Abstract
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 Zn<sup>2+</sup> binding sites based on peptide studies. However, Zn<sup>2+</sup> binding to natural αC and fibrin (ogen) remains to be demonstrated. The objective of this study was to directly demonstrate Zn<sup>2+</sup> 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 Zn<sup>2+</sup> binding, chelated Zn<sup>2+</sup> in the form of Zn<sup>2+</sup>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 Zn<sup>2+</sup> 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 Zn<sup>2+</sup> binding and demonstrate the utility of the ZnTAP complex with tracer for fibrinogen binding interactions.
Highlights
Fibrinogen functions in hemostasis as the precursor to fibrin blood clots
Zn2+nitrilotriacetic acid linked to alkaline phosphatase tracer (ZnTAP) was prepared for the purpose of directly demonstrating Zn2+ binding to fibrinogen and to the αC region of fibrinogen
To prepare ZnTAP, EDTA was added to Ni-NTA-alkaline phosphatase to remove Ni2+
Summary
Fibrinogen functions in hemostasis as the precursor to fibrin blood clots. It is an elongated molecule with two sets of three protein chains (Aα, Bβ and γ)2 [1]. From E the two sets of chains extend in opposing directions, leading to two D regions [3, 4]. The Aα chains split away from D regions as a connector sequence leading to the αC domains. Fibrinogen is converted to fibrin monomers as thrombin cleaves peptides from the Aα and Bβ chains. The αC domains are released from their intramolecular docking sites with thrombin cleavage of Aα and Bβ chains, and they form intermolecular associations with other αC domains during polymer assembly. The transglutaminase, factor XIII, catalyzes covalent cross linking between γ chains and among αC domains to strengthen and stabilize the fibrin clot [5]
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