ABSTRACT
Homozygous plasminogen activator inhibitor-1 (PAI-1)-deficient (PAI-1-/-) mice were generated by homologous recombination in D3 embryonic stem cells. Deletion of the genomic sequences encompassing the transcription initiation site and the entire coding regions of murine PAI-1 was demonstrated by Southern blot analysis. A 3.0-kb PAI-1-specific mRNA was identified by Northern blot analysis in liver from PAI-1 wild type (PAI-1+/+) but not from PAI-1-/- mice. Plasma PAI-1 levels, measured 2-4 h after endotoxin (2.0 mg/kg) injection were 63 +/- 2 ng/ml, 30 +/- 10 ng/ml, and undetectable (< 2 ng/ml) in PAI-1+/+, heterozygous (PAI-1+/-) and PAI-1-/- mice, respectively (mean +/- SEM, n = 4-11). PAI-1-specific immunoreactivity was demonstrable in kidneys of PAI-1+/+ but not of PAI-1-/- mice. SDS-gel electrophoresis of plasma incubated with 125I-labeled recombinant human tissue-type plasminogen activator revealed an approximately 115,000-M(r) component with plasma from endotoxin-stimulated (0.5 mg/kg) PAI-1+/+ but not from PAI-1-/- mice, which could be precipitated with a polyclonal anti-PAI-1 antiserum. PAI-1-/- mice were viable, produced similar sizes of litters as PAI-1+/+ mice, and showed no apparent macroscopic or microscopic histological abnormalities.
Subject(s)
Kidney/metabolism , Plasminogen Activator Inhibitor 1/deficiency , Plasminogen Activator Inhibitor 1/genetics , Recombination, Genetic , Animals , Base Sequence , Blotting, Southern , Cells, Cultured , Codon/metabolism , DNA/isolation & purification , DNA/metabolism , DNA Primers , DNA, Complementary/metabolism , Embryo, Mammalian , Female , Gene Deletion , Genomic Library , Homozygote , Kidney/cytology , Kidney Glomerulus/cytology , Kidney Glomerulus/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred Strains , Mice, Transgenic , Molecular Sequence Data , Plasminogen Activator Inhibitor 1/biosynthesis , Polymerase Chain Reaction , Restriction Mapping , Stem Cells/metabolism , Transcription, Genetic , TransfectionABSTRACT
Chimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, but only partial fibrin-binding properties of t-PA (Nelles et al., J Biol Chem 1987; 262: 10855-62). The following domain deletion and/or duplication mutants of such a t-PA/u-PA chimera were constructed, purified and characterized: rt-PA-delta FE/u-PA, with deletion of the finger-like (F) and epidermal growth factor-like (E) domains, rt-PA-delta K1 delta K2/u-PA, with kringle 1 (K1) replaced by a second copy of kringle 2 (K2), and rt-PA-delta FEK1 delta K2/u-PA, with F and E domain deletions in rt-PA-delta K1 delta K2/u-PA. The specific activities on fibrin plates of the single-chain (sc) chimeras ranged between 68,000 IU/mg for rt-PA-delta K1 delta K2/scu-PA and 200,000 IU/mg for rt-PA-delta FEK1 delta K2/scu-PA, as compared to 120,000 IU/mg for rscu-PA. The specific activities of their plasmin-generated two-chain (tc) derivatives ranged between 120,000 IU/mg for rt-PA-delta K1 delta K2/tcu-PA and 240,000 IU/mg for rt-PA-delta FEK1 delta K2/tcu-PA, as compared to 100,000 IU/mg for rtcu-PA. All two-chain chimeras activated plasminogen following Michaelis-Menten kinetics, with catalytic efficiencies between 0.072 microM-1s-1 for rt-PA-delta K1 delta K2/tcu-PA and 0.081 microM-1 s-1 for rt-PA-delta FEK1 delta K2/tcu-PA, as compared to 0.088 microM-1 s-1 for rtcu-PA.(ABSTRACT TRUNCATED AT 250 WORDS)
