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 plasminoge n activator (t-PA) comprising only the kringle-2 and proteinase domain s. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer t han 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 o f t-PA. In this study the inactivation of BM 06.022 in plasma was furt her investigated. Varying concentrations of BM 06.022 were incubated i n plasma for 0-150 min. Activity assays on serial samples showed a dos e-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 mu g/ml to 38 min at 10 mu g/ml. SDS-polyacrylamide gel electr ophoresis (SDS-PAGE) followed by fibrin autography showed the generati on of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against C1-inactivator, alpha(2)-antipla smin and alpha(1)-antitrypsin. During the incubation of BM 06.022 in p lasma, plasmin was generated dose-dependently as revealed by varying d egrees of alpha(2)-antiplasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was ra pidly (i.e, within 45 min) converted into its two-chain form at concen trations of 5 mu g/ml BM 06.022 and higher. In conclusion, BM 06.022 a t therapeutic concentrations in plasma was inactivated by C1-inactivat or, alpha(2)-antiplasmin and alpha(1)-antitrypsin. The half-life of th e activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inac tivated faster than the single-chain form.