A steady-state template model that describes the kinetics of fibrin-stimulated [Glu1]- and [Lys78]plasminogen activation by native tissue-type plasminogen activator and variants that lack either the finger or kringle-2 domain.
The kinetics of activation of both [Glu1]- and [Lys78]Plg(S741C-fluorescein by native (recombinant) tissue-type plasminogen activator and its deletion variants lacking either the finger or kringle-2 domain were measured by fluorescence within fully polymerized fibrin clots. The kinetics conform to the Michaelis-Menten equation at any fixed fibrin concentration so long as the plasminogen concentration is expressed as either the free or fibrin-bound, but not the total. The apparent kcat and Km values both vary systematically with the concentration of fibrin. Competition kinetics disclosed an active site-dependent interaction between t-Pa and [Glu1]Plg(S741C-fluorescein) in the presence, but not the absence, of fibrin. A steady-state template model having the rate equation v/[A]o = kcat(app).[Plg]/(Km(app) + [Plg]) was derived and used to interpret the data. The model indicates that catalytic efficiency is determined by the stability of the ternary activator-fibrin-plasminogen complex rather than the binding of the activator or plasminogen to fibrin. This implies that efforts to improve the enzymatic properties of t-PA might be more fruitfully directed at enhancing the stability of the ternary complex rather than fibrin binding.
Horrevoets
A J
AJ
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Pannekoek
H
H
Nesheim
M E
ME
D000465:Algorithms; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D018082:Kringles; D008956:Models, Chemical; D010446:Peptide Fragments / Q000378:metabolism*; D010958:Plasminogen / Q000378:metabolism*; D013698:Templates, Genetic; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D016335:Zinc Fingers
D010446:Peptide Fragments; C018998:lysyl-plasminogen; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator