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 b een associated with impaired fibrin-mediated activation of plasminogen (Pg) by its activator tPA, suggesting the involvement of these domains in fibri nolysis. To test this suggestion, we expressed in E. coil the alphaC-fragme nt (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 a lpha 392-610) and tested their effects on activation of Pg and their intera ction with Pg and tPA, When the activation was monitored by cleavage of a c hromogenic substrate with newly formed plasmin, the reaction was much more efficient in the presence of the alphaC-fragment, This stimulation was abol ished upon digestion of the alphaC-fragment with plasmin. In surface plasmo n resonance experiments, both tPA and Pg bound to the immobilized CIC-fragm ent with K(d)s Of 33 and 32 nM, respectively. Similar results were obtained by ELISA. This binding occurred via independent sites since saturating amo unts of Pg did not prevent binding of tPA and vice versa, Both sites were l ocalized in the COOH-terminal half of the alphaC-domain since the A alpha 3 92-610 fragment bound both tPA and Pg and was an effective stimulator where as A alpha 221-.391 was inactive. These results indicate that the fibrinoge n alphaC-domains contain novel high-affinity tPA- and Pg-binding sites that play an important role in the regulation of fibrinolysis.