Linhardt Research Labs Papers


Recent Submissions

Now showing 1 - 5 of 1022
  • Item
    A System for Heparin Removal
    (1982) Langer, R.; Linhardt, Robert J.; Klein, M.; Galliher, P.M.; Cooney C.L.; Flanagan, M.M.
    Extracorporeal medical machines rely on systemic heparinization to improve blood compatibility. However, heparin can lead to serious complications such as hemorrhaging. We propose a new approach to control heparin levels by employing a blood filter containing immobilized heparinase. Such a filter could potentially enable heparinization of an extracorporeal circuit without simultaneous heparinization of the patient. The principal findings of our work thus far include (1) increasing volumetric enzyme production over 1000-fold from previously published procedures; (2) purifying heparinase by over 1000-fold from the crude cell extracts; (3) characterizing the biochemical properties of heparinase; (4) isolating the first heparinase inhibitors; (5) immobilizing heparinase with 91% activity recovery and excellent stability; and (6) demonstrating that columns as small as 1.5 mL can remove clinically used quantities of heparin in aqueous medium and in blood.
  • Item
    Mechanism of Diacyl Peroxide Decomposition
    (1982) Linhardt, Robert J.; Murr, B.L.; Montgomery, E.; Osby J.; Sherbine, J.
    The presence of ion-pair intermediates in diacyl peroxide decomposition has been established. Various substituted (4-X·pOOiyl)phenylaoetyl Y·benzoyl peroxides and three corresponding 4·X-bemhydryl-4·nitrobenzolc carbonic anhydride& (X • CH3, H, CU were prepared. All compounds decompo1ed in 90% acetone-water (v/ v), giving the ionic product. eater, alcohol, and acid. The fraction of ester (R) was similar to that found in the solvolysis of substituted benzhydryl·N·nitroeoe.mides, indicating a similar spectrum of ion-pair intermediates. The yield of ester product could be increased markedly by the addition of common ion. The mixed carbonic carboxylic an.hydrides were not productl of peroxide decomposition in either nucleophillc or nonnucleophiUc aolventa and abowtd a lowe1 deoompoei;ion raU. than the peroxide. Lastly, both the peroxide and the mixed carbonic carboxylic Anhydride decompoMd in chloroform with net retention. Neither CIDNP nor any radical abstraction product was detected.
  • Item
    Heparinase Production by Flavobacterium heparinum
    (1981) Galliher, P.M.; Cooney, C.L.; Langer R.; Linhardt, Robert J.
    Heparinase production by Flavobacterium heparinum in complex protein digest medium, with heparin employed as the inducer, has been studied and improved. The maximum productivity of heparinase has been increased 156-fold over that achieved by previously published methods to 375 U/liter per h in the complex medium. Rapid deactivation of heparinase activity, both specific and total, was observed at the onset of the stationary phase. Nutritional studies on growth and heparinase production showed an obligate requirement for L-histidine and no vitamin requirement. L-Methionine partially relieved the L-histidine requirement. A defined medium containing glucose, ammonium sulfate, basal salts, L-methionine, and L-histidine was developed for growth and heparinase production. The growth rate in this medium was 0.21 h-1, which is 40%, higher than that in complex medium. The maximum volumetric productivity of heparinase in the defined medium was increased to 1,475 U/liter per h, providing a 640-fold increase over that achieved by previously published methods. No rapid deactivation was observed. An examination of alternate inducers for heparinase showed that heparin degradation products, hyaluronic acid, heparin monosulfate, N-acetyl-D-glucosamine, and maltose, induce heparinase in complex medium. An Azure A assay was modified and fully developed to measure the heparin concentration during fermentation and the heparinase specific activity of crude extracts of F. heparinum obtained from sonication, thus negating the need for further purification to measure activity."
  • Item
    In Vivo Activity of Microbial Heparinase
    (1982) Langer, R.; Linhardt, Robert J.; Larsen, A.K.; Cooney, C.L.; Tapper, D.; Klein, M.
    Heparin, prepared from mammalian intestinal mucosa, is the most widely used anticoagulant. It has been estimated, however, that the use of heparin leads to complications such as bleeding 8-33% of the time1. Of all prescription drugs, heparin is the drug responsible for the greatest number of deaths in otherwise healthy patients 2. For these reasons, it would be extremely useful to have a method to control blood heparin levels. We propose that this might be accomplished by using heparinase, an enzyme which specifically degrades the anticoagulant activity of heparin. In this report, we discuss the in vivo activity of both free and immobilized heparinase.
  • Item
    Heparinase: In Vivo Activity and Immunogenicity in Rabbits
    (1983) Klein, M.D.; Drongowski, R.A.; Linhardt, Robert J.; Cooney, C.L.; Langer, R.S.
    Anticoagulation with heparin is required during extracorporeal circulation for hemodialysis and cardiopulmonary bypass as well as during vascular surgery. Reversal of anticoagulation with protamine may be associated with hypotension and rebound anticoagulation and requires stoichiometric doses. Heparinase from Flavobacterium heparinum catalytically degrades heparin and reverses its anticoagulant effect. Heparin was administered to New Zealand White rabbits and plasma levels were assayed with the APTT anticoagulant assay and the azure A chemical assay. Heparinase actively degraded heparin both in vitro in rabbit plasma and in vivo in rabbit blood as determined by both the anticoagulant and chemical assays when compared to control heparin disappearance curves. Antibodies to heparinase were demonstrated by the ELISA technique in rabbits receiving i.v. heparinase. These antibodies, however, did not effect the activity of the enzyme in vitro or in vivo. No toxic effects of heparinase were noted in observations of the animals or in blood and histologic studies. Heparinase, either free or immobilized, may be a useful heparin-reversing agent without the drawbacks of protamine.