PMID- 29968421 OWN - NLM STAT- MEDLINE DCOM- 20191223 LR - 20230829 IS - 1538-7836 (Electronic) IS - 1538-7836 (Linking) VI - 16 IP - 9 DP - 2018 Sep TI - Chemical and pharmacological aspects of neutralization of heparins from different animal sources by protamine. PG - 1789-1799 LID - 10.1111/jth.14221 [doi] AB - Essentials Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity. Protamine-neutralization was evaluated on a variety of physical-chemical methods. HBI requires more protamine than HPI to fully neutralize its anticoagulant activity. Protamine preferentially removes higher-sulfated chains of HBI while HPI is evenly precipitated. SUMMARY: Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated alpha-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated alpha-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated alpha-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure. CI - (c) 2018 International Society on Thrombosis and Haemostasis. FAU - Glauser, B F AU - Glauser BF AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. FAU - Santos, G R C AU - Santos GRC AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. AD - Laboratorio de Apoio ao Desenvolvimento Tecnologico, Laboratorio Brasileiro de Controle de Dopagem, Instituto de Quimica, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. FAU - Silva, J D AU - Silva JD AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. FAU - Tovar, A M F AU - Tovar AMF AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. FAU - Pereira, M S AU - Pereira MS AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. FAU - Vilanova, E AU - Vilanova E AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. FAU - Mourao, P A S AU - Mourao PAS AD - Laboratorio de Tecido Conjuntivo, Hospital Universitario Clementino Fraga Filho and Instituto de Bioquimica Medica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Cidade Universitaria, Rio de Janeiro, Brazil. LA - eng PT - Comparative Study PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20180813 PL - England TA - J Thromb Haemost JT - Journal of thrombosis and haemostasis : JTH JID - 101170508 RN - 0 (Anticoagulants) RN - 0 (Disaccharides) RN - 0 (Heparin Antagonists) RN - 0 (Protamines) RN - 70FD1KFU70 (Sulfur) RN - 9005-49-6 (Heparin) SB - IM MH - Animals MH - Anticoagulants/chemistry/isolation & purification/*pharmacology MH - Biological Assay MH - Cattle MH - Chemical Precipitation MH - Chromatography, Affinity MH - Disaccharides/chemistry MH - Dose-Response Relationship, Drug MH - Heparin/chemistry/isolation & purification/*pharmacology MH - Heparin Antagonists/*pharmacology MH - Intestinal Mucosa/chemistry MH - Mass Spectrometry MH - Nuclear Magnetic Resonance, Biomolecular MH - Partial Thromboplastin Time MH - Protamines/chemistry/*pharmacology MH - Rats MH - Species Specificity MH - Sulfur/analysis MH - Swine OTO - NOTNLM OT - anticoagulants OT - blood coagulation tests OT - hemorrhage OT - heparin antagonists OT - thrombosis EDAT- 2018/07/04 06:00 MHDA- 2019/12/24 06:00 CRDT- 2018/07/04 06:00 PHST- 2018/02/14 00:00 [received] PHST- 2018/07/04 06:00 [pubmed] PHST- 2019/12/24 06:00 [medline] PHST- 2018/07/04 06:00 [entrez] AID - S1538-7836(22)02358-3 [pii] AID - 10.1111/jth.14221 [doi] PST - ppublish SO - J Thromb Haemost. 2018 Sep;16(9):1789-1799. doi: 10.1111/jth.14221. Epub 2018 Aug 13.