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In vitro stabililty of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma

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Author: Rijken, D.C. · Groeneveld, E. · Barrett-Bergshoeff, M.M.
Institution: Gaubius Laboratory TNO Preventie en Gezondheid
Source:Thrombosis and Haemostasis, 6, 72, 906-911
Identifier: 232792
Keywords: Biology · alpha 1 antitrypsin · alpha 2 antiplasmin · alteplase · fibrinogen · plasmin · reteplase · tissue plasminogen activator · fibrinolytic agent · proteinase inhibitor · recombinant protein · dose response · drug activity · drug half life · drug inactivation · drug stability · immunoblotting · plasma · polyacrylamide gel electrophoresis · blood · genetics · half life time · mutation · Drug Stability · Fibrinolytic Agents · Half-Life · Human · Mutation · Protease Inhibitors · Recombinant Proteins · Tissue Plasminogen Activator


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