Structure, Stability, and Interaction of Fibrin αC-Domain Polymers

Abstract

Abstract Title of the thesis: Structure, Stability, and Interaction of Fibrin αC-Domain Polymers Ariza Mahid, Master of Science, 2011 Thesis directed by: Leonid Medved Ph.D., Sc.D., Professor, Department of Biochemistry and Molecular Biology, and Center for Vascular and Inflammatory Diseases Fibrinogen is a multidomain plasma protein that after conversion into polymeric fibrin protects damaged vasculature from blood loss and subsequently participates in wound healing through the interaction of its individual domains with various plasma proteins and cellular receptors. Fibrin(ogen)αC-domains formed by the C-terminal portions of its Aαchains (amino acid residues Aα392-610) play an important role in fibrin assembly, fibrinolysis, and angiogenesis. These domains are inert in fibrinogen and highly reactive in fibrin suggesting that their structure in the latter, in which they form αC polymers, differs from that in fibrinogen. While the structure of the isolated αC-domain has been established, the structure of the αC-domains in fibrin αC polymers remains to be determined. The major goals of the present study were to clarify the structure of fibrin αC-domain polymers and to test our hypothesis that polymerization of the αC-domains results in the exposure of their binding sites. To accomplish these goals, we prepared a recombinant αC region (Aα221-610) including the αC-domain, demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and covalently cross-linked such oligomers with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear arrays when analyzed by electron microscopy. Spectral studies revealed that theαC-domains in such oligomers were folded into compact structures with significant amount ofα-sheets and exhibited high thermal stability. These results indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of αC polymers in fibrin. Binding studies performed by ELISA and SPR revealed that, in contrast to the monomeric αC-domain, the cross-linked oligomers exhibited prominent binding to tPA and plasminogen indicating that their tPA- and plasminogen-binding sites are exposed. In agreement, these oligomers drastically increased the rate of plasminogen activation in the chromogenic substrate assay. Thus, our study with cross-linked Aα221-610 oligomers clarified the structure of the αC-domains in fibrin αC polymers and confirmed our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for future structural and functional studies of fibrin αC-domains.