Identification and Characterization of Novel tPA- and Plasminogen-Binding Sites within Fibrin(ogen) αC-Domains

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Abstract
Molecular defects in the αC-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 αC-fragment (residues Aα221−610) corresponding to the entire αC-domain as well as its NH2- and COOH-terminal halves (residues Aα221−391 and Aα392−610) 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 αC-fragment. This stimulation was abolished upon digestion of the αC-fragment with plasmin. In surface plasmon resonance experiments, both tPA and Pg bound to the immobilized αC-fragment with Kds 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 αC-domain since the Aα392−610 fragment bound both tPA and Pg and was an effective stimulator whereas Aα221−391 was inactive. These results indicate that the fibrinogen αC-domains contain novel high-affinity tPA- and Pg-binding sites that play an important role in the regulation of fibrinolysis.