Identification and characterization of novel tPA- and plasminogen-binding sites within fibrin(ogen) alpha C-domains.
Molecular defects in the alphaC-domains of some abnormal fibrinogens have been associated with impaired fibrin-mediated activation of plasminogen (Pg) by its activator tPA, suggesting the involvement of these domains in fibrinolysis. To test this suggestion, we expressed in E. coli the alphaC-fragment (residues Aalpha221-610) corresponding to the entire alphaC-domain as well as its NH(2)- and COOH-terminal halves (residues Aalpha221-391 and Aalpha392-610) and tested their effects on activation of Pg and their interaction with Pg and tPA. When the activation was monitored by cleavage of a chromogenic substrate with newly formed plasmin, the reaction was much more efficient in the presence of the alphaC-fragment. This stimulation was abolished upon digestion of the alphaC-fragment with plasmin. In surface plasmon resonance experiments, both tPA and Pg bound to the immobilized alphaC-fragment with K(d)s of 33 and 32 nM, respectively. Similar results were obtained by ELISA. This binding occurred via independent sites since saturating amounts of Pg did not prevent binding of tPA and vice versa. Both sites were localized in the COOH-terminal half of the alphaC-domain since the Aalpha392-610 fragment bound both tPA and Pg and was an effective stimulator whereas Aalpha221-391 was inactive. These results indicate that the fibrinogen alphaC-domains contain novel high-affinity tPA- and Pg-binding sites that play an important role in the regulation of fibrinolysis.
Tsurupa
G
G
Department of Biochemistry, The American Red Cross Holland Laboratory, Rockville, Maryland 20855, USA.
Medved
L
L
D001667:Binding, Competitive / Q000235:genetics; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D010958:Plasminogen / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000494:pharmacology; D020349:Surface Plasmon Resonance; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; C098000:fibrinogen alphaC; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D011503:Transglutaminases; D010450:Endopeptidases; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin