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In vitro stability of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma
D C Rijken1, E Groeneveld, M M Barrett-Bergshoeff
1Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
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 microgram/ml to 38 min at 10 micrograms/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 Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin. During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of alpha 2-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 micrograms/ml BM 06.022 and higher. In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, alpha 2-antiplasmin and alpha 1-antitrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
BM 06.022, a tissue-type plasminogen activator (t-PA) mutant, is rapidly inactivated in plasma by protease inhibitors. This study details the inactivation mechanisms of BM 06.022, revealing its interaction with key plasma proteins.
Area of Science:
- Biochemistry
- Pharmacology
- Proteolysis
Background:
- BM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) with a longer in vivo half-life.
- The in vitro activity half-life of BM 06.022 in plasma is shorter than that of t-PA.
Purpose of the Study:
- To investigate the inactivation mechanisms of BM 06.022 in human plasma.
- To characterize the kinetics and molecular interactions involved in BM 06.022 clearance.
Main Methods:
- Incubation of varying BM 06.022 concentrations in plasma.
- Activity assays, SDS-PAGE, fibrin autography, and immunoblotting.
- Quantification of inhibitor consumption and substrate degradation.
Main Results:
- BM 06.022 activity declined dose-dependently, with half-lives ranging from 72 to 38 minutes.
- Formation of BM 06.022 complexes with Cl-inactivator, alpha 2-antiplasmin, and alpha 1-antitrypsin.
- Plasmin generation and conversion of single-chain to two-chain BM 06.022 were observed.
Conclusions:
- BM 06.022 is inactivated in plasma by Cl-inactivator, alpha 2-antiplasmin, and alpha 1-antitrypsin.
- The inactivation process involves complex formation and plasmin generation.
- Understanding these mechanisms is crucial for the therapeutic application of BM 06.022.