Print Email Facebook Twitter In vitro stabililty of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma Title In vitro stabililty of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma Author Rijken, D.C. Groeneveld, E. Barrett-Bergshoeff, M.M. Gaubius Laboratory TNO Preventie en Gezondheid Publication year 1994 Abstract BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated. Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against C1-inactivator, α2-antiplasmin and α1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of α2-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i.e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by C1-inactivator, α2-antiplasmin and α1-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form. Chemicals/CAS: alpha 1 antitrypsin, 9041-92-3; alteplase, 105857-23-6; fibrinogen, 9001-32-5; plasmin, 9001-90-5, 9004-09-5; reteplase, 133652-38-7; tissue plasminogen activator, 105913-11-9; proteinase inhibitor, 37205-61-1; BM 06.022; Fibrinolytic Agents; Protease Inhibitors; Recombinant Proteins; Tissue Plasminogen Activator, EC 126.96.36.199 Subject Biologyalpha 1 antitrypsinalpha 2 antiplasminalteplasefibrinogenplasminreteplasetissue plasminogen activatorfibrinolytic agentproteinase inhibitorrecombinant proteindose responsedrug activitydrug half lifedrug inactivationdrug stabilityimmunoblottingplasmapolyacrylamide gel electrophoresisbloodgeneticshalf life timemutationDrug StabilityFibrinolytic AgentsHalf-LifeHumanMutationProtease InhibitorsRecombinant ProteinsTissue Plasminogen Activator To reference this document use: http://resolver.tudelft.nl/uuid:516e6a47-470f-4713-bb6f-ac06ea63057b TNO identifier 232792 ISSN 0340-6245 Source Thrombosis and Haemostasis, 72 (6), 906-911 Bibliographical note p.908 ontbreekt Document type article Files To receive the publication files, please send an e-mail request to TNO Library.