Fibrinogen is a multidomain polyfunctional plasma protein that after thrombin-mediated conversion into fibrin plays a prominent role in a number of important physiological and pathological processes, including hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and atherogenesis. Better understanding of the mechanisms underlying these processes requires establishment of the structure and interactions of this complex molecule. The long-term objectives of our research are to generate comprehensive knowledge on the structure of fibrinogen and fibrin and to establish the molecular mechanisms of their multiple interactions with various proteins and cell types. Significant progress has been made in understanding the fibrinogen structure in the last decade. However, the structural organization of the C-terminal portions of its A1 chains (1C- domains) and the N-terminal portions of its B2 chains (B2N-domains) is still unclear. Numerous data suggest that the conformation of the 1C-domains in fibrinogen may differ from that in fibrin, in which they form 1C- polymers and become highly reactive. Still, the molecular mechanisms of 1C-polymer formation remain to be established. This application focuses on the 1C-domains, which are involved in fibrin assembly, fibrin- dependent fibrinolysis, angiogenesis, atherogenesis, and renal amyloidosis. The first Specific Aim is to establish the NMR solution structure of the isolated fibrinogen 1C-domain, to elucidate the structural organization of this domain in fibrin 1C-polymers, and to clarify the structural basis for hereditary renal amyloidosis caused by mutations in the 1C-domain region. The second Specific Aim is to elucidate the molecular mechanisms of the intra- and intermolecular interactions of the 1C-domains, which are responsible for the formation of physiologically active 1C-polymers in fibrin, and for the pathological consequences of their acquired and congenital defects.
These aims will be accomplished by preparation of recombinant 1C-domain fragments and their truncated variants, as well as recombinant and proteolitically obtained fragments that represent the central region of fibrin(ogen) with the (B)2N-domains, and studying their structure and numerous interactions by biochemical and biophysical methods. The proposed studies will generate basic knowledge that will have an impact on our understanding of the molecular mechanisms underlying formation of fibrin clots in normal and pathological conditions and ability to control the above mentioned fibrin-dependent processes. Because acquired and congenital defects in the 1C-domains may cause severe pathological consequences, including hemorrhage, various thrombotic disorders, and renal amyloidosis, the proposed studies will also further clarify the molecular mechanisms underlying these pathologies, and thereby contribute to the development of their therapies.
The proposed studies will generate basic knowledge about the structure and function of individual domains of fibrin(ogen), the major component of the blood coagulation cascade. Such knowledge is required to better understand and control the molecular mechanisms underlying the fibrin(ogen)-dependent processes, such as blood clotting, fibrinolysis, inflammation, and angiogenesis. Because congenital and acquired defects in these domains may cause severe pathological consequences, including hemorrhage, various thrombotic disorders, and renal amyloidosis, the proposed studies will further clarify the molecular mechanisms underlying these pathologies, and thereby contribