Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli.
The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis.
Wilhelm
O G
OG
Lilly Laboratory for Clinical Research, Eli Lilly and Company, Indianapolis, Indiana.
Jaskunas
S R
SR
Vlahos
C J
CJ
Bang
N U
NU
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013462:Sulfur Radioisotopes; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D008239:Lysine