarditis); when immediate efficacy is required, rather than the delayed action of warfarin (onset of atrial fibrillation); and when warfarin is contraindicated (early pregnancy). Regardless of whether unfractionated or low molecular-weight heparin (LMWH) is used, therapeutic doses must be prescribed: continuously perfused intravenous and subcutaneous injections (t.i.d.) with repeated biological monitoring for the former, or subcutaneous injections (b.i.d.) with initial biological controls preferred and repeated in elderly subjects or those suffering from renal insufficiency. International guidelines have specified the respective roles of heparin in general, and each preparation individually with an ever-increasing use of LMWH, the efficacy of which has been proven in the majority of common thromboembolic pathologies and in pregnant women.

  14. The Use of Heparin during Endovascular Peripheral Arterial Interventions: A Synopsis

    Directory of Open Access Journals (Sweden)

    Arno M. Wiersema


    Full Text Available A large variety exists for many aspects of the use of heparin as periprocedural prophylactic antithrombotics (PPAT during peripheral arterial interventions (PAI. This variation is present, not only within countries, but also between them. Due to a lack of (robust data, no systematic review on the use of heparin during PAI could be justified. A synopsis of all available literature on heparin during PAI describes that heparin is used on technical equipment to reduce the thrombogenicity and in the flushing solution with saline. Heparin could have a cumulative anticoagulant effect when used in combination with ionic contrast medium. No level-1 evidence exists on the use of heparin. A measurement of actual anticoagulation status by means of an activated clotting time should be mandatory.

  15. Low-dose versus high-dose heparinization during arteriovenous carbon dioxide removal. (United States)

    Murphy, J A; Savage, C M; Alpard, S K; Deyo, D J; Jayroe, J B; Zwischenberger, J B


    The purpose of this study was to compare low-dose (LD) and high-dose (HD) systemic heparinization in a prospective randomized study of arteriovenous carbon dioxide removal (AVCO2R) during acute respiratory distress syndrome, using a commercially available heparin-coated oxygenator. Adult sheep (n = 13) received an LD50 smoke inhalation and 40% TBSA third degree cutaneous flame burn injury. At 40-48 h post-injury, animals underwent cannulation of the carotid artery and jugular vein and were then randomized to HD heparin (activated clotting time, ACT > 300s, n = 6) and LD heparin (ACT heparin (ACT heparin-coated oxygenator does not increase thrombogenicity during AVCO2R for smoke/burn-induced severe lung injury in sheep.

  16. Single-step synthesis of heparin-doped polypyrrole nanoparticles for delivery of angiogenic factor. (United States)

    Xiong, Gordon M; Yap, Yi Zhen; Choong, Cleo


    To perform one-pot synthesis of heparin-immobilized polypyrrole (PPy) nanoparticles and evaluate the use of these nanoparticles for the delivery of VEGF. Heparin-stabilized synthesis of PPy nanoparticles was performed via oxidative polymerization. VEGF-bound PPy-heparin nanoparticles were delivered to endothelial cells and bioactivity of VEGF was assessed by Matrigel tube formation. Size-controllable synthesis of heparin-doped PPy nanoparticles was achieved, and heparin promoted the conjugation of VEGF. Angiogenic activity of the VEGF-conjugated PPy nanoparticles was verified. Heparin-doped PPy nanoparticles can be synthesized using one-pot reaction and provide a delivery platform by which VEGF can be conjugated onto.

  17. The glmS Ribozyme Cofactor is a General Acid-Base Catalyst (United States)

    Viladoms, Julia; Fedor, Martha J.


    The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The D-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (β ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst. PMID:23113700

  18. Effects of the cofactor binding sites on the activities of secondary alcohol dehydrogenase (SADH). (United States)

    Wang, Tao; Chen, Xiangjun; Han, Jun; Ma, Sichun; Wang, Jianmei; Li, Xufeng; Zhang, Hui; Liu, Zhibin; Yang, Yi


    SADHs from Thermoanaerobacter ethanolicus are enzymes that, together with various cofactors, catalyze the reversible reduction of carbonyl compounds to their corresponding alcohols. To explore how cofactors bind to SADH, TeSADH was cloned in this study, and Ser(199) and Arg(200) were replaced by Tyr and Asp, respectively. Both sites were expected to be inside or adjacent to the cofactor-binding domain according to computational a prediction. Analysis of TeSADH activities revealed that the enzymatic efficiency (kcat/Km) of the S199Y mutant was noticeably enhanced using by NADH, NADPH as cofactors, and similar with that of wild-type using by NADP(+), NAD(+). Conversely, the activity of the R200D mutant significantly decreased with all cofactors. Furthermore, in yeast, the S199Y mutant substantially elevated the ethanol concentration compared with the wild type. Molecular dynamics simulation results indicated the H-bonding network between TeSADH and the cofactors was stronger for the S199Y mutant and the binding energy was simultaneously increased. Moreover, the fluorescence results indicated the S199Y mutant exhibited an increased preference for NAD(P)H, binding with NAD(P)H more compactly compared with wild type. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules


    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Rehault-Godbert, Sophie


    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ova...

  20. Aplikace nízkomolekulárních heparinů


    Maršíková, Miroslava


    This master's thesis focuses on subcutaneous administration of low molecular weight heparins. The theoretical part is focused on anticoagulation treatment. It specifies aspects in which treatment with low molecular weight heparins is more beneficial than standard heparin treatment. It describes subcutaneous administration techniques referred to in the scientific literature in the Czech Republic since the year 1995. It also provides descriptions of subcutaneous administration for the most freq...

  1. Inactivation of human antithrombin by neutrophil elastase. Kinetics of the heparin-dependent reaction. (United States)

    Jordan, R E; Nelson, R M; Kilpatrick, J; Newgren, J O; Esmon, P C; Fournel, M A


    Human neutrophil elastase catalyzes the inactivation of antithrombin by a specific and limited proteinolytic cleavage. This inactivation reaction is greatly accelerated by an active anticoagulant heparin subfraction with high binding affinity for antithrombin. A potentially complex reaction mechanism is suggested by the binding of both neutrophil elastase and antithrombin to heparin. The in vitro kinetic behavior of this system was examined under two different conditions: 1) at a constant antithrombin concentration in which the active anticoagulant heparin was varied from catalytic to saturating levels; and 2) at a fixed, saturating heparin concentration and variable antithrombin levels. Under conditions of excess heparin, the inactivation could be continuously monitored by a decrease in the ultraviolet fluorescence emission of the inhibitor. A Km of approximately 1 microM for the heparin-antithrombin complex and a turnover number of approximately 200/min was estimated from these analyses. Maximum acceleratory effects of heparin on the inactivation of antithrombin occur at heparin concentrations significantly lower than those required to saturate antithrombin. The divergence in acceleratory effect and antithrombin binding contrasts with the anticoagulant functioning of heparin in promoting the formation of covalent antithrombin-enzyme complexes and is likely to derive from the fact that neutrophil elastase is not consumed in the inactivation reaction. A size dependence was observed for the heparin effect since an anticoagulantly active octasaccharide fragment of heparin, with avid antithrombin binding activity, was without effect on the inactivation of antithrombin by neutrophil elastase. Despite the completely nonfunctional nature of elastase-cleaved antithrombin and the altered physical properties of the inhibitor as indicated by fluorescence and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the inactivated inhibitor exhibited a circulating half

  2. Effects of a supersulfated low molecular weight heparin (IK-SSH) on different hemostatic parameters. (United States)

    Glusa, E; Barthel, W; Schenk, J; Radziwon, P; Butti, A; Markwardt, F; Breddin, K H


    In a phase I trial effects of a new supersulfated low molecular weight heparin (IK-SSH) on different hemostatic parameters were investigated in healthy volunteers. Parameters studied were activated partial thromboplastin time (aPTT), thrombin time, Heptest, anti-activated factor II (anti-FIIa) and anti-activated factor X (anti-FXa) activity, platelet adhesion, platelet count, platelet-induced thrombin generation time (PITT), bleeding time, antithrombin III, fibrinogen and several safety parameters. After single intravenous (i.v.) injections of IK-SSH (0.14, 0.33 and 0.66 mg/kg) aPTT, Heptest and PITT were strongly and dose-dependently prolonged. After ascending subcutaneous (s.c.) doses of IK-SSH (0.33, 0.66 and 1 mg/kg) aPTT, Heptest and PITT were prolonged in a dose-dependent manner. Repeat s.c. injections of 1 mg/kg IK-SSH for 5 days markedly prolonged aPTT, Heptest and PITT. No cumulative effects were observed. Anti-FIIa and anti-FXa activity were not or only slightly increased. Bleeding time, thrombin time and platelet adhesion were not significantly changed after i.v. and s.c. injections of IK-SSH. However, tissue factor pathway inhibitor (TFPI) concentration was markedly increased after each injection of IK-SSH and returned to the preinjection value 24 h later. IK-SSH prolongs aPTT, Heptest and PITT in a similar manner as other low molecular weight heparins but without significantly affecting thrombin time, FIIa and FXa activity. The release of TFPI may well be responsible for the prolongation of aPTT, Heptest and PITT. IK-SSH may be further developed as an antithrombotic agent.

  3. Effect of low-dose heparin on urinary albumin excretion in insulin-dependent diabetes mellitus

    DEFF Research Database (Denmark)

    Myrup, B; Hansen, P M; Jensen, T


    We investigated the effect of heparin on urinary albumin excretion in patients with insulin-dependent diabetes mellitus. 39 patients with persistent urinary albumin excretion of 30-300 mg/24 h were randomly treated for 3 months with subcutaneous injections twice daily of isotonic saline, 5000 IU...... unfractionated heparin, or 2000 anti-Xa IU low-molecular-weight heparin. Unfractionated and low-molecular-weight heparin induced a significant reduction in urinary albumin excretion (p = 0.04 and p = 0.004). The mechanism and clinical relevance is unknown but deserve further attention....

  4. Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization (United States)

    Bao, Ji; Wu, Qiong; Sun, Jiu; Zhou, Yongjie; Wang, Yujia; Jiang, Xin; Li, Li; Shi, Yujun; Bu, Hong


    Whole-liver perfusion-decellularization is an attractive scaffold–preparation technique for producing clinical transplantable liver tissue. However, the scaffold’s poor hemocompatibility poses a major obstacle. This study was intended to improve the hemocompatibility of perfusion-decellularized porcine liver scaffold via immobilization of heparin. Heparin was immobilized on decellularized liver scaffolds (DLSs) by electrostatic binding using a layer-by-layer self-assembly technique (/h-LBL scaffold), covalent binding via multi-point attachment (/h-MPA scaffold), or end-point attachment (/h-EPA scaffold). The effect of heparinization on anticoagulant ability and cytocompatibility were investigated. The result of heparin content and release tests revealed EPA technique performed higher efficiency of heparin immobilization than other two methods. Then, systematic in vitro investigation of prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT), platelet adhesion and human platelet factor 4 (PF4, indicates platelet activation) confirmed the heparinized scaffolds, especially the /h-EPA counterparts, exhibited ultralow blood component activations and excellent hemocompatibility. Furthermore, heparin treatments prevented thrombosis successfully in DLSs with blood perfusion after implanted in vivo. Meanwhile, after heparin processes, both primary hepatocyte and endothelial cell viability were also well-maintained, which indicated that heparin treatments with improved biocompatibility might extend to various hemoperfusable whole-organ scaffolds’ preparation. PMID:26030843

  5. Hemocompatibility improvement of perfusion-decellularized clinical-scale liver scaffold through heparin immobilization

    National Research Council Canada - National Science Library

    Bao, Ji; Wu, Qiong; Sun, Jiu; Zhou, Yongjie; Wang, Yujia; Jiang, Xin; Li, Li; Shi, Yujun; Bu, Hong


    .... However, the scaffold's poor hemocompatibility poses a major obstacle. This study was intended to improve the hemocompatibility of perfusion-decellularized porcine liver scaffold via immobilization of heparin...

  6. Heparin and insulin in the management of hypertriglyceridemia-associated pancreatitis: case series and literature review. (United States)

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


    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.

  7. Increased accuracy in heparin and protamine administration decreases bleeding: a pilot study

    DEFF Research Database (Denmark)

    Runge, Marx; Møller, Christian H; Steinbrüchel, Daniel A


    of the study was to evaluate whether a heparin-protamine titration system, Hemochron RxDx, could reduce postoperative bleeding and blood transfusion. Fifty-three patients were included prospectively over a 6-month period. The test group (RxDx group; 28 patients) received heparin and protamine doses calculated...... using the Hemochron RxDx system, which performs a baseline activated clotting time (ACT) value together with a heparin response test. An accurate heparin dose was calculated based on the Bull dose/response curve. Protamine doses were calculated by the same method. In the control group (25 patients...

  8. A pyrene-based fluorescent sensor for ratiometric detection of heparin and its complex with heparin for reversed ratiometric detection of protamine in aqueous solution (United States)

    Gong, Weiwei; Wang, Shihuai; Wei, Yuting; Ding, Liping; Fang, Yu


    An imidazolium-modified pyrene derivative, IPy, was used for ratiometric detection of heparin, and its complex with heparin was used for reversed ratiometric detection of protamine in both aqueous solution and serum samples. The cationic fluorescent probe could interact with anionic heparin via electrostatic interaction to bring about blue-to-green fluorescence changes as monomer emission significantly decreases and excimer increases. The binary combination of IPy and heparin could be further used for green-to-blue detection of protamine since heparin prefers to bind to protamine instead of the probe due to its stronger affinity with protamine. The cationic probe shows high sensitivity to heparin with a low detection limit of 8.5 nM (153 ng/mL) and its combination with heparin displays high sensitivity to protamine with a detection limit as low as 15.4 nM (107.8 ng/mL) according to the 3σ IUPAC criteria. Moreover, both sensing processes are fast and can be performed in serum solutions, indicating possibility for practical applications.

  9. Taspase1 processing alters TFIIA cofactor properties in the regulation of TFIID. (United States)

    Malecová, Barbora; Caputo, Valentina S; Lee, Diane F; Hsieh, James J; Oelgeschläger, Thomas


    TFIIA is an important positive regulator of TFIID, the primary promoter recognition factor of the basal RNA polymerase II transcription machinery. TFIIA antagonises negative TFIID regulators such as negative cofactor 2 (NC2), promotes specific binding of the TBP subunit of TFIID to TATA core promoter sequence elements and stimulates the interaction of TBP-associated factors (TAFs) in the TFIID complex with core promoter elements located downstream of TATA, such as the initiator element (INR). Metazoan TFIIA consists of 3 subunits, TFIIAα (35 kDa), β (19 kDa) and γ (12 kDa). TFIIAα and β subunits are encoded by a single gene and result from site-specific cleavage of a 55 kDa TFIIA(α/β) precursor protein by the protease Taspase1. Metazoan cells have been shown to contain variable amounts of TFIIA (55/12 kDa) and Taspase1-processed TFIIA (35/19/12 kDa) depending on cell type, suggesting distinct gene-specific roles of unprocessed and Taspase1-processed TFIIA. How precisely Taspase1 processing affects TFIIA functions is not understood. Here we report that Taspase1 processing alters TFIIA interactions with TFIID and the conformation of TFIID/TFIIA promoter complexes. We further show that Taspase1 processing induces increased sensitivity of TFIID/TFIIA complexes to the repressor NC2, which is counteracted by the presence of an INR core promoter element. Our results provide first evidence that Taspase1 processing affects TFIIA regulation of TFIID and suggest that Taspase1 processing of TFIIA is required to establish INR-selective core promoter activity in the presence of NC2.

  10. Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Amit Ghosh

    Full Text Available Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.

  11. Comparative study of heparin-binding proteins profile of Murrah buffalo (Bubalus bubalis semen

    Directory of Open Access Journals (Sweden)

    S. S. Ramteke


    Full Text Available Aim: The experiment was conducted to study the total seminal plasma protein (TSPP and heparin-binding proteins (HBPs in relation to initial semen quality of buffalo bull. Materials and Methods: Semen from two Murrah buffalo bulls (bull no. 605 and 790 with mass motility of ≥3+ were used for the study and categorized into three groups (Group I- Mass motility 3+, Group II- Mass motility 4+ and Group III- Mass motility 5+. Seminal plasma from semen was separated by centrifugation. HBPs was isolated and purified from heparin-agarose affinity column by modified elution buffer. TSPP and isolated HBPs concentration was estimated by Lowry’s method. The purified HBPs were resolved on Sodium dodecyl sulfate polyacrylamide gel electrophoresis to check the protein profile of two bulls. Results: The mean values of TSPP concentrations in bull no. 605 and 790 in Group I, II and III were 30.64±0.12, 31.66±0.09, 32.53±0.19 and 28.51±0.09, 29.49±0.15, 30.45±0.17 mg/mL, respectively. The mean values of HBPs concentrations in bull no. 605 and 790 in Group I, II and III were 3.11±0.07, 3.32±0.06, 3.46±0.08 and 2.51±0.08, 2.91±0.05, 3.10±0.03 mg/mL, respectively. Both the values of TSPP and HBPs were significantly higher (p<0.01 in bull no. 605 when compared to 790 in all the three groups. 31 kDa HBP was more intensely present in bull no. 605, thus may indicate its superiority over bull no. 790 in relation to fertility potential. Conclusion: TSPP and HBPs shows variation in concentration with respect to initial semen quality. Furthermore, presence of fertility related 31 kDa HBPs in one of the bull may be an indication of high fertility of a bull. In future, in-vivo and in-vitro correlative study on larger basis is needed for the establishment of fertility-related HBPs in semen which might establish criteria for selection of buffalo bull with high fertility potential.

  12. An Unreported Cause of Buccal Mucosal Hematoma: A Rare Complication After Heparin Therapy in a Patient with Chronic Renal Failure

    Directory of Open Access Journals (Sweden)

    Mustafa Yıldız


    Full Text Available Patients receiving anti-coagulants such as heparin can suffer from significant complications of these medications. Herein, we report a buccal mucosal hematoma in a heparinized patient with chronic renal failure.

  13. Nebulized heparin is associated with fewer days of mechanical ventilation in critically ill patients: a randomized controlled trial

    NARCIS (Netherlands)

    Dixon, Barry; Schultz, Marcus J.; Smith, Roger; Fink, James B.; Santamaria, John D.; Campbell, Duncan J.


    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

  14. Nebulised heparin is associated with fewer days of mechanical ventilation in critically ill patients: a randomised controlled trial

    NARCIS (Netherlands)

    Dixon, B.; Schultz, M.J.; Smith, R.; Fink, J.B.; Santamaria, J.D.; Campbell, D.J.


    INTRODUCTION: 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 nebulised heparin improved lung function in

  15. Heparin-binding protein (HBP): an early marker of respiratory failure after trauma? (United States)

    Johansson, J; Brattström, O; Sjöberg, F; Lindbom, L; Herwald, H; Weitzberg, E; Oldner, A


    Trauma and its complications contribute to morbidity and mortality in the general population. Trauma victims are susceptible to acute respiratory distress syndrome (ARDS) and sepsis. Polymorphonuclear leucocytes (PMNs) are activated after trauma and there is substantial evidence of their involvement in the development of ARDS. Activated PMNs release heparin-binding protein (HBP), a granule protein previously shown to be involved in acute inflammatory reactions. We hypothesised that there is an increase in plasma HBP content after trauma and that the increased levels are related to the severity of the trauma or later development of severe sepsis and organ failure (ARDS). We investigated HBP in plasma samples within 36 h from trauma in 47 patients admitted to a level one trauma centre with a mean injury severity score (ISS) of 26 (21-34). ISS, admission sequential organ failure assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE) II scores were recorded at admission. ARDS and presence of severe sepsis were determined daily during intensive care. We found no correlation between individual maximal plasma HBP levels at admission and ISS, admission SOFA or APACHE II. We found, however, a correlation between HBP levels and development of ARDS (P = 0.026, n = 47), but not to severe sepsis. HBP is a potential biomarker candidate for early detection of ARDS development after trauma. Further research is required to confirm a casual relationship between plasma HBP and the development of ARDS. © 2013 The Acta Anaesthesiologica Scandinavica Foundation.

  16. Generation and characterization of conditional heparin-binding EGF-like growth factor knockout mice.

    Directory of Open Access Journals (Sweden)

    Atsushi Oyagi

    Full Text Available Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b altered dopamine and serotonin levels in the brain, (c decreases in spine density in neurons of the prefrontal cortex, (d reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA receptor and post-synaptic protein-95 (PSD-95, (e decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.

  17. Thrombin-inhibitory activity of whale heparin oligosaccharides. (United States)

    Ototani, N; Kodama, C; Kikuchi, M; Yosizawa, Z


    Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase. The analytical data indicated that the proportions of trisulfated disaccharide (IdUA(2S)alpha 1----4GlcNS(6S)) and disulfated disaccharide (UA1----4GlcNS(6S)) increased with the manifestation of high thrombin-inhibitory activity, while that of monosulfated disaccharide (UA1----4GlcNS) decreased. The present observations, together with those so far reported, suggest that the presence of the former structural elements, specifically IdUA(2S)alpha 1----4GlcNS(6S), as well as the antithrombin III-binding pentasaccharide at the proper positions in the molecules of whale heparin oligosaccharides is essential for the manifestation of high inhibitory activity for thrombin in the presence of antithrombin III. The structural bases for the manifestation of the anticoagulant activity of whale and porcine heparins and their oligosaccharides are also discussed.

  18. p53 transactivation and the impact of mutations, cofactors and small molecules using a simplified yeast-based screening system.

    Directory of Open Access Journals (Sweden)

    Virginia Andreotti

    Full Text Available BACKGROUND: The p53 tumor suppressor, which is altered in most cancers, is a sequence-specific transcription factor that is able to modulate the expression of many target genes and influence a variety of cellular pathways. Inactivation of the p53 pathway in cancer frequently occurs through the expression of mutant p53 protein. In tumors that retain wild type p53, the pathway can be altered by upstream modulators, particularly the p53 negative regulators MDM2 and MDM4. METHODOLOGY/PRINCIPAL FINDINGS: Given the many factors that might influence p53 function, including expression levels, mutations, cofactor proteins and small molecules, we expanded our previously described yeast-based system to provide the opportunity for efficient investigation of their individual and combined impacts in a miniaturized format. The system integrates i variable expression of p53 proteins under the finely tunable GAL1,10 promoter, ii single copy, chromosomally located p53-responsive and control luminescence reporters, iii enhanced chemical uptake using modified ABC-transporters, iv small-volume formats for treatment and dual-luciferase assays, and v opportunities to co-express p53 with other cofactor proteins. This robust system can distinguish different levels of expression of WT and mutant p53 as well as interactions with MDM2 or 53BP1. CONCLUSIONS/SIGNIFICANCE: We found that the small molecules Nutlin and RITA could both relieve the MDM2-dependent inhibition of WT p53 transactivation function, while only RITA could impact p53/53BP1 functional interactions. PRIMA-1 was ineffective in modifying the transactivation capacity of WT p53 and missense p53 mutations. This dual-luciferase assay can, therefore, provide a high-throughput assessment tool for investigating a matrix of factors that can influence the p53 network, including the effectiveness of newly developed small molecules, on WT and tumor-associated p53 mutants as well as interacting proteins.

  19. Anti-Platelet Factor 4/Heparin Antibody Formation Occurs Endogenously and at Unexpected High Frequency in Polycythemia Vera

    Directory of Open Access Journals (Sweden)

    Sara C. Meyer


    Full Text Available Background. Myeloproliferative neoplasms (MPN encounter thromboses due to multiple known risk factors. Heparin-induced thrombocytopenia (HIT is a thrombotic syndrome mediated by anti-platelet factor 4 (PF4/heparin antibodies with undetermined significance for thrombosis in MPN. We hypothesized that anti-PF4/heparin Ab might occur in MPN and promote thrombosis. Methods. Anti-PF4/heparin antibodies were analyzed in 127 MPN patients including 76 PV and 51 ET. Screening, validation testing, and isotype testing of anti-PF4/heparin Ab were correlated with disease characteristics. Results. Anti-PF4/heparin antibodies were detected in 21% of PV and 12% of ET versus 0.3–3% in heparin-exposed patients. Validation testing confirmed anti-PF4/heparin immunoglobulins in 15% of PV and 10% of ET. Isotype testing detected 9.2% IgG and 5.3% IgM in PV and exclusively IgM in ET. IgG-positive PV patients encountered thromboses in 57.1% suggesting anti-PF4/heparin IgG may contribute to higher risk for thrombosis in MPN. Overall, 45% of PV patients experienced thromboses with 11.8% positive for anti-PF4/heparin IgG versus 7.1% in PV without thrombosis. Conclusion. Anti-PF4/heparin antibodies occur endogenously and more frequently in MPN than upon heparin exposure. Thrombotic risk increases in anti-PF4/heparin IgG-positive PV reflecting potential implications and calling for larger, confirmatory cohorts. Anti-PF4/heparin IgG should be assessed upon thrombosis in PV to facilitate avoidance of heparin in anti-PF4/heparin IgG-positive PV.

  20. Poly(vinyl alcohol)-heparin hydrogels as sensor catheter membranes

    NARCIS (Netherlands)

    Brinkman, E.; van der Does, L.; Bantjes, A.


    Poly(vinyl alcohol)-heparin hydrogels with varying water content were synthesized for use as sensor catheter membranes. Films were cast from aqueous mixtures of poly(viny) alcohol) (PVA), a photosensitive cross-linker p-diazonium diphenyl amine polymer (PA), glutaraldehyde (GA) and heparin. After


    NARCIS (Netherlands)


    Heparins blunt the development of glomerulosclerosis in several disease models in the rat and this protective effect may be related to suppression of glomerular cell proliferation. In this study the direct effect of heparins on another key event in glomerulosclerosis, extracellular matrix (ECM)

  2. Lipoprotein lipase deficiency due to long-term heparinization presenting as severe hypertriglyceridaemia in pregnancy.


    Watts, G F; Cameron, J; Henderson, A; Richmond, W.


    A case of severe hypertriglyceridaemia presenting in the third trimester of pregnancy in a woman on long-term heparin prophylaxis is described. The hypertriglyceridaemia was attributed to impaired clearance of triglyceride-rich lipoprotein particles secondary to heparin-induced reduction in the activity of the lipolytic enzyme, lipoprotein lipase.

  3. Prophylactic heparin in patients with severe sepsis treated with drotrecogin alfa (activated)

    NARCIS (Netherlands)

    Levi, Marcel; Levy, Mitchell; Williams, Mark D.; Douglas, Ivor; Artigas, Antonio; Antonelli, Massimo; Wyncoll, Duncan; Janes, Jonathan; Booth, Frank V.; Wang, Dazhe; Sundin, David P.; Macias, William L.


    RATIONALE: Patients with severe sepsis frequently receive prophylactic heparin during drotrecogin alfa (activated) (DrotAA) treatment due to risk of venous thromboembolic events (VTEs). Biological plausibility exists for heparin to reduce DrotAA efficacy and/or increase bleeding. OBJECTIVES:

  4. Low molecular weight heparin as an anticoagulation strategy for left-sided ablation procedures. (United States)

    Hinsley, Karen; Evans-Langhorst, Margaret; Porter, Courtney; Chandler, Stephanie; VanderPluym, Christina; Triedman, John; Bezzerides, Vassilios J


    This quality improvement study was implemented to demonstrate consistent and reliable post procedure anticoagulation for patients undergoing left-sided ablations. We evaluated the safety and efficacy of anticoagulation practice during a transition from anticoagulation with overnight infusion of unfractionated heparin to a single subcutaneous injection of low molecular weight heparin. Outcomes for patients who received unfractionated heparin from January 2014 to October 2014, were compared with outcomes of patients who received low molecular weight heparin from October 2014 to October 2015. Complications prepractice and postpractice change were documented and compared to establish confidence in the practice change and confirm the safety of the anticoagulation therapy management. There were no differences in the type or frequency of complications/adverse events demonstrated between the patients who had received unfractionated heparin for anticoagulation prophylaxis and those who received low molecular weight heparin. No thromboembolic events were reported or documented with either anticoagulation strategy. After confidence in the safety and efficacy of the practice change was established, a decision was made to discharge patients home the same day as there procedure, effectively reducing inpatient bed days and overall costs. Administration of low molecular weight heparin provides predictable anticoagulation and equally safe as unfractionated heparin when administered to patients post left-sided ablation. A secondary gain has been reduction of procedural costs by elimination of the previously required inpatient observation stay. © 2017 Wiley Periodicals, Inc.

  5. Poly(dimethylsiloxane)-poly(ethyleneoxide)-heparin block copolymers. I. Synthesis and characterization

    NARCIS (Netherlands)

    Grainger, D.W.; Kim, S.W.; Feijen, Jan


    Amphiphilic block copolymers containing poly(dimethylsiloxane), poly(ethylene oxide), and heparin (PDMS-PEO-Hep) have been prepared via a series of coupling reactions using functionalized prepolymers, diisocyanates, and derivatized heparins. All intermediate steps of the synthesis yield quantifiable

  6. Covalently bound conjugates of albumin and heparin: Synthesis, fractionation and characterization

    NARCIS (Netherlands)

    Hennink, Wim E.; Feijen, Jan; Ebert, Charles D.; Kim, Sung Wan


    Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10–16 weight % of heparin) were

  7. In vitro Heparin Precipitation in the Plasma of Euthyroid women with ...

    African Journals Online (AJOL)

    Antimicrosomal autoantibody (Anti-TPO) ELISA assay was determined. The plasma treatment at the different PH with heparin and aspirin in physiological buffers were carried out. RESULT: At PH5.0 (i.e. the Uterus physiological PH) using 100i.u/ml heparin in 0.2M acetate buffer yielded 70% precipitate compared to ...

  8. Heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep. (United States)

    Murakami, Kazunori; McGuire, Roy; Cox, Robert A; Jodoin, Jeffrey M; Bjertnaes, Lars J; Katahira, Jiro; Traber, Lillian D; Schmalstieg, Frank C; Hawkins, Hal K; Herndon, David N; Traber, Daniel L


    Pseudomonas pneumonia is a common complication of smoke inhalation injury. Airway casts formed from clotted mucous occur frequently in this condition. A recent report shows that intravenous heparin improves oxygenation and reduces lung damage in a sheep model of smoke inhalation. We hypothesized that nebulized heparin could be an effective means of reducing cast formation. Female sheep (n = 19) were surgically prepared for a study of acute lung injury (ALI). After a tracheotomy, 48 breaths of cotton smoke (heparin-nebulized group (n = 5; animals received aerosolized heparin [10,000 I.U.] 1 h after the bacterial instillation and subsequently every 4 h thereafter), an intravenous heparin group (n = 5,300 U/kg/23 h, infusion was started 1 h after the injury), a saline-nebulization group (n = 5; animals received inhaled nebulized saline), and a sham injury group (n = 4, treated in the same fashion, but no injury). The animals were sacrificed after 24 h of mechanical ventilation, and lung samples were harvested. Sheep exposed to lung injury presented with typical hyperdynamic cardiovascular changes and a corresponding drop in PaO2. These changes were significantly attenuated in the heparin groups. Histological changes consisting of cellular infiltrates, lung edema, congestion, and cast formation were reduced by heparin. These data suggest that nebulized inhaled heparin is a beneficial therapy for sepsis-induced ALI.

  9. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity. (United States)

    Li, Binghan; Lu, Dan; Chen, Yuqing; Zhao, Minghui; Zuo, Li


    Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin's effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4) and pre-osteoclasts (RAW264.7). In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand), M-CSF (macrophage colony-stimulating factor), and OPG (osteoprotegerin), which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG). However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  10. Design of a new type of coating for the controlled release of heparin

    NARCIS (Netherlands)

    Hinrichs, W.L.J.; Hinrichs, W.L.J.; ten Hoopen, Hermina W.M.; Wissink, M.J.B.; Engbers, G.H.M.; Feijen, Jan


    Thrombus formation at the surface of blood contacting devices can be prevented by local release of heparin. Preferably, the release rate should be constant for prolonged periods of time. The minimum heparin release rate to achieve thromboresistance will be different for various applications and

  11. A genome-wide association study of heparin-induced thrombocytopenia using an electronic medical record

    DEFF Research Database (Denmark)

    Karnes, Jason H; Cronin, Robert M; Rollin, Jerome


    of GWAS-associated single nucleotide polymorphisms (SNPs) on PF4/heparin antibodies in non-heparin treated individuals. In a recessive model, we observed significant SNP associations (odds ratio [OR] 18.52; 95% confidence interval [CI] 6.33-54.23; p=3.18×10(-9)) with HIT near the T-Cell Death...

  12. Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin. (United States)

    Baik, Jong Youn; Dahodwala, Hussain; Oduah, Eziafa; Talman, Lee; Gemmill, Trent R; Gasimli, Leyla; Datta, Payel; Yang, Bo; Li, Guoyun; Zhang, Fuming; Li, Lingyun; Linhardt, Robert J; Campbell, Andrew M; Gorfien, Stephen F; Sharfstein, Susan T


    Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 μg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. A randomized trial of heparin plus ursodiol vs. heparin alone to prevent hepatic veno-occlusive disease after hematopoietic stem cell transplantation

    National Research Council Canada - National Science Library

    Park, S H; Lee, M H; Lee, H; Kim, H S; Kim, K; Kim, W S; Jung, C W; Im, Y H; Yoon, S S; Kang, W K; Park, K; Park, C H; Kim, S W


    ...). There is no safe and proven therapy for established VOD, and focus has been on its prevention. Previous studies have shown that a continuous infusion of unfractionated heparin or ursodiol may reduce the incidence of VOD...

  14. The US regulatory and pharmacopeia response to the global heparin contamination crisis. (United States)

    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


    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.

  15. Complete Penile Necrosis in a Patient With Heparin-induced Thrombocytopenia: A Case Report

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Blais


    Full Text Available Penile necrosis is a rare condition that has been mostly described in association with diabetes mellitus and end-stage renal disease. We report an unusual case of acute penile necrosis because of heparin-induced thrombocytopenia. A 75-year-old man presented with acute renal failure and experienced cardiac complications during the hospitalization. The patient was treated twice with intravenous heparin. He developed symptoms of penile necrosis 4 days after the reintroduction of heparin. At that moment, the platelet count dropped by 61%, and the analysis of heparin-pf4 antibodies was positive for heparin-induced thrombocytopenia. The patient underwent a total penectomy and a perineal urethrostomy.

