Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator.
TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.
Stewart
R J
RJ
Hamilton Civic Hospitals Research Centre and McMaster University, Hamilton, Ontario L8V 1C3, Canada.
Fredenburgh
J C
JC
Leslie
B A
BA
Keyt
B A
BA
Rischke
J A
JA
Weitz
J I
JI
D000409:Alanine; D019943:Amino Acid Substitution; D005337:Fibrin / Q000378:metabolism*; D014644:Genetic Variation; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism*
D011994:Recombinant Proteins; D005337:Fibrin; C104096:TNK-tissue plasminogen activator; D010959:Tissue Plasminogen Activator; D000409:Alanine