Zinc(II) Chelate Binds to Fibrinogen and Its 伪C Region

摘要

Fibrinogen is the immediate precursor of fibrin blood clots. When fibrinogen is cleaved by thrombin, it assembles into fibrin polymers through intermolecular interactions where calcium and zinc ions may both be involved. Fibrinogen’s αC domains play a major role in fibrin polymer assembly, and they contain a Zn2+?binding sites based on peptide studies. However, Zn2+?binding to natural αC and fibrin (ogen) remains to be demonstrated. The of this study was to directly demonstrate Zn2+?interactions with purified fibrinogen and its αC region. Both human and bovine αC regions were investigated due to the significant differences in their protein sequences. To detect Zn2+?binding, chelated Zn2+?in the form of Zn2+nitrilotriacetic acid linked to alkaline phosphatase tracer (ZnTAP) was prepared and used in ELISA and Western blot style methods. Gel electrophoresis was used to monitor changes to fibrinogen after reaction with plasmin. Human and bovine αC regions were obtained by limited plasmin digests of fibrinogen and purified by Con-A and Zn-immobilized metal ion chromatography. The results showed ZnTAP binding to fibrinogen in solution and inhibition of binding to fibrinogen coated microplates by ELISA. Inhibition concentrations (IC50) of 0.58 μM for human fibrinogen and 0.26 μM for bovine fibrinogen were determined. Blot probes revealed ZnTAP binding to fibrinogen with disulfide bonds intact and to the Aα and Bβ chains with disulfide bonds reduced. Reduced Aα chain reaction was less than that of intact fibrinogen and the reduced Bβ chain was only weakly reactive. With plasmin treatment, ZnTAP binding decreased by ELISA in parallel with the degradation of αC region by gel electrophoresis analysis. Purified αC bound ZnTAP in ELISA and on Western blots. In this study we demonstrated that Zn2+?binds to fibrinogen and its Aα and Bβ chains, and to the αC region of the Aα chain. The results support a role for protein conformation in Zn2+?binding and demonstrate the utility of the ZnTAP complex with tracer for fibrinogen binding interactions.