Identification and characterization of novel tPA- and plasminogen-binding sites within fibrin(ogen) αC-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 A alpha 221-610) corresponding to the entire alphaC-domain as well as its NH2- and COOH-terminal halves (residues A alpha 221-391 and A alpha 392-610) 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 CIC-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 A alpha 392-610 fragment bound both tPA and Pg and was an effective stimulator whereas A alpha 221-.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.