Effect of fibrin-like stimulators on the activation of plasminogen by tissue-type plasminogen activator (t-PA)--studies with active site mutagenized plasminogen and plasmin resistant t-PA.
The activation of plasminogen by t-PA was measured in the presence and absence of fibrin stimulation, using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant plasminogen with the active site Ser740 mutagenized to Ala. Recombinant wild type t-PA (rt-PA) was used as well as rt-PA-Glu275, a recombinant single chain t-PA in which the Arg of the plasmin sensitive Arg275-Ile276 peptide bond was substituted with Glu. Conversion of 125I-labeled single chain plasminogen to two-chain plasmin by wild-type or mutant t-PA, was quantitated by SDS gel electrophoresis and radioisotope counting of gel slices, and expressed as initial activation rates (v0 in pM s-1) per 1 nM enzyme. In the absence of fibrin stimulation, the v0 for the activation of nPlg and rPlg-Ala740 with the single chain forms of both t-PAs were comparable (0.6 to 2.7 pM s-1) but were lower than with the corresponding two-chain forms (5.3 to 23 pM s-1). In the presence of 1 microM soluble fibrin monomer (desAAfibrin), the v0 for nPlg and rPlg-Ala740 by single chain rt-PA was also comparable (24 and 33 pM s-1 respectively), whereas with 1 microM CNBr-digested fibrinogen, the v0 for nPlg with single chain rt-PA was about 20-fold higher than that of rPlg-Ala740 (135 and 7.5 pM s-1 respectively). In contrast, the v0 for nPlg and rPlg-Ala740 by single chain rt-PA-Glu275, two-chain rt-PA-Glu275 or two-chain rt-PA were comparable in the presence of either desAAfibrin or CNBr-digested fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Lijnen
H R
HR
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Van Hoef
B
B
De Cock
F
F
Collen
D
D
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D003488:Cyanogen Bromide; D005337:Fibrin / Q000494:pharmacology*; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000187:drug effects* / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000502:physiology*
D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D003488:Cyanogen Bromide