In vitro stability of a tissue-type plasminogen activator mutant, BM 06.022, in human plasma.
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)
Rijken
D C
DC
Gaubius Laboratory, TNO-PG, Leiden, The Netherlands.
Groeneveld
E
E
Barrett-Bergshoeff
M M
MM
D004355:Drug Stability; D005343:Fibrinolytic Agents / Q000097:blood* / Q000493:pharmacokinetics; D006207:Half-Life; D006801:Humans; D009154:Mutation; D011480:Protease Inhibitors / Q000097:blood; D011994:Recombinant Proteins / Q000097:blood / Q000493:pharmacokinetics; D010959:Tissue Plasminogen Activator / Q000097:blood* / Q000235:genetics* / Q000493:pharmacokinetics
D005343:Fibrinolytic Agents; D011480:Protease Inhibitors; D011994:Recombinant Proteins; C087896:reteplase; D010959:Tissue Plasminogen Activator