  16. Facile immobilization of heparin on bioabsorbable iron via mussel adhesive protein (MAPs

    Directory of Open Access Journals (Sweden)

    Xuchen Xu


    Full Text Available Motivated by adhesive proteins in mussels, strategies using dopamine to modified surface have become particularly attractive. In the present work, we developed a novel and convenient method to modify the biodegradable Fe plates with heparin. Iron was first treated by a facile one-step pH-induced polymerization of dopamine, and then a high density heparin was successfully grafted onto the surface via coupling with polydopamine (PDA active layer. Heparin immobilization contributed much longer blood clotting coagulation time than the pure Fe sample, and hence reduced the risk of thrombosis. Cell viability tests suggested that the heparin modified Fe plates were more favorable to the proliferation of ECV304 cells. In summary, the heparin modified Fe plates with good anti-thrombus properties and inhibiting the proliferation of VSMC cells provide great prospects for biodegradable iron.

  17. Heparin-Induced Cardiac Tamponade and Life-Threatening Hyperkalemia in a Patient with Chronic Hemodialysis

    Directory of Open Access Journals (Sweden)

    Ho-Ming Su


    Full Text Available Heparin, a commonly used anticoagulant agent, is frequently used in patients undergoing hemodialysis. As with most medications, heparin has a significant side effect profile. Two of its most important side effects, major bleeding and hyperkalemia, may be devastating without immediate diagnosis and treatment. Major bleeding such as gastrointestinal, genitourinary or intracranial bleeding is occasionally encountered and rarely neglected. However, heparin-induced cardiac tamponade is rarely encountered and may be easily overlooked. Another side effect, heparin-induced hyperkalemia, an unusual but well-described side effect, is frequently forgotten until life-threatening arrhythmia has occurred. We report a case involving a 40-year-old male patient with uremia, who had received heparin for 10 days for deep vein thrombosis in the left lower extremity. Hemopericardium with cardiac tamponade and life-threatening hyperkalemia were both noted in this patient.

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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 bulls 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.

  19. Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells. (United States)

    Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju


    Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts. (United States)

    Prier, Christopher K; Arnold, Frances H


    Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

  1. 1-Year Results of a Multicenter Randomized Controlled Trial Comparing Heparin-Bonded Endoluminal to Femoropopliteal Bypass. (United States)

    Reijnen, Michel M P J; van Walraven, Laurens A; Fritschy, Wilbert M; Lensvelt, Mare M A; Zeebregts, Clark J; Lemson, M Suzanna; Wikkeling, Otmar R M; Smeets, Luuk; Holewijn, Suzanne


    This study sought to compare heparin-bonded endografts with femoropopliteal bypass, including quality of life, using general health and disease-specific questionnaires as well as patency rates. Endovascular treatment continues to advance and is gaining acceptance as primary treatment for long occlusive or stenotic lesions in the superficial femoral artery. Heparin-bonded expanded polytetrafluoroethylene endografts have been related to outcomes comparable to bypass surgery, but this has not been tested in a randomized fashion. A multicenter randomized-controlled trial was performed comparing femoropopliteal bypass with heparin-bonded expanded polytetrafluoroethylene endografts. Data were analyzed on an intention-to-treat and per-protocol manner. A total of 129 patients were randomized and 125 patients were treated, 63 in the endoluminal and 62 in the surgical group (42 venous, 20 prosthetic). Enrollment was terminated before reaching the predefined target number for patency. Baseline characteristics and anatomical data were similar. Patients were treated for critical limb ischemia in 38.1% and 32.2% in the endoluminal and surgical arms, respectively. Mean lesion length was 23 cm in both groups and lesions were largely TransAtlantic Inter-Society Consensus II D. Hospitalization time and 30-day morbidity were significantly lower in the endoluminal group, without differences in serious adverse events (n = 5 each; surgical: 4 venous and 1 polytetrafluoroethylene bypass). There were no significant differences in Rutherford category between groups at any time point. At 30 days the endoluminal group showed a greater improvement in quality-of-life scores. At 1 year, these differences had largely disappeared and no differences in primary (endoluminal: 64.8%; surgical: 63.6%), assisted primary (endoluminal: 78.1%; surgical: 79.8%), secondary patency (endoluminal: 85.9%; surgical: 83.3%), and target vessel revascularization (endoluminal: 72.1%; surgical: 71.0%) were observed

  2. Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli. (United States)

    Reschke, Stefan; Sigfridsson, Kajsa G V; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke


    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

  3. Identification of a Bis-molybdopterin Intermediate in Molybdenum Cofactor Biosynthesis in Escherichia coli* (United States)

    Reschke, Stefan; Sigfridsson, Kajsa G. V.; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke


    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo–S and Mo–O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme. PMID:24003231

  4. Heparin-induced platelet aggregation (H-IPA): dose/response relationship for two low molecular weight (LMW) heparin preparations (CY 216 and CY 222)

    Energy Technology Data Exchange (ETDEWEB)

    Brace, L.D.; Fareed, J.


    The authors have previously demonstrated that heparin and a LMW heparin derivative (PK 10169) causes platelet aggregation in a dose-dependent manner that can be inhibited by antagonists of the thromboxane pathway. Using fractions of these agents separated on the basis of molecular weight (MW) by gel permeation chromatography, the authors showed that H-IPA was directly dependent upon the MW of the agents tested. In order to further examine this MW dependence, the authors tested two other LMW heparin preparations, CY 216 and CY 222 and subfractions of these agents separated on the basis of MW. Citrate anticoagulated whole blood was drawn from drug-free normal healthy donors whose platelets aggregated when heparin was added to their platelet-rich plasma (PRP). PRP was prepared, various concentrations of the agents or their subfractions were added and aggregation was monitored for 40 minutes at 37/sup 0/C. The results demonstrate that like heparin and PK 10169, CY 216 and CY 222 caused platelet aggregation in a dose and MW dependent manner. Fractions with MW less than 2500 daltons caused aggregation only at concentrations exceeding the therapeutic range of the agents. The authors conclude that the ability to cause H-IPA is an inherent property of heparin and its fractions.

  5. Low-molecular weight heparin increases circulating sFlt-1 levels and enhances urinary elimination.

    Directory of Open Access Journals (Sweden)

    Henning Hagmann

    Full Text Available RATIONALE: Preeclampsia is a devastating medical complication of pregnancy which leads to maternal and fetal morbidity and mortality. While the etiology of preeclampsia is unclear, human and animal studies suggest that excessive circulating levels of soluble fms-like tyrosine-kinase-1 (sFlt-1, an alternatively spliced variant of VEGF-receptor1, contribute to the signs and symptoms of preeclampsia. Since sFlt-1 binds to heparin and heparan sulfate proteoglycans, we hypothesized that the anticoagulant heparin, which is often used in pregnancy, may interfere with the levels, distribution and elimination of sFlt-1 in vivo. OBJECTIVE: We systematically determined serum and urine levels of angiogenic factors in preeclamptic women before and after administration of low molecular weight heparin and further characterized the interaction with heparin in biochemical studies. METHODS AND RESULTS: Serum and urine samples were used to measure sFlt-1 levels before and after heparin administration. Serum levels of sFlt-1 increased by 25% after heparin administration in pregnant women. The magnitude of the increase in circulating sFlt-1 correlated with initial sFlt-1 serum levels. Urinary sFlt-1 levels were also elevated following heparin administration and levels of elimination were dependent on the underlying integrity of the glomerular filtration barrier. Biochemical binding studies employing cation exchange chromatography revealed that heparin bound sFlt-1 had decreased affinity to negatively charged surfaces when compared to sFlt-1 alone. CONCLUSION: Low molecular weight heparin administration increased circulating sFlt1 levels and enhanced renal elimination. We provide evidence that both effects may be due to heparin binding to sFlt1 and masking the positive charges on sFlt1 protein.

  6. [Heparin in coronary angioplasty. Randomized study in cases with low risk of acute occlusion]. (United States)

    Tanajura, L F; Sousa, A G; Pinto, I M; Chaves, A J; Centemero, M P; Feres, F; Mattos, L A; Cano, M N; Maldonado, G A; Sousa, J E


    To assess the efficacy of heparin in preventing the abrupt closure after coronary angioplasty in low risk patients for this phenomenon. In the last 4 years, 525 patients successfully dilated were randomized to receive intravenous heparin (n = 264) or not (n = 261) after the angioplasty. The excluding criteria were contraindications for heparin and risk for abrupt closure (refractory unstable angina, primary coronary angioplasty in acute myocardial infarction, evidence of intracoronary thrombus, intimal tear after the procedure and cases of chronic total occlusions). Both heparin and non heparin groups were similar in respect to female sex (15% x 17%; p = NS), age over 70 years old (7% x 9%; p = NS), previous myocardial infarction (26% x 24%; p = NS), multi-vessel procedures (4% x 7%; p = NS, stable angina (40% x 46%; p = NS), unstable angina (52% x 48%; p = NS) and angioplasty after thrombolytic therapy (8% x 6%; p = NS). The overall incidence of abrupt closure was 2/525 (0.4%), with one case (0.4%) in each group. The in-hospital mortality was 1/525 (0.2%), which occurred in a non-heparin patient, due to a anterior myocardial infarction. Major complications occurred similarly in heparin and non-heparin groups (0.4%). Bleeding complications were observed more frequently in the heparin group (7% x 2%; p = 0.002). All of them were in the catheterization site and none required blood transfusion. Severe systemic bleeding were not observed. In patients regarded as low risk for abrupt closure, the incidence of this complication was really low (0.4%) and heparin probably do not prevent it.

  7. Heparin Binds Lamprey Angiotensinogen and Promotes Thrombin Inhibition through a Template Mechanism. (United States)

    Wei, Hudie; Cai, Haiyan; Wu, Jiawei; Wei, Zhenquan; Zhang, Fei; Huang, Xin; Ma, Lina; Feng, Lingling; Zhang, Ruoxi; Wang, Yunjie; Ragg, Hermann; Zheng, Ying; Zhou, Aiwu


    Lamprey angiotensinogen (l-ANT) is a hormone carrier in the regulation of blood pressure, but it is also a heparin-dependent thrombin inhibitor in lamprey blood coagulation system. The detailed mechanisms on how angiotensin is carried by l-ANT and how heparin binds l-ANT and mediates thrombin inhibition are unclear. Here we have solved the crystal structure of cleaved l-ANT at 2.7 Å resolution and characterized its properties in heparin binding and protease inhibition. The structure reveals that l-ANT has a conserved serpin fold with a labile N-terminal angiotensin peptide and undergoes a typical stressed-to-relaxed conformational change when the reactive center loop is cleaved. Heparin binds l-ANT tightly with a dissociation constant of ∼10 nm involving ∼8 monosaccharides and ∼6 ionic interactions. The heparin binding site is located in an extensive positively charged surface area around helix D involving residues Lys-148, Lys-151, Arg-155, and Arg-380. Although l-ANT by itself is a poor thrombin inhibitor with a second order rate constant of 500 m(-1) s(-1), its interaction with thrombin is accelerated 90-fold by high molecular weight heparin following a bell-shaped dose-dependent curve. Short heparin chains of 6-20 monosaccharide units are insufficient to promote thrombin inhibition. Furthermore, an l-ANT mutant with the P1 Ile mutated to Arg inhibits thrombin nearly 1500-fold faster than the wild type, which is further accelerated by high molecular weight heparin. Taken together, these results suggest that heparin binds l-ANT at a conserved heparin binding site around helix D and promotes the interaction between l-ANT and thrombin through a template mechanism conserved in vertebrates. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. How much heparin do we really need to go on pump? A rethink of current practices.

    LENUS (Irish Health Repository)

    Shuhaibar, M N


    OBJECTIVES: Patients undergoing myocardial revascularisation using extracorporeal circulation require heparin anticoagulation. We aimed to evaluate the effect of reducing heparin dosage on target activated clotting time (ACT) and postoperative blood loss. METHODS: In a prospective randomised trial, 195 patients undergoing isolated primary CABG were randomised into four groups A, B, C, and D receiving an initial heparin dosage of 100, 200, 250 and 300 iu\\/kg, respectively. Extra incremental heparin (50 iu\\/kg) was added if required to achieve a target ACT of 480 s before initiating cardiopulmonary bypass. Postoperative blood loss was measured from the time of heparin reversal to drain removal 24h later. RESULTS: Target ACT was achieved in 0, 63, 68.3 and 82.4% of patients in groups A, B, C and D, respectively, after the initial dose of heparin. In group B, of those not achieving target act a single increment of heparin was sufficient to achieve target ACT in further 18.6%. The mean ACT after the initial dose in groups B, C and D was 482.9, 519 and 588 s, respectively (P<0.05). Postoperative blood loss in millilitre per kilogram was directly proportional to preoperative heparin dose. CONCLUSIONS: Patients receiving lower dose of heparin has lower postoperative blood loss. Of those achieving the target ACT, group B was significantly the closest to the target ACT. A starting dose of 200 iu\\/kg of heparin and if necessary one 50 iu\\/kg increment achieved target ACT in 81.5% of patients. The added benefit of significant drop in postoperative blood loss is evident.

  9. Heparin improves oxygenation and minimizes barotrauma after severe smoke inhalation in an ovine model. (United States)

    Cox, C S; Zwischenberger, J B; Traber, D L; Traber, L D; Haque, A K; Herndon, D N


    Inhalation injury is one of the main causes of mortality in burn victims. The tracheobronchial epithelium sloughs and combines with a protein rich exudate to form casts of the airways that can lead to obstruction. We studied the effects of a continuous infusion of heparin on the acute pulmonary injury that occurs after smoke inhalation injury in sheep. Twelve ewes with vascular catheters received a standardized smoke inhalation injury and mechanical ventilation according to protocol for 72 hours. The heparin group (n = 6) received a 400 unit per kilogram bolus of heparin followed by a continuous infusion to maintain the activated clotting time between 250 to 300 seconds. The control group (n = 6) received a saline solution vehicle. Hemodynamics, blood gases and plasma samples for conjugated dienes were taken every six hours. At necropsy, pulmonary tissue was collected for histologic findings, polymorphonuclear neutrophil leukosequestration, wet-to-dry weight ratios and conjugated dienes. PaO2 to FIO2 ratios were improved in the heparin group compared with the control group at 12 to 72 hours after injury, and peak airway pressures were higher in the control group compared with the heparin group. Positive end expiratory pressure requirements were higher in the control group compared with the heparin group. There were significantly fewer airway tracheobronchial casts as determined by our tracheobronchial casts scoring system (2.4 +/- 0.4 versus 0.67 +/- 0.21) and confirmed by histologic examination. Pulmonary blood-free wet-to-dry weight ratios were higher in the control group compared with the heparin group (6.4 +/- 0.5 versus 5.2 +/- 0.1; p heparin. Heparin decreases tracheobronchial cast formation, improves oxygenation, minimizes barotrauma and reduces pulmonary edema in an ovine model of severe smoke inhalation injury. Heparin does not reduce oxygen free radical activity after smoke inhalation injury.

  10. Stability of Ceftazidime and Heparin in Four Different Types of Peritoneal Dialysis Solutions. (United States)

    Kandel, Surendra; Zaidi, Syed Tabish R; Wanandy, S Troy; Ming, Long C; Castelino, Ronald L; Sud, Kamal; Patel, Rahul P


    Intraperitoneal (IP) administration of ceftazidime is recommended for the treatment of peritoneal dialysis-associated peritonitis (PDAP) from Pseudomonas. Patients with PDAP may also need IP heparin to overcome problems with drainage of turbid peritoneal dialysis (PD) fluids and blockage of catheters with fibrin. Physico-chemical stability of ceftazidime and heparin, and biological stability of heparin in many types of PD solutions is unknown. Therefore, we investigated the stability of ceftazidime and heparin in 4 types of PD solutions. A total of 12 PD bags (3 for each type of solution) containing ceftazidime and heparin were prepared and stored at 4°C for 120 hours, and then at 25°C for 6 hours, and finally at 37°C for 12 hours. An aliquot was withdrawn after predefined time points and analyzed for the concentration of ceftazidime and heparin using high-performance liquid-chromatography (HPLC). Samples were assessed for pH, color changes, particle content, and anticoagulant activity of heparin. Ceftazidime and heparin retained more than 91% of their initial concentration when stored at 4°C for 120 hours followed by storage at 25°C for 6 hours and then at 37°C for 12 hours. Heparin retained more than 95% of its initial activity throughout the study period. Particle formation was not detected at any time under the storage conditions. The pH and color remained essentially unchanged throughout the study. Ceftazidime-heparin admixture retains its stability over long periods of storage at different temperatures, allowing its potential use for PDAP treatment in outpatient and remote settings. Copyright © 2018 International Society for Peritoneal Dialysis.

  11. Postoperative Bleeding After Change in Heparin Supplier: A Cardiothoracic Center Experience. (United States)

    Bojan, Mirela; Fischer, Andreas; Narayanasamy, Ashok; Yea, Paul; Dunnett, Eleanor; Kelleher, Andrea


    Unfractionated heparin is a mixture of glycosaminoglycans with different pharmacologic and pharmacokinetic properties. The literature suggests that blood loss after cardiac surgery is related to both elevated postoperative heparin concentrations and the potency of different heparin brands. An audit of the observed increase in the incidence of cardiac surgery-related bleeding after change in heparin supplier. Patient characteristics were compared between groups before and after a change in heparin brands. Tertiary cardiothoracic center. All patients undergoing cardiac surgery between August 1, 2011, and April 30, 2012. None. Two hundred eighty patients underwent surgery before a change in heparin brands and 216 after a change. Their preoperative and intraoperative characteristics were similar. Postoperative chest tube drainages and blood transfusions were significantly greater after the change in heparin brands (postoperative chest drainage 476.8 ± 393.1 v 344.8 ± 323.2 mL/6 h and 1,062.2 ± 738.8 v 841.8 ± 567.4 mL/24 h, respectively; both p brand. The likelihood ratio chi-square test for nested models identified an added predictive value of the heparin brand when included as a predictor of bleeding (chest drainage >800 mL/6 h) in a model comprising recirculation, assessed using either an elevated anti-factor X activity or ratio between nonheparinase R and heparinase-modified R. It is likely that the observed increase in postoperative bleeding was related to the pharmacologic properties of the new heparin brand rather than a higher incidence of heparin recirculation. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Multifunctional silk-heparin biomaterials for vascular tissue engineering applications. (United States)

    Seib, F Philipp; Herklotz, Manuela; Burke, Kelly A; Maitz, Manfred F; Werner, Carsten; Kaplan, David L


    Over the past 30 years, silk has been proposed for numerous biomedical applications that go beyond its traditional use as a suture material. Silk sutures are well tolerated in humans, but the use of silk for vascular engineering applications still requires extensive biocompatibility testing. Some studies have indicated a need to modify silk to yield a hemocompatible surface. This study examined the potential of low molecular weight heparin as a material for refining silk properties by acting as a carrier for vascular endothelial growth factor (VEGF) and improving silk hemocompatibility. Heparinized silk showed a controlled VEGF release over 6 days; the released VEGF was bioactive and supported the growth of human endothelial cells. Silk samples were then assessed using a humanized hemocompatibility system that employs whole blood and endothelial cells. The overall thrombogenic response for silk was very low and similar to the clinical reference material polytetrafluoroethylene. Despite an initial inflammatory response to silk, apparent as complement and leukocyte activation, the endothelium was maintained in a resting, anticoagulant state. The low thrombogenic response and the ability to control VEGF release support the further development of silk for vascular applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Treatment of mechanical aortic valve thrombosis with heparin and eptifibatide. (United States)

    Vora, Amit N; Gehrig, Thomas; Bashore, Thomas M; Kiefer, Todd L


    A 75-year old woman with a history of coronary disease status post 3-vessel coronary artery bypass grafting (CABG) 8 years ago and a repeat one-vessel CABG 2 years ago in the setting of aortic valve replacement with a #19 mm St. Jude bileaflet mechanical valve for severe aortic stenosis presented with two to three weeks of progressive dyspnea and increasing substernal chest discomfort. Echocardiography revealed a gradient to 31 mmHg across her aortic valve, increased from a baseline of 13 mmHg five months previously. Fluoroscopy revealed thrombosis of her mechanical aortic valve. She was not a candidate for surgery given her multiple comorbidities, and fibrinolysis was contraindicated given a recent subdural hematoma 1 year prior to presentation. She was treated with heparin and eptifibatide and subsequently demonstrated resolution of her aortic valve thrombosis. We report the first described successful use of eptifibatide in addition to unfractionated heparin for the management of subacute valve thrombosis in a patient at high risk for repeat surgery or fibrinolysis.

  14. Arterial indications for the low molecular weight heparins

    Directory of Open Access Journals (Sweden)

    Ageno Walter


    Full Text Available Abstract Antithrombotic treatment is of proven importance in patients with acute coronary syndromes. There is now accumulating evidence from several clinical trials in patients with unstable angina pectoris that the low molecular weight heparins (LMWHs are at least as effective as unfractionated heparin. The LMWHs are easier to use, with the potential to facilitate long-term outpatient treatment. The results of the trials have actually failed to show any clear advantage, however, of the LMWHs over the standard antiplatelet treatment, despite the evidence of a sustained hypercoagulability. Potentially, the use of higher doses of LMWHs could improve the outcomes, but this is as yet unproven and could be associated with unacceptably increased risk of bleeding. During the acute phase of a stroke, aspirin is the first choice of antithrombotic drug because it reduces the risk of recurrent stroke. LMWH cannot be recommended as an antithrombotic agent for the acute treatment of stroke. Prophylactic use of low dose LMWH for the prevention of venous thromboembolism should be considered in every patient with a stroke.

  15. Intravitreal low molecular weight heparin in PVR surgery.

    Directory of Open Access Journals (Sweden)

    Kumar Atul


    Full Text Available Purpose: To evaluate the efficacy of low molecular weight heparin (LMWH in prevention of postoperative fibrin formation following vitreoretinal surgery with proliferative vitreoretinopathy (PVR. Material and Methods: Thirty consecutive patients of retinal detachment with advanced PVR were enrolled in the study. They were randomised to study and control groups (n = 15 each. Study group patients received vitreoretinal surgery with 5 IU/cc of LMWH in vitrectomy infusion fluid. The control group patients received vitroretinal surgery without heparin in the infusion fluid. Patients were followed up at 1 week, 1 month and 3 months after surgery. Postoperative bleeding, media clarity, best-corrected visual acuity and success of the surgery at the end of 3 months were compared between the two groups. Results: At each follow-up visit, the study group showed a better media clarity, which was statistically significant ( P = 0.0042. The study group had a 50% better chance of retinal reattachment compared to the control group. Five patients had intraoperative bleeding in the study group (33% compared to 3 patients in the control group (20%. Conclusion: Use of intravitreal LMWH prevents postoperative fibrin formation and is beneficial in repair of retinal detachments with PVR.

  16. Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome. (United States)

    Kim, Kyumin; Heo, Dong-Hyuk; Kim, Iktae; Suh, Jeong-Yong; Kim, Minkyu


    The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. A manganese(IV)/iron(IV) intermediate in assembly of the manganese(IV)/iron(III) cofactor of Chlamydia trachomatis ribonucleotide reductase. (United States)

    Jiang, Wei; Hoffart, Lee M; Krebs, Carsten; Bollinger, J Martin


    We recently showed that the class Ic ribonucleotide reductase from the human pathogen Chlamydia trachomatis uses a Mn(IV)/Fe(III) cofactor to generate protein and substrate radicals in its catalytic mechanism [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. Here, we have dissected the mechanism of formation of this novel heterobinuclear redox cofactor from the Mn(II)/Fe(II) cluster and O2. An intermediate with a g = 2 EPR signal that shows hyperfine coupling to both 55Mn and 57Fe accumulates almost quantitatively in a second-order reaction between O2 and the reduced R2 complex. The otherwise slow decay of the intermediate to the active Mn(IV)/Fe(III)-R2 complex is accelerated by the presence of the one-electron reductant, ascorbate, implying that the intermediate is more oxidized than Mn(IV)/Fe(III). Mössbauer spectra show that the intermediate contains a high-spin Fe(IV) center. Its chemical and spectroscopic properties establish that the intermediate is a Mn(IV)/Fe(IV)-R2 complex with an S = 1/2 electronic ground state arising from antiferromagnetic coupling between the Mn(IV) (S(Mn) = 3/2) and high-spin Fe(IV) (S(Fe) = 2) sites.

  18. Preparation of Low Molecular Weight Heparin by Microwave Discharge Electrodeless Lamp/TiO2 Photo-Catalytic Reaction. (United States)

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


    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.

  19. Audit of the peri-delivery use of unfractionated heparin in women on therapeutic low-molecular weight heparin. (United States)

    Lambert, J R; Austin, S K; Peebles, D; Cohen, H


    There is no evidence-based approach for the optimal management of peri-delivery anticoagulation in women receiving therapeutic dose of low-molecular weight heparin (LMWH) during pregnancy. Nevertheless, the maintenance of anticoagulation for the maximal period peri-delivery appears appropriate in women considered to be at high risk of venous or arterial thromboembolism. We developed a regimen based on fixed thromboprophylactic dose of unfractionated heparin (UFH) peri-delivery and undertook an audit to evaluate the use and feasibility of this approach and any adverse events. Fixed intravenous thromboprophylactic dose of UFH (15,000 units/24 h) was commenced on the evening prior to a planned delivery [induction of labour or elective caesarean section (CS)], stopped 4 h predelivery and restarted 2-6 h postdelivery. Compliance was good with 32/38 consecutive deliveries managed according to the regimen. There were no cases of postpartum haemorrhage and no thrombosis associated with these 32 deliveries. Twenty-one patients were delivered by CS (11 elective) and eight patients received epidural/spinal anaesthesia without complication. In conclusion, the fixed thromboprophylactic dose UFH regimen provided maintenance of anticoagulation except for a matter of hours without excessive bleeding risk (conducive to neuroaxial anaesthesia) and was simple, flexible and acceptable to staff and patients.

  20. Improvement in physical and biological properties of chitosan/soy protein films by surface grafted heparin. (United States)

    Wang, Xiaomei; Hu, Ling; Li, Chen; Gan, Li; He, Meng; He, Xiaohua; Tian, Weiqun; Li, Mingming; Xu, Li; Li, Yinping; Chen, Yun


    A series of chitosan/soy protein isolate (SPI) composite films (CS-n, n=0, 10 and 30, corresponding to SPI content in the composites) were prepared. Heparin was grafted onto the surface of CS-n to fabricate a series of heparinized films (HCS-n). CS-n and HCS-n were characterized by ATR-Fourier transform infrared spectroscopy and water contact angle. The surface heparin density was measured by toluidine blue assay. The results showed that heparin has been successfully grafted onto the surface of CS-n. Heparin evenly distributed on the surface of the films and the heparin content increased with the increase of SPI content, and the hydrophilicity of the films was enhanced due to the grafted heparin. The cytocompatibility and hemocompatibility of CS-n and HCS-n were evaluated by cell culture (MTT assay, live/dead assay, cell morphology and cell density observation), platelet adhesion test, plasma recalcification time (PRT) measurement, hemolysis assay and thrombus formation test. HCS-n showed higher cell adhesion rate and improved cytocompatibility compared to the corresponding CS-n. HCS-n also exhibited lower platelet adhesion, longer PRT, higher blood anticoagulant indexes (BCI) and lower hemolysis rate than the corresponding CS-n. The improved cytocompatibility and hemocompatibility of HCS-n would shed light on the potential applications of chitosan/soy protein-based biomaterials that may come into contact with blood. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Non-Anticoagulant Heparins Are Hepcidin Antagonists for the Treatment of Anemia

    Directory of Open Access Journals (Sweden)

    Maura Poli


    Full Text Available The peptide hormone hepcidin is a key controller of systemic iron homeostasis, and its expression in the liver is mainly regulated by bone morphogenetic proteins (BMPs, which are heparin binding proteins. In fact, heparins are strong suppressors of hepcidin expression in hepatic cell lines that act by inhibiting the phosphorylation of SMAD1/5/8 proteins elicited by the BMPs. The inhibitory effect of heparins has been demonstrated in cells and in mice, where subcutaneous injections of non-anticoagulant heparins inhibited liver hepcidin expression and increased iron bioavailability. The chemical characteristics for high anti-hepcidin activity in vitro and in vivo include the 2O-and 6O-sulfation and a molecular weight above 7 kDa. The most potent heparins have been found to be the super-sulfated ones, active in hepcidin suppression with a molecular weight as low as 4 kDa. Moreover, the alteration of endogenous heparan sulfates has been found to cause a reduction in hepcidin expression in vitro and in vivo, indicating that heparins act by interfering with the interaction between BMPs and components of the complex involved in the activation of the BMP/SMAD1/5/8 pathway. This review summarizes recent findings on the anti-hepcidin activity of heparins and their possible use for the treatment of anemia caused by hepcidin excess, including the anemia of chronic diseases.

  2. Unfractionated Heparin Promotes Osteoclast Formation in Vitro by Inhibiting Osteoprotegerin Activity

    Directory of Open Access Journals (Sweden)

    Binghan Li


    Full Text Available Heparin has been proven to enhance bone resorption and induce bone loss. Since osteoclasts play a pivotal role in bone resorption, the effect of heparin on osteoclastogenesis needs to be clarified. Since osteocytes are the key modulator during osteoclastogenesis, we evaluated heparin’s effect on osteoclastogenesis in vitro by co-culturing an osteocyte cell line (MLO-Y4 and pre-osteoclasts (RAW264.7. In this co-culture system, heparin enhanced osteoclastogenesis and osteoclastic bone resorption while having no influence on the production of RANKL (receptor activator of NFκB ligand, M-CSF (macrophage colony-stimulating factor, and OPG (osteoprotegerin, which are three main regulatory factors derived from osteocytes. According to previous studies, heparin could bind specifically to OPG and inhibit its activity, so we hypothesized that this might be a possible mechanism of heparin activity. To test this hypothesis, osteoclastogenesis was induced using recombinant RANKL or MLO-Y4 supernatant. We found that heparin has no effect on RANKL-induced osteoclastogenesis (contains no OPG. However, after incubation with OPG, the capacity of MLO-Y4 supernatant for supporting osteoclast formation was increased. This effect disappeared after OPG was neutralized and reappeared after OPG was replenished. These results strongly suggest that heparin promotes osteocyte-modulated osteoclastogenesis in vitro, at least partially, through inhibiting OPG activity.

  3. A photoacoustic tool for therapeutic drug monitoring of heparin (Conference Presentation) (United States)

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


    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.

  4. Heparin-induced thrombocytopenia: a review of concepts regarding a dangerous adverse drug reaction. (United States)

    Junqueira, Daniela Rezende Garcia; Carvalho, Maria das Graças; Perini, Edson


    Heparin is a natural agent with antithrombotic action, commercially available for therapeutic use as unfractionated heparin and low molecular weight heparin. Heparin-induced thrombocytopenia (HIT) is a serious adverse reaction to heparin that promotes antibody-mediated platelet activation. HIT is defined as a relative reduction in platelet count of 50% (even when the platelet count at its lowest level is above>150 x 10(9)/L) occurring within five to 14 days after initiation of the therapy. Thrombocytopenia is the main feature that directs the clinical suspicion of the reaction and the increased risk of thromboembolic complications is the most important and paradoxical consequence. The diagnosis is a delicate issue, and requires a combination of clinical probability and laboratory tests for the detection of platelet activation induced by HIT antibodies. The absolute risk of HIT has been estimated between 1% and 5% under treatment with unfractionated heparin, and less than 1% with low molecular weight heparin. However, high-quality evidence about the risk of HIT from randomized clinical trials is scarce. In addition, information on the frequency of HIT in developing countries is not widely available. This review aims to provide a better understanding of the key features of this reaction and updated information on its frequency to health professionals and other interested parties. Knowledge, familiarity, and access to therapeutic options for the treatment of this adverse reaction are mandatory to minimize the associated risks, improving patient safety. Copyright © 2013 Elsevier Editora Ltda. All rights reserved.

  5. Identification and Characterization of the Novel p97 co-factors, Rep8 and ASPL

    DEFF Research Database (Denmark)

    Klausen, Louise Kjær

    The highly conserved and ubiquitin-specific AAA ATPase p97 acts on ubiquitylated substrates in diverse cellular mechanisms such as chromatin-associated degradation, fusion of homotypic membranes and ER-associated degradation. Different p97 cofactors associate with the ATPase, thereby constituting...

  6. Biochemical and genetic characterization of three molybdenum cofactor hydroxylases in Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Hoff, Tine; Frandsen, Gitte Inselmann; Rocher, Anne


    Aldehyde oxidases and xanthine dehydrogenases/oxidases belong to the molybdenum cofactor dependent hydroxylase class of enzymes. Zymograms show that Arabidopsis thaliana has at least three different aldehyde oxidases and one xanthine oxidase. Three different cDNA clones encoding putative aldehyde...

  7. Time-resolved fluorescence analysis of the mobile flavin cofactor in ...

    Indian Academy of Sciences (India)


    and facilitates efficient flavin reduction. It has been suggested that mobility of the flavin ring also plays an important role in the catalysis of the related en- zyme phenol hydroxylase. 45. Optimal regulation of catalysis in these redox enzymes requires strict regu- lation of the cofactor mobility. Specific (transient) interactions with ...

  8. Time-resolved fluorescence analysis of the mobile flavin cofactor in ...

    Indian Academy of Sciences (India)

    Conformational heterogeneity of the FAD cofactor in -hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations ...

  9. Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

    NARCIS (Netherlands)

    Huijbers, Mieke M.E.; Martínez-Júlvez, Marta; Westphal, Adrie H.; Delgado-Arciniega, Estela; Medina, Milagros; Berkel, Van Willem J.H.


    Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin

  10. Cellular Cofactors of Lentiviral Integrase: From Target Validation to Drug Discovery

    Directory of Open Access Journals (Sweden)

    Oliver Taltynov


    Full Text Available To accomplish their life cycle, lentiviruses make use of host proteins, the so-called cellular cofactors. Interactions between host cell and viral proteins during early stages of lentiviral infection provide attractive new antiviral targets. The insertion of lentiviral cDNA in a host cell chromosome is a step of no return in the replication cycle, after which the host cell becomes a permanent carrier of the viral genome and a producer of lentiviral progeny. Integration is carried out by integrase (IN, an enzyme playing also an important role during nuclear import. Plenty of cellular cofactors of HIV-1 IN have been proposed. To date, the lens epithelium-derived growth factor (LEDGF/p75 is the best studied cofactor of HIV-1 IN. Moreover, small molecules that block the LEDGF/p75-IN interaction have recently been developed for the treatment of HIV infection. The nuclear import factor transportin-SR2 (TRN-SR2 has been proposed as another interactor of HIV IN-mediating nuclear import of the virus. Using both proteins as examples, we will describe approaches to be taken to identify and validate novel cofactors as new antiviral targets. Finally, we will highlight recent advances in the design and the development of small-molecule inhibitors binding to the LEDGF/p75-binding pocket in IN (LEDGINs.

  11. Role of XDHC in Molybdenum Cofactor Insertion into Xanthine Dehydrogenase of Rhodobacter capsulatus (United States)

    Leimkühler, Silke; Klipp, Werner


    Rhodobacter capsulatus xanthine dehydrogenase (XDH) is composed of two subunits, XDHA and XDHB. Immediately downstream of xdhB, a third gene was identified, designated xdhC, which is cotranscribed with xdhAB. Interposon mutagenesis revealed that the xdhC gene product is required for XDH activity. However, XDHC is not a subunit of active XDH, which forms an α2β2 heterotetramer in R. capsulatus. It was shown that XDHC neither is a transcriptional regulator for xdh gene expression nor influences XDH stability. To analyze the function of XDHC for XDH in R. capsulatus, inactive XDH was purified from an xdhC mutant strain. Analysis of the molybdenum cofactor content of this enzyme demonstrated that in the absence of XDHC, no molybdopterin cofactor MPT is present in the XDHAB tetramer. In contrast, absorption spectra of inactive XDH isolated from the xdhC mutant revealed the presence of iron-sulfur clusters and flavin adenine dinucleotide, demonstrating that XDHC is not required for the insertion of these cofactors. The absence of MPT from XDH isolated from an xdhC mutant indicates that XDHC either acts as a specific MPT insertase or might be a specific chaperone facilitating the insertion of MPT and/or folding of XDH during or after cofactor insertion. PMID:10217763

  12. Effects of surface-bound and intravenously administered heparin on cell-surface interactions: inflammation and coagulation. (United States)

    Johnson, G; Curry, B; Cahalan, L; Prater, R; Biggerstaff, J; Hussain, A; Gartner, M; Cahalan, P


    Intravenous administration of heparin and heparin-bonded extracorporeal circuits are frequently used to mitigate the deleterious effects of blood contact with synthetic materials. The work described here utilized human blood in a micro-perfusion circuit to experimentally examine the effects of intravenous and surface-bound heparin on cellular activation. Activation markers of coagulation and of the inflammatory response were examined using flow cytometry; specifically, markers of platelet, monocyte, polymorphonuclear leukocyte (PMN), and lymphocyte activation were quantified. The results indicate that surface-bound heparin reduces the inflammatory response whereas systemically administered heparin does not. This finding has important implications for blood-contacting devices, particularly within the context of recently elucidated connections between inflammation pathways and coagulation disorders. Data presented indicate that surface-bound heparin and intravenously administered heparin play distinct, but vital roles in rendering biomaterial surfaces compatible with blood.

  13. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

    Directory of Open Access Journals (Sweden)

    Zvonimir Marelja


    Full Text Available Iron sulfur (Fe-S clusters and the molybdenum cofactor (Moco are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii increased iron transiently displaces manganese on superoxide

  14. The blood and vascular cell compatibility of heparin-modified ePTFE vascular grafts (United States)

    Hoshi, Ryan A.; Van Lith, Robert; Jen, Michele C.; Allen, Josephine B.; Lapidos, Karen A.; Ameer, Guillermo


    Prosthetic vascular grafts do not mimic the antithrombogenic properties of native blood vessels and therefore have higher rates of complications that involve thrombosis and restenosis. We developed an approach for grafting bioactive heparin, a potent anticoagulant glycosaminoglycan, to the lumen of ePTFE vascular grafts to improve their interactions with blood and vascular cells. Heparin was bound to aminated poly(1,8-octanediol-co-citrate) (POC) via its carboxyl functional groups onto POC-modified ePTFE grafts. The bioactivity and stability of the POC-immobilized heparin (POC–Heparin) were characterized via platelet adhesion and clotting assays. The effects of POC–Heparin on the adhesion, viability and phenotype of primary endothelial cells (EC), blood outgrowth endothelial cells (BOECs) obtained from endothelial progenitor cells (EPCs) isolated from human peripheral blood, and smooth muscle cells were also investigated. POC–Heparin grafts maintained bioactivity under physiologically relevant conditions in vitro for at least one month. Specifically, POC–Heparin-coated ePTFE grafts significantly reduced platelet adhesion and inhibited whole blood clotting kinetics. POC–Heparin supported EC and BOEC adhesion, viability, proliferation, NO production, and expression of endothelial cell-specific markers von Willebrand factor (vWF) and vascular endothelial-cadherin (VE-cadherin). Smooth muscle cells cultured on POC–Heparin showed increased expression of α-actin and decreased cell proliferation. This approach can be easily adapted to modify other blood contacting devices such as stents where antithrombogenicity and improved endothelialization are desirable properties. PMID:23069711

  15. The methanogenic redox cofactor F420is widely synthesized by aerobic soil bacteria. (United States)

    Ney, Blair; Ahmed, F Hafna; Carere, Carlo R; Biswas, Ambarish; Warden, Andrew C; Morales, Sergio E; Pandey, Gunjan; Watt, Stephen J; Oakeshott, John G; Taylor, Matthew C; Stott, Matthew B; Jackson, Colin J; Greening, Chris


    F 420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F 420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F 420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F 420 , with oligoglutamate sidechains of different lengths. To understand the evolution of F 420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the F o precursor to F 420 originated in methanogens, F 420 itself was first synthesized in an ancestral actinobacterium. F 420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

  16. Determination of cofactors in marine sediments; Bestimmung von Kofaktoren im marinen Sediment

    Energy Technology Data Exchange (ETDEWEB)

    Huschek, G.; Kayser, A.


    In respect to a sieving study of sediments in three different sediment locations (Wadden Sea (A), river Elbe-Aestuar (B), Baltic Sea (C)) a determination of selected organic compounds were carried out to study the influence of the sieving process on the variance of cofactors and analytical results. The organic pollutants PCB, Organochlorpesticides (OCP) and PAH were investigated in three fine sediment fractions <2 mm, > and < 63 {mu}m of the locations by accredited and standardised methods. At the same time the cofactor Organic Carbon (TOC) as well as Total Carbon (TC) and Total Nitrogen (TN) were determined in the fine sediment fractions. The endocrine disruptor Bisphenol A could be detected in the sediment locations B and C. The comparison of TOC, TC and TN average values by t-test showed significant differences between the fine fractions of location B and C. Because of low TOC values no signification was detectable in sediment A. The results show that the TOC values in fine sediments are affected by sieving. An enrichment of carbon was detected in the <63 {mu}m fraction in consideration of variance. A correlation between the cofactor TOC and the organic pollutant values could only be detected in the fine fractions of location B. The enrichment of pollutant values in the fine fractions was here analogue of TOC. For a comprehensive evaluation of the cofactor TOC no sufficient sieving sample materials were available in this study for location A and C. So a final statement regarding TOC in fine sediment fractions as a cofactor was not possible. The results show that a determination of organic contaminants in the fine sediment fraction <63 {mu}m make sense because of their enrichment in fine fractions in respect to the TOC values. (orig.)

  17. Mechanism of assembly of the dimanganese-tyrosyl radical cofactor of class Ib ribonucleotide reductase: enzymatic generation of superoxide is required for tyrosine oxidation via a Mn(III)Mn(IV) intermediate. (United States)

    Cotruvo, Joseph A; Stich, Troy A; Britt, R David; Stubbe, JoAnne


    Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y(•)) generated by oxidation of a reduced dinuclear metal cluster. The Fe(III)2-Y(•) cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from Fe(II)2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a Mn(III)2-Y(•) cofactor in their NrdF subunit. Mn(II)2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the Mn(III)2-Y(•) cofactor. Here we investigate the mechanism of assembly of the Mn(III)2-Y(•) cofactor in Bacillus subtilis NrdF. Cluster assembly from Mn(II)2-NrdF, NrdI(hq), and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdI(hq) reduces O2 to O2(•-) (40-48 s(-1), 0.6 mM O2), the O2(•-) channels to and reacts with Mn(II)2-NrdF to form a Mn(III)Mn(IV) intermediate (2.2 ± 0.4 s(-1)), and the Mn(III)Mn(IV) species oxidizes tyrosine to Y(•) (0.08-0.15 s(-1)). Controlled production of O2(•-) by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the Mn(II)2 cluster with O2 and satisfies the requirement for an "extra" reducing equivalent in Y(•) generation.

  18. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; McCarthy, J B; Furcht, L T


    Cell adhesion to extracellular matrix molecules such as fibronectin involves complex transmembrane signaling processes. Attachment and spreading of primary fibroblasts can be promoted by interactions of cell surface integrins with RGD-containing fragments of fibronectin, but the further process......, as synthetic peptides coupled to ovalbumin, can support cell attachment. Only three of these sequences can promote focal adhesion formation when presented as multicopy complexes, and only one of these (WQPPRARI) retains this activity as free peptide. The major activity of this peptide resides in the sequence...... PRARI. The biological response to this peptide and to the COOH-terminal fragment may be mediated through cell surface heparan sulfate proteoglycans because treatment of cells with heparinase II and III, or competition with heparin, reduces the response. Treatment with chondroitinase ABC or competition...

  19. Obstetric outcome with low molecular weight heparin therapy during pregnancy.

    LENUS (Irish Health Repository)

    Donnelly, J


    This was a prospective study of women attending a combined haematology\\/obstetric antenatal clinic in the National Maternity Hospital (2002-2008). Obstetric outcome in mothers treated with low molecular weight heparin (LMWH) was compared to the general obstetric population of 2006. There were 133 pregnancies in 105 women. 85 (63.9%) received prophylactic LMWH and 38 (28.6%) received therapeutic LMWH in pregnancy. 10 (7.5%) received postpartum prophylaxis only. The perinatal mortality rate was 7.6\\/1000 births. 14 (11.3%) women delivered preterm which is significantly higher than the hospital population rate (5.7%, p<0.05). Despite significantly higher labour induction rates (50% vs 29.2% p<0.01), there was no difference in CS rates compared to the general hospital population (15.4% vs 18.9%, NS). If carefully managed, these high-risk women can achieve similar vaginal delivery rates as the general obstetric population.

  20. [Oxygenator thrombosis without heparin resistance in polycythemia vera]. (United States)

    Lehot, J-J; Was, B; Dendeleu, L; Jegaden, O


    A 55-year-old male with a history of positive HIV serology and polycythemia vera underwent coronary artery bypass graft surgery with normothermic extracorporeal circulation. Following heparin administration the activated clotting time (ACT) was 633 seconds (Hemocron with kaolin). Lower than expected arterial and venous oxygen partial pressures together with high pressure (350 mmHg) in the arterial line upstream of the oxygenator were observed. Because of these signs the oxygenator was changed during the procedure. The outcome was uneventful. Electronic microscopic examination of the oxygenator membrane and thermic exchanger revealed fibrin and platelet deposits. Similar cases are described in the literature during polycythemia vera. Therefore the prevention might be a preoperative treatment with antiplatelet therapy in polycytemia vera. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  1. Aspirin vs Heparin for the Prevention of Preeclampsia. (United States)

    Katsi, Vasiliki; Kanellopoulou, Theoni; Makris, Thomas; Nihoyannopoulos, Petros; Nomikou, Efrosyni; Tousoulis, Dimitrios


    Preeclampsia is a hypertensive disorder of pregnancy that remains a significant cause of maternal morbidity and mortality worldwide. Preeclampsia can be resolved by delivery, and most of the proposed preventive treatment approaches are based on processes involved in placental development in early pregnancy. Yet, none of these has been established in clinical practice. Low-dose aspirin is the most promising candidate, nevertheless; while some individual randomized controlled trials showed minimal or no statistically significant benefit, recent metanalyses showed that early initiation before 16 weeks of gestation is associated with prevention of early-onset preeclampsia and reduction in prevalence of perinatal death or morbidity of pregnant women. Heparin could be an alternative antithrombotic and anti-inflammatory median to prevent preeclampsia either alone or in combination with aspirin; however, results are conflicting concerning efficacy.

  2. Cell-instructive starPEG-heparin-collagen composite matrices. (United States)

    Binner, Marcus; Bray, Laura J; Friedrichs, Jens; Freudenberg, Uwe; Tsurkan, Mikhail V; Werner, Carsten


    Polymer hydrogels can be readily modulated with regard to their physical properties and functionalized to recapitulate molecular cues of the extracellular matrix (ECM). However, they remain structurally different from the hierarchical supramolecular assemblies of natural ECM. Accordingly, we herein report a set of hydrogel composite materials made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I that combine semisynthetic and ECM-derived components. Collagen fibrillogenesis was controlled by temperature and collagen concentration to form collagen microstructures which were then homogeneously distributed within the 3D composite matrix during hydrogel formation. The collagen-laden hydrogel materials showed a heterogeneous local variation of the stiffness and adhesion ligand density. Composite gels functionalized with growth factors and cell adhesive peptides (RGDSP) supported the growth of embedded human umbilical cord vein endothelial cells (HUVECs) and induced the alignment of embedded bone marrow-derived human mesenchymal stem cells (MSCs) to the collagen microstructures in vitro. The introduced composite hydrogel material is concluded to faithfully mimic cell-instructive features of the ECM. Cell-instructive materials play an important role in the generation of both regenerative therapies and advanced tissue and disease models. For that purpose, biofunctional polymer hydrogels recapitulating molecular cues of the extracellular matrix (ECM) were successfully applied in various different studies. However, hydrogels generally lack the hierarchical supramolecular structure of natural ECM. We have therefore developed a hydrogel composite material made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I fibrils. The collagen-laden scaffolds showed a heterogeneous local variation in the stiffness of the material. The composite gels were successfully tested in culture experiments with human umbilical

  3. Argatroban in the management of heparin-induced thrombocytopenia

    Directory of Open Access Journals (Sweden)

    Luciano Babuin


    Full Text Available Luciano Babuin, Vittorio PengoClinical Cardiology, Department of Cardiac Thoracic and Vascular Sciences, University of Padua School of Medicine, Padova, ItalyAbstract: Heparin-induced thrombocytopenia (HIT is an immunoglobulin-mediated serious complication of heparin therapy characterized by thrombocytopenia and high risk for venous and arterial thrombosis: HIT and thrombosis syndrome (HITTS. Argatroban, a direct thrombin ­inhibitor, is indicated as the anticoagulant for the treatment and prophylaxis of thrombosis in patients with HIT and in patients undergoing percutaneous coronary intervention (PCI who have HIT. The aim of this review is to examine the pharmacological characteristics and the clinical efficacy and safety of this drug in adults with HIT, including those undergoing PCI. Briefly, 2 prospective multicenter, nonrandomized, open-label studies evaluated the efficacy and safety of argatroban as an anticoagulant in patients with HIT or HITTS. Both studies showed that the incidence of the primary efficacy end point, a composite of all-cause death, all-cause amputation, or new thrombosis, was reduced in argatroban-treated patients vs control subjects with HIT or HITTS. In both studies, bleeding rates were similar between the groups. Argatroban was evaluated as the anticoagulant therapy in 3 prospective, multicenter, open-label studies in HIT patients who underwent PCI. The studies were similar in design with respect to patient inclusion and exclusion criteria, the argatroban dosing regimen, and primary efficacy outcomes. The investigators performed a pooled analysis of these studies, which showed that most (≥95% patients achieved a satisfactory outcome from the procedure and adequate ­anticoagulation (coprimary end points.Keywords: argatroban, thrombocytopenia, thrombosis

  4. Effect of catheter-lock solutions on catheter-related infection and inflammatory syndrome in hemodialysis patients: heparin versus citrate 46% versus heparin/gentamicin. (United States)

    Venditto, Marcia; du Montcel, Sophie Tezenas; Robert, Jérôme; Trystam, David; Dighiero, Jean; Hue, Danièle; Bessette, Christelle; Deray, Gilbert; Mercadal, Lucile


    Prevention strategies are emerging with the use of catheter-lock solutions (CLS) to prevent catheter-related infections. We compared 3 CLS: heparin, citrate (46%) and heparin/gentamicin (H/G). Three periods of 6 months using the three CLS were compared. 265 catheters were studied. The CRI rate per 1,000 catheter-days was 2.9 for heparin, 3.4 for citrate and 0.4 for H/G. The free-infection catheter survival tended to be higher with H/G (log-rank test, p = 0.06) and the CRP had a significant decreasing course (p = 0.03). Since 2006 H/G was used as CLS in our dialysis unit. The resistance to gentamicin of Enterobacteriaceae increased in the nephrology department and in the entire hospital. On the other hand, the resistance of Staphylococcus aureus to gentamicin dropped to nil. CLS with heparin/gentamicin tended to decrease CRI compared to citrate 46% and heparin and frankly improved the CRP course after catheter insertion. Gentamicin resistance should be monitored.

  5. The use of fondaparinux for the treatment of venous thromboembolism in a patient with heparin-induced thombocytopenia and thrombosis caused by heparin flushes

    Directory of Open Access Journals (Sweden)

    Alex C Spyropoulos


    Full Text Available Alex C Spyropoulos1, Sharyl Magnuson1, Sei Keng Koh21Clinical Thrombosis Center, Lovelace Medical Center, Albuquerque, NM, USA; 2Department of Pharmacy, Singapore General Hospital, SingaporeAbstract: Heparin-induced thrombocytopenia (HIT is an immunologic drug reaction characterized by paradoxical association with venous and arterial thrombosis. The syndrome is caused by IgG antibodies that are reactive against complexes of platelet factor 4 and heparin. Fondparinux does not bind to platelet factor 4, is structurally too short to induce an antibody response, and could in theory be a useful agent to treat HIT. A 69-year-old white female presented with a lower extremity extensive iliofemoral deep vein thrombosis after a right total knee arthroplasty and was subsequently found to have a pulmonary embolism. The patient was noted to have heparin flushes during her operation. Her platelet drop decreased >50% from baseline during initiation of antithrombotic therapy. She was started on subcutaneous fondaparinux 7.5 mg once daily injection. Her serotonin release assay and enzyme-linked immunosorbent assay for heparin antibodies were positive for HIT. Her platelet count nadir was 60 × 103/mm3 on day 5 and the platelet count rebounded after 8 days of fondaparinux therapy. No recurrent thrombotic or bleeding events were noted throughout her therapy. Anecdotal reports have shown that fondaparinux can be a useful agent to treat HIT with or without thrombosis.Keywords: fondaparinux, heparin-induced thrombocytopenia with thrombosis (HITT

  6. Rapid and durable response to intravenous immunoglobulin in delayed heparin-induced thrombocytopenia: a case report. (United States)

    Lei, Brandon Z; Shatzel, Joseph J; Sendowski, Merav


    Heparin-induced thrombocytopenia (HIT) results in platelet consumption and a virulent thrombotic state, which generally responds to cessation of heparin and initiation of anticoagulation. Rarely, delayed HIT can occur and/or persist after heparin is discontinued. A 47-year-old male developed delayed HIT with severe thrombocytopenia and thrombosis after cardiac surgery. Thrombocytopenia developed and persisted after heparin cessation and did not improve despite sequential use of argatroban followed by bivalirudin. Treatment with intravenous immunoglobulin (IVIg) was well tolerated and resulted in rapid resolution of thrombocytopenia. There are few case reports on the management of delayed HIT with severe and prolonged thrombocytopenia. The risk for thrombosis and bleeding in the setting of an undefined time course increases uncertainty in management. This case, along with others accumulating in the literature, suggest that IVIg may be effective in treating delayed HIT with persistent thrombocytopenia. © 2016 AABB.

  7. Low-molecular-weight or unfractionated heparin in venous thromboembolism: the influence of renal function. (United States)

    Trujillo-Santos, Javier; Schellong, Sebastian; Falga, Conxita; Zorrilla, Vanessa; Gallego, Pedro; Barrón, Manuel; Monreal, Manuel


    In patients with acute venous thromboembolism and renal insufficiency, initial therapy with unfractionated heparin may have some advantages over low-molecular-weight heparin. We used the Registro Informatizado de la Enfermedad TromboEmbólica (RIETE) Registry data to evaluate the 15-day outcome in 38,531 recruited patients. We used propensity score matching to compare patients treated with unfractionated heparin with those treated with low-molecular-weight heparin in 3 groups stratified by creatinine clearance levels at baseline: >60 mL/min, 30 to 60 mL/min, or 60 mL/min (n = 1598 matched pairs), 30 to 60 mL/min (n = 277 matched pairs), and 60 mL/min or <30 mL/min, but not in those with levels between 30 and 60 mL/min. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Structure-Activity Relationships of Bioengineered Heparin/Heparan Sulfates Produced in Different Bioreactors

    Directory of Open Access Journals (Sweden)

    Ha Na Kim


    Full Text Available Heparin and heparan sulfate are structurally-related carbohydrates with therapeutic applications in anticoagulation, drug delivery, and regenerative medicine. This study explored the effect of different bioreactor conditions on the production of heparin/heparan sulfate chains via the recombinant expression of serglycin in mammalian cells. Tissue culture flasks and continuously-stirred tank reactors promoted the production of serglycin decorated with heparin/heparan sulfate, as well as chondroitin sulfate, while the serglycin secreted by cells in the tissue culture flasks produced more highly-sulfated heparin/heparan sulfate chains. The serglycin produced in tissue culture flasks was effective in binding and signaling fibroblast growth factor 2, indicating the utility of this molecule in drug delivery and regenerative medicine applications in addition to its well-known anticoagulant activity.

  9. Outcomes Following the Use of Nebulized Heparin for Inhalation Injury (HIHI Study). (United States)

    McIntire, Allyson M; Harris, Serena A; Whitten, Jessica A; Fritschle-Hilliard, Andrew C; Foster, David R; Sood, Rajiv; Walroth, Todd A

    Inhalation injury (IHI) causes significant morbidity and mortality in burn victims due to both local and systemic effects. Nebulized heparin promotes improvement in lung function and decreased mortality in IHI by reducing the inflammatory response and fibrin cast formation. The study objective was to determine if nebulized heparin 10,000 units improves lung function and decreases mechanical ventilation duration, mortality, and hospitalization length in IHI with minimal systemic adverse events. This retrospective, case-control study evaluated efficacy and safety of nebulized heparin administered to mechanically ventilated adults admitted within 48 hr of confirmed IHI. Nebulized heparin 10,000 units was administered Q4H for 7 days, or until extubation if sooner, alternating with albuterol and a mucolytic. Patients were matched on a case-by-case basis based on percent TBSA burn and age to patients from a historical group with IHI before heparin protocol implementation. The primary outcome was duration of mechanical ventilation. Secondary outcomes included lung injury score, ventilator-free days during the first 28 days, 28-day mortality, hospitalization length, ventilator-associated pneumonia incidence, bronchoscopy incidence, and bleeding events. Data were collected in 72 patients, 36 of which received nebulized heparin and 36 historical controls. Two patients from the heparin group and three patients from the control group died/were discharged while on the ventilator. Data were analyzed separately with 1) all subjects included and 2) with subjects who died/were discharged on the ventilator excluded. In the latter comparison, patients receiving nebulized heparin demonstrated a statistically significant decrease in median (interquartile range) duration of initial mechanical ventilation compared with controls [7.0 (4.0, 13.5) vs. 14.5 (5.3, 22.3) days; P = .044]. Patients in the heparin group had a significantly increased number of median (interquartile range

  10. The molybdenum cofactor biosynthesis protein MobA from Rhodobacter capsulatus is required for the activity of molybdenum enzymes containing MGD, but not for xanthine dehydrogenase harboring the MPT cofactor. (United States)

    Leimkühler, S; Klipp, W


    The requirement of MobA for molybdoenzymes with different molybdenum cofactors was analyzed in Rhodobacter capsulatus. MobA is essential for DMSO reductase and nitrate reductase activity, both enzymes containing the molybdopterin guanine dinucleotide cofactor (MGD), but not for active xanthine dehydrogenase, harboring the molybdopterin cofactor. In contrast to the mob locus of Escherichia coli and R. sphaeroides, the mobB gene is not located downstream of mobA in R. capsulatus. The mobA gene is expressed constitutively at low levels and no increase in mobA expression could be observed even under conditions of high MGD demand.

  11. A Polymer Therapeutic Having Universal Heparin Reversal Activity: Molecular Design and Functional Mechanism. (United States)

    Kalathottukaren, Manu Thomas; Abbina, Srinivas; Yu, Kai; Shenoi, Rajesh A; Creagh, A Louise; Haynes, Charles; Kizhakkedathu, Jayachandran N


    Heparins are widely used to prevent blood clotting during surgeries and for the treatment of thrombosis. However, bleeding associated with heparin therapy is a concern. Protamine, the only approved antidote for unfractionated heparin (UFH) could cause adverse cardiovascular events. Here, we describe a unique molecular design used in the development of a synthetic dendritic polycation named as universal heparin reversal agent (UHRA), an antidote for all clinically used heparin anticoagulants. We elucidate the mechanistic basis for the selectivity of UHRA to heparins and its nontoxic nature. Isothermal titration calorimetry based binding studies of UHRAs having different methoxypolyethylene glycol (mPEG) brush structures with UFH as a function of solution conditions, including ionic strength, revealed that mPEG chains impose entropic penalty to the electrostatic binding. Binding studies confirm that, unlike protamine or N-UHRA (a truncated analogue of UHRA with no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity toward heparins through a combined steric repulsion and Donnan shielding effect (a balance of Fel and Fsteric). Clotting assays reveal that UHRA with mPEG chains did not adversely affect clotting, and neutralized UFH over a wide range of concentrations. Conversely, N-UHRA and protamine display intrinsic anticoagulant activity and showed a narrow concentration window for UFH neutralization. In addition, we found that mPEG chains regulate the size of antidote-UFH complexes, as revealed by atomic force microscopy and dynamic light scattering studies. UHRA molecules with mPEG chains formed smaller complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes to neutralize heparin's activity.

  12. Release of proteins via ion exchange from albumin-heparin microspheres


    Kwon, Glen S.; Bae, You Han; Cremers, H.F.M.; Cremers, Harry; Feijen, Jan; Kim, Sung Wan


    Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg lysozyme on albumin-heparin microspheres was linear until saturation was abruptly reached, The adsorption isotherms of human lysozyme at low and high ionic strength were typical of adsorption isoth...

  13. An Antithrombin-Heparin Complex Increases the Anticoagulant Activity of Fibrin Clots

    Directory of Open Access Journals (Sweden)

    Lesley J. Smith


    Full Text Available Clotting blood contains fibrin-bound thrombin, which is a major source of procoagulant activity leading to clot extension and further activation of coagulation. When bound to fibrin, thrombin is protected from inhibition by antithrombin (AT + heparin but is neutralized when AT and heparin are covalently linked (ATH. Here, we report the surprising observation that, rather than yielding an inert complex, thrombin-ATH formation converts clots into anticoagulant surfaces that effectively catalyze inhibition of thrombin in the surrounding environment.

  14. Anticoagulation and endothelial cell behaviors of heparin-loaded graphene oxide coating on titanium surface

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    Pan, Chang-Jiang, E-mail: [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an 223003 (China); Pang, Li-Qun [Department of General Surgery, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an 223300 (China); Gao, Fei [Zhejiang Zylox Medical Devices Co., Ltd., Hangzhou 310000 (China); Wang, Ya-Nan; Liu, Tao; Ye, Wei; Hou, Yan-Hua [Jiangsu Provincial Key Laboratory for Interventional Medical Devices, Huaiyin Institute of Technology, Huai' an 223003 (China)


    Owing to its unique physical and chemical properties, graphene oxide (GO) has attracted tremendous interest in many fields including biomaterials and biomedicine. The purpose of the present study is to investigate the endothelial cell behaviors and anticoagulation of heparin-loaded GO coating on the titanium surface. To this end, the titanium surface was firstly covered by the polydopamine coating followed by the deposition of the GO coating. Heparin was finally loaded on the GO coating to improve the blood compatibility. The results of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) indicated that the heparin-loaded GO coating was successfully created on the titanium surface. The scanning electron microscopy (SEM) images indicated that a relative uniform GO coating consisting of multilayer GO sheets was formed on the substrate. The hydrophilicity of the titanium surface was enhanced after the deposition of GO and further improved significantly by the loading heparin. The GO coating can enhance the endothelial cell adhesion and proliferation as compared with polydopamine coating and the blank titanium. Loading heparin on the GO coating can significantly reduce the platelet adhesion and prolong the activated partial thromboplastin time (APTT) while not influence the endothelial cell adhesion and proliferation. Therefore, the heparin-loaded GO coating can simultaneously enhance the cytocompatibility to endothelial cells and blood compatibility of biomaterials. Because the polydopamine coating can be easily prepared on most of biomaterials including polymer, ceramics and metal, thus the approach of the present study may open up a new window of promising an effective and efficient way to promote endothelialization and improve the blood compatibility of blood-contact biomedical devices such as intravascular stents. - Highlights: • Heparin-loaded graphene oxide coating was

  15. Purification of foot-and-mouth disease virus by heparin as ligand for certain strains. (United States)

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


    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.

  16. Does a Nebulized Heparin/N-acetylcysteine Protocol Improve Outcomes in Adult Smoke Inhalation?


    Natalie S. Kashefi, MS; Jonathan I. Nathan, MD, MBA; Sharmila Dissanaike, MD, FACS


    Background: Smoke inhalation is a major source of morbidity and mortality. Heparin and N-acetylcysteine treatment has potential efficacy in inhalation injury. We investigated the impact of a heparin/N-acetylcysteine/albuterol nebulization protocol in adult patients with inhalation injury. Methods: A retrospective review was performed of adult inhalation injury patients, admitted to a regional burn center between January 2011 and July 2012, who underwent a protocol of alternating treatments of...

  17. Nebulised heparin: a new approach to the treatment of acute lung injury?


    Suter, Peter M


    The administration of heparin by nebulisation has been proposed for the 'local' treatment of pulmonary coagulation disturbances in acute lung injury (ALI). Alveolar and lung micro-vascular fibrin accumulation and breakdown inhibition indeed play a central role in the development and clinical course of this disease. Preclinical studies provide some evidence of the beneficial effects of heparin inhalation in several animal models of ALI. Clinical investigations are sparse, and trials such as th...

  18. Low-dose intravenous heparin infusion in patients with aneurysmal subarachnoid hemorrhage: a preliminary assessment (United States)

    Simard, J. Marc; Aldrich, E. Francois; Schreibman, David; James, Robert F.; Polifka, Adam; Beaty, Narlin


    Object Aneurysmal subarachnoid hemorrhage (aSAH) predisposes to delayed neurological deficits, including stroke and cognitive and neuropsychological abnormalities. Heparin is a pleiotropic drug that antagonizes many of the pathophysiological mechanisms implicated in secondary brain injury after aSAH. Methods The authors performed a retrospective analysis in 86 consecutive patients with Fisher Grade 3 aSAH due to rupture of a supratentorial aneurysm who presented within 36 hours and were treated by surgical clipping within 48 hours of their ictus. Forty-three patients were managed postoperatively with a low-dose intravenous heparin infusion (Maryland low-dose intravenous heparin infusion protocol: 8 U/kg/hr progressing over 36 hours to 10 U/kg/hr) beginning 12 hours after surgery and continuing until Day 14 after the ictus. Forty-three control patients received conventional subcutaneous heparin twice daily as deep vein thrombosis prophylaxis. Results Patients in the 2 groups were balanced in terms of baseline characteristics. In the heparin group, activated partial thromboplastin times were normal to mildly elevated; no clinically significant hemorrhages or instances of heparin-induced thrombocytopenia or deep vein thrombosis were encountered. In the control group, the incidence of clinical vasospasm requiring rescue therapy (induced hypertension, selective intraarterial verapamil, and angioplasty) was 20 (47%) of 43 patients, and 9 (21%) of 43 patients experienced a delayed infarct on CT scanning. In the heparin group, the incidence of clinical vasospasm requiring rescue therapy was 9% (4 of 43, p = 0.0002), and no patient suffered a delayed infarct (p = 0.003). Conclusions In patients with Fisher Grade 3 aSAH whose aneurysm is secured, postprocedure use of a low-dose intravenous heparin infusion may be safe and beneficial. PMID:24032706

  19. A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli* (United States)

    Jiang, Ming; Chen, Minjiao; Guo, Zu-Feng; Guo, Zhihong


    1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases. PMID:20643650

  20. Vascular immunotargeting to endothelial determinant ICAM-1 enables optimal partnering of recombinant scFv-thrombomodulin fusion with endogenous cofactor.

    Directory of Open Access Journals (Sweden)

    Colin F Greineder

    Full Text Available The use of targeted therapeutics to replenish pathologically deficient proteins on the luminal endothelial membrane has the potential to revolutionize emergency and cardiovascular medicine. Untargeted recombinant proteins, like activated protein C (APC and thrombomodulin (TM, have demonstrated beneficial effects in acute vascular disorders, but have failed to have a major impact on clinical care. We recently reported that TM fused with an scFv antibody fragment to platelet endothelial cell adhesion molecule-1 (PECAM-1 exerts therapeutic effects superior to untargeted TM. PECAM-1 is localized to cell-cell junctions, however, whereas the endothelial protein C receptor (EPCR, the key co-factor of TM/APC, is exposed in the apical membrane. Here we tested whether anchoring TM to the intercellular adhesion molecule (ICAM-1 favors scFv/TM collaboration with EPCR. Indeed: i endothelial targeting scFv/TM to ICAM-1 provides ~15-fold greater activation of protein C than its PECAM-targeted counterpart; ii blocking EPCR reduces protein C activation by scFv/TM anchored to endothelial ICAM-1, but not PECAM-1; and iii anti-ICAM scFv/TM fusion provides more profound anti-inflammatory effects than anti-PECAM scFv/TM in a mouse model of acute lung injury. These findings, obtained using new translational constructs, emphasize the importance of targeting protein therapeutics to the proper surface determinant, in order to optimize their microenvironment and beneficial effects.

  1. Development of CHARMM-compatible force-field parameters for cobalamin and related cofactors from quantum mechanical calculations. (United States)

    Pavlova, Anna; Parks, Jerry M; Gumbart, James C


    Corrinoid cofactors such as cobalamin are used by many enzymes and are essential for most living organisms. Therefore, there is broad interest in investigating cobalamin-protein interactions with molecular dynamics simulations. Previously developed parameters for cobalamins are based mainly on crystal structure data. Here, we report CHARMM-compatible force field parameters for several corrinoids developed from quantum mechanical calculations. We provide parameters for corrinoids in three oxidation states, Co3+, Co2+ and Co1+, and with various axial ligands. Lennard-Jones parameters for the cobalt center in the Co(II) and Co(I) states were optimized using a helium atom probe, and partial atomic charges were obtained with a combination of natural population analysis (NPA) and restrained electrostatic potential (RESP) fitting approaches. The Force Field Toolkit was used to optimize all bonded terms. The resulting parameters, determined solely from calculations of cobalamin alone or in water, were then validated by assessing their agreement with DFT geometries and by analyzing molecular dynamics simulation trajectories of several corrinoid proteins for which X-ray crystal structures are available. In each case, we obtained excellent agreement with the reference data. In comparison to previous CHARMM-compatible parameters for cobalamin we observe a better agreement for the fold angle and lower RMSD in the cobalamin binding site. The approach described here is readily adaptable for developing CHARMM-compatible force-field parameters of other corrinoids or large biomolecules.

  2. Electrodeposition of nanostructured bioactive hydroxyapatite-heparin composite coatings on titanium for dental implant applications. (United States)

    Bozzini, Benedetto; Barca, Amilcare; Bogani, Francesco; Boniardi, Marco; Carlino, Paolo; Mele, Claudio; Verri, Tiziano; Romano, Alessandro


    In this paper we describe the one-pot fabrication of hydroxyapatite (HA)-heparin composites by electrodeposition onto Ti substrates and their characterisation in terms of structure, morphology, heparin content and bioactivity. HA coatings are well known and widely applied osteointegration enhancers, but post-implant healing rate in dental applications is still suboptimal: e.g. coagulation control plays a key role and the incorporation of an anticoagulant is considered a highly desirable option. In this study, we have developed an improved, simple and robust growth procedure for single-phase, pure HA-heparin films of thickness 1/3 μm. HA-heparin, forming nanowires, has the ideal morphology for bone mineralisation. Staining assays revealed homogeneous incorporation of sizable amounts of heparin in the composite films. The bioactivities of the HA and HA-heparin coatings on Ti were compared by HeLa cell proliferation/viability tests and found to be enhanced by the presence of the anticoagulant.

  3. Heparin concentration is critical for cell culture with human platelet lysate. (United States)

    Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang


    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.

  4. Heparinized and Saline Solutions in the Maintenance of Arterial and Central Venous Catheters After Cardiac Surgery. (United States)

    Ziyaeifard, Mohsen; Alizadehasl, Azin; Aghdaii, Nahid; Sadeghi, Ali; Azarfarin, Rasoul; Masoumi, Gholamreza; Golbargian, Ghodrat


    Heparinized saline solution is used to prevent occlusion in the arterial catheters and central venous pressure monitoring catheters. Even at low dose, heparin administration can be associated with serious complications. Normal saline solution can maintain patency of arterial catheters and central venous pressure monitoring catheters. The current study aimed to compare the efficacy of normal saline with that of heparinized one to maintain patency of arterial and central venous catheters after cardiac surgery. In the current randomized controlled trial, 100 patients, with an age range of 18 - 65 years of valve and coronary artery surgery were studied in Rajaie heart center, Tehran, Iran. Patients were randomized to receive either heparinized saline (n = 50) or normal saline flush solutions (n = 50). In the study, arterial catheters and central venous pressure monitoring catheters were daily checked for any signs of occlusion in three postoperative days as primary end-point of the study. According to the information obtained from the study, four (8%) arterial catheters in the saline group (P value: 0.135) and three (6%) arterial catheters in the heparin group (P value = 0.097) were obstructed. Statistical analysis showed that the incidence of obstruction and changes in all other parameters between the two groups during the three-day follow-up was not significant (all P values > 0.05). It seems that there is no difference in the use of heparinized and normal saline solutions to prevent catheter occlusion of arterial and central venous pressure.

  5. Citrate versus heparin anticoagulation in continuous renal replacement therapy in small children. (United States)

    Raymakers-Janssen, Paulien A M A; Lilien, Marc; van Kessel, Ingrid A; Veldhoen, Esther S; Wösten-van Asperen, Roelie M; van Gestel, Josephus P J


    Citrate is preferred over heparin as an anticoagulant in adult continuous renal replacement therapy (CRRT). However, its potential adverse effects and data on use in CRRT in infants and toddlers is limited. We conducted a prospective study on using citrate in CRRT in critically ill small children. Children who underwent CRRT with the smallest filter in our PICU between November 2011 and November 2016 were included. Both heparin and citrate were applied according to a strict protocol. Our primary outcome was circuit survival time. Secondary outcomes were alkalosis, citrate toxicity, and number of red blood cell transfusions. Heparin was used in six patients (121 circuits, total CRRT time 3723 h). Citrate was used in 14 patients (105 circuits, total CRRT time 4530 h). Median circuit survival time with heparin was 21 h (IQR 14.5-27.5) compared to 45.2 h (IQR 37.5-52.8) with citrate (p CRRT regimes. In the heparin group, a median of 6.5 units of red blood cells (IQR 1.5-23.8) were given during CRRT, compared to three in the citrate group (IQR 2.0-5.0, p = 0.12). Use of regional citrate significantly prolongs circuit survival time and thereby should increase CRRT efficiency when compared to heparin. In addition, citrate appears safe for CRRT in critically ill small children.

  6. Shape-persistent and adaptive multivalency: rigid transgeden (TGD) and flexible PAMAM dendrimers for heparin binding. (United States)

    Bromfield, Stephen M; Posocco, Paola; Fermeglia, Maurizio; Tolosa, Juan; Herreros-López, Ana; Pricl, Sabrina; Rodríguez-López, Julián; Smith, David K


    This study investigates transgeden (TGD) dendrimers (polyamidoamine (PAMAM)-type dendrimers modified with rigid polyphenylenevinylene (PPV) cores) and compares their heparin-binding ability with commercially available PAMAM dendrimers. Although the peripheral ligands are near-identical between the two dendrimer families, their heparin binding is very different. At low generation (G1), TGD outperforms PAMAM, but at higher generation (G2 and G3), the PAMAMs are better. Heparin binding also depends strongly on the dendrimer/heparin ratio. We explain these effects using multiscale modelling. TGD dendrimers exhibit "shape-persistent multivalency"; the rigidity means that small clusters of surface amines are locally well optimised for target binding, but it prevents the overall nanoscale structure from rearranging to maximise its contacts with a single heparin chain. Conversely, PAMAM dendrimers exhibit "adaptive multivalency"; the flexibility means individual surface ligands are not so well optimised locally to bind heparin chains, but the nanostructure can adapt more easily and maximise its binding contacts. As such, this study exemplifies important new paradigms in multivalent biomolecular recognition. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. A model ternary heparin conjugate by direct covalent bond strategy applied to drug delivery system. (United States)

    Wang, Ying; Xin, Dingcheng; Hu, Jiawen; Liu, Kaijian; Pan, Jiangao; Xiang, Jiannan


    A model ternary heparin conjugate by direct covalent bond strategy has been developed, in which modified heparin using active mix anhydride as intermediate conjugates with model drug molecule and model specific ligand, respectively. Designed ester bonds between model drug and heparin facilitate hydrolysis kinetics research. The strategy can be extended to design and synthesize a targeted drug delivery system. The key point is to use mixed anhydride groups as activating intermediates to mediate the synthesis of the ternary heparin conjugate. Formation of mixed anhydride is detected by the conductimetry experiment. The ternary heparin conjugate is characterized by (13)C NMR, FT-IR and GPC, respectively. The decreased trend on degree of substitution (DS) is consistent with that of introduced anticancer drug and specific ligand in drug delivery system. Moreover, their anticoagulant activity is evaluated by measuring activated partial thromboplastin time (APTT) and anti-factor Xa activity. The results show that model ternary heparin conjugate with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.

  8. Increased unfractionated heparin requirements with decreasing body mass index in pregnancy. (United States)

    Patil, Avinash S; Clapp, Tracy; Gaston, Piyamas K; Kuhl, David; Rinehart, Eliza; Meyer, Norman L


    Pregnant women receiving low-molecular-weight heparin for therapeutic anticoagulation are often converted to unfractionated heparin in anticipation of labor. We aim to characterize the impact of maternal body mass index on attainment of target anticoagulation during the conversion process. We conducted a five-year retrospective study of a pregnancy cohort converted from low-molecular-weight heparin to unfractionated heparin in the third trimester. Patient demographics, anticoagulation regimens, and clinical outcomes were extracted from the medical record. Nonparametric statistical methods were used for analysis by body mass index (35). Thirty-one subjects were evenly distributed by body mass index (p = 0.97). Linear regression revealed an inverse correlation between patient body mass index and unfractionated heparin dose needed to achieve therapeutic anticoagulation (p = 0.04). Subjects with body mass index > 35 attained therapeutic activated partial thromboplastin time levels at 18 U (Units)/kg/h, while subjects with body mass index body mass index < 30 during pregnancy. This paradoxical relationship may be explained by physiologic characteristics that increase unfractionated heparin elimination, including diminished adiposity and increased renal clearance.

  9. Short-and long-term neural biocompatibility of heparin coated sapphire implants

    Energy Technology Data Exchange (ETDEWEB)

    Wang Anfeng [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); McAllister, James P. [Department of Neurological Surgery, Wayne State University, 4201 Antoine Street, UHC-6E, Detroit, MI 48201 (United States); Finlayson, Paul [Department of Otolaryngology, Wayne State University, 550 E Canfield, Lande Room 327, Detroit, MI 48201 (United States); Li, Jie [Department of Neurological Surgery, Wayne State University, 4201 Antoine Street, UHC-6E, Detroit, MI 48201 (United States); Brabant, Kelley [Department of Neurological Surgery, Wayne State University, 4201 Antoine Street, UHC-6E, Detroit, MI 48201 (United States); Tang Haiying [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); Black, Carolyn [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); Department of Neurological Surgery, Wayne State University, 4201 Antoine Street, UHC-6E, Detroit, MI 48201 (United States); Cao Ting [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); Liang Xuemei [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); Salley, Steven O. [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); Auner, Gregory W. [Department of Electrical and Computer Engineering, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States); Ng, K.Y. Simon [Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, MI 48202 (United States)]. E-mail:


    Sapphire is one of the most promising materials for the development of implantable biomedical devices due to its exceptional chemical, mechanical, electrical, thermal and optical properties. Silicon has also been widely used to manufacture neuroprosthetic devices in the past. However, both of these materials have been found to cause the most severe tissue reactions while implanted in vivo in the rat brain, compared with other biomaterials. In order to enhance the biocompatibility of sapphire and silicon, their surfaces were modified by depositing a self-assembled monolayer (SAM) of octadecyltrichlorosilane (OTS), followed by the photo-immobilization of heparin. To comprehensively evaluate the short- and long-term neural biocompatibility, sapphire and silicon wafers (2.5 mm dia x 0.25 mm thick) with and without heparin coating were implanted on the surface of adult rat's cortex for 10, 28 and 90 days. Specific evaluations of the cell types that contribute to an inflammatory response were performed. The histological results indicate that the biocompatibility of sapphire is dramatically improved by heparin immobilization, while this dramatic improvement is not observed on heparin coated silicon. The failure to improve the biocompatibility of silicon by heparin immobilization can be attributed to the corrosion of the silicon surface in vivo, which was confirmed by atomic force microscopy (AFM). Meanwhile, no corrosion was observed on heparin coated sapphire surfaces and a very thin layer of proteins or extracellular matrix was deposited on the surfaces.

  10. Update on the clinical use of the low-molecular-weight heparin, parnaparin

    Directory of Open Access Journals (Sweden)

    Giuseppe Camporese


    Full Text Available Giuseppe Camporese1, Enrico Bernardi2, Franco Noventa31Unit of Angiology and 3Department of Clinical and Experimental Medicine, Clinical Epidemiology Group, University Hospital of Padua, Italy; 2Department of Emergency and Accident Medicine, Hospital of Conegliano Veneto, ItalyAbstract: Parnaparin is a low-molecular-weight heparin that has widely shown its efficacy and safety in prevention of venous thromboembolism, in the treatment of chronic venous disorders, and in the treatment of venous and arterial (stable and unstable angina, acute ST-segment elevation myocardial infarction thrombosis. Parnaparin at the respective dosages of 3200, 4250, 6400, or 12800 IUaXa for a period ranging from 3 to 5 days to 6 months, is usually administered subcutaneously by means of once-daily regimen and is better tolerated than unfractionated heparin at the injection site. In the variety of commercially available low-molecular-weight heparins, parnaparin represents a useful therapeutic option, even though little evidence is available comparing the superiority or the equivalent efficacy and safety of parnaparin to that of the unfractionated heparin or placebo. This review summarizes the available literature on the use of parnaparin in different settings of cardiovascular diseases, including papers published during the past year and ongoing studies.Keywords: low-molecular-weight heparin, heparin, parnaparin, acute coronary syndromes, venous thromboembolism

  11. Development and evaluation of a fluorescence microplate assay for quantification of heparins and other sulfated carbohydrates. (United States)

    Lühn, Susanne; Schrader, Thomas; Sun, Wei; Alban, Susanne


    Due to their complex composition, quantification of heparins is difficult. On the one hand there are many biological tests, which only indirectly detect effects of the antithrombin-binding material. On the other hand direct quantitative methods are available but they are often insensitive, challenging, time-consuming or expensive. The aim of this study was to develop a sensitive, rapid, simple as well as inexpensive direct quantification assay suitable for routine analysis. Based on Polymer-H, a novel heparin complexing, fluorescent labeled synthetic polymer (lambda((ex)) 320nm, lambda((em)) 510nm), a microplate assay was developed and optimized. The specificity of the assay was evaluated by structure-assay response relationships studies using structurally defined glucan sulfates, heparins, and other natural and synthetic sulfated carbohydrates. The fluorescence intensity of Polymer-H (7.5microg/ml) showed to be concentration-dependently amplified by heparins as well as by other sulfated carbohydrates. The best sensitivity, accuracy and linearity were observed in a range from 0.63 to 5.0microg/ml heparins. No differences in the fluorescence between various heparins were observed, so that only one calibration curve is needed. In addition, all types of carbohydrates with a degree of sulfation (DS)> approximately 1.2 and a M(r)>3000 can be quantified as well. By own calibration curves also other sulfated carbohydrates like fondaparinux or other glycosaminoglycans (DS>0.4) can be determined. Copyright 2009 Elsevier B.V. All rights reserved.

  12. Structure of a bacterial microcompartment shell protein bound to a cobalamin cofactor. (United States)

    Thompson, Michael C; Crowley, Christopher S; Kopstein, Jeffrey; Bobik, Thomas A; Yeates, Todd O


    The EutL shell protein is a key component of the ethanolamine-utilization microcompartment, which serves to compartmentalize ethanolamine degradation in diverse bacteria. The apparent function of this shell protein is to facilitate the selective diffusion of large cofactor molecules between the cytoplasm and the lumen of the microcompartment. While EutL is implicated in molecular-transport phenomena, the details of its function, including the identity of its transport substrate, remain unknown. Here, the 2.1 Å resolution X-ray crystal structure of a EutL shell protein bound to cobalamin (vitamin B12) is presented and the potential relevance of the observed protein-ligand interaction is briefly discussed. This work represents the first structure of a bacterial microcompartment shell protein bound to a potentially relevant cofactor molecule.

  13. DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes. (United States)

    Cruz-Reyes, Jorge; Mooers, Blaine H M; Abu-Adas, Zakaria; Kumar, Vikas; Gulati, Shelly

    Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100-400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans.

  14. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor. (United States)

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung; van Nocker, Steven


    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.

  15. Evidence That the [beta] Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(IV)/Iron(III) Cofactor in Site 1

    Energy Technology Data Exchange (ETDEWEB)

    Dassama, Laura M.K.; Boal, Amie K.; Krebs, Carsten; Rosenzweig, Amy C.; Bollinger, Jr., J. Martin (NWU); (Penn)


    The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the {beta} subunit oxidizes a cysteine residue {approx}35 {angstrom} away in the {alpha} subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn{sup IV} ion of a Mn{sup IV}/Fe{sup III} cluster, which assembles in a reaction between O{sub 2} and the Mn{sup II}/Fe{sup II} complex of {beta}. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn{sup IV} ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn{sup IV} ion most likely resides in this site (i.e., {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}), but a subsequent computational study favored its occupation of site 2 ({sup 1}Fe{sup III}/{sup 2}Mn{sup IV}). In this work, we have sought to resolve the location of the Mn{sup IV} ion in Ct RNR-{beta} by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn{sup IV}/Fe{sup III} clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both {sup 1}Mn{sup II}/{sup 2}Fe{sup II} and {sup 1}Fe{sup II}/{sup 2}Mn{sup II} complexes are competent to react with O{sub 2} to produce the corresponding oxidized states. However, with diminished Mn{sup II} loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 'low-Mn' samples on a per-Mn basis implies that the {sup 1}Mn{sup IV}/{sup 2}Fe{sup III}-{beta} is at least the more active of the two oxidized forms and may be the only active form.

  16. CoFactor: Folate Requirement for Optimization of 5-Fluouracil Activity in Anticancer Chemotherapy

    Directory of Open Access Journals (Sweden)

    Muhammad Wasif Saif


    Full Text Available Intracellular reduced folate exists as a “pool” of more than 6 interconvertable forms. One of these forms, 5,10 methylenetetrahydrofolic acid (CH2THF, is the key one-carbon donor and reduced folate substrate for thymidylate synthase (TS. This pathway has been an important target for chemotherapy as it provides one of the necessary nucleotide substrates for DNA synthesis. The fluoropyrimidine 5-fluorouracil (5-FU exerts its main cytotoxic activity through TS inhibition. Leucovorin (5-formyltetrahydrofolate; LV has been used to increase the intracellular reduced folate pools and enhance TS inhibition. However, it must be metabolized within the cell through multiple intracellular enzymatic steps to form CH2THF. CoFactor (USAN fotrexorin calcium, (dl-5,10,-methylenepteroyl-monoglutamate calcium salt is a reduced folate that potentiates 5-FU cytotoxicity. According to early clinical trials, when 5-FU is modulated by CoFactor instead of LV, there is greater anti-tumor activity and less toxicity. This review presents the emerging role of CoFactor in colorectal and nongastrointestinal malignancies.

  17. A network analysis of cofactor-protein interactions for analyzing associations between human nutrition and diseases. (United States)

    Scott-Boyer, Marie Pier; Lacroix, Sébastien; Scotti, Marco; Morine, Melissa J; Kaput, Jim; Priami, Corrado


    The involvement of vitamins and other micronutrients in intermediary metabolism was elucidated in the mid 1900's at the level of individual biochemical reactions. Biochemical pathways remain the foundational knowledgebase for understanding how micronutrient adequacy modulates health in all life stages. Current daily recommended intakes were usually established on the basis of the association of a single nutrient to a single, most sensitive adverse effect and thus neglect interdependent and pleiotropic effects of micronutrients on biological systems. Hence, the understanding of the impact of overt or sub-clinical nutrient deficiencies on biological processes remains incomplete. Developing a more complete view of the role of micronutrients and their metabolic products in protein-mediated reactions is of importance. We thus integrated and represented cofactor-protein interaction data from multiple and diverse sources into a multi-layer network representation that links cofactors, cofactor-interacting proteins, biological processes, and diseases. Network representation of this information is a key feature of the present analysis and enables the integration of data from individual biochemical reactions and protein-protein interactions into a systems view, which may guide strategies for targeted nutritional interventions aimed at improving health and preventing diseases.

  18. Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions. (United States)

    Anderson, Lindsey N; Koech, Phillip K; Plymale, Andrew E; Landorf, Elizabeth V; Konopka, Allan; Collart, Frank R; Lipton, Mary S; Romine, Margaret F; Wright, Aaron T


    The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chloroflexus aurantiacus J-10-fl, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.

  19. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    Energy Technology Data Exchange (ETDEWEB)

    Fielding,E.; Widboom, P.; Bruner, S.


    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  20. Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics. (United States)

    Senger, Moritz; Mebs, Stefan; Duan, Jifu; Wittkamp, Florian; Apfel, Ulf-Peter; Heberle, Joachim; Haumann, Michael; Stripp, Sven Timo


    The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H2-forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN(-)) ligands in the active oxidized state (Hox) and one additional CO ligand in the inhibited state (Hox-CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fourier-transform infrared spectroscopy. Combination of (13)CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 Hox and 16 Hox-CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For Hox-CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN(-) at the distal iron ion of the cofactor as opposed to an apical CO. For Hox, two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective (13)CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.

  1. Stalled flavodoxin binds its cofactor while fully exposed outside the ribosome. (United States)

    Houwman, Joseline A; Westphal, Adrie H; van Berkel, Willem J H; van Mierlo, Carlo P M


    Correct folding of proteins is crucial for cellular homeostasis. More than thirty percent of proteins contain one or more cofactors, but the impact of these cofactors on co-translational folding remains largely unknown. Here, we address the binding of flavin mononucleotide (FMN) to nascent flavodoxin, by generating ribosome-arrested nascent chains that expose either the entire protein or C-terminally truncated segments thereof. The native α/β parallel fold of flavodoxin is among the most ancestral and widely distributed folds in nature and exploring its co-translational folding is thus highly relevant. In Escherichia coli (strain BL21(DE3) Δtig::kan) FMN turns out to be limiting for saturation of this flavoprotein on time-scales vastly exceeding those of flavodoxin synthesis. Because the ribosome affects protein folding, apoflavodoxin cannot bind FMN during its translation. As a result, binding of cofactor to released protein is the last step in production of this flavoprotein in the cell. We show that once apoflavodoxin is entirely synthesized and exposed outside the ribosome to which it is stalled by an artificial linker containing the SecM sequence, the protein is natively folded and capable of binding FMN. Copyright © 2015. Published by Elsevier B.V.

  2. Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor. (United States)

    Huijbers, Mieke M E; Martínez-Júlvez, Marta; Westphal, Adrie H; Delgado-Arciniega, Estela; Medina, Milagros; van Berkel, Willem J H


    Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered.

  3. The mechanism of assembly and cofactor insertion into Rhodobacter capsulatus xanthine dehydrogenase. (United States)

    Schumann, Silvia; Saggu, Miguel; Möller, Nadine; Anker, Stefan D; Lendzian, Friedhelm; Hildebrandt, Peter; Leimkühler, Silke


    Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alphabeta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of cofactor insertion and subunit assembly of R. capsulatus XDH, using engineered subunits with appropriate substitutions in the interfaces. In an (alphabeta) heterodimeric XDH containing the XdhA and XdhB subunits, the molybdenum cofactor (Moco) was shown to be absent, indicating that dimerization of the (alphabeta) subunits has to precede Moco insertion. In an (alphabeta)(2) XDH heterotetramer variant, including only one active Moco-center, the active (alphabeta) site of the chimeric enzyme was shown to be fully active, revealing that the two subunits act independent without cooperativity. Amino acid substitutions at two cysteine residues coordinating FeSI of the two [2Fe-2S] clusters of the enzyme demonstrate that an incomplete assembly of FeSI impairs the formation of the XDH (alphabeta)(2) heterotetramer and, thus, insertion of Moco into the enzyme. The results reveal that the insertion of the different redox centers into R. capsulatus XDH takes place sequentially. Dimerization of two (alphabeta) dimers is necessary for insertion of sulfurated Moco into apo-XDH, the last step of XDH maturation.

  4. Systematic discovery of cofactor motifs from ChIP-seq data by SIOMICS. (United States)

    Ding, Jun; Dhillon, Vikram; Li, Xiaoman; Hu, Haiyan


    Understanding transcriptional regulatory elements and particularly the transcription factor binding sites represents a significant challenge in computational biology. The chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) experiments provide an unprecedented opportunity to study transcription factor binding sites on the genome-wide scale. Here we describe a recently developed tool, SIOMICS, to systematically discover motifs and binding sites of transcription factors and their cofactors from ChIP-seq data. Unlike other tools, SIOMICS explores the co-binding properties of multiple transcription factors in short regions to predict motifs and binding sites. We have previously shown that the original SIOMICS method predicts motifs and binding sites of more cofactors in more accurate and time-effective ways than two popular methods. In this paper, we present the extended SIOMICS method, SIOMICS_Extension, and demonstrate its usage for systematic discovery of cofactor motifs and binding sites. The SIOMICS tool, including SIOMICS and SIOMICS_Extension, are available at Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Non-covalent synthesis of metal oxide nanoparticle-heparin hybrid systems: a new approach to bioactive nanoparticles. (United States)

    Vismara, Elena; Valerio, Antonio; Coletti, Alessia; Torri, Giangiacomo; Bertini, Sabrina; Eisele, Giorgio; Gornati, Rosalba; Bernardini, Giovanni


    Heparin has been conjugated to Fe3O4, Co3O4, and NiO nanoparticles (NPs) through electrostatic interactions, producing colloidal suspensions of hybrid metal oxide heparin NPs that are stable in water. Negative zeta potentials and retention of heparin's ability to capture toluidine blue indicate that heparin's negative charges are exposed on the surface of the coated NPs. IR results confirmed the formation of nanohybrids as did NMR experiments, which were also interpreted on the basis of toluidine blue tests. Transmission electron microscopy results revealed that the heparin coating does not modify the shape or dimension of the NPs. Dynamic light scattering and negative zeta potential measurements confirmed that heparin surface functionalisation is an effective strategy to prevent NP aggregation.

  6. Host co-factors of the retrovirus-like transposon Ty1

    Directory of Open Access Journals (Sweden)

    Risler Jenni K


    Full Text Available Abstract Background Long-terminal repeat (LTR retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. Results To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E -10. The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. Conclusion Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are potential, or in a few cases, presumptive HIV-1 co-factors in human cells. RHF genes whose absence results in decreased Ty1 c

  7. Host co-factors of the retrovirus-like transposon Ty1 (United States)


    Background Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event. Results To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ. Conclusion Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are potential, or in a few cases, presumptive HIV-1 co-factors in human cells. RHF genes whose absence results in decreased Ty1 cDNA include characterized RNA metabolism and modification genes, consistent with their having roles in early

  8. Manipulating the surface active and anticoagulant properties of heparin through amphiphilic molecular constructs (United States)

    Mintz, Rosita Candida

    Cardiovascular devices implanted within the vasculature are subjected to non-specific adsorption of plasma proteins. This initiates the blood coagulation cascade and platelet adhesion and activation, leading to thrombus formation. In this thesis Heparin Alkyl Diblock (HAD) surfactants were developed to improve the blood compatibility of cardiovascular biomaterials. The material designs involved using heparin, a natural anticoagulant, to inhibit coagulation pathway enzymes and mimic the cell glycocalyx to provide a repulsive force field to inhibit non-specific protein adsorption. Type AB linear (HAD Cn, n = 6,10,12,18) and branched (HAD nx 18, n = 2,3,4) heparin surfactants were synthesized by end point coupling primary and secondary alkyl amines to heparin via reductive amination. Surfactant yields (83--4%) and anticoagulant activity (149.8 +/- 3.7--39.6 +/- 0.6 IU/mg) decreased with increased branching and hydrocarbon number. Surfactant adsorption, self assembly and molecular packing of HAD surfactants at the air/liquid and liquid/solid interface were a function of the number of hydrocarbons in the surfactant alkyl segment and the presence or absence of an ionic liquid phase. Increased molecular packing was observed at the air/PBS and PBS/graphite interface, relative to aqueous interfaces, resulting from buffer cations shielding heparin's negatively charged sulfate and carboxyl groups. At the PBS/graphite interface, the surfactant's apparent heparin head group cross section decreased in diameter (1.84 to 1.05 nm) and increased in tilt angle (75.7 to 81.9°) with increasing alkyl carbon number (n = 6 to 18). The heparin head group reached a minimum diameter, equivalent to the surfactant's diameter at the air/PBS interface (0.57 nm) just prior to 36 hydrocarbons in the surfactant. For surfactants with 36 to 78 hydrocarbons, the surfactant's heparin head group oriented normal to the graphite surface and alkyl overlap or interdigitation increased (0.02 to 0.59 nm

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

    Directory of Open Access Journals (Sweden)

    Sabrina Bertini


    Full Text Available 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.

  10. New technology: heparin and antimicrobial-coated catheters. (United States)

    Ibeas-Lopez, Jose


    Although tunneled hemodialysis catheter must be considered the last option for vascular access, it is necessary in some circumstances in the dialysis patient. Thrombosis and infections are the main causes of catheter-related comorbidity. Fibrin sheath, intimately related with the biofilm, is the precipitating factor of this environment, determining catheter patency and patient morbidity. Its association with bacterial overgrowth and thrombosis has led to the search of multiple preventive measures. Among them is the development of catheter coatings to prevent thrombosis and infections. There are two kinds of treatments to cover the catheter surface: antithrombotic and antimicrobial coatings. In nondialysis-related settings, mainly in intensive care units, both have been shown to be efficient in the prevention of catheter-related infection. This includes heparin, silver, chlorhexidine, rifampicine and minocycline. In hemodialysis population, however, few studies on surface-treated catheters have been made and they do not provide evidence that shows complication reduction. The higher effectiveness of coatings in nontunneled catheters may depend on the short average life of these devices. Hemodialysis catheters need to be used over long periods of time and require clinical trials to show effectiveness of coatings over long periods. This also means greater knowledge of biofilm etiopathogeny and fibrin sheath development.

  11. Low-molecular-weight heparins: pharmacologic profile and product differentiation. (United States)

    Fareed, J; Jeske, W; Hoppensteadt, D; Clarizio, R; Walenga, J M


    The interchangeability of low-molecular-weight heparins (LMWHs) has been the subject of discussion since these products were first introduced for the prophylaxis of deep vein thrombosis. Experimental evidence now exists to show that LMWHs differ from each other in a number of characteristics. Products have been differentiated on the basis of molecular weight and biologic properties, but only limited information derived from the clinical setting is available. Potency has been described on the basis of anti-Factor Xa activity, but at equivalent anti-Xa activities, the anti-Factor IIa activity of different products shows marked variations. At the relatively small doses used for the management of postsurgical deep vein thrombosis, the effect of these interproduct differences may be relatively minor, but as LMWHs are developed for therapeutic use at much higher doses, such differences may become clinically important. Variations in safety and efficacy reported in clinical trials of LMWHs may reflect the known differences in their molecular composition and pharmacologic properties.

  12. Spectrofluorimetric determination of heparin using doxycycline-europium probe

    Energy Technology Data Exchange (ETDEWEB)

    Li Jing [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Liu Jinkai [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Zhu Xiaojing [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Peng Qian [Department of Chemistry, Shandong Normal University, Jinan 250014 (China); Jiang Chongqiu [Department of Chemistry, Shandong Normal University, Jinan 250014 (China)]. E-mail:


    A new spectrofluorimetric method was developed for the determination of the trace amount of heparin (Hep). Using doxycycline (DC)-europium ion (Eu{sup 3+}) as a fluorescent probe, in the buffer solution of pH=8.9, Hep can remarkably enhance the fluorescence intensity of the DC-Eu{sup 3+} complex at {lambda}=612 nm and the enhanced fluorescence intensity of Eu{sup 3+} ion is in proportion to the concentration of Hep. Optimum conditions for the determination of Hep were also investigated. The linear range and detection limit for the determination of Hep are 0.04-0.8 {mu}g/mL and 19.7 ng/mL, respectively. This method is simple, practical, and relatively free of interference from coexisting substances and can be successfully applied to assess Hep in biological samples. By the Rosenthal graphic method, the association constant and binding numbers of Hep with the probe are 6.60x10{sup 4} L/mol and 33.9. Moreover, the enhancement mechanism of the fluorescence intensity in the DC-Eu{sup 3+} system and the DC-Eu{sup 3+}-Hep-CTMAB system have been also discussed.

  13. Combined metabolic engineering of precursor and co-factor supply to increase α-santalene production by Saccharomyces cerevisiae

    National Research Council Canada - National Science Library

    Scalcinati, Gionata; Partow, Siavash; Siewers, Verena; Schalk, Michel; Daviet, Laurent; Nielsen, Jens


    ...-santalene production. A multistep metabolic engineering strategy targeted to increase precursor and cofactor supply was employed to manipulate the yeast metabolic network in order to redirect carbon toward the desired product...

  14. Presentation, management and outcome of heparin-induced thrombocytopenia after valvular heart surgery. (United States)

    Arangalage, Dimitri; Lepage, Laurent; Faille, Dorothée; Cimadevilla, Claire; Dilly, Marie-Pierre; Papy, Emmanuelle; Alhenc-Gelas, Martine; Ghodbane, Walid; Nataf, Patrick; Iung, Bernard; Steg, Philippe Gabriel; Vahanian, Alec; Ajzenberg, Nadine; Messika-Zeitoun, David


    The use of heparin exposes patients to heparin-induced thrombocytopenia, which is a challenging issue for both diagnosis and patient management. We sought to describe the clinical presentation, management and outcome of a series of patients diagnosed with heparin-induced thrombocytopenia after heart valve surgery. All consecutive patients diagnosed with heparin-induced thrombocytopenia during the postoperative period of heart valve surgery over a 6-year period were prospectively enrolled in a single-centre registry. Clinical and biological data were collected. In-hospital and mid-term outcomes were assessed. Information regarding the occurrence of all medical events including death, recurrence of thromboembolic events and/or thrombocytopenia was collected. We identified 93 patients (incidence proportion = 2.8%). Most patients (82%) were asymptomatic with isolated thrombocytopenia at the time of diagnosis. The other main circumstance of diagnosis was the occurrence of thromboembolic events in 17 patients (6 strokes, 10 prosthetic valve thrombosis and 1 peripheral embolic event). The in-hospital mortality rate was 1%. No thrombolysis, interventional procedure or redo surgery was performed. Danaparoid sodium was used as heparin replacement therapy in most cases (96%) and leading to complete and uneventful thrombus resolution in all cases with only one possibly related major bleeding complication. During a mean follow-up of 36 ± 20 months, no patient presented recurrence of any heparin-induced thrombocytopenia-related complication. In this contemporary series of patients, heparin-induced thrombocytopenia incidence was low and isolated thrombocytopenia was the most frequent presentation. Conservative management with early diagnosis and substitutive anticoagulation therapy introduction was associated with a low rate of clinical events and a remarkably good outcome with a low mortality rate. © The Author 2016. Published by Oxford University Press on behalf of the


    Shao, Zhengyi


    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.

  16. Voltammetric determination of heparin based on its interaction with malachite green

    Directory of Open Access Journals (Sweden)

    Xueliang Niu


    Full Text Available In this paper malachite green (MG was used as a bioprobe to determine heparin concentration by linear sweep voltammetry on the dropping mercury working electrode (DME. In Britton-Robinson (B-R buffer solution of pH 1.5, MG had a well-defined second order derivative linear sweep voltammetric reductive peak at –0.618 V (vs. SCE. After the addition of heparin into the MG solution, the reductive peak current decreased apparently without the movement of peak potential. Based on the difference of the peak current, a new voltammetric method for the determination of heparin was established. The conditions for the binding reaction and the electrochemical detection were optimized. Under the selected experimental conditions the difference of peak current was directly proportional to the concentration of heparin in the range from 0.3 to 10.0 mg/L with the linear regression equation as ∆ip″ (nA = 360.19 C (mg/L + 178.88 (n = 15, γ = 0.998 and the detection limit as 0.28 mg/L (3σ. The effects of coexisting substances such as metal ions, amino acids on the determination of heparin were investigated and the results showed that this method had good selectivity. This method was further applied to determine the heparin content in heparin sodium injection samples with satisfactory results and good recovery. The stoichiometry of the biocomplex was calculated by the electrochemical method and the binding mechanism was further discussed.

  17. Effects of heparin on platelet aggregation and release and thromboxane A2 production

    Energy Technology Data Exchange (ETDEWEB)

    Mohammad, S.F.; Anderson, W.H.; Smith, J.B.; Chuang, H.Y.; Mason, R.G.


    Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with /sup 3/H-arachidonic acid and /sup 14/C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of /sup 14/C-serotonin and /sup 3/H-arachidonic acid metabolites, it appeared that either release of /sup 14/C and /sup 3/H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both /sup 14/C and /sup 3/H.

  18. Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction. (United States)

    Craig, P A; Olson, S T; Shore, J D


    The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the

  19. A water-forming NADH oxidase from Lactobacillus pentosus and its potential application in the regeneration of synthetic biomimetic cofactors

    Directory of Open Access Journals (Sweden)

    Claudia eNowak


    Full Text Available The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox. Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13 % FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyse the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as a by-product.

  20. Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules. (United States)

    Guyot, Nicolas; Labas, Valérie; Harichaux, Grégoire; Chessé, Magali; Poirier, Jean-Claude; Nys, Yves; Réhault-Godbert, Sophie


    The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

  1. The role of heparin in sepsis: much more than just an anticoagulant. (United States)

    Li, Xu; Ma, Xiaochun


    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.

  2. Effect of heparin calcium different concentrations on some physical properties and structure in polyacrylamide matrix

    Energy Technology Data Exchange (ETDEWEB)

    Abdelrazek, E.M., E-mail: [Physics Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt. (Egypt); Ibrahim, Hosam S. [Biophysics Division, Physics Department, Faculty of Science, Mansoura University, Mansoura 35516 (Egypt)


    Films of polyacrylamide (PAAm) doped with different concentrations of heparin calcium, from 0.0 to 8 wt%, have been prepared by casting method. Studies were carried out utilizing X-ray, FT-IR, UV/VIS, DSC and DC electrical conduction to characterize the structural, optical and thermal properties of the films. Results revealed that the structural and chemical characterizations of PAAm films are affected by the addition of heparin calcium content. XRD spectra revealed that the amorphous phases increase with increase in filling levels of heparin (FLs). FT-IR analysis revealed that incorporation of heparin calcium leads to a small modification in the spectra of films. The optical absorption spectra in the UV/VIS region revealed structural variation increases with increase in concentration, which is reflected in the form of decrease in the energy band gap E{sub g}. Significant changes of DSC curves of the films suggest that strong interaction established between heparin calcium and PAAm molecules. The DC electric conduction data were interpreted on the basis of an intrachain one-dimensional interpolaron hopping model of Kuivalainen.

  3. Immediate Type Hypersensitivity to Heparins: Two Case Reports and a Review of the Literature. (United States)

    Cesana, Philipp; Scherer, Kathrin; Bircher, Andreas J


    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.

  4. Eosinophil cationic protein (ECP) can bind heparin and other glycosaminoglycans through its RNase active site. (United States)

    Torrent, Marc; Nogués, M Victòria; Boix, Ester


    The eosinophil cationic protein (ECP) is an eosinophil-secreted RNase involved in the immune host defense, with a cytotoxic activity against a wide range of pathogens. During inflammation and eosinophilia disorders, ECP is secreted to the inflammation area, where it would contribute to the immune response. ECP secretion causes also severe damage to the host own tissues. ECP presents a high affinity for heparin and this property might be crucial for its immunomodulating properties, antipathogen action, and its toxicity against eukaryotic cells. ECP, also known as human RNase 3, belongs to the mammalian RNase A superfamily and its RNase activity is required for some of its biological properties. We have now proven that ECP heparin binding affinity depends on its RNase catalytic site, as the enzymatic activity is blocked by heparin. We have applied molecular modeling to analyze ECP binding to heparin representative probes, and identified protein residues at the catalytic and substrate binding sites that could contribute to the interaction. ECP affinity for heparin and other negatively charged glycosaminoglycans (GAGs) can explain not only its binding to the eukaryote cells glycocalix but also the reported high affinity for the specific carbohydrates at bacteria cell wall, promoting its antimicrobial action. 2010 John Wiley & Sons, Ltd.

  5. Manufacture and property research of heparin grafted electrospinning PCU artificial vascular scaffolds. (United States)

    Li, Qing; Mu, Lanlan; Zhang, Fenghua; Mo, Zhichao; Jin, Chuanyu; Qi, Weiguo


    PCU (polycarbonate polyurethane) is supposed to be an ideal elastomer for manufacturing artificial vessel scaffold with perfect mechanical strength and biocompatibility. Surface grafting by heparin sodium can increase its anticoagulant hemorrhagic, achieving a better application in artificial vessels. Artificial vessels were preliminarily prepared by electrostatic spinning, treated by NH3 plasma and cross-linked with the anticoagulant heparin sodium chemically. Performances of the PCU-Hep (heparin sodium grafted purethane artificial vessels) artificial vessel were calculated through the physical and chemical property tests, evaluation of blood and biocompatibility. Results manifested that heparin sodium was successfully grafted to the vascular surface, porosity, pore diameter and water permeability of the vascular prosthesis fitted the requirements of artificial vessels, the blood test results demonstrated that the vascular material had a low hemolysis, in vitro cytotoxicity experiment and animal experiments proved an excellent biocompatibility. Thus the heparin sodium grafted electrospinning vessels could reduce intravascular thrombus and had potential clinical application. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Evaluation of the activated partial thromboplastin time (APTT) sensitivity to heparin using five commercial reagents: implications for therapeutic monitoring. (United States)

    Manzato, F; Mengoni, A; Grilenzoni, A; Lippi, G


    Heparin is an effective drug for prevention and treatment of thromboembolic conditions. Although several biological assays have been proposed for monitoring unfractionated heparin therapy, the measurement of the activated partial thromboplastin time (APTT) is the most widely employed test, and the overall risk of thromboembolic episodes was markedly reduced by maintaining APTT ratios above 1.5. However, the adjustment of the heparin therapy on the basis of APTT presents several questions which are still unresolved. Major discrepancies were found in APTTs performed using different reagents in both ex vivo and in vitro heparinized samples and occasionally with different lots of the same reagents; poor correlation was observed between APTT values and plasma heparin concentrations. In order to gain further insights into this phenomenon, we analysed the sensitivity to heparin of five commercial reagents for APTT measurement in 19 ex vivo heparinized samples. Differences were observed; correlation coefficients ranged from 0.820 to 0.985 and slopes of linear regressions from 0.26 to 1.14. Moreover, unsatisfactory correlations were obtained when APTT ratios were compared with heparin plasma concentrations in the same patients' samples. In the heparin therapeutic range of 0.35 - 0.70 U/ml, reagent-dependent differences were observed in the corresponding APTT values. These results point out a critical role of the assay methodology in monitoring heparin therapy by APTT. We suggest that reference materials and methods should be urgently identified, a universally agreed scale for reporting results should be established and reference ranges for the unfractionated heparin therapy should be reconsidered taking on account the reagent employed.

  7. Comparison of low-molecular-weight heparin with unfractionated heparin acutely and with placebo for 6 weeks in the management of unstable coronary artery disease - Fragmin in unstable coronary artery disease study (FRIC)

    NARCIS (Netherlands)

    Klein, W; Buchwald, A; Hillis, SE; Monrad, S; Sanz, G; Turpie, AGG; van der Meer, Jan; Olaisson, E; Undeland, S; Ludwig, K


    Background Low-molecular-weight heparin has a number of pharmacological and pharmacokinetic advantages over unfractionated heparin that make it potentially suitable, when used in combination with aspirin, for the treatment of unstable coronary artery disease. Method and Results Patients with

  8. Low Molecular Weight Heparin Overdose: A 10 Year Case Series

    Directory of Open Access Journals (Sweden)

    Adeline Ngo


    Full Text Available Background: Low molecular weight heparin (LMWH is used for the treatment and prevention of coagulative disorders. Few patients receiving therapeutic doses of LMWH develop major hemorrhage. Currently there are few reports in the literature on acute overdose on adults.  In this study, clinical profile, treatment and outcome of 21 patients who acutely overdosed enoxaparin are described. Methods: A retrospective chart review of California Poison Control System (CPCS database: Visual Dot Lab during 1997 to 2007 was obtained. All patients with a definite reported overdose of subcutaneous injection of LMWH were included. Results: In total, 21 patients who were all exposed to enoxaparin were studied. The reasons for overdose included medical miscalculation (3 cases, all infants, intentional misuse (2 patients, accidental overdose (7 cases, suicidal attempt (7 cases and unknown in 2 patients. 7 cases were documented to have overdosed more than 2 times the therapeutic dose. The overdose ranged from 50 mg to 1300mg (0.1-80 times the therapeutic range. No patients were documented to experience bleeding or have thrombocytopenia although complete follow-up was only available for 11 patients. Reassurance was given to patients with less than 0.14 times the therapeutic dose. The 2 patients who received protamine were overdosed with more than 2.5 times the therapeutic dose of enoxaparin. Conclusion: Most patients had no complications and were not treated with protamine. This study suggests that a large dosage of LMWH is unlikely to result in any life threatening complications, though further studies are needed to certainly conclude about this. The use of protamine in LMWH overdose seems to remain controversial.

  9. IVIg for Treatment of Severe Refractory Heparin-Induced Thrombocytopenia. (United States)

    Padmanabhan, Anand; Jones, Curtis G; Pechauer, Shannon M; Curtis, Brian R; Bougie, Daniel W; Irani, Mehraboon S; Bryant, Barbara J; Alperin, Jack B; Deloughery, Thomas G; Mulvey, Kevin P; Dhakal, Binod; Wen, Renren; Wang, Demin; Aster, Richard H


    Heparin-induced thrombocytopenia (HIT) complicated by severe thrombocytopenia and thrombosis can pose significant treatment challenges. Use of alternative anticoagulants in this setting may increase bleeding risks, especially in patients who have a protracted disease course. Additional therapies are lacking in this severely affected patient population. We describe three patients with HIT who had severe thromboembolism and prolonged thrombocytopenia refractory to standard treatment but who achieved an immediate and sustained response to IVIg therapy. The mechanism of action of IVIg was evaluated in these patients and in five additional patients with severe HIT. The impact of a common polymorphism (H/R 131) in the platelet IgG receptor FcγRIIa on IVIg-mediated inhibition of platelet activation was also examined. At levels attained in vivo, IVIg inhibits HIT antibody-mediated platelet activation. The constant domain of IgG (Fc) but not the antigen-binding portion (Fab) is required for this effect. Consistent with this finding, IVIg had no effect on HIT antibody binding in a solid-phase HIT immunoassay (platelet factor 4 enzyme-linked immunoassay). The H/R131 polymorphism in FcγRIIa influences the susceptibility of platelets to IVIg treatment, with the HH131 genotype being most susceptible to IVIg-mediated inhibition of antibody-induced activation. However, at high doses of IVIg, activation of platelets of all FcγRIIa genotypes was significantly inhibited. All three patients did well on long-term anticoagulation therapy with direct oral anticoagulants. These studies suggest that IVIg treatment should be considered in patients with HIT who have severe disease that is refractory to standard therapies. Copyright © 2017 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  10. Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

    LENUS (Irish Health Repository)

    Harmon, Shona


    Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

  11. A membrane-, mediator-, cofactor-less glucose/oxygen biofuel cell. (United States)

    Coman, Vasile; Vaz-Domínguez, Cristina; Ludwig, Roland; Harreither, Wolfgang; Haltrich, Dietmar; De Lacey, Antonio L; Ruzgas, Tautgirdas; Gorton, Lo; Shleev, Sergey


    We report the fabrication and characterisation of a non-compartmentalised, mediator and cofactor free glucose-oxygen biofuel cell based on adsorbed enzymes exhibiting direct bioelectrocatalysis, viz. cellobiose dehydrogenase from Dichomera saubinetii and laccase from Trametes hirsuta as the anodic and cathodic bioelements, respectively, with the following characteristics: an open-circuit voltage of 0.73 V; a maximum power density of 5 microW cm(-2) at 0.5 V of the cell voltage and an estimated half-life of > 38 h in air-saturated 0.1 M citrate-phosphate buffer, pH 4.5 containing 5 mM glucose.

  12. Characterization of a "TRAMP-like" co-factor of the human RNA exosome

    DEFF Research Database (Denmark)

    Christensen, Marianne Skovgaard; Kristiansen, Maiken Søndergaard; Lubas, Michal Szymon

    exosome, the major 3’-5’ exonuclease complex in human cells. PROMPTs have a lot in common with the yeast Cryptic Unstable Transcripts (CUTs), which are degraded by the concerted effort of the exosome, and its co-factor complex TRAMP (Trf4p/Air1p/Mtr4p). We have identified human proteins with functional...... similarities to components of the yeast TRAMP complex, and show that these are involved in the degradation of PROMPTs. While, these proteins form transient complexes with the exosome, our preliminary results also indicate that complex formation can occur directly with catalytic components of the exosome...

  13. Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB


    Simon Arragain; Emilio Jiménez-Vicente; Scandurra, Alessandro A.; Stefan Burén; Rubio, Luis M.; Carlos Echavarri-Erasun


    One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases. Here we study the diversity and phylogeny of naturally occurring NifB proteins, th...

  14. Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB


    Stefan Burén; Xi Jiang; Gema López-Torrejón; Carlos Echavarri-Erasun; Rubio, Luis M.


    Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium Azotobacter vinelandii and the thermo...

  15. Cofactor of BRCA1: A new genetic marker for common malignant liver cancer


    Editorial Office


    A new study has identified a vital gene in the pathogenesis and progression of liver cancer hepatocellular carcinoma (HCC), according to a team of biotechnology researchers at The American University in Cairo, Egypt, in a scientific paper published recently by AMOR. The study on human gene ‘Cofactor of BRCA1’ (dubbed COBRA1) and its potential role as a reliable cancer predictor for HCC is especially important due to the disease’s grim outlook. HCC is “ranked as the second most common cause of...

  16. End-label free-solution electrophoresis of the low molecular weight heparins. (United States)

    Sudor, J; Novotny, M V


    The intact heparins are highly charged oligosaccharides. Their charge polydispersity and the possible occurrence of numerous isomers complicate the analysis of these biomedically important glycoconjugates. After unsuccessful attempts to resolve the low molecular weight heparins in entangled matrixes, or through the use of counterions (Stefansson, M.; Novotny, M. V. Anal. Chem. 1994, 66, 3466-3471), we have designed a unique end-label reagent to incorporate both a fluorescent moiety and a desirable frictional increment to the analyte molecules. The resolution of small oligomers was improved dramatically following this approach. We also propose a scheme, based on the end-label free-solution electrophoresis model (Mayer, P.; Slater, G. W.; Drouin, G. Anal. Chem. 1994, 66, 1777-1780), that could potentially predict the migration times of some oligomers of complex heparin mixtures.

  17. Use of heparin for cytapheresis and plasmapheresis in a continuous flow centrifuge. (United States)

    Morales, M; Pizzuto, J; Reyna, M; Ambriz, R; Avilés, A; Conte, G; Sinco, A


    We evaluated the use of heparin in continuous flow centrifugation by continuous infusion. Doses were modified by assessment of the anticoagulant effect by the thrombin time dilution test (TTDT). Heparin is an efficient anticoagulant in continuous flow centrifugation and the TTDT is an effective and reliable method for control. The initial dose in leukapheresis is one unit per milliliter of blood during the first hour, then one-half the dose during the next hour, and then a one-quarter of the dose until the procedure is completed. A TTDT performed every 30 to 60 minutes will indicate whether the heparin dose should be modified. For plasmapheresis, it is necessary to determine the specific dose for each patient. There was no case of bleeding or extracorporeal coagulation of the blood.

  18. Safety and Efficacy of Argatroban in the Management of Heparin-Induced Thrombocytopenia

    Directory of Open Access Journals (Sweden)

    Bernd Saugel


    Full Text Available Heparin-induced thrombocytopenia (HIT is a life-threatening adverse reaction to heparin therapy that is characterized by thrombocytopenia and an increased risk of venous and arterial thrombosis. According to guidelines, in patients with strongly suspected or confirmed HIT all sources of heparin have to be discontinued and an alternative, nonheparin anticoagulant for HIT treatment must immediately be started. For both the prophylaxis of thrombembolic events in HIT and the treatment of HIT with thrombosis the direct thrombin inhibitor argatroban is approved in the United States. The objective of this review is to describe the mechanism of action and the pharmacokinetic profile of argatroban, to characterize argatroban regarding its safety and therapeutic efficacy and to discuss its place in therapy in HIT.

  19. Nasal administration of heparin-loaded microspheres based on poly(lactic acid). (United States)

    Yildiz, Ayca; Okyar, Alper; Baktir, Gül; Araman, Ahmet; Ozsoy, Yildiz


    In this study, heparin-loaded microspheres having smooth surface and small particle size were designed in order to provide the absorption of heparin through nasal mucosa. For this purpose, microspheres at different polymer/drug ratios (1:10, 1:2.5 and 1:1) and at different concentrations of polyvinyl alcohol, emulsifying agent (1.5% and 2.5% w/v) were prepared by solvent evaporation method with poly(lactic acid). The microspheres were for evaluated shape and surface properties, particle size, production yield, encapsulation efficiency and in vitro drug release. Based on the in vitro data, selected microspheres were applied by nasal route to Wistar albino rats. According to in vivo studies, heparin-loaded microspheres may be used by nasal route as an alternative to parenteral route.

  20. Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space. (United States)

    Stegemann, Björn; Klebe, Gerhard


    Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

  1. Immobilization of heparin on the surface of polypropylene non-woven fabric for improvement of the hydrophilicity and blood compatibility. (United States)

    Li, Rong; Wang, Hengdong; Wang, Wenfeng; Ye, Yin


    A polypropylene non-woven fabric (PPNWF) was exposed to oxygen plasma to produce peroxides on its surface. These peroxides were used to initiate graft polymerization of acrylic acid (AA) on the surface of PPNWF. Direct heparinization was accomplished via a reaction between heparin and PP-PAA (AA grafted PPNWF) which was activated by EDC (N-ethyl-N'-[3-(dimethylamino)propyl] carbodiimide). Indirect heparinized PPNWF was prepared by grafting poly(ethylene oxide) (PEO) on a PP-PAA surface to form PP-PAA-PEO, followed by reaction with heparin which was activated by EDC before use. The surface modified PPNWFs were characterized by attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectroscopy, electron spectroscopy for chemical analysis (ESCA) and contact angle goniometry. It was found that hydrophilicity was greatly improved, as indicated by the decrease of the water contact angle from 142 to 33°. In vitro blood compatibility evaluation of modified PPNWFs, including hemolysis rate, platelet adhesion, plasma protein adsorption and activated partial thromboplastin time (APTT) was investigated. The results suggested that both heparinized PPNWFs showed lower hemolysis rates and better platelet anti-adhesion than non-heparinized controls. Furthermore, PPNWF obtained via indirect immobilization of heparin showed better hydrophilicity and blood compatibility than direct heparinization of PPNWF.

  2. A common standard is inappropriate for determining the potency of ultra low molecular weight heparins such as semuloparin and bemiparin

    National Research Council Canada - National Science Library

    Jeske, Walter P; Hoppensteadt, Debra; Gray, Angel; Walenga, Jeanine M; Cunanan, Josephine; Myers, Lauren; Fareed, Jawed; Bayol, Alain; Rigal, Hélène; Viskov, Christian


    ... units within a certain chain length that impart the various and heterogeneous biological activities of heparin. The most notable illustration of the heterogeneous sulfation pattern is the presence of a particular 3-O sulfate group required for specific interaction of heparin with antithrombin (AT) [2] . This 3-O sulfate group is present on approximately o...

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

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


    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.

  4. The effect of heparin on pregnancy associated plasma protein-A concentration in healthy, non-pregnant individuals

    DEFF Research Database (Denmark)

    Jespersen, Camilla H B; Vestergaard, Kirstine R; Schou, Morten


    OBJECTIVES: The objective of this study was to determine the differences in pregnancy associated plasma protein-A (PAPP-A) concentrations in heparin naive and heparin treated healthy men and non-pregnant women, to find a possible difference in different age groups, and to determine the response...... in PAPP-A concentration to repeated injections of unfractionated heparin. DESIGN AND METHODS: Twenty-five healthy, non-pregnant volunteers divided into five groups (determined by gender and age) received 5000 IU unfractionated heparin intravenously. Five young men received an additional 5000 IU after 90...... and 180 min. Blood samples to determine PAPP-A concentration and APTT were drawn at different time points. RESULTS: Injection of heparin elicited increase in and rapid normalization of PAPP-A concentrations in all subjects. The group of 20-30-year-old never-pregnant women had lower responses than...

  5. A new approach for heparin standardization: combination of scanning UV spectroscopy, nuclear magnetic resonance and principal component analysis.

    Directory of Open Access Journals (Sweden)

    Marcelo A Lima

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

  6. The physiologic anticoagulant and anti-inflammatory role of heparins and their utility in the prevention of pregnancy complications. (United States)

    Mastrolia, Salvatore Andrea; Mazor, Moshe; Holcberg, Gershon; Leron, Elad; Beharier, Ofer; Loverro, Giuseppe; Erez, Offer


    Accumulating evidence supports the concept of increased thrombin generation, placental vascular lesions, and inflammation as crucial points in the development of the great obstetrical syndromes [preeclampsia, intrauterine growth restriction (IUGR), preterm labor (PTL), preterm prelabor rupture of membranes (PROM), fetal demise and recurrent abortions]. In light of this, the role of heparins for primary or secondary prevention of these syndromes is becoming more and more apparent, mainly due to the antithrombotic and anti-inflammatory effects of heparins. There is agreement regarding the use of heparin in the prevention of gestational complications in patients with antiphospholipid syndrome, while its use for other obstetrical complications is under debate. In the present review we will describe the physiologic role of heparins on coagulation and inflammation and we will discuss current evidence regarding the use of heparins for the prevention/treatment of obstetrical syndromes.

  7. Sperm in poor quality semen from bulls during heat stress have a lower affinity for binding hydrogen-3 heparin

    Energy Technology Data Exchange (ETDEWEB)

    Ax, R.L.; Gilbert, G.R.; Shook, G.E.


    Binding assays with (/sup 3/H) heparin were performed using spermatozoa collected prior to, during, and following summer heat stress to dairy bulls. Ejaculates collected in August 1983 after a period of ambient temperatures exceeding 29.4/sup 0/C exhibited a high frequency of abnormal sperm, and motility was reduced in some samples. Sperm in samples collected during heat stress possessed dissociation constants for binding (/sup 3/H) heparin ranging from 134.5 to 163.2 nmol. In contrast, sperm in semen collected prior to and after heat stress had significantly lower dissociation constants (higher affinity) for (/sup 3/H)heparin, 12.9 to 56.4 nmol. The number of binding sites for (/sup 3/H) heparin on sperm did not change among collection periods. It was concluded that the binding affinity for (/sup 3/H) heparin may reflect membrane integrity of bull sperm.

  8. Additive effect of heparin on the photoinactivation of Escherichia coli using tricationic P-porphyrins. (United States)

    Matsumoto, Jin; Suzuki, Kou; Uezono, Hidekazu; Watanabe, Kaho; Yasuda, Masahide


    Polycationic porphyrins have received substantial attention in developing singlet oxygen-sensitizers for biological use such as in the photoinactivation of bacteria and photodynamic therapy (PDT) of tumor cells because they have strong binding affinities for DNA and proteins. However, these strong cellular interactions can retard elimination of the drug after PDT. Therefore, the studies on the interactions of porphyrins with other molecules present much interest, in order to modulate the sensitizers' activity or even remove them from the human body after PDT. Here, we studied the additive effect of heparin on the photoinactivation by polycationic porphyrins using Escherichia coli as a model cell. Tricationic P-porphyrin sensitizers substituted with an N-alkylpyridinium group (alkyl = pentyl (1a), hexyl (1b), and heptyl (1c)) or N-hexylammonium (1d) as the axial ligand were used. Additionally, dicationic Sb-porphyrin substituted with an N-hexylpyridinium group (1e) was prepared. We studied the additive effect of heparin on the photoinactivation of E. coli by 1a-1e. The bactericidal activities were evaluated using the half-life (T1/2 in min) of E. coli and the minimum effective concentrations ([P]) of the porphyrin sensitizers. In the absence of heparin, the [P] values were determined to be 0.4-0.5 μM for 1a-1c and 2.0 μM for 1d-1e. The bactericidal activity of 1a-1c was completely retarded by the addition of heparin (1.0 μM). However, the addition of heparin (1.0 μM) could not completely retard the bactericidal activity of 1d-1e whose [P] values were relatively large. It is suggested that tricationic 1a-1c adsorbed onto the anionic heparin through electrostatic interactions. The adsorption of 1 on heparin disturbs the uptake of 1 into E. coli cells. Thus, the addition of heparin was found to be a useful method for retarding photoinactivation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Venous thrombosis after abdominal surgery. A comparison between subcutaneous heparin and antithrombotic stockings, or both

    DEFF Research Database (Denmark)

    Rasmussen, A; Hansen, P T; Lindholt, J


    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...... of the lower limbs as a test for deep vein thrombosis. There were 29.7% 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....

  10. The physiologic and therapeutic role of heparin in implantation and placentation. (United States)

    Quaranta, Michela; Erez, Offer; Mastrolia, Salvatore Andrea; Koifman, Arie; Leron, Elad; Eshkoli, Tamar; Mazor, Moshe; Holcberg, Gershon


    Implantation, trophoblast development and placentation are crucial processes in the establishment and development of normal pregnancy. Abnormalities of these processes can lead to pregnancy complications known as the great obstetrical syndromes: preeclampsia, intrauterine growth restriction, fetal demise, premature prelabor rupture of membranes, preterm labor, and recurrent pregnancy loss. There is mounting evidence regarding the physiological and therapeutic role of heparins in the establishment of normal gestation and as a modality for treatment and prevention of pregnancy complications. In this review, we will summarize the properties and the physiological contributions of heparins to the success of implantation, placentation and normal pregnancy.

  11. The physiologic and therapeutic role of heparin in implantation and placentation

    Directory of Open Access Journals (Sweden)

    Michela Quaranta


    Full Text Available Implantation, trophoblast development and placentation are crucial processes in the establishment and development of normal pregnancy. Abnormalities of these processes can lead to pregnancy complications known as the great obstetrical syndromes: preeclampsia, intrauterine growth restriction, fetal demise, premature prelabor rupture of membranes, preterm labor, and recurrent pregnancy loss. There is mounting evidence regarding the physiological and therapeutic role of heparins in the establishment of normal gestation and as a modality for treatment and prevention of pregnancy complications. In this review, we will summarize the properties and the physiological contributions of heparins to the success of implantation, placentation and normal pregnancy.

  12. Effects of Cofactors on Conformation Transition of Random Peptides Consisting of a Reduced Amino Acid Alphabet. (United States)

    Lu, Ming-Feng; Xie, Ying; Zhang, Yue-Jie; Xing, Xue-Yan


    This study aims to explore the structure characteristic of random polypeptides constructed by origin early amino acid alphabet, as well as the effects of cofactors on conformation transition of random peptides. DNA library R8-4 encoding VNM random peptides were constructed by small cassette strategy. Subsequently, a random polypeptide library was constructed using in vitro translation. Expression and purification of VNM random peptides were also performed by a conventional method of recombinant. CD spectrum analysis indicated that VNM random polypeptides have a secondary structure characteristic of protein, such as the content of α-helix is greater than 60%, random coil is about 20% β sheet, and β turn is less than 10%. CD spectrum changed with the addition of 10-40 µM ATP and NADP, but slightly changed by NAD; no influence was observed with MgSO4. Bis-ANS binding assay indicated that fluorescent intensity of bis-ANS was strengthened slightly by 10 VNM random peptides. Fluorescent intensity was strengthened fourfold by adding 10-40 µM ATP, NAD, and NADH, whereas the inducing effect of NADPH and MgSO4 were negligible. VNM random peptides have a classic secondary structure and hydrophobic domain in water solution. Moreover, conformation transition and hydrophobic domain could be induced by cofactor, indicating the preliminary evidence for the hypothesis that "the origin of primitive protein was induced by small molecule."

  13. Effect of mitochondrial cofactors and antioxidants supplementation on cognition in the aged canine. (United States)

    Snigdha, Shikha; de Rivera, Christina; Milgram, Norton W; Cotman, Carl W


    A growing body of research has focused on modifiable risk factors for prevention and attenuation of cognitive decline in aging. This has led to an unprecedented interest in the relationship between diet and cognitive function. Several preclinical and epidemiologic studies suggest that dietary intervention can be used to improve cognitive function but randomized controlled trials are increasingly failing to replicate these findings. Here, we use a canine model of aging to evaluate the effects of specific components of diet supplementation which contain both antioxidants and a combination of mitochondrial cofactors (lipoic acid [LA] and acetyl-l-carnitine) on a battery of cognitive functions. Our data suggest that supplementation with mitochondrial cofactors, but not LA or antioxidant alone, selectively improve long-term recall in aged canines. Furthermore, we found evidence that LA alone could have cognitive impairing effects. These results contrast to those of a previous longitudinal study in aged canine. Our data demonstrate that one reason for this difference may be the nutritional status of animals at baseline for the 2 studies. Overall, this study suggests that social, cognitive, and physical activity together with optimal dietary intake (rather than diet alone) promotes successful brain aging. Published by Elsevier Inc.

  14. In Search of Novel Targets for Heart Disease: Myocardin and Myocardin-Related Transcriptional Cofactors

    Directory of Open Access Journals (Sweden)

    Alexander T. Mikhailov


    Full Text Available Growing evidence suggests that gene-regulatory networks, which are responsible for directing cardiovascular development, are altered under stress conditions in the adult heart. The cardiac gene regulatory network is controlled by cardioenriched transcription factors and multiple-cell-signaling inputs. Transcriptional coactivators also participate in gene-regulatory circuits as the primary targets of both physiological and pathological signals. Here, we focus on the recently discovered myocardin-(MYOCD related family of transcriptional cofactors (MRTF-A and MRTF-B which associate with the serum response transcription factor and activate the expression of a variety of target genes involved in cardiac growth and adaptation to stress via overlapping but distinct mechanisms. We discuss the involvement of MYOCD, MRTF-A, and MRTF-B in the development of cardiac dysfunction and to what extent modulation of the expression of these factors in vivo can correlate with cardiac disease outcomes. A close examination of the findings identifies the MYOCD-related transcriptional cofactors as putative therapeutic targets to improve cardiac function in heart failure conditions through distinct context-dependent mechanisms. Nevertheless, we are in support of further research to better understand the precise role of individual MYOCD-related factors in cardiac function and disease, before any therapeutic intervention is to be entertained in preclinical trials.

  15. Crystallization and preliminary crystallographic analysis of molybdenum-cofactor biosynthesis protein C from Thermus thermophilus

    Energy Technology Data Exchange (ETDEWEB)

    Kanaujia, Shankar Prasad; Ranjani, Chellamuthu Vasuki; Jeyakanthan, Jeyaraman; Baba, Seiki; Chen, Lirong; Liu, Zhi-Jie; Wang, Bi-Cheng; Nishida, Masami; Ebihara, Akio; Shinkai, Akeo; Kuramitsu, Seiki; Shiro, Yoshitsugu; Sekar, Kanagaraj; Yokoyama, Shigeyuki (IIS); (SP8); (Georgia)


    The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 {angstrom}, {beta} = 104.9{sup o}; the crystal diffracted to a resolution of 1.9 {angstrom}. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 {angstrom}, and diffracted to 1.75 {angstrom} resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P2{sub 1} and R32, respectively.

  16. Polyphosphate is a cofactor for the activation of factor XI by thrombin (United States)

    Choi, Sharon H.; Smith, Stephanie A.


    Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, β-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis. PMID:21976677

  17. Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Lindsey N.; Koech, Phillip K.; Plymale, Andrew E.; Landorf, Elizabeth V.; Konopka, Allan; Collart, Frank; Lipton, Mary S.; Romine, Margaret F.; Wright, Aaron T.


    The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are critically needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically-predicted functions. Of particular importance are transport mechanisms, used to shuttle nutrients and metabolites across cell mem-branes, such as B vitamins, which are indispensable to metabolic reactions crucial to the survival of diverse microbes ranging from members of environmental microbial communities to human pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, and characterization of intra-cellular enzyme-cofactor/nutrient associations are needed to enable a significantly improved understanding of microbial biochemis-try and physiology, how microbes associate with others, and how they sense and respond to environmental perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular protein-cofactor associations through live cell labeling of the filamentous anoxygenic pho-toheterotroph, Chloroflexus aurantiacus J-10-fl, known for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by iden-tifying B vitamin transport and disposition mechanisms required for survival.

  18. Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA. (United States)

    Guse, Annika; Stevenson, Clare E M; Kuper, Jochen; Buchanan, Grant; Schwarz, Gunter; Giordano, Gerard; Magalon, Axel; Mendel, Ralf R; Lawson, David M; Palmer, Tracy


    Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.

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

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


    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.

  20. NarJ chaperone binds on two distinct sites of the aponitrate reductase of Escherichia coli to coordinate molybdenum cofactor insertion and assembly. (United States)

    Vergnes, Alexandra; Pommier, Janine; Toci, René; Blasco, Francis; Giordano, Gérard; Magalon, Axel


    Understanding when and how metal cofactor insertion occurs into a multisubunit metalloenzyme is of fundamental importance. Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme. In the assembly pathway of the multisubunit molybdoenzyme, membrane-bound nitrate reductase A from Escherichia coli, a NarJ-assisted molybdenum cofactor (Moco) insertion step, must precede membrane anchoring of the apoenzyme. Here, we have shown that the NarJ chaperone interacts at two distinct binding sites of the apoenzyme, one interfering with its membrane anchoring and another one being involved in molybdenum cofactor insertion. The presence of the two NarJ-binding sites within NarG is required to ensure productive formation of active nitrate reductase. Our findings supported the view that enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.

  1. Chaperonin cofactors, Cpn10 and Cpn20, of green algae and plants function as hetero-oligomeric ring complexes. (United States)

    Tsai, Yi-Chin C; Mueller-Cajar, Oliver; Saschenbrecker, Sandra; Hartl, F Ulrich; Hayer-Hartl, Manajit


    The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

  2. Effect of low-dose heparin on urinary albumin excretion in insulin-dependent diabetes mellitus

    NARCIS (Netherlands)

    Myrup, B.; Hansen, P.M.; Jensen, T.; Kofoed-Enevoldsen, A.; Feldt-Rasmussen, B.; Gram, J.; Kluft, C.; Jespersen, J.; Deckert, T.


    We investigated the effect of heparin on urinary albumin excretion in patients with insulin-dependent diabetes mellitus. 39 patients with persistent urinary albumin excretion of 30-300 mg/24 h were randomly treated for 3 months with subcutaneous injections twice daily of isotonic saline, 5000 IU

  3. Assessment of HIT Antibody Complex in Hip Fracture Patients Receiving Enoxaparin or Unfractionated Heparin

    DEFF Research Database (Denmark)

    Griffin, Justin W; Hopkinson, William J; Rud-Lassen, Michael


    Thromboembolic disease is a common complication of hip fracture in the elderly. Anticoagulants represent a standard of care in preventing postoperative thrombotic complications following surgical fixation. We asked whether levels of antibody to heparin-platelet factor 4 (PF4) complex were differe...

  4. Pericardial fluid and serum VEGF in response to different types of heparin treatment. (United States)

    Gerrah, Rabin; Tshori, Sagi; Gilon, Dan


    Heparin is an important medication in the treatment of patients with unstable angina pectoris. We designed an observational study to compare the effects of standard heparin (SH) with low molecular weight heparin (LMWH) on vascular endothelial growth factor (VEGF) levels in patients undergoing coronary artery bypass grafting (CABG). Thirty-two patients with unstable angina pectoris undergoing CABG were prospectively categorized into two groups according to the type of heparin administration before surgery. VEGF levels determined by enzyme linked immunosorbent assay (ELISA) were compared between the two groups' blood samples obtained before the surgery and pericardial fluid after pericardial opening. There was no difference in preoperative characteristics between the two groups. Serum VEGF levels were similar (P=0.3) in patients treated by SH (85+/-55 pg/ml) compared to those treated with LMWH (105+/-64 pg/ml). VEGF levels in the pericardial fluid were significantly raised (P<0.0001) in patients of LMWH group (36+/-13 pg/ml) compared to SH group (13+/-6 pg/ml). A good correlation was observed between VEGF in the serum and platelet count in both SH group (r=0.8) and LMWH group (r=0.7). Local response of the ischemic myocardium, as expressed by VEGF levels, differs in patients treated with SH compared to patients treated with LMWH. VEGF levels in pericardial fluid of patients receiving LMWH were 2-3-folds higher than patients in SH group.

  5. The impact of heparin-coated circuits on hemodynamics during and after cardiopulmonary bypass

    NARCIS (Netherlands)

    de Vroege, R; Huybregts, R; van Oeveren, W; van Klarenbosch, J; Linley, G; Mutlu, J; Jansen, E; Hack, E; Eijsman, L; Wildevuur, C

    This study was performed to investigate if heparin-coated extracorporeal circuits can reduce the systemic inflammatory reaction with the subsequent release of vasoactive substances during and after cardiopulmonary bypass. Fifty-one patients scheduled for coronary artery bypass grafting were perfused

  6. Mechanical fibrinogen-depletion supports heparin-free mesenchymal stem cell propagation in human platelet lysate. (United States)

    Laner-Plamberger, Sandra; Lener, Thomas; Schmid, Doris; Streif, Doris A; Salzer, Tina; Öller, Michaela; Hauser-Kronberger, Cornelia; Fischer, Thorsten; Jacobs, Volker R; Schallmoser, Katharina; Gimona, Mario; Rohde, Eva


    Pooled human platelet lysate (pHPL) is an efficient alternative to xenogenic supplements for ex vivo expansion of mesenchymal stem cells (MSCs) in clinical studies. Currently, porcine heparin is used in pHPL-supplemented medium to prevent clotting due to plasmatic coagulation factors. We therefore searched for an efficient and reproducible medium preparation method that avoids clot formation while omitting animal-derived heparin. We established a protocol to deplete fibrinogen by clotting of pHPL in medium, subsequent mechanical hydrogel disruption and removal of the fibrin pellet. After primary culture, bone-marrow and umbilical cord derived MSCs were tested for surface markers by flow cytometry and for trilineage differentiation capacity. Proliferation and clonogenicity were analyzed for three passages. The proposed clotting procedure reduced fibrinogen more than 1000-fold, while a volume recovery of 99.5 % was obtained. All MSC types were propagated in standard and fibrinogen-depleted medium. Flow cytometric phenotype profiles and adipogenic, osteogenic and chondrogenic differentiation potential in vitro were independent of MSC-source or medium type. Enhanced proliferation of MSCs was observed in the absence of fibrinogen but presence of heparin compared to standard medium. Interestingly, this proliferative response to heparin was not detected after an initial contact with fibrinogen during the isolation procedure. Here, we present an efficient, reproducible and economical method in compliance to good manufacturing practice for the preparation of MSC media avoiding xenogenic components and suitable for clinical studies.

  7. Low dose intravesical heparin as prophylaxis against recurrent noninvasive (stage Ta) bladder cancer

    DEFF Research Database (Denmark)

    Bitsch, M; Hermann, G G; Andersen, J P


    A controlled randomized clinical trial was conducted to examine the efficacy of topical low dose heparin (0.125 gm./l., 25,000 units per l.) as prophylaxis against recurrent noninvasive (stage Ta) transitional cell bladder cancer. Transurethral tumor resection was done with irrigation fluid......) bladder cancer....

  8. Post-transplant IVC occlusion and thrombosis treated with tPA, heparin, and sharp recanalization. (United States)

    Mindikoglu, Ayse L; Miller, Jonathan S; Borge, Marc A; Van Thiel, David H


    Complete inferior vena cava (IVC) thrombosis can be a lethal complication in a liver transplant recipient. The case of a 52-year-old liver transplant recipient, who developed complete IVC as well as left iliofemoral thrombosis, is reported. After treatment with combined tissue plasminogen activator (tPA) and heparin, the IVC was successfully recanalized with sharp dissection, balloon dilatation, and stent placement.

  9. Surface heparin treatment of the decellularized porcine heart valve : Effect on tissue calcification

    NARCIS (Netherlands)

    Yang, Min; Lin, Yang-Hua; Shi, Wei-Ping; Shi, Hong-Can; Gu, Y. John; Shu, Yu-Sheng

    Tissue calcification is a major cause of failure of bioprosthetic heart valves. Aim of this study was to examine whether surface heparin treatment of the decellularized porcine heart valve reduces tissue calcification. Fresh porcine aortic heart valves were dissected as tissue discs and divided into

  10. Citrate versus heparin anticoagulation in continuous renal replacement therapy in small children

    NARCIS (Netherlands)

    Raymakers-Janssen, Paulien A M; Lilien, Marc; van Kessel, Ingrid A.; Veldhoen, Esther S.; Wösten-van Asperen, Roelie M.; van Gestel, Josephus P.J.


    Background: Citrate is preferred over heparin as an anticoagulant in adult continuous renal replacement therapy (CRRT). However, its potential adverse effects and data on use in CRRT in infants and toddlers is limited. We conducted a prospective study on using citrate in CRRT in critically ill small

  11. Heparin improves organ microcirculatory disturbances in caerulein-induced acute pancreatitis in rats. (United States)

    Dobosz, Marek; Mionskowska, Lucjanna; Hac, Stanislaw; Dobrowolski, Sebastian; Dymecki, Dariusz; Wajda, Zdzislaw


    Microcirculatory disturbances are important early pathophysiological events in various organs during acute pancreatitis. The aim of the study was to evaluate changes in microperfusion of the pancreas, liver, kidney, stomach, colon, skeletal muscle, and to investigate the influence of heparin on the organ microcirculation in caerulein-induced experimental acute pancreatitis. Acute pancreatitis was induced by 4 intraperitoneal injections of caerulein (Cn) (15 microg/kg). The organ microcirculation was measured by laser Doppler flowmetry. Serum interleukin 6 and hematocrit levels were analysed. Acute pancreatitis resulted in a significant drop of microperfusion in all examined organs. Heparin administration (2 x 2.5 mg/kg) improved the microcirculation in pancreas (36.9 +/- 4% vs 75.9 +/- 10%), liver (56.6 +/- 6% vs 75.2 +/- 16%), kidney (45.1 +/- 6% vs 79.3 +/- 5%), stomach (65.2 +/- 8% vs 78.1 +/- 19%), colon (69.8 +/- 6% vs 102.5 +/- 19%), and skeletal muscle (59.2 +/- 6% vs 77.9 +/- 13%). Heparin treatment lowered IL-6 (359.0 +/- 66 U/mL vs 288.5 +/- 58 U/mL) and hematocrit level (53 +/- 4% vs 46 +/- 3%). Heparin administration has a positive influence on organ microcirculatory disturbances accompanying experimental Cn-induced acute pancreatitis. Copyright 2004 The WJG Press ISSN

  12. Aspirin plus Heparin or Aspirin Alone in Women with Recurrent Miscarriage

    NARCIS (Netherlands)

    Kaandorp, Stef P.; Goddijn, Mariette; van der Post, Joris A. M.; Hutten, Barbara A.; Verhoeve, Harold R.; Hamulyak, Karly; Mol, Ben Willem; Folkeringa, Nienke; Nahuis, Marleen; Papatsonis, Dimitri N. M.; Buller, Harry R.; van der Veen, Fulco; Middeldorp, Saskia


    BACKGROUND Aspirin and low-molecular-weight heparin are prescribed for women with unexplained recurrent miscarriage, with the goal of improving the rate of live births, but limited data from randomized, controlled trials are available to support the use of these drugs. METHODS In this randomized

  13. In vitro Heparin Precipitation in the Plasma of Euthyroid women with ...

    African Journals Online (AJOL)

    And history of recurrent miscarriage associated with auto antibodies have had a high rate of life births in subsequent pregnancies when they were treated with low dose aspirin together with low dose heparin. An in-vitro assay thus provide basis for the intricate interaction. DESIGN: Two hundred and ten (210) healthy ...

  14. Aspirin plus heparin or aspirin alone in women with recurrent miscarriage

    NARCIS (Netherlands)

    Kaandorp, Stef P.; Goddijn, Mariëtte; van der Post, Joris A. M.; Hutten, Barbara A.; Verhoeve, Harold R.; Hamulyák, Karly; Mol, Ben Willem; Folkeringa, Nienke; Nahuis, Marleen; Papatsonis, Dimitri N. M.; Büller, Harry R.; van der Veen, Fulco; Middeldorp, Saskia


    Aspirin and low-molecular-weight heparin are prescribed for women with unexplained recurrent miscarriage, with the goal of improving the rate of live births, but limited data from randomized, controlled trials are available to support the use of these drugs. In this randomized trial, we enrolled 364

  15. Risk of postpartum hemorrhage in women receiving therapeutic doses of low-molecular-weight heparin

    NARCIS (Netherlands)

    Roshani, S.; Cohn, D.M.; Stehouwer, A.; Hamers, S.; Wolf, H.; Van Der Post, J.M.; Büller, H.R.; Kamphuisen, P.W.; Middeldorp, S.


    Introduction: Low-molecular-weight heparin (LMWH) is considered safe in pregnancy, but the optimal dose for prevention of venous thrombosis is unclear. Prophylactic doses may not prevent DVT adequately while higher doses may induce postpartum hemorrhage (PPH). We studied whether use of therapeutic

  16. Impact of autoclave sterilization on the activity and structure of formulated heparin. (United States)

    Beaudet, Julie M; Weyers, Amanda; Solakyildirim, Kemal; Yang, Bo; Takieddin, Majde; Mousa, Shaker; Zhang, Fuming; Linhardt, Robert J


    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.

  17. Improved synthesis of polystyrene-poly(ethylene oxide)-heparin block copolymers

    NARCIS (Netherlands)

    Vulic, I.; Loman, A.J.B.; Feijen, Jan; Okano, T.; Kim, S.W.


    A novel procedure for the synthesis of block copolymers composed of a hydrophobic block of polystyrene, a hydrophilic block of poly(ethylene oxide) and a bioactive block of nitrous acid-degraded heparin was developed. Amino-semitelechelic polystyrene was prepared by anionic polymerization of styrene

  18. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

    DEFF Research Database (Denmark)

    Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa


    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin...

  19. Blood interaction with a Bioline heparin coated HIA-VAD : A study on calves

    NARCIS (Netherlands)

    vanderKamp, KWHJ; Magielse, CPE; Elstrodt, JM; vanderMeer, J; vanOeveren, W; Rakhorst, G

    The blood compatibility of ventricular assist devices developed by the Helmholtz institute Aachen (HA-VAD's) was tested on calves. Seven calves received a non-coated HIA-VAD (control) and three a Bioline heparin coated device. The circulatory support of these HIA-VAD's lasted one week. Mechanical

  20. Extrapulmonary colony formation after intravenous injection of tumour cells into heparin treated animals

    NARCIS (Netherlands)

    Maat, B.


    Recent data on extrapulmonary colony formation after heparin administration are inconclusive. A systemic study of this topic was undertaken with 4 experimental tumour systems and 2 distinct periods of reduced clotting capacity in rats and mice. I.v. injection of various numbers of tumour cells into


    NARCIS (Netherlands)



    Adriamycin-loaded albumin-heparin conjugate microspheres (ADR-AHCMS) were evaluated as possible intraperitoneal (i.p.) delivery systems for site-specific cytotoxic action. The biocompatibility of the microspheres after intraperitoneal injection was tested first. 1 day after i.p. administration of

  2. Adriamycin-loaded albumin-heparin conjugate microspheres for intraperitoneal chemotherapy

    NARCIS (Netherlands)

    Cremers, Harry; Cremers, H.F.M.; Seymour, L.W.; Lam, K.; Los, G.; van Vugt, M.; Kwon, G.; Bae, Y.H.; Kim, S.W.; Feijen, Jan


    Adriamycin-loaded albumin-heparin conjugate microspheres (ADR-AHCMS) were evaluated as possible intraperitoneal (i.p.) delivery systems for site-specific cytotoxic action. The biocompatibility of the microspheres after intraperitoneal injection was tested first. 1 day after i.p. administration of

  3. Set up of a protocol for heparin use in special patients

    Directory of Open Access Journals (Sweden)

    N. Manresa Ramón


    Full Text Available Low-molecular weight (LMW heparins bring a series of advantages as compared to non-fractionated heparin (NFH, such as safety, efficacy, bioavailability, fewer monitoring, and persistent anti-coagulant response. There exist, however, a concern about their use in particular patients that may require a special control, such as those with renal failure, age over 75 years, obesity, and pregnancy. The aim of this study was the set up between the department of Pharmacy, Hematology, and Internal Medicine of a consensus protocol for the follow-up ad monitoring of LMWH in patients requiring a special control. For this purpose, we carried out a bibliographical review of the different heparins used under de above mentioned conditions. Based on the evidence available and the consensus among the members of the working group, we established a protocol that contained recommendations on prophylaxis, management and monitoring by means of the determination of anti-Xa factor. Besides, we included some clues on the therapeutic figures of anti-Xa and administration schedules for obtaining anti-Xa values within the range. Enoxaparin was the selected heparin given the evidence and its availability at our center.

  4. Viscoelastic blood coagulation measurement with Sonoclot predicts postoperative bleeding in cardiac surgery after heparin reversal. (United States)

    Bischof, Dominique B; Ganter, Michael T; Shore-Lesserson, Linda; Hartnack, Sonja; Klaghofer, Richard; Graves, Kirk; Genoni, Michele; Hofer, Christoph K


    The aim of the study was to determine if Sonoclot with its sensitive glass bead-activated, viscoelastic test can predict postoperative bleeding in patients undergoing cardiac surgery at predefined time points. A prospective, observational clinical study. A teaching hospital, single center. Consecutive patients undergoing cardiac surgery (N = 300). Besides routine laboratory coagulation studies and heparin management with standard (kaolin) activated clotting time, additional native blood samples were analyzed on a Sonoclot using glass bead-activated tests. Glass bead-activated clotting time, clot rate, and platelet function were recorded immediately before anesthesia induction and at the end of surgery after heparin reversal but before chest closure. Primary outcome was postoperative blood loss (chest tube drainage at 4, 8, and 12 hours postoperatively). Secondary outcome parameters were transfusion requirements, need for surgical re-exploration, time of mechanical ventilation, length of intensive care unit and hospital stay, and hospital morbidity and mortality. Patients were categorized into "bleeders" and "nonbleeders." Patient characteristics, operations, preoperative standard laboratory parameters, and procedural times were comparable between bleeders and nonbleeders except for sex and age. Bleeders had higher rates of transfusions, surgical re-explorations, and complications. Only glass bead measurements by Sonoclot after heparin reversal before chest closure but not preoperatively were predictive for increased postoperative bleeding. Sonoclot with its glass bead-activated tests may predict the risk for postoperative bleeding in patients undergoing cardiac surgery at the end of surgery after heparin reversal but before chest closure. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Simultaneous immobilization of heparin and gentamicin on polypropylene textiles: a dual therapeutic activity. (United States)

    Degoutin, Stéphanie; Jimenez, Maude; Chai, Feng; Pinalie, Thibaut; Bellayer, Severine; Vandenbossche, Marianne; Neut, Christel; Blanchemain, Nicolas; Martel, Bernard


    The aim of this work was to prepare a nonwoven polypropylene (PP) textile functionalized with bioactive molecules in order to improve simultaneously anticoagulation and antibacterial properties. The immobilization of either heparin (anticoagulation agent) or gentamicin (aminoglycoside class antibiotic) alone has already been proven to be effective on PP nonwoven textiles. In this work, we managed to go further, by immobilizing both heparin and gentamicin at the same time on one unique textile. A successive immersion in different heparin and gentamicin bathes successfully led to a dual drug coated textile, as confirmed by several characterization techniques (Fourier transform infrared-attenuated total reflectance, X-ray photoelectron spectroscopy, and scanning electron microscopy). The immobilization times were varied in order to determine the best compromise between cytocompatibility, anticoagulant effect, and antimicrobial activity. Short immersion times in gentamicin solutions confer very good antimicrobial activity to the textile and avoid cytotoxicity, whereas long immersion times in heparin solution were necessary to observe a significant anticoagulant effect. © 2013 Wiley Periodicals, Inc.

  6. High-dose heparin fails to improve acute lung injury following smoke inhalation in sheep. (United States)

    Murakami, Kazunori; Enkhbaatar, Perenlei; Shimoda, Katsumi; Mizutani, Akio; Cox, Robert A; Schmalstieg, Frank C; Jodoin, Jeffrey M; Hawkins, Hal K; Traber, Lillian D; Traber, Daniel L


    Thrombin is involved in various inflammatory responses. In sepsis, coagulation abnormalities are major complications. Acute lung injury is one of the most life-threatening problems that can result from sepsis. We hypothesized that high-dose heparin might be effective in attenuating acute lung injury in our sepsis model. Female sheep ( n =16) were surgically prepared for the study. After a tracheotomy, 48 breaths of cotton smoke (heparin infusion group ( n =6), a Ringer's lactate infusion group ( n =6), and a sham-injury group ( n =4; surgically prepared in the same fashion but receiving no inhalation injury or bacteria). The treatment was started 1 h after the insult, and was continued thereafter for 24 h. The dose of heparin was adjusted by monitoring to target an activated clotting time of between 300 and 400 s (baseline=approx. 150 s). Sheep exposed to lung injury presented with typical hyperdynamic cardiovascular changes, including an increased cardiac output and a fall in systemic vascular resistance. There was a decrease in the arterial partial pressure of O(2). In conclusion, high-dose heparin did not prevent lung dysfunction in this model, in which acute lung injury was induced by combined smoke and septic challenge.

  7. Nebulised heparin: a new approach to the treatment of acute lung injury? (United States)

    Suter, Peter M


    The administration of heparin by nebulisation has been proposed for the 'local' treatment of pulmonary coagulation disturbances in acute lung injury (ALI). Alveolar and lung micro-vascular fibrin accumulation and breakdown inhibition indeed play a central role in the development and clinical course of this disease. Preclinical studies provide some evidence of the beneficial effects of heparin inhalation in several animal models of ALI. Clinical investigations are sparse, and trials such as the one presented by Dixon and colleagues in a recent issue of Critical Care are welcome as they provide insight into the possible clinical use of nebulised heparin in this situation. This phase 1 trial involved 16 patients with early ALI, and showed the feasibility of the approach. In addition, non-significant changes in respiratory functions and systemic anticoagulant effects were documented with the four doses tested. The study of Dixon and colleagues adds to data that helps pave the way towards a possible clinical use of heparin by nebulisation in ALI. It remains to be clarified in which clinical situations, at what time points and with which dosages the best chances exist for a beneficial effect on the prognosis of these patients.

  8. Does a Nebulized Heparin/N-acetylcysteine Protocol Improve Outcomes in Adult Smoke Inhalation?

    Directory of Open Access Journals (Sweden)

    Natalie S. Kashefi, MS


    Conclusions: The implementation of a heparin/N-acetylcysteine/albuterol protocol did not reduce mortality or duration of mechanical ventilation in this cohort of adults with inhalation injury and resulted in a significant increase in pneumonia rates. Larger prospective studies are necessary, with close attention paid to minimizing the infection risk incurred from frequent administration of nebulized medications.

  9. Release of proteins via ion exchange from albumin-heparin microspheres

    NARCIS (Netherlands)

    Kwon, Glen S.; Bae, You Han; Cremers, H.F.M.; Cremers, Harry; Feijen, Jan; Kim, Sung Wan


    Albumin-heparin and albumin microspheres were prepared as ion exchange gels for the controlled release of positively charged polypeptides and proteins. The adsorption isotherms of chicken egg and human lysozyme, as model proteins, on microspheres were obtained. An adsorption isotherm of chicken egg

  10. Synthesis and characterization of polystyrene-poly(ethylene oxide)-heparin block copolymers

    NARCIS (Netherlands)

    Vulić, I.; Okano, T.; Kim, S.W.; Feijen, Jan


    A procedure for the preparation of new block copolymers composed of a hydrophobic block of polystyrene, a hydrophilic spacer-block of poly(ethylene oxide) and a bioactive block of heparin was investigated. Polystyrene with one amino group per chain was synthesized by free radical oligomerization of

  11. Influence of heparin on the assay of amitriptyline, clomipramine, and their metabolites

    NARCIS (Netherlands)

    Levering, S.C.M.; Oostelbos, M.C.J.M.; Toll, P.J.M.M.; Loonen, A.J.M.


    In this study the effect of the use of lithium heparin containers on the plasma levels of amitriptyline, clomipramine, and their metabolites was investigated. Twenty-five patients (10 men and 15 women, mean age 51.8 ± 14.9 years) taking either amitriptyline or clomipramine in a daily dosage varying

  12. Electron transfer between the heme bound oxygen and the tetrahydrobiopterin cofactor of nitric oxide synthase: a DFT study (United States)

    Menyhárd, Dóra K.


    Nitric oxide is synthesized from L-Arg by nitric oxide synthases (NOSs). DFT calculations carried out in the present study demonstrate that there is direct coupling between the heme bound oxygen and the tetrahydrobiopterin (H 4B) cofactor in the activated state of NOS. Results indicate that radicalization of H 4B causes the coupled reduction of heme bound oxygen. In our model system H 3B rad radical formation is prompted by proton dissociation from the N5 site of the cofactor; spin density is transferred to the heme bound oxygen, which we found in an orientation preconditioned for H abstraction from the substrate.

  13. Influence of spacer length on heparin coupling efficiency and fibrinogen adsorption of modified titanium surfaces

    Directory of Open Access Journals (Sweden)

    Gbureck Uwe


    Full Text Available Abstract 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-(Trimethoxysilylpropyl]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

  14. Effect of routine heparins treatment in acute coronary syndrome on serum pregnancy-associated plasma protein a concentration. (United States)

    Wang, Guodong; Zhang, Aiyuan; Han, Xiaoli; Zhang, Jungang; Zhang, Guangfang; Sun, Liping


    Pregnancy-associated plasma protein A (PAPP-A) has been suggested as a useful marker of acute coronary syndrome (ACS). Serum PAPP-A concentrations are affected by unfractionated heparin (UFH) in ACS population, and we tried to investigate the time profile of effects of routine heparins treatment on serum PAPP-A concentrations in ACS population thoroughly and give advice to sample collection of related study. Twenty cases were involved in this study: ten patients with acute myocardial infarction received subcutaneous low molecular weight heparin (LMWH) twice a day (group A), and the other ten percutaneous coronary intervention patients with stenting received intravenous UFH (group B). Samples were collected before and after heparin administration and serum PAPP-A concentrations were analyzed in these samples. Serum PAPP-A concentration increased in both group A and B. In group A, PAPP-A concentration elevated gradually (14.5 to 29.4 mIU/L, PPAPP-A peak was induced by additional heparin administration. Heparins-induced increase in serum PAPP-A concentration lasted until 48h after drug use was discontinued. We recommend that samples from these patients for PAPP-A measurement should be collected at least 48h after the last administration if its not available before the administration of heparins.

  15. Specific binding of the glycosaminoglycan /sup 3/H-heparin to bull, monkey, and rabbit spermatozoa in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Handrow, R.R.; Boehm, S.K.; Lenz, R.W.; Robinson, J.A.; Ax, R.L.

    In Vitro binding and some binding parameters of the glycosaminoglycan heparin to viable epididymal or ejaculated bull spermatozoa, ejaculated rabbit spermatozoa, and frozen-thawed rhesus monkey spermatozoa were investigated. Nonspecific binding was affected only by the concentration of /sup 3/H-heparin, whereas specific binding was saturable, reversible, and dependent on the pH, temperature, and calcium concentration of the incubation medium. Magnesium concentration dependence was observed in the presence of calcium but could not be detected in the absence of calcium. Bound /sup 3/H-heparin was displaced by several orders of magnitude greater concentrations of chondroitin sulfate. Scatchard plot analysis suggested multiple binding affinities for /sup 3/H-heparin to spermatozoa. /sup 3/H-heparin was shown to bind to sperm heads and flagella. Fluorescein-labeled heparin bound to acrosomal, postacrosomal, and flagellar membranes. It was concluded that the specific binding of heparin involved a proteinaceous component on, or intercalated with, spermatozoal membranes. Thus, glycosaminoglycans present in the female reproductive tract may contribute to sperm capacitation and enhance the likelihood of successful fertilization in mammals.

  16. Chemically modified, non-anticoagulant heparin derivatives are potent galectin-3 binding inhibitors and inhibit circulating galectin-3-promoted metastasis. (United States)

    Duckworth, Carrie A; Guimond, Scott E; Sindrewicz, Paulina; Hughes, Ashley J; French, Neil S; Lian, Lu-Yun; Yates, Edwin A; Pritchard, D Mark; Rhodes, Jonathan M; Turnbull, Jeremy E; Yu, Lu-Gang


    Concentrations of circulating galectin-3, a metastasis promoter, are greatly increased in cancer patients. Here we show that 2- or 6-de-O-sulfated, N-acetylated heparin derivatives are galectin-3 binding inhibitors. These chemically modified heparin derivatives inhibited galectin-3-ligand binding and abolished galectin-3-mediated cancer cell-endothelial adhesion and angiogenesis. Unlike standard heparin, these modified heparin derivatives and their ultra-low molecular weight sub-fractions had neither anticoagulant activity nor effects on E-, L- or P-selectin binding to their ligands nor detectable cytotoxicity. Intravenous injection of such heparin derivatives (with cancer cells pre-treated with galectin-3 followed by 3 subcutaneous injections of the derivatives) abolished the circulating galectin-3-mediated increase in lung metastasis of human melanoma and colon cancer cells in nude mice. Structural analysis using nuclear magnetic resonance and synchrotron radiation circular dichroism spectroscopies showed that the modified heparin derivatives bind to the galectin-3 carbohydrate-recognition domain. Thus, these chemically modified, non-anticoagulant, low-sulfated heparin derivatives are potent galectin-3 binding inhibitors with substantial potential as anti-metastasis/cancer drugs.

  17. Homozygous antithrombin deficiency type II causing neonatal thrombosis. (United States)

    Swoboda, Vanessa; Zervan, Katharina; Thom, Katharina; Mannhalter, Christine; Quehenberger, Peter; Pabinger, Ingrid; Male, Christoph


    We report four children from different families with homozygous antithrombin (AT) deficiency type II affecting the heparin binding site (p.Leu131Phe mutation). All children had severe spontaneous venous and/or arterial thromboembolic events shortly after birth. This report intends to raise awareness among clinicians about this rare but severe condition. When thrombosis occurs in an otherwise healthy newborn, a severe congenital thrombophilic disorder should be considered. In homozygous AT deficiency type II, AT activity is typically reduced but may also be in the normal range, posing a diagnostic challenge. Rapid diagnosis is important to initiate appropriate therapy. Standard anticoagulation with heparin may prove ineffective in severe AT deficiency, requiring substitution of AT concentrate and early switch to alternative anticoagulants such as vitamin K antagonists. Copyright © 2017. Published by Elsevier Ltd.

  18. Anti-PF4/heparin antibodies are associated with arteriovenous fistula thrombosis in non-diabetic hemodialysis patients. (United States)

    Tsai, Yueh-Feng; Chen, Chien-An; Kuo, Chieh; Lin, Kao-Chang


    Anti-platelet factor 4/heparin complex antibodies (anti-PF4/heparin Ab) have been found to cause heparin-induced thrombocytopenia (HIT), a clinical syndrome thrombocytopenia and thrombosis. There is still controversy as to whether the presence of anti-PF4/heparin antibodies in hemodialysis patients augments clot formation in access fistula thrombosis, peripheral artery disease (PAD), and coronary heart disease (CHD). We enrolled 111 non-diabetic hemodialysis patients without liver cirrhosis and without an ankle-brachial index (ABI) ≥1.3 (arterial calcification). ABI measurements were performed and patients with an ABI ≤0.9 were defined as having PAD and included in the PAD group. ELISA was used for determination of anti-PF4/heparin Ab. Correlation factors include PAD, native arteriovenous fistula (AVF) thrombosis, platelet count, and CHD. Thirty-seven of 111 patients (33.3%) presented with anti-PF4/heparin Ab. Thirty-eight of 111 patients (34%) had PAD; fourteen of these patients (36.8%) and 23/73 of patients without PAD (31.5%) were anti-PF4/heparin Ab-positive (P = 0.57). Fifty-two of 111 patients (46.8%) had AVF thrombosis; twenty-three of these patients (44.2%) and 14/59 of patients without AVF thrombosis (23.7%) were anti-PF4/heparin Ab-positive (P = 0.02). The odds ratio for AVF thrombosis was 2.55 (95% CI 1.14-5.71) for anti-PF4/heparin Ab-positive patients. Thirty-two of 111 patients (28.8%) had thrombocytopenia (platelet count hemodialysis patients.

  19. Inhaled unfractionated heparin improves abnormalities of alveolar coagulation, fibrinolysis and inflammation in endotoxemia-induced lung injury rats. (United States)

    Wang, Zong-Yu; Wu, Sheng-Nan; Zhu, Zhao-Zhong; Yang, Ba-Xian; Zhu, Xi


    Acute lung injury/acute respiratory distress syndrome presents with not only local inflammation, but also pulmonary coagulopathy which is characterized by an alveolar procoagulant response, anticoagulant inhibition, fibrinolytic supression and fibrin deposition. We thus had hypothesized that if aerosolized unfractionated heparin was inhaled into alveolar spaces, it could block the procoagulant tendency, lessen depletion of coagulation factors, and even influence the inflammatory response. We also assessed the effects of different administration regimens of heparin. Male Wistar rats were given inhaled heparin starting 30 minutes before (prophylactic heparin) or 2 hours after (therapeutic heparin) intravenous lipopolysaccharide (LPS) was administered at 6-hour intervals; control groups received inhaled normal saline with or without being exposed to LPS. Thrombin-antithrombin complexes, activated protein C, tissue type and urokinase type plasminogen activators (t-PA/u-PA), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α, interleukin-6 in bronchoalveolar lavage, and lung tissue myeloperoxidase activity, and histology score were measured at three time-points. PAI-1/(t-PA + u-PA) was calculated based on the before-mentioned parameters. Statistical analysis was made using one-way analysis of variance (ANOVA) with post hoc test or Student's t test in the case of heterogeneity of variance. An alveolar procoagulant reaction, depressed fibrinolysis, and inflammatory response occurred in endotoxemia-induced lung injury. Local prophylactic application of heparin attenuated coagulation and early inflammation, promoted fibrinolysis, and reduced the histology score. Therapeutic application of heparin had similar, but weaker effects. Intrapulmonary application of unfractionated heparin by inhalation might inhibit alveolar procoagulant reaction and the early inflammatory response, promote fibrinolysis, and alleviate pulmonary pathology in endotoxemia-induced lung

  20. The effect of electrostatic heparin/collagen layer-by-layer coating degradation on the biocompatibility (United States)

    Chen, Jialong; Huang, Nan; Li, Quanli; Chu, Chun H.; Li, Jun; Maitz, Manfred F.


    Electrostatic layer-by-layer coatings of heparin and collagen have been suggested before to improve the biocompatibility of blood-contacting devices. However, to our knowledge, there have been no systematic studies about the effect of degradation of this coating on its biocompatibility, anticoagulant properties and the cyto-compatibility. The purpose of this study was to design an in vitro experiment in this regard that can assess the degradation behavior and the biocompatibility change of the coating. The coating degradation in physiological saline (PS) under static and dynamic condition was monitored by DR-FTIR, SEM, AFM and water contact angle, moreover, heparin densities on the topmost surface and the release heparin every day were measured by toluidine blue O (TBO) assay. The results showed that the degradation rate of the coating in is much faster under flow and shear conditions than during static incubation, and only very limited collagen and heparin remain on the surface after 15 days incubation in dynamic condition. With the degradation, the hemocompatibility of the coating got worse, especially when incubated under dynamic conditions. The degradation products of the coating do not lead to coagulation but behave -as heparin- anticoagulant. The compatibility of the coating to endothelial cells improved within 15d incubation in static medium, but it for degradation under dynamic conditions, it improved for 5d but at 15d incubation, it was almost as low as for the bare substrate. These results highlight the necessity for appropriate testing of newly developed coatings not only in the initial state but also after extended exposure to a physiological ambient.

