Mechanisms of activation of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase.
The molecular basis of the marked interspecies variability in the response of plasma fibrinolytic systems to activation by streptokinase (SK) or recombinant staphylokinase (STAR) was studied using highly purified plasminogens and alpha 2-antiplasmins from five representative species (man, baboon, rabbit, dog and cow). Human plasminogen reacted rapidly and stoichiometrically with both SK and STAR to yield potent plasminogen activators (catalytic efficiencies, kcat/Km, of 1.0 microM-1 x s-1 and 0.3 microM-1 x s-1, respectively). The complex with SK was insensitive to alpha 2-antiplasmin, which, however, rapidly inhibited the complex with STAR (second-order rate constant, k1,app of 8 x 10(6) M-1 x s-1). In a system composed of a 0.06-ml 125I-fibrin-labeled plasma clot submerged in 0.30 ml plasma, both SK and STAR had potent fibrinolytic properties, causing 50% clot lysis in 2 h (EC50), with 120 nM and 13 nM, respectively. Clot lysis with SK was non-fibrin specific (residual fibrinogen < 10%), whereas lysis with STAR was highly fibrin specific (residual fibrinogen 76%). Canine plasminogen reacted avidly with SK, but SK was rapidly degraded; it reacted rapidly and quantitatively with STAR to form a potent plasminogen-activating complex (kcat/Km of 0.4 microM-1 x s-1) which was sensitive to neutralization by alpha 2-antiplasmin (k1,app of 6 x 10(5) M-1 x s-1). In a canine plasma milieu, SK was relatively potent (EC50 200 nM) and fibrin specific, whereas STAR was very potent (EC50 1.3 nM) but poorly fibrin specific. Baboon and rabbit plasminogen did not form stable stoichiometric complexes with SK, but reacted stoichiometrically and quantitatively with STAR. The complexes with STAR, however, had low catalytic efficiencies for the activation of their autologous plasminogens (kcat/Km 0.02 microM-1 x s-1) and reacted more slowly with alpha 2-antiplasmin (k1,app 5-10 x 10(5) M-1 x s-1). Bovine plasminogen was virtually unreactive towards both SK and STAR as well as to their complexes with human plasminogen, as monitored by measurement of the initial activation rates. The resistance to fibrinogen degradation with STAR observed in the human system could be transferred to the canine system by reconstituting canine plasma, depleted of plasminogen and alpha 2-antiplasmin, with the human proteins. Conversely, the sensitivity to fibrinogen degradation of the canine system could be transferred to the human system by reconstituting depleted plasma with canine plasminogen and alpha 2-antiplasmin. It is concluded that the variability in the response of mammalian plasma fibrinolytic systems to activation with SK or STAR is determined mainly by the extent of complex formation of these compounds with plasminogen, by the catalytic efficiencies of the complexes for the activation of autologous plasminogen and by the rate of inhibition of these complexes by alpha 2-antiplasmin.
Collen
D
D
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Van Hoef
B
B
Schlott
B
B
Hartmann
M
M
Gührs
K H
KH
Lijnen
H R
HR
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D010215:Papio; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000494:pharmacology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D013045:Species Specificity; D013300:Streptokinase / Q000378:metabolism*; D000979:alpha-2-Antiplasmin / Q000378:metabolism* / Q000494:pharmacology
D011994:Recombinant Proteins; D000979:alpha-2-Antiplasmin; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D013300:Streptokinase; D010960:Plasminogen Activators; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus