On the molecular interactions between fibrin, tissue-type plasminogen activator and plasminogen.
The molecular interactions involved in the fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA) were studied using natural human plasminogen (nPlg) and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active site Ser740 to Ala. Using this rPlg-Ala740 moiety, the dissociation constant of the interaction between plasminogen and CNBr-digested fibrinogen was determined to be 0.40 microM. In addition, conversion of 125I-labeled single chain plasminogen to two chain plasmin by single chain recombinant t-PA (rt-PA) in the absence or the presence of CNBr-digested fibrinogen was quantitated on reduced SDS-gel electrophoresis, combined with autoradiography and radioisotope counting of gel bands. In the absence of fibrin, the activation rate of nPlg and rPlg-Ala740 by single-chain rt-PA was comparable. In the presence of fibrin, however, the activation rate of rPlg-Ala740 was about 20-fold lower than that of nPlg. These results with rPlg-Ala740 may be explained by an impaired formation of the stable cyclic ternary complex between plasminogen, t-PA and fibrin, which mediates the fibrin stimulation of plasminogen activation by t-PA or, alternatively, by impaired conversion of single chain rt-PA to two chain rt-PA at the fibrin surface.
Lijnen
H R
HR
Center for Thrombosis and Vascular Research, K.U. Leuven, Belgium.
Van Hoef
B
B
Collen
D
D
D000818:Animals; D002478:Cells, Cultured; D003488:Cyanogen Bromide; D005337:Fibrin / Q000378:metabolism*; D007700:Kinetics; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000378:metabolism* / Q000494:pharmacology
D011994:Recombinant Proteins; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D003488:Cyanogen Bromide