  1. The role of VLA-4 binding for experimental melanoma metastasis and its inhibition by heparin. (United States)

    Schlesinger, Martin; Roblek, Marko; Ortmann, Katrin; Naggi, Annamaria; Torri, Giangiacomo; Borsig, Lubor; Bendas, Gerd


    Heparin is known to efficiently attenuate metastasis in various tumour models by different mechanisms including inhibition of tumour cell contacts with soluble and cellular components such as inhibition of heparanase or P- and L-selectin. We recently showed that heparin efficiently binds to VLA-4 integrin in melanoma cells in vitro. Here we describe VLA-4 integrin as a mediator of melanoma metastasis that is inhibited by the low molecular weight heparin (LMWH) Tinzaparin. sh-RNA-mediated knock-down of VLA-4 integrin in B16F10 murine melanoma cells (B16F10-VLA-4kd) was performed and cell binding characteristics were investigated in vitro. Experimental metastasis of B16F10-VLA-4kd and B16F10 cells and interference by Tinzaparin were analysed in mice. VLA-4 knock-down of B16F10 cells resulted in loss of VCAM-1 binding, but preserved the capacity to bind platelets through P-selectin. The observed reduced metastasis of B16F10-VLA-4kd cells confirmed the role of VLA-4 in this process. However, loss of melanoma VLA-4 function hardly further affected reduction of metastasis in P-selectin deficient mice. Tinzaparin treatment of mice injected with B16F10 and B16F10-VLA-4kd cells significantly reduced metastasis suggesting its potential to block both P- and L-selectin and VLA-4 in vivo. The use of N-acetylated heparin, which has no VLA-4 binding activity but blocks P- and L-selectin was less efficient than Tinzaparin in mice injected with B16F10 cells and B16F10-VLA-4kd cells. These findings provide evidence that heparin inhibits experimental melanoma metastasis primarily by blocking VLA-4 and P-selectin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. P-selectin, carcinoma metastasis and heparin: novel mechanistic connections with therapeutic implications

    Directory of Open Access Journals (Sweden)

    Varki A.


    Full Text Available Metastasis is a multistep cascade initiated when malignant cells penetrate the tissue surrounding the primary tumor and enter the bloodstream. Classic studies indicated that blood platelets form complexes around tumor cells in the circulation and facilitate metastases. In other work, the anticoagulant drug heparin diminished metastasis in murine models, as well is in preliminary human studies. However, attempts to follow up the latter observation using vitamin K antagonists failed, indicating that the primary mechanism of heparin action was unrelated to its anticoagulant properties. Other studies showed that the overexpression of sialylated fucosylated glycans in human carcinomas is associated with a poor prognosis. We have now brought all these observations together into one mechanistic explanation, which has therapeutic implications. Carcinoma cells expressing sialylated fucosylated mucins can interact with platelets, leukocytes and endothelium via the selectin family of cell adhesion molecules. The initial organ colonization of intravenously injected carcinoma cells is attenuated in P-selectin-deficient mice, in mice receiving tumor cells pretreated with O-sialoglycoprotease (to selectively remove mucins from cell surfaces, or in mice receiving a single dose of heparin prior to tumor cell injection. In each case, we found that formation of a platelet coating on cancer cells was impeded, allowing increased access of leukocytes to the tumor cells. Several weeks later, all animals showed a decrease in the extent of established metastasis, indicating a long-lasting effect of the short-term intervention. The absence of obvious synergism amongst the three treatments suggests that they all act via a common pathway. Thus, a major mechanism of heparin action in cancer may be inhibition of P-selectin-mediated platelet coating of tumor cells during the initial phase of the metastatic process. We therefore suggest that heparin use in cancer be re

  3. Acidosis induced by carbon dioxide insufflation decreases heparin potency: a risk factor for thrombus formation. (United States)

    Gorter, Karin A M; Stehouwer, Marco C; Van Putte, Bart P; Vlot, Eline A; Urbanus, Rolf T


    Since the introduction of CO2 insufflation during open heart surgery in our hospital, we incidentally observed thrombus formation in the dissected heart, in the pericardium and in the cardiotomy reservoir of the cardiopulmonary bypass system. Furthermore, we measured very high levels of pCO2, causing severe acidosis, in stagnant blood in the pericardium and cardiotomy reservoir. In this in vitro study, we assessed the influence of acidosis and hypothermia on heparin potency and thrombin formation. We assessed heparin potency in function of pH (pH 5.0-7.4) and temperature (24-37°C) by comparing the activated partial thromboplastin time in platelet-poor plasma between samples with and without unfractionated heparin. We measured thrombin formation in platelet-poor plasma by means of fluorescent, calibrated, automated thrombography in function of pH (pH 5.0-7.4) and temperature (24-37°C). The parameters of interest were the endogenous thrombin potential and the peak amount of thrombin generation. The major finding of this study is the significant decrease in the efficiency of unfractionated heparin in delaying thrombus formation at acidotic (pH 5.0-7.0) conditions (p=0.034-0.05). Furthermore, we found that thrombin formation is significantly increased at hypothermic (24-34°C) conditions (p=acidosis may lead to a decreased heparin potency. Acidosis, as induced by CO2 insufflation, may predispose patients to incidental thrombus formation in stagnant blood in the open thorax and in the cardiotomy reservoir. Hypothermia might further increase this risk. Therefore, we recommend reconsidering the potential advantages and disadvantages of using CO2 insufflation during cardiopulmonary bypass.

  4. Prevention of venous thromboembolism and safe use of heparin in Spanish hospitals. (United States)

    Saturno, Pedro J; Gama, Zenewton A S; Fonseca, Yadira A


    To assess compliance with basic and actionable indicators in relation to prevention of venous thromboembolism (VTE) and safe use of heparin. We built, pilot tested and measured a set of evidence-based structure (existence of guidelines) and process (risk assessment for VTE, and dose adjustment to patient weight and renal function when prescribing heparin) indicators in a nation-wide random sample of 22 hospitals. Compliance with process indicators is estimated at national level and by groups of hospitals (stratified by size). At hospital level, compliance is assessed with Lot Quality Acceptance Sampling, for 85% compliance standard (α ≤ 0.05), 55% threshold (β ≤ 0.10). Contents of existing guidelines are analyzed, and their influence on performance is assessed using logistic regression. Acute care hospitals in Spain. None Problem identification through indicators assessment. Less than half of hospitals have guidelines and their contents are very variable and incomplete. No hospital complies with the standard for VTE prevention and only one for heparin dose adjustment. Nationally, VTE risk assessment is performed in 5.8% of patients (95% CI: 5.6-6.0), and heparin dose is explicitly adjusted in 17.5% (95% CI: 16.8-18.2). Performance is relatively higher in large hospitals and it is associated with the existence of guidelines for VTE prevention (OR: 8.3; 95% CI: 2.1-32.1). We have identified some actionable contributing factors to safety problems using evidence-based structure and process indicators. Explicit process design and key clinical interventions (risk assessment for VTE and heparin dose adjustment) should be addressed to improve the current situation.

  5. Substrate- and Cofactor-independent Inhibition of Histone Demethylase KDM4C

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Lohse, Brian; Rand, Kasper Dyrberg


    Inhibition of histone demethylases has within recent years advanced into a new strategy for treating cancer and other diseases. Targeting specific histone demethylases can be challenging as the active sites of KDM1A-B and KDM-4A-D histone demethylases, respectively, are highly conserved. Most...... inhibitors developed up-to-date target either the cofactor- or substrate-binding sites of these enzymes, resulting in a lack of selectivity and off-target effects. This study describes the discovery of the first peptide-based inhibitors of KDM4 histone demethylases that do not share the histone peptide...... sequence, or inhibit through substrate competition. Through screening of DNA-encoded peptide libraries against KDM1 and -4 histone demethylases by phage display, two cyclic peptides targeting the histone demethylase KDM4C were identified and developed as inhibitors by amino acid replacement, truncation...

  6. Co-factors necessary for PPAR mediated transactivation of endogenous target genes

    DEFF Research Database (Denmark)

    Grøntved, Lars; Nielsen, Ronni; Stunnenberg, Henk

    physiological scenarios. PPARa and PPARd are transcriptional regulators of fatty acid oxidation and ketogenesis, whereas PPAR? controls genes involved in lipid storage. Consequently, there must be PPAR subtype specific molecular determinants that secure PPAR selective recognition and activation of target...... promoters in a given cell type. In vitro experiments suggest that the different PPAR subtypes might have dissimilar binding preference for some PPAR target sites and may also have different affinity for some transcriptional co-factors. However the molecular mechanisms behind PPAR subtype specific activation...... of endogenous target gene in different cell types are elusive. To mutually compare the ability of the PPAR subtypes to activate endogenous target genes in a given cell, PPARa, PPARb/d and PPARg2 were HA tagged and rapidly, equally and synchronously expressed using adenoviral delivery. Within a few hours after...

  7. Optimizing Cofactor Specificity of Oxidoreductase Enzymes for the Generation of Microbial Production Strains—OptSwap

    DEFF Research Database (Denmark)

    King, Zachary A.; Feist, Adam


    Central oxidoreductase enzymes (eg, dehydrogenases, reductases) in microbial metabolism often have preferential binding specificity for one of the two major currency metabolites NAD(H) and NADP(H). These enzyme specificities result in a division of the metabolic functionality of the currency...... metabolites: enzymes reducing NAD+ to NADH drive oxidative phosphorylation, and enzymes reducing NADP+ to NADPH drive anabolic reactions. In this work, we introduce the computational method OptSwap, which predicts bioprocessing strain designs by identifying optimal modifications of the cofactor binding...... specificities of oxidoreductase enzyme and complementary reaction knockouts. Using the Escherichia coli genome-scale metabolic model iJO1366, OptSwap predicted eight growth-coupled production designs with significantly greater product yields or substrate-specific productivities than designs predicted with gene...

  8. Regulatory Enhancer-Core-Promoter Communication via Transcription Factors and Cofactors. (United States)

    Zabidi, Muhammad A; Stark, Alexander


    Gene expression is regulated by genomic enhancers that recruit transcription factors and cofactors to activate transcription from target core promoters. Over the past years, thousands of enhancers and core promoters in animal genomes have been annotated, and we have learned much about the domain structure in which regulatory genomes are organized in animals. Enhancer-core-promoter targeting occurs at several levels, including regulatory domains, DNA accessibility, and sequence-encoded core-promoter specificities that are likely mediated by different regulatory proteins. We review here current knowledge about enhancer-core-promoter targeting, regulatory communication between enhancers and core promoters, and the protein factors involved. We conclude with an outlook on open questions that we find particularly interesting and that will likely lead to additional insights in the upcoming years. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Income poverty, poverty co-factors, and the adjustment of children in elementary school. (United States)

    Ackerman, Brian P; Brown, Eleanor D


    Since 1990, there have been great advances in how developmental researchers construct poverty. These advances are important because they may help inform social policy at many levels and help frame how American culture constructs poverty for children, both symbolically and in the opportunities children and families get to escape from poverty. Historically, developmental perspectives have embodied social address and main effects models, snapshot views of poverty effects at single points in time, and a rather narrow focus on income as the symbolic marker of the ecology of disadvantage. More recent views, in contrast, emphasize the diverse circumstances of disadvantaged families and diverse outcomes of disadvantaged children, the multiple sources of risk and the multiple determinants of poor outcomes for these children, dynamic aspects of that ecology, and change as well as continuity in outcome trajectories. The advances also consist of more powerful frames for understanding the ecology of disadvantage and the risk it poses for child outcomes. Most developmental researchers still tend to frame causal variables ultimately in terms of the dichotomy between social causation and social selection views, with a primary emphasis on the former. In part, this framing has reflected limitations of sample size and design, because the theoretical and empirical power of reciprocal selection models is clear (Kim et al., 2003). The conceptual advances that prompt such models include widespread acknowledgement of third variable problems in interpreting effects, of the clear need for multivariate approaches, and the need to pursue mechanisms and moderators of the relations between causal candidates and child outcomes. In the context of these advances, one of the core goals of our research program has been to construct robust representations of environmental adversity for disadvantaged families. Most of our research focuses on contextual co-factors at a family level (e.g., maternal

  10. CD63 is an essential cofactor to leukocyte recruitment by endothelial P-selectin. (United States)

    Doyle, Emily L; Ridger, Victoria; Ferraro, Francesco; Turmaine, Mark; Saftig, Paul; Cutler, Daniel F


    The activation of endothelial cells is critical to initiating an inflammatory response. Activation induces the fusion of Weibel-Palade Bodies (WPB) with the plasma membrane, thus transferring P-selectin and VWF to the cell surface, where they act in the recruitment of leukocytes and platelets, respectively. CD63 has long been an established component of WPB, but the functional significance of its presence within an organelle that acts in inflammation and hemostasis was unknown. We find that ablating CD63 expression leads to a loss of P-selectin-dependent function: CD63-deficient HUVECs fail to recruit leukocytes, CD63-deficient mice exhibit a significant reduction in both leukocyte rolling and recruitment and we show a failure of leukocyte extravasation in a peritonitis model. Loss of CD63 has a similar phenotype to loss of P-selectin itself, thus CD63 is an essential cofactor to P-selectin.

  11. Characterization of water-forming NADH oxidases for co-factor regeneration

    DEFF Research Database (Denmark)

    Rehn, Gustav; Pedersen, Asbjørn Toftgaard; J. Charnock, Simon

    an environmentaland economic perspective [1]. Alcohol dehydrogenases (ADH) offer one such alternative. However, the reaction requires the oxidized nicotinamide co-factor (NAD+) that must be recycled due to its high cost contribution. One regeneration method that offers certain advantages is the oxidation of NADH......Traditional chemical methods for alcohol oxidation are often associated with issues such as high consumption of expensive oxidizing agents, generation of metal waste and the use of environmentally undesirable organic solvents. Developing green, selective catalysts is therefore important from...... using water forming NADH oxidases (NOX-2). The implementation of the ADH/NOX system for alcohol oxidation, however, requires consideration of several different issues. Enzyme activity and stability at relevant pH and temperature conditions, but also the tolerance to the substrates and products present...

  12. Constrained spin-density dynamics of an iron-sulfur complex: ferredoxin cofactor. (United States)

    Ali, Md Ehesan; Nair, Nisanth N; Staemmler, Volker; Marx, Dominik


    The computation of antiferromagnetic exchange coupling constants J by means of efficient density-based approaches requires in practice to take care of both spin projection to approximate the low spin ground state and proper localization of the magnetic orbitals at the transition metal centers. This is demonstrated here by a combined approach where the extended broken-symmetry (EBS) technique is employed to include the former aspect, while spin density constraints are applied to ensure the latter. This constrained EBS (CEBS) approach allows us to carry out ab initio molecular dynamics on a spin-projected low spin potential energy surface that is generated on-the-fly by propagating two coupled determinants and thereby accessing the antiferromagnetic coupling along the trajectory. When applied to the prototypical model of the oxidized [2Fe-2S] cofactor in Ferredoxins, [Fe(2)S(2)(SH)(4)](2-), at room temperature, CEBS leads to remarkably good results for geometrical structures and coupling constants J.

  13. Esmond E. Snell--the pathfinder of B vitamins and cofactors. (United States)

    Hayashi, Hideyuki; Tanase, Sumio; Yagi, Toshiharu


    Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

  14. Structure and stability of an azoreductase with an FAD cofactor from the strict anaerobe Clostridium perfringens. (United States)

    Morrison, Jessica; Dai, Shuo; Ren, Jie; Taylor, Amanda; Wilkerson, Mitchell; John, Gilbert; Xie, Aihua


    Azoreductase enzymes present in many microorganisms exhibit the ability to reduce azo dyes, an abundant industrial pollutant, to produce carcinogenic metabolites that threaten human health. All biochemically-characterized azoreductases, around 30 to date, have been isolated from aerobic bacteria, except for AzoC, the azoreductase of Clostridium perfringens, which is from a strictly anaerobic bacterium. AzoC is a recently biochemically-characterized azoreductase. The lack of structural information on AzoC hinders the mechanistic understanding of this enzyme. In this paper, we report on the biophysical characterization of the structure and thermal stability of AzoC by using a wide range of biophysical tools: Liquid Chromatography-Mass Spectrometry (LC-MS), Circular Dichroism Spectroscopy, Fourier-transform Infrared (FTIR) Spectroscopy, SDS-PAGE, Size Exclusion Chromatography, MALDI-TOF and UV-visible spectroscopy. We found that the flavin cofactor of AzoC is FAD, while all other structurally-known azoreductases employ FMN as a cofactor. The secondary structure of AzoC has 16% less α-helix structures, 5% more β-sheet structures and 11% more turn and unordered than the average of structurally-known azoreductase that have 10-14% sequence similarities with AzoC. We also found that oxidized AzoC is trimeric, which is unique amongst structurally known azoreductases. In contrast, reduced AzoC is monomeric, despite similarities in catalytic activity and thermal stability of oxidized and reduced AzoC. Our results show that the use of FTIR spectroscopy is crucial for characterization of the β-sheet content in AzoC, illustrating the need for complementary biophysical tools for secondary structural characterization of proteins.

  15. Molybdenum cofactor and isolated sulphite oxidase deficiencies: Clinical and molecular spectrum among Egyptian patients. (United States)

    Zaki, Maha S; Selim, Laila; El-Bassyouni, Hala T; Issa, Mahmoud Y; Mahmoud, Iman; Ismail, Samira; Girgis, Mariane; Sadek, Abdelrahim A; Gleeson, Joseph G; Abdel Hamid, Mohamed S


    Molybdenum cofactor deficiency (MoCD) and Sulfite oxidase deficiency (SOD) are rare autosomal recessive conditions of sulfur-containing amino acid metabolism with overlapping clinical features and emerging therapies. The clinical phenotype is indistinguishable and they can only be differentiated biochemically. MOCS1, MOCS2, MOCS3, and GPRN genes contribute to the synthesis of molybdenum cofactor, and SUOX gene encodes sulfite oxidase. The aim of this study was to elucidate the clinical, radiological, biochemical and molecular findings in patients with SOD and MoCD. Detailed clinical and radiological assessment of 9 cases referred for neonatal encephalopathy with hypotonia, microcephaly, and epilepsy led to a consideration of disorders of sulfur-containing amino acid metabolism. The diagnosis of six with MoCD and three with SOD was confirmed by biochemical tests, targeted sequencing, and whole exome sequencing where suspicion of disease was lower. Novel SUOX mutations were detected in 3 SOD cases and a novel MOCS2 mutation in 1 MoCD case. Most patients presented in the first 3 months of life with intractable tonic-clonic seizures, axial hypotonia, limb hypertonia, exaggerated startle response, feeding difficulties, and progressive cystic encephalomalacia on brain imaging. A single patient with MoCD had hypertrophic cardiomyopathy, hitherto unreported with these diseases. Our results emphasize that intractable neonatal seizures, spasticity, and feeding difficulties can be important early signs for these disorders. Progressive microcephaly, intellectual disability and specific brain imaging findings in the first year were additional diagnostic aids. These clinical cues can be used to minimize delays in diagnosis, especially since promising treatments are emerging for MoCD type A. Copyright © 2016 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  16. Increased isobutanol production in Saccharomyces cerevisiae by eliminating competing pathways and resolving cofactor imbalance. (United States)

    Matsuda, Fumio; Ishii, Jun; Kondo, Takashi; Ida, Kengo; Tezuka, Hironori; Kondo, Akihiko


    Isobutanol is an important target for biorefinery research as a next-generation biofuel and a building block for commodity chemical production. Metabolically engineered microbial strains to produce isobutanol have been successfully developed by introducing the Ehrlich pathway into bacterial hosts. Isobutanol-producing baker's yeast (Saccharomyces cerevisiae) strains have been developed following the strategy with respect to its advantageous characteristics for cost-effective isobutanol production. However, the isobutanol yields and titers attained by the developed strains need to be further improved through engineering of S. cerevisiae metabolism. Two strategies including eliminating competing pathways and resolving the cofactor imbalance were applied to improve isobutanol production in S. cerevisiae. Isobutanol production levels were increased in strains lacking genes encoding members of the pyruvate dehydrogenase complex such as LPD1, indicating that the pyruvate supply for isobutanol biosynthesis is competing with acetyl-CoA biosynthesis in mitochondria. Isobutanol production was increased by overexpression of enzymes responsible for transhydrogenase-like shunts such as pyruvate carboxylase, malate dehydrogenase, and malic enzyme. The integration of a single gene deletion lpd1Δ and the activation of the transhydrogenase-like shunt further increased isobutanol levels. In a batch fermentation test at the 50-mL scale from 100 g/L glucose using the two integrated strains, the isobutanol titer reached 1.62 ± 0.11 g/L and 1.61 ± 0.03 g/L at 24 h after the start of fermentation, which corresponds to the yield at 0.016 ± 0.001 g/g glucose consumed and 0.016 ± 0.0003 g/g glucose consumed, respectively. These results demonstrate that downregulation of competing pathways and metabolic functions for resolving the cofactor imbalance are promising strategies to construct S. cerevisiae strains that effectively produce isobutanol.

  17. Dissecting Torsin/cofactor function at the nuclear envelope: a genetic study. (United States)

    Laudermilch, Ethan; Tsai, Pei-Ling; Graham, Morven; Turner, Elizabeth; Zhao, Chenguang; Schlieker, Christian


    The human genome encodes four Torsin ATPases, the functions of which are poorly understood. In this study, we use CRISPR/Cas9 engineering to delete all four Torsin ATPases individually and in combination. Using nuclear envelope (NE) blebbing as a phenotypic measure, we establish a direct correlation between the number of inactivated Torsin alleles and the occurrence of omega-shaped herniations within the lumen of the NE. A similar, although not identical, redundancy is observed for LAP1 and LULL1, which serve as regulatory cofactors for a subset of Torsin ATPases. Unexpectedly, deletion of Tor2A in a TorA/B/3A-deficient background results in a stark increase of bleb formation, even though Tor2A does not respond to LAP1/LULL1 stimulation. The robustness of the observed phenotype in Torsin-deficient cells enables a structural analysis via electron microscopy tomography and a compositional analysis via immunogold labeling. Ubiquitin and nucleoporins were identified as distinctively localizing components of the omega-shaped bleb structure. These findings suggest a functional link between the Torsin/cofactor system and NE/nuclear pore complex biogenesis or homeostasis and establish a Torsin-deficient cell line as a valuable experimental platform with which to decipher Torsin function. © 2016 Laudermilch et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (

  18. Clinical effects of low-molecular-weight heparin combined with ...

    African Journals Online (AJOL)

    The treatment lasted for 2 weeks. Clinical parameters, laboratory test indices, Acute Physiology and Chronic Health Evaluation (APACHE II) score, and computed tomography score of pancreatic necrosis (CTSPN) were assessed in both groups. Results: On admission, no significant differences were noted in clinical features, ...

  19. The History of Antithrombotic Therapy: The Discovery of Heparin, the Vitamin K Antagonists, and the Utility of Aspirin. (United States)

    Handin, Robert I


    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.

  20. Effect of heparin and related glycosaminoglycan on PDGF-induced lung fibroblast proliferation, chemotactic response and matrix metalloproteinases activity

    Directory of Open Access Journals (Sweden)

    Masahiro Sasaki


    Full Text Available Fibroblast migration, proliferation, extacellular matrix protein synthesis and degradation are the key events in various biological and pathological processes in pulmonary fibrosis. In addition, biopsy specimens from the lungs of patients with plumomary fibrosis show increased numbers of mast cells which have metachromatic granules containing heparin, histamin and proteases. Little is known about how these products influence pulmonary fibrosis. In the present study, we investigated the effect of heparin and related glycosaminoglycans on PDGF-induced lung fibroblast proliferation and chemotactic response in vitro. In addition, we examined the effect of heparin on both the induction of matorix metalloproteinases (MMPs and MMPs activity in lung fibroblasts in vitro.

  1. Distribution of metallothionein I + II and vesicular zinc in the developing central nervous system: correlative study in the rat

    DEFF Research Database (Denmark)

    Penkowa, M; Nielsen, H; Hidalgo, J


    Because zinc (Zn) is a co-factor in enzymes and participates in neurotransmission, it is essential for brain development. However, because excess Zn may cause neuronal injury, cerebral mechanisms for Zn regulation must operate. The metallothionein isoforms I and II (MT I + II) are putative candid...

  2. Comparison of anti-thrombotic strategies using Bivalirudin, Heparin plus Glycoprotein IIb/IIIa inhibitors and Unfractionated Heparin Monotherapy for patients undergoing percutaneous coronary intervention - A single centre observational study. (United States)

    Kaul, Upendra; Dua, Ajay; Sethi, Arvind K; Arambam, Priyadarshini; Seth, Ashok


    The study was planned to compare Anti-thrombotic strategies for patients undergoing PCI in a real world population with an emphasis on occurrence of major bleeding, composite ischemic end points and economic outcomes. The present study is a single center, prospective, observational study in consecutive patients undergoing PCI at Fortis Escorts Heart Institute (FEHI) and describes Authors' experience with three different Anti-Thrombotic Strategies in a real world population. Patients were consecutively enrolled in the study and the choice of Anti-thrombotic strategy was left to individual operator(s) based on their own clinical judgment and patient's affordability. No specific inclusion/exclusion criteria were specified on the choice of Anti-Thrombotic Strategy. A total 1453 patients were consecutively enrolled into the study and were followed telephonically after 30 days. 252 patients were treated with Bivalirudin (Angiomax) during PCI (17.3%), 430 (29.6%) patients were treated with Heparin plus GPI & remaining 771 (53.1%) were treated with Heparin monotherapy. Incidence of major bleeding was lowest in patients treated with Bivalirudin (1.59%) when compared to Heparin plus GPI (3.49%) and Heparin monotherapy (5.97%), p = 0.005 Bivalirudin vs. Heparin Monotherapy, and p = 0.145, Bivalirudin vs. Heparin + GPI. No bleeding was observed in STEMI patients treated with Bivalirudin compared to 7.4% in patients treated with GPI and 14.3% in patients treated with UFH. Similarly non-access site bleeding was lowest in patients treated with Bivalirudin. Only 4 patients (1.6%) treated with Bivalirudin required Blood transfusion compared to 25 in Heparin plus GPI (5.8%) and 38 (5%) in Heparin Monotherapy arm. In Composite Ischemic end-points, no "All-cause Mortality" was observed in Bivalirudin group compared to 2.8% in Heparin plus GPI. Early stent thrombosis was seen in 1 patient with Heparin plus GPI and none with Heparin monotherapy and Bivalirudin group. None of the

  3. Separation of xylose and glucose using an integrated membrane system for enzymatic cofactor regeneration and downstream purification

    DEFF Research Database (Denmark)

    Morthensen, Sofie Thage; Sigurdardóttir, Sigyn Björk; Meyer, Anne S.


    Mixtures of xylose, glucose and pyruvate were fed to a membrane bioreactor equipped with a charged NF membrane (NTR 7450). Value-added products were obtained in the reactor via enzymatic cofactor-dependent catalysis of glucose to gluconic acid and pyruvate to lactic acid, respectively. The initial...


    Directory of Open Access Journals (Sweden)

    Barbara Petschacher


    Full Text Available Soluble water-forming NAD(PH oxidases constitute a promising NAD(P+ regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(PH oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 93R94H showed comparable high rates and low Km values for NADPH (kcat 20 s−1, Km 6 μM and NADH (kcat 25 s−1, Km 9 μM with retention of 70 % of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2-heptanone production.

  5. Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation, Kinetics and Evolutionary Studies. (United States)

    Fuentealba, Matias; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana; Cabrera, Ricardo


    Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2

  6. Comparison of anti-thrombotic strategies using Bivalirudin, Heparin plus Glycoprotein IIb/IIIa inhibitors and Unfractionated Heparin Monotherapy for patients undergoing percutaneous coronary intervention – A single centre observational study

    Directory of Open Access Journals (Sweden)

    Upendra Kaul


    Conclusion: Bivalirudin use during PCI is associated with a distinct advantage of having lower access site and non-access site bleeding without compromising on the efficacy. We observed a reduction in blood transfusions, hospital stay and mortality for patients treated with Bivalirudin compared with Heparin plus GPI or Heparin Monotherapy. Bivalirudin can be safely adopted into our Institutional protocol for the treatment of high risk PCI such as STEMI, ACS, and Complex elective PCI.

  7. Isolation of vascular smooth muscle cell cultures with altered responsiveness to the antiproliferative effect of heparin. (United States)

    Caleb, B L; Hardenbrook, M; Cherington, V; Castellot, J J


    Smooth muscle cell (SMC) hyperplasia in the arterial wall is an important component of both atherogenesis and post-vascular surgical restenosis. One naturally-occurring group of molecules which can suppress SMC proliferation in animal models and in cell culture systems are the complex carbohydrates of the heparan sulfate class, including heparin. In this communication, we have used retrovirus vectors to introduce several oncogenes into SMC: SV40 Large T antigen (SVLT), polyoma virus Large T antigen (PyLT), v-myc, and adenovirus E1a. We analyzed a total of 11 cultures. A combination of Western blot analysis, immunoprecipitation, and indirect immunofluorescence confirmed the expression of the infected oncogenic protein in each culture we isolated. All four oncogenes permitted the maintenance of a normal SMC phenotype, as assessed by the general morphology of cells in the light microscope and the presence of SMC-specific alpha-actin in an immunofluorescence assay. Doubling times in infected cells ranged from 20 to 33 hr, and final cell densities in infected cultures ranged from 4 x 10(4) to 5 x 10(5) cells per cm2. By comparison, the parent line had a doubling time of 30 hr and reached a final cell density of 1 x 10(5) cells per cm2. Despite the differences sometimes observed in these proliferation parameters, neither one was strongly correlated with heparin responsiveness. PyLT, v-myc, and E1a all produced SMC cultures or lines which retained sensitivity to the antiproliferative activity of heparin (ED50 = 50 micrograms/ml). In contrast, SVLT expression yielded SMC lines which were highly resistant to heparin (ED50 > 300 micrograms/ml). These results suggest that altered responsiveness to heparin is dependent upon which oncogenic protein is being expressed in the cells. The availability of cloned, immortal SMC lines with a wide range of heparin responsiveness should aid in the understanding of the cellular and molecular mechanism of action of this potentially

  8. Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis. (United States)

    Chen, Cynthia; Lodish, Harvey F


    Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis. Copyright © 2014 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  9. Mapping the heparin-binding site of the osteoinductive protein NELL1 by site-directed mutagenesis. (United States)

    Takahashi, Kaneyoshi; Imai, Arisa; Iijima, Masumi; Yoshimoto, Nobuo; Maturana, Andrés D; Kuroda, Shun'ichi; Niimi, Tomoaki


    Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Fondaparinux (ARIXTRA) as an alternative anti-thrombotic prophylaxis when there is hypersensitivity to low molecular weight and unfractionated heparins. (United States)

    Parody, Rocio; Oliver, Arturo; Souto, Juan Carlos; Fontcuberta, Jordi


    During the last decade, new anticoagulant drugs with anti-factor-Xa properties have been described (1, 2). Among them is fondaparinux that has been licensed recently. It is a pentasaccharide mimicking the site where heparin binds to antithrombin III (1). This new drug has produced very promising clinical results in the prophylaxis of venous thrombosis after orthopedic surgery (3). Here we report two different clinical situations in which fondaparinux has yielded a successful outcome: first, a patient with repeated cutaneus reaction to several different low molecular weight heparins (LMWH), and second, a patient with severe heparin-induced thrombocytopenia (HIT). We decided to use fondaparinux in both cases since it is commercially available in Spain and mostly because the absence of in vitro cross-reaction with heparins, as discussed later.

  11. Non-Covalent Synthesis of Metal Oxide Nanoparticle–Heparin Hybrid Systems: A New Approach to Bioactive Nanoparticles

    Directory of Open Access Journals (Sweden)

    Giorgio Eisele


    Full Text Available Heparin has been conjugated to Fe3O4, Co3O4, and NiO nanoparticles (NPs through electrostatic interactions, producing colloidal suspensions of hybrid metal oxide heparin NPs that are stable in water. Negative zeta potentials and retention of heparin’s ability to capture toluidine blue indicate that heparin’s negative charges are exposed on the surface of the coated NPs. IR results confirmed the formation of nanohybrids as did NMR experiments, which were also interpreted on the basis of toluidine blue tests. Transmission electron microscopy results revealed that the heparin coating does not modify the shape or dimension of the NPs. Dynamic light scattering and negative zeta potential measurements confirmed that heparin surface functionalisation is an effective strategy to prevent NP aggregation.

  12. [Fondaparinux as an alternative anticoagulant in heparin-induced thrombocytopenia in the patient with a ventricular assist device]. (United States)

    Cegarra-Sanmartín, V; Paniagua, P; Galán, J; Muñoz, C; Moral, M V


    Heparin-induced thrombocytopenia is a reaction associated with the use of this drug. It occurs in up to 3% of patients treated for at least 5 days. Its treatment is to stop the heparin, and according to patient needs, replace it with another anticoagulant. We present a patient who, after a heart transplant, and the need for a ventricular assist device, required anticoagulation. The patient developed heparin-induced thrombocytopenia. Heparin was stopped and anticoagulation was replaced by fondaparinux. The peri-operative complications and the management of the coagulation are described. Copyright © 2012 Sociedad Española de Anestesiología, Reanimación y Terapéutica del Dolor. Published by Elsevier España. All rights reserved.

  13. Validation of quantitative polymerase chain reaction methodology for monitoring DNA as a surrogate marker for species material contamination in porcine heparin. (United States)

    Auguste, C; Dereux, S; Rousset, M; Anger, P


    Heparin is a widely used intravenous anticoagulant comprising of a very complex mixture of glycosaminoglycan chains, mainly derived from porcine intestinal mucosa. The species of origin and the absence of contaminants from other species are important determinants of the different physicochemical characteristics of heparin. They also determine the potential for introducing infectious and adventitious agents into heparin batches destined for medicinal use. We perform routine quantitative polymerase chain reaction (Q-PCR) release tests to confirm the quality of all crude heparin batches, including those used for the manufacture of enoxaparin sodium. Here we further demonstrate that the assessment of the DNA content in crude heparin is a good surrogate marker of contamination at the mucosa level. After spiking porcine mucosa with ovine mucosa and processing this material to form crude heparin, we were able to observe similar ratios of species-specific DNA in both the starting and end products. Experiments performed with 3,000 and 1,500 ppm contamination found these concentrations to be well above the detection limit for our assay of heparin batches. Additionally this Q-PCR method can be used to detect contamination in mucosa, thus providing a tool capable of monitoring for contaminants throughout the crude heparin manufacturing process. Q-PCR analysis of industrial crude heparin samples has confirmed over time the value of this method to assess the pure porcine origin of heparin.

  14. Heparin-binding protein: an early indicator of critical illness and predictor of outcome in cardiac arrest. (United States)

    Dankiewicz, Josef; Linder, Adam; Annborn, Martin; Rundgren, Malin; Friberg, Hans


    To investigate plasma levels of the neutrophil-borne heparin-binding protein (HBP) in patients with induced hypothermia after cardiac arrest (CA), and to study any association to severity of organ failure, incidence of infection and neurological outcome. This study included 84 patients with CA of mixed origin who were treated with hypothermia. Plasma samples from 7 time points during the first 72 h after return of spontaneous circulation (ROSC) were collected and analyzed for HBP with an ELISA. Outcomes were dichotomized: a cerebral performance category scale (CPC) of 1-2 at 6 months follow-up was considered a good outcome, a CPC of 3-5, a poor outcome. Patient data, including APACHE II and SOFA-scores were retrieved from the computerized system for quality assurance for intensive care. At 6 h and 12 h after CA, plasma levels of HBP were significantly higher among patients with a poor outcome. A receiver operated characteristics (ROC)-analysis yielded respective areas under curve (AUC) values of 0.68 and 0.70. This was similar to APACHE II and SOFA-score AUC values. There was a significant correlation between early elevated HBP-values and time to ROSC. HBP-levels were not higher in patients with infections at any time. Elevated HBP is an early indicator of organ failure and poor neurological outcome after CA, independent of microbial infection, and should be further evaluated in prospective trials. The temporal profile of HBP is suggestive of a role in the pathogenesis of critical illness after CA. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. The effect of heparin on pregnancy associated plasma protein-A concentration in healthy, non-pregnant individuals. (United States)

    Jespersen, Camilla H B; Vestergaard, Kirstine R; Schou, Morten; Teisner, Børge; Iversen, Kasper


    The objective of this study was to determine the differences in pregnancy associated plasma protein-A (PAPP-A) concentrations in heparin naive and heparin treated healthy men and non-pregnant women, to find a possible difference in different age groups, and to determine the response in PAPP-A concentration to repeated injections of unfractionated heparin. Twenty-five healthy, non-pregnant volunteers divided into five groups (determined by gender and age) received 5000 IU unfractionated heparin intravenously. Five young men received an additional 5000 IU after 90 and 180 min. Blood samples to determine PAPP-A concentration and APTT were drawn at different time points. Injection of heparin elicited increase in and rapid normalization of PAPP-A concentrations in all subjects. The group of 20-30-year-old never-pregnant women had lower responses than the individuals of the four other groups. The difference was not significant (p > 0.05). Repeated injections of heparin caused additional peaks in PAPP-A concentration of about the same sizes as the first peak. We observed an increase in time to normalization of PAPP-A concentration (from 75-90 min to 90-150 min) and APTT levels with repeated injections. We observed a rapid normalization of PAPP-A. Our result has a great similarity to the half-life of unfractionated heparin. This result combined with the finding of equally sized peaks in PAPP-A concentration, and that all of this was found in healthy, non-pregnant individuals, suggests that heparin might compete for a binding-site on PAPP-A or with PAPP-A itself for a common receptor in healthy arterial vessels. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Taurolidine lock is superior to heparin lock in the prevention of catheter related bloodstream infections and occlusions.

    Directory of Open Access Journals (Sweden)

    Evelyn D Olthof

    Full Text Available BACKGROUND AND AIMS: Patients on home parenteral nutrition (HPN are at risk for catheter-related complications; mainly infections and occlusions. We have previously shown in HPN patients presenting with catheter sepsis that catheter locking with taurolidine dramatically reduced re-infections when compared with heparin. Our HPN population therefore switched from heparin to taurolidine in 2008. The aim of the present study was to compare long-term effects of this catheter lock strategy on the occurrence of catheter-related bloodstream infections and occlusions in HPN patients. METHODS: Data of catheter-related complications were retrospectively collected from 212 patients who received HPN between January 2000 and November 2011, comprising 545 and 200 catheters during catheter lock therapy with heparin and taurolidine, respectively. We evaluated catheter-related bloodstream infection and occlusion incidence rates using Poisson-normal regression analysis. Incidence rate ratios were calculated by dividing incidence rates of heparin by those of taurolidine, adjusting for underlying disease, use of anticoagulants or immune suppressives, frequency of HPN/fluid administration, composition of infusion fluids, and duration of HPN/fluid use before catheter creation. RESULTS: Bloodstream infection incidence rates were 1.1/year for heparin and 0.2/year for taurolidine locked catheters. Occlusion incidence rates were 0.2/year for heparin and 0.1/year for taurolidine locked catheters. Adjusted incidence ratios of heparin compared to taurolidine were 5.9 (95% confidence interval, 3.9-8.7 for bloodstream infections and 1.9 (95% confidence interval, 1.1-3.1 for occlusions. CONCLUSIONS: Given that no other procedural changes than the catheter lock strategy were implemented during the observation period, these data strongly suggest that taurolidine decreases catheter-related bloodstream infections and occlusions in HPN patients compared with heparin.

  17. Heparin-induced conformational changes of fibronectin within the extracellular matrix promote hMSC osteogenic differentiation. (United States)

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


    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.

  18. Nebulized Heparin With N-Acetylcysteine and Albuterol Reduces Duration of Mechanical Ventilation in Patients With Inhalation Injury. (United States)

    McGinn, Kaitlin A; Weigartz, Katie; Lintner, Alicia; Scalese, Michael J; Kahn, Steven A


    Nebulized heparin has been proposed to improve pulmonary function in patients with inhalation injuries. The purpose of this study was to evaluate the impact of nebulized heparin with N-acetylcysteine (NAC) and albuterol on the duration of mechanical ventilation in burn patients. This is a retrospective study evaluating mechanically ventilated adult patients admitted to a regional burn center with inhalation injury. Outcomes were compared between patients who were prescribed a combination of nebulized heparin with NAC and albuterol versus similar patients who did not. A total of 48 patients met inclusion criteria (heparin n = 22; nonheparin n = 26). Patients in the nonheparin group had higher percentage of total body surface area (TBSA) burned (29.00 [5.75-51.88] vs 5.25 [0.50-13.25] %TBSA; P = .009), longer duration of mechanical ventilation (6.50 [2.75-17.00] vs 3.00 [1.00-8.25] days; P = .022), and longer intensive care unit length of stay (LOS) (3.00 [3.00-28.75] vs 5.50 days [2.00-11.25]; P = .033). Upon regression, use of heparin was the only variable associated with reducing the duration of mechanical ventilation ( P = .039). Nebulized heparin in combination with NAC and albuterol was associated with a significant reduction in the duration of mechanical ventilation.

  19. Heparin/N-acetylcysteine: an adjuvant in the management of burn inhalation injury: a study of different doses. (United States)

    Elsharnouby, Noha M; Eid, Hala E A; Abou Elezz, Nahla F; Aboelatta, Yasser A


    Nebulized heparin may reduce fibrin cast formation and reduce the degree of airway obstruction in burn inhalation injury. Twenty-nine patients admitted to burn intensive care unit (ICU) within 24 hours of burn inhalation injury were included in this prospective double-blinded randomized study. Group H5 received nebulized heparin sulfate 5,000 IU, and group H10 received nebulized heparin sulfate 10,000 IU. Heparin was given in alternation with N-acetylcysteine every 2 hours. Lung injury score assessed daily for 7 days was the primary outcome. Duration of mechanical ventilation, coagulation profile, length of ICU stay, and mortality were the secondary outcomes. Median lung injury scores were significantly lower in group H10 on days 5 (1.9 vs 1), 6 (1.4 vs 0.5), and 7 (1.3 vs 0.5). Group H10 had also a lower duration of mechanical ventilation than did group H5 (P = .037). The groups had no significant difference in coagulation parameters, length of ICU stay (P = .17), and mortality (P = .6). Nebulized heparin 10,000 IU decreased lung injury scores and duration of mechanical ventilation but had no effect on length of ICU stay and mortality. Moreover, nebulized heparin 10,000 IU was safe and had no effect on coagulation parameters. © 2013.

  20. Heparin-induced thrombocytopenia presenting as unilateral lower limb paralysis following lumbar spine surgery: case report. (United States)

    Smith, Brandon W; Joseph, Jacob R; Park, Paul


    Heparin-induced thrombocytopenia (HIT) is a state of thrombocytopenia with a paradoxically elevated thrombotic potential after exposure to heparin. Severe cases can present with multiorgan involvement with direct and secondary effects. Although HIT has been reported following other surgeries, to the authors' knowledge there has not been a report of HIT after spinal surgery. The present case details the course of a patient who underwent elective lumbar surgery followed by delayed presentation of shortness of breath due to multiple pulmonary embolisms and right lower-extremity paralysis due to extensive iliofemoral clot burden with acute compartment syndrome. The patient was treated with intravenous argatroban for extensive thrombosis and also required open thrombectomy and fasciotomies for treatment of compartment syndrome. Although the patient eventually experienced motor recovery, residual sensory deficits persisted at last follow-up. In this report, the pathophysiology, clinical presentation, and treatment of HIT are reviewed.

  1. Structural Characterization of the Low-Molecular-Weight Heparin Dalteparin by Combining Different Analytical Strategies

    Directory of Open Access Journals (Sweden)

    Antonella Bisio


    Full Text Available A number of low molecular weight heparin (LMWH products are available for clinical use and although all share a similar mechanism of action, they are classified as distinct drugs because of the different depolymerisation processes of the native heparin resulting in substantial pharmacokinetic and pharmacodynamics differences. While enoxaparin has been extensively investigated, little information is available regarding the LMWH dalteparin. The present study is focused on the detailed structural characterization of Fragmin® by LC-MS and NMR applied both to the whole drug and to its enzymatic products. For a more in-depth approach, size homogeneous octasaccharide and decasaccharide components together with their fractions endowed with high or no affinity toward antithrombin were also isolated and their structural profiles characterized. The combination of different analytical strategies here described represents a useful tool for the assessment of batch-to-batch structural variability and for comparative evaluation of structural features of biosimilar products.

  2. Venous thromboembolism in pregnancy: prophylaxis and treatment with low molecular weight heparin

    DEFF Research Database (Denmark)

    Andersen, Anita Sylvest; Berthelsen, Jørgen G.; Bergholt, Thomas


    side effects of treatment and no osteoporotic fractures or episodes of heparin-induced thrombocytopenia. The 166 pregnancies resulted in 159 live infants. There was a significantly higher risk of preterm delivery (13% vs. 6%) and intrauterine growth restriction (4.4% vs. 3.5%). Delivery by cesarean......-induced thrombocytopenia. The 166 pregnancies resulted in 159 live infants. There was a significantly higher risk of preterm delivery (13% vs. 6%) and intrauterine growth restriction (4.4% vs. 3.5%). Delivery by cesarean section was more common in these high-risk LMWH-treated pregnancies (33.1%) than in untreated......OBJECTIVE: To evaluate the safety of individually dosed low molecular weight heparin (LMWH) for prophylaxis and treatment of thromboembolic complications in pregnancy. DESIGN: Cohort study with a chronologic register-based control group. SETTING: Department of Obstetrics and Gynecology, Hiller...

  3. Venous thromboembolism in pregnancy: prophylaxis and treatment with low molecular weight heparin

    DEFF Research Database (Denmark)

    Andersen, Anita Sylvest; Berthelsen, Jørgen G; Bergholt, Thomas


    side effects of treatment and no osteoporotic fractures or episodes of heparin-induced thrombocytopenia. The 166 pregnancies resulted in 159 live infants. There was a significantly higher risk of preterm delivery (13% vs. 6%) and intrauterine growth restriction (4.4% vs. 3.5%). Delivery by cesarean......OBJECTIVE: To evaluate the safety of individually dosed low molecular weight heparin (LMWH) for prophylaxis and treatment of thromboembolic complications in pregnancy. DESIGN: Cohort study with a chronologic register-based control group. SETTING: Department of Obstetrics and Gynecology, Hillerød...... Hospital, Denmark. POPULATION: All 166 women treated with LMWH in pregnancy between 1 January 2001 and 31 December 2005. METHODS: Women treated with LMWH in pregnancy were identified and individual case records reviewed retrospectively. General data on the LMWH-treated women were compared to the 18...

  4. Bioinspired Heparin Nanosponge Prepared by Photo-crosslinking for Controlled Release of Growth Factors

    DEFF Research Database (Denmark)

    Choi, Won Il; Sahu, Abhishek; Vilos, Cristian


    Growth factors have great therapeutic potential for various disease therapy and tissue engineering applications. However, their clinical efficacy is hampered by low bioavailability, rapid degradation in vivo and non-specific biodistribution. Nanoparticle based delivery systems are being evaluated...... factor binding ability. Four different growth factors, bFGF, VEGF, BMP-2, and HGF were successfully encapsulated into Hep-NS. In vitro studies showed sustained release of all the growth factors for almost 60 days and the rate of release was directly dependent on the amount of heparin in Hep......-NS. The released growth factors retained their bioactivity as assessed by a cell proliferation assay. This heparin nanosponge is therefore a promising nanocarrier for the loading and controlled release of growth factors....

  5. Heparin Assisted Photochemical Synthesis of Gold Nanoparticles and Their Performance as SERS Substrates (United States)

    Rodríguez-Torres, Maria del Pilar; Díaz-Torres, Luis Armando; Romero-Servin, Sergio


    Reactive and pharmaceutical-grade heparins were used as biologically compatible reducing and stabilizing agents to photochemically synthesize colloidal gold nanoparticles. Aggregates and anisotropic shapes were obtained photochemically under UV black-light lamp irradiation (λ = 366 nm). Heparin-functionalized gold nanoparticles were characterized by Scanning Electron Microscopy and UV-Vis spectroscopy. The negatively charged colloids were used for the Surface Enhanced Raman Spectroscopy (SERS) analysis of differently charged analytes (dyes). Measurements of pH were taken to inspect how the acidity of the medium affects the colloid-analyte interaction. SERS spectra were taken by mixing the dyes and the colloidal solutions without further functionalization or addition of any aggregating agent. PMID:25342319

  6. Comparison of Efficacy Compressive Stockings with Heparin in Prevention of Deep Vein Thrombosis in Stroke Patients

    Directory of Open Access Journals (Sweden)

    Nastaran Majdi-Nasab


    Full Text Available Background: The present study is carried out to make a comparison between two pharmacological (heparin and physical (compression stockings in the prevention of deep vein thrombosis in lower limb of the patients suffered from acute stroke. Materials and Methods: In this investigation as a clinical trial, the effectiveness of the above methods on 100 patients with the stroke was compared in two groups of 50 persons. Results: Three patients in physical group and two patients in pharmacological group got deep vein thrombosis that showed no significant difference between two groups.Conclusion: In spite of no significant relationship and due to less incurrence of thrombosis in heparin group, it is more reasonable to use pharmacological methods.

  7. Heparin Assisted Photochemical Synthesis of Gold Nanoparticles and Their Performance as SERS Substrates

    Directory of Open Access Journals (Sweden)

    Maria del Pilar Rodríguez-Torres


    Full Text Available Reactive and pharmaceutical-grade heparins were used as biologically compatible reducing and stabilizing agents to photochemically synthesize colloidal gold nanoparticles. Aggregates and anisotropic shapes were obtained photochemically under UV black-light lamp irradiation (λ = 366 nm. Heparin-functionalized gold nanoparticles were characterized by Scanning Electron Microscopy and UV-Vis spectroscopy. The negatively charged colloids were used for the Surface Enhanced Raman Spectroscopy (SERS analysis of differently charged analytes (dyes. Measurements of pH were taken to inspect how the acidity of the medium affects the colloid-analyte interaction. SERS spectra were taken by mixing the dyes and the colloidal solutions without further functionalization or addition of any aggregating agent.

  8. Human Endothelial Cells: Use of Heparin in Cloning and Long-Term Serial Cultivation (United States)

    Thornton, Susan C.; Mueller, Stephen N.; Levine, Elliot M.


    Endothelial cells from human blood vessels were cultured in vitro, with doubling times of 17 to 21 hours for 42 to 79 population doublings. Cloned human endothelial cell strains were established for the first time and had similar proliferative capacities. This vigorous cell growth was achieved by addition of heparin to culture medium containing reduced concentrations of endothelial cell growth factor. The routine cloning and long-term culture of human endothelial cells will facilitate studying the human endothelium in vitro.

  9. Heparins crossover in percutaneous coronary interventions: a real issue with increasing rate of transradial procedures? (United States)

    Sciahbasi, Alessandro; Rigattieri, Stefano; Calcagno, Simone; Mancone, Massimo; Pendenza, Gianluca; Cera, Maria; Danza, Aurora Ilaria; Di Russo, Cristian; Bruno, Pasqualina; Fedele, Silvio; Pugliese, Francesco Rocco; Sardella, Gennaro


    Current guidelines give a class III recommendation to the intraprocedural use of unfractionated heparin (UFH) in patients pretreated with enoxaparin. The aim of our study was to evaluate bleeding complications in patients who underwent percutaneous coronary interventions (PCIs) performed using intraprocedural crossover of heparin therapy. We retrospectively evaluated all PCIs performed at two Italian hospitals since January 2011 to December 2013. After a propensity-matched analysis, patients were divided into two groups (with a ratio 1 : 2) according to intraprocedural crossover of heparins (from enoxaparin to UFH) (Group 1) or intraprocedural UFH alone (Group 2). The primary end-point was a haemoglobin drop of at least 3 g/dl within 48 h after the procedure. During the 3 years analysed, 3224 patients underwent PCI, and after the propensity analysis, 309 patients were considered eligible for our study: 104 patients in Group 1 (69 ± 12 years, 78% men) and 205 patients in Group 2 (69 ± 13 years, 80% men, P = NS). There were no significant differences between the two groups for BMI, periprocedural use of antiplatelet therapy, baseline haemoglobin haematocrit or platelets levels. The primary end-point did not differ between the two groups (2.9% in Group 1 and 3.4% in Group 2, P = 0.550). Also, nadir of haematocrit or haemoglobin levels did not differ between the two groups. Finally, in hospital, major adverse cardio-cerebrovascular events did not differ between the two groups (1.9% in Group 1 and 3.9% in Group 2, P = 0.50). In this retrospective analysis of a large PCI database, the 'heparins crossover' during PCI was not associated with increased bleeding risk.

  10. Preparation and evaluation of a novel oral delivery system for low molecular weight heparin


    Lavanya, Nallaguntla; Muzib, Yallamalli Indira; Aukunuru, Jithan; Balekari, Umamahesh


    Objective: The objective of the present work was to prepare and evaluate a novel oral formulation for systemic delivery of low molecular weight heparin (LMWH). The formulation consisted of Eudragit S 100-coated positively charged liposomes encapsulating LMWH and a penetration enhancer. Materials and Methods: Positively charged liposomes were first prepared by the thin film hydration method using lipid (soy phosphotidylcholine and cholesterol) and stearyl amine (SA) in the optimum ratio of 16:...

  11. Heparin-Related Thrombocytopenia Triggered by Severe Status of Systemic Lupus Erythematosus and Bacterial Infection

    Directory of Open Access Journals (Sweden)

    Satoshi Suzuki


    Full Text Available A patient with severe lupus nephritis developed thrombocytopenia during treatment with high-dose steroids. In addition to viral- or disease-induced cytopenia, the pathology was believed to arise from diverse contributing factors, such as thrombotic microangiopathy and heparin-related thrombocytopenia (HIT. By combining plasma exchange therapy and intravenous cyclophosphamide, we successfully controlled the SLE activity and improved the thrombocytopenia. An antecedent bacterial infection or SLE activity is believed to have contributed to the concurrent HIT.

  12. Incomplete filling of lithium heparin tubes affects the activity of creatine kinase and gamma-glutamyltransferase. (United States)

    Lippi, G; Avanzini, P; Cosmai, M; Aloe, R; Ernst, D


    This study aims to assess whether or not incomplete filling of primary lithium heparin tubes may influence the activity of creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT). Blood was drawn from 20 healthy volunteer using an identical sequence of tubes. First, a 6 mL, 13 x 100 mm 14 unit/mL lithium heparin Vacuette was filled and discharged. Then, three identical lithium heparin Vacuette tubes were filled, one to the nominal volume (i.e., full-draw tube), another with half of the nominal volume (half-draw tube) and the last with one-third of the nominal volume (low-draw tube). The plasma was separated and tested for CK (non-activated by N-acetylcysteine), AST and ALT on a Beckman Coulter Unicel DxC 800. Tests for CK were performed with a different reagent on a Beckman Coulter AU5800 (activated by N-acetylcysteine). Although the concentrations of ASL and ALT measured on the Unicel DxC and that of CK measured on the AU5800 did not change significantly across the different specimens, those of CK and GGT measured on the Unicel DxC 800 were significantly increased in the half-draw and low-draw tubes. The percentage bias of CK on the Unicel DxC 800 (using Bland Altman plots) was 3.3% and 7.9% for the half-draw and low-draw tubes, respectively, whereas that of GGT was 10.3% and 16.6% for the half-draw and low-draw tubes, respectively. These results suggest that short-draw lithium heparin tubes might be unsuitable for testing GGT and CK using specific combinations of reagents and instrumentation.

  13. Subcutis calcinosis caused by injection of calcium-containing heparin in a chronic kidney injury patient

    Directory of Open Access Journals (Sweden)

    Lilia Ben Fatma


    Full Text Available Subcutis calcinosis, characterized by abnormal calcium deposits in the skin, is a rare complication of using calcium-containing heparin occurring in patients with advanced renal failure. We report the case of an 83-year-old female, a known case of chronic kidney disease (CKD for four years with recent worsening of renal failure requiring hospitalization and hemodialysis. She developed subcutis calcinosis following injection of calcium-containing heparin. Biochemical tests showed serum parathormone level at 400 pg/dL, hypercalcemia, elevated calcium-phosphate product and monoclonal gammopathy related to multiple myeloma. She developed firm subcu-taneous nodules in the abdomen and the thighs, the injection sites of Calciparin ® (calcium nadroparin that was given as a preventive measure against deep vein thrombosis. The diagnosis of subcutis calcinosis was confirmed by the histological examination showing calcium deposit in the dermis and hypodermis. These lesions completely disappeared after discontinuing calcium nadro-parin injections. Subcutis calcinosis caused by injections of calcium-containing heparin is rare, and, to the best our knowledge, not more than 12 cases have been reported in the literature. Pathogenesis is not well established but is attributed to the calcium disorders usually seen in advanced renal failure. Diagnosis is confirmed by histological tests. Outcome is mostly favorable. The main differential diagnosis is calciphylaxis, which has a poor prognosis. Even though rarely reported, we should be aware that CKD patients with elevated calcium-phosphorus product can develop subcutis calcinosis induced by calcium-containing heparin. When it occurs, fortunately and unlike calci-phylaxis, outcome is favorable.

  14. Risk of Intraatrial Thrombi After Thoracoscopic Ablation in Absence of Heparin and Appendage Closure. (United States)

    Budera, Petr; Osmancik, Pavel; Herman, Dalibor; Talavera, David; Petr, Robert; Straka, Zbynek


    Catheter and surgical ablation of atrial fibrillation (AF) can be associated with a risk of thromboembolic events. The goal of this study was to assess optimal anticoagulation management during thoracoscopic ablation of AF. Fifty-two patients with persistent or long-standing persistent AF underwent hybrid ablation consisting of thoracoscopic ablation followed by electrophysiologic (EP) evaluation and consecutive ablation if indicated. The thoracoscopic ablation was performed using three different anticoagulation protocols: (1) without periprocedural heparin and without occlusion of the left atrial appendage; (2) with periprocedural heparin but without left atrial appendage occlusion; and (3) with periprocedural heparin and left atrial appendage occlusion. Transesophageal echocardiography (TEE) was obligatorily used to screen for intraatrial thrombi before the surgical and EP procedure and before hospital discharge for patients in protocols 2 and 3. In group 1 (n = 20), 1 patient (5%) had a postoperative stroke with persistent neurologic deficit, and 6 other patients (30%) had a new thrombus in the left atrial appendage seen on the pre-EP TEE. In group 2 (n = 6), 3 left atrial appendage thrombi occurred (50%; 2 on predischarge TEE and 1 on pre-EP TEE). In group 3 (n = 26), no intracardiac thrombi were found on predischarge and pre-EP TEE, and there were no strokes in this group of patients, namely, the rates of thrombus or stroke were significantly reduced when compared with groups 1 and 2 (p = 0.001). Thoracoscopic ablation of AF can be associated with a risk of left atrial appendage thrombus formation and possibly also stroke. With administration of heparin during the ablation, followed by occlusion of the left atrial appendage as a part of the procedure, this risk can be effectively reduced. Copyright © 2017 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.

  15. Heparin or bivalirudin for non-primary PCI: Beware of neat and simple answers…. (United States)

    Deliargyris, Efthymios N; Kimmelstiel, Carey


    The debate regarding the choice of heparin or bivalirudin as the preferred anticoagulant in PCI is still ongoing Nonrandomized registry data are severely limited for comparative analyses and should therefore always be interpreted with caution Clinicians should resist simplistic data interpretations or populist cries relating to cost, but rather focus on valid benefit:risk analyses for their clinical decision making. © 2017 Wiley Periodicals, Inc.

  16. Relationship between heparin anticoagulation and clinical outcomes in coronary stent intervention: observations from the ESPRIT trial. (United States)

    Tolleson, Thaddeus R; O'Shea, J Conor; Bittl, John A; Hillegass, William B; Williams, Kathryn A; Levine, Glenn; Harrington, Robert A; Tcheng, James E


    We evaluated the relationship between the degree of heparin anticoagulation and clinical efficacy and bleeding in patients undergoing contemporary percutaneous coronary intervention (PCI) with stent implantation. Despite universal acceptance of heparin anticoagulation as a standard of care in PCI, considerable controversy still exists regarding the appropriate dosing of heparin. The study population (n = 2,064) comprised all patients enrolled in the Enhanced Suppression of the Platelet IIb/IIIa Receptor with Integrilin Therapy (ESPRIT) trial. The index activated clotting time (ACT) was defined as the ACT measured after the last heparin dose and before first device activation and was correlated with outcome and bleeding events. No association was observed between decreasing ACT levels and the rate of ischemic events in the treatment or placebo arms. The incidence of the primary composite end point (death, myocardial infarction, urgent target vessel revascularization, and thrombotic bailout glycoprotein IIb/IIIa inhibitor therapy at 48 h) was actually lowest in the lowest ACT tertile for both the placebo (10.0%) and treatment groups (6.1%). When analyzed by tertile, major bleeding rates did not increase in the lowest ACT tertile in patients given placebo (0.6%) versus those receiving eptifibatide (0.7%). Major bleeding rates increased as the ACT increased in the eptifibatide-treated patients. Ischemic end points in patients undergoing contemporary PCI with stent placement do not increase by decreasing ACT levels, at least to a level of 200 s. Bleeding events do increase with increasing ACT levels and are enhanced with eptifibatide treatment. An ACT of 200 to 250 s is reasonable in terms of efficacy and safety with the use of contemporary technology and pharmacotherapy.

  17. Subcutis calcinosis caused by injection of calcium-containing heparin in a chronic kidney injury patient. (United States)

    Fatma, Lilia Ben; El Ati, Zohra; Azzouz, Haifa; Rais, Lamia; Krid, Madiha; Smaoui, Wided; Maiz, Hédi Ben; Béji, Soumaya; Zouaghi, Karim; Zitouna, Moncef; Moussa, Fatma Ben


    Subcutis calcinosis, characterized by abnormal calcium deposits in the skin, is a rare complication of using calcium-containing heparin occurring in patients with advanced renal failure. We report the case of an 83-year-old female, a known case of chronic kidney disease (CKD) for four years with recent worsening of renal failure requiring hospitalization and hemodialysis. She developed subcutis calcinosis following injection of calcium-containing heparin. Biochemical tests showed serum parathormone level at 400 pg/dL, hypercalcemia, elevated calcium-phosphate product and monoclonal gammopathy related to multiple myeloma. She developed firm subcutaneous nodules in the abdomen and the thighs, the injection sites of Calciparin ® (calcium nadroparin) that was given as a preventive measure against deep vein thrombosis. The diagnosis of subcutis calcinosis was confirmed by the histological examination showing calcium deposit in the dermis and hypodermis. These lesions completely disappeared after discontinuing calcium nadroparin injections. Subcutis calcinosis caused by injections of calcium-containing heparin is rare, and, to the best our knowledge, not more than 12 cases have been reported in the literature. Pathogenesis is not well established but is attributed to the calcium disorders usually seen in advanced renal failure. Diagnosis is confirmed by histological tests. Outcome is mostly favorable. The main differential diagnosis is calciphylaxis, which has a poor prognosis. Even though rarely reported, we should be aware that CKD patients with elevated calcium-phosphorus product can develop subcutis calcinosis induced by calcium-containing heparin. When it occurs, fortunately and unlike calciphylaxis, outcome is favorable.

  18. Nebulized heparin for patients under mechanical ventilation: an individual patient data meta-analysis. (United States)

    Glas, Gerie J; Serpa Neto, Ary; Horn, Janneke; Cochran, Amalia; Dixon, Barry; Elamin, Elamin M; Faraklas, Iris; Dissanaike, Sharmila; Miller, Andrew C; Schultz, Marcus J


    Pulmonary coagulopathy is a characteristic feature of lung injury including ventilator-induced lung injury. The aim of this individual patient data meta-analysis is to assess the effects of nebulized anticoagulants on outcome of ventilated intensive care unit (ICU) patients. A systematic search of PubMed (1966-2014), Scopus, EMBASE, and Web of Science was conducted to identify relevant publications. Studies evaluating nebulization of anticoagulants in ventilated patients were screened for inclusion, and corresponding authors of included studies were contacted to provide individual patient data. The primary endpoint was the number of ventilator-free days and alive at day 28. Secondary endpoints included hospital mortality, ICU- and hospital-free days at day 28, and lung injury scores at day seven. We constructed a propensity score-matched cohort for comparisons between patients treated with nebulized anticoagulants and controls. Data from five studies (one randomized controlled trial, one open label study, and three studies using historical controls) were included in the meta-analysis, compassing 286 patients. In all studies unfractionated heparin was used as anticoagulant. The number of ventilator-free days and alive at day 28 was higher in patients treated with nebulized heparin compared to patients in the control group (14 [IQR 0-23] vs. 6 [IQR 0-22]), though the difference did not reach statistical significance (P = 0.459). The number of ICU-free days and alive at day 28 was significantly higher, and the lung injury scores at day seven were significantly lower in patients treated with nebulized heparin. In the propensity score-matched analysis, there were no differences in any of the endpoints. This individual patient data meta-analysis provides no convincing evidence for benefit of heparin nebulization in intubated and ventilated ICU patients. The small patient numbers and methodological shortcomings of included studies underline the need for high

  19. Does a Nebulized Heparin/N-acetylcysteine Protocol Improve Outcomes in Adult Smoke Inhalation? (United States)

    Kashefi, Natalie S; Nathan, Jonathan I; Dissanaike, Sharmila


    Smoke inhalation is a major source of morbidity and mortality. Heparin and N-acetylcysteine treatment has potential efficacy in inhalation injury. We investigated the impact of a heparin/N-acetylcysteine/albuterol nebulization protocol in adult patients with inhalation injury. A retrospective review was performed of adult inhalation injury patients, admitted to a regional burn center between January 2011 and July 2012, who underwent a protocol of alternating treatments of heparin and N-acetylcysteine/albuterol nebulization every 4 hours. The study cohort was matched 1:1 by age, sex, and burn size to a control cohort admitted within 5 years before protocol implementation. The study (n = 20) and control cohorts (n = 20) were well matched, with nearly identical age (50 vs 49 years), sex distribution (70% male), burn size (total body surface area, 22% vs 21%), and inhalation injury, except grade I injuries (79% vs 47%, P = 0.01). The protocol did not change mortality (30% vs 25%, P = 0.72) or duration of mechanical ventilation (8.5 vs 8.8 days, P = 0.9). There was no difference in development of sepsis (40% vs 33%, P = 0.7) or acute respiratory distress syndrome (15% vs 10%, P = 1); however, those who received the protocol were more likely to develop pneumonia (45% vs 11%, P = 0.03). The implementation of a heparin/N-acetylcysteine/albuterol protocol did not reduce mortality or duration of mechanical ventilation in this cohort of adults with inhalation injury and resulted in a significant increase in pneumonia rates. Larger prospective studies are necessary, with close attention paid to minimizing the infection risk incurred from frequent administration of nebulized medications.

  20. Polyguluronate sulfate and its oligosaccharides but not heparin promotes FGF19/FGFR1c signaling (United States)

    Lan, Ying; Zeng, Xuan; Guo, Zhihua; Zeng, Pengjiao; Hao, Cui; Zhao, Xia; Yu, Guangli; Zhang, Lijuan


    Fibroblast growth factor 19(FGF19) functions as a hormone by affecting glucose metabolism. FGF19 improves glucose tolerance when overexpressed in mice with impaired glucose tolerance or diabetes. A functional cellular FGF19 receptor consists of FGF receptor (FGFR) and glycosaminoglycan complexed with either α Klotho or β Klotho. Interestingly, in mice with diet-induced diabetes, a single injection of FGF1 is enough to restore blood sugar levels to a healthy range. FGF1 binds heparin with high affinity whereas FGF19 does not, indicating that polysaccharides other than heparin might enhance FGF19/FGFR signaling. Using a FGFs/FGFR1c signaling-dependent BaF3 cell proliferation assay, we discovered that polyguluronate sulfate (PGS) and its oligosaccharides, PGS12 and PGS25, but not polyguluronate (PG), a natural marine polysaccharide, enhanced FGF19/FGFR1c signaling better than that of heparin based on 3H-thymidine incorporation. Interestingly, PGS6, PGS8, PGS10, PGS12, PGS25, and PGS, but not PG, had comparable FGF1/FGFR1c signal-stimulating activity compared to that of heparin. These results indicated that PGS and its oligosaccharides were excellent FGF1/FGFR1c and FGF19/FGFR1c signaling enhancers at cellular level. Since the inexpensive PGS and PGS oligosaccharides can be absorbed through oral route, these seaweed-derived compounds merit further investigation as novel agents for the treatment of type 2 diabetes through enhancing FGF1/FGFR1c and FGF19/FGFR1c signaling in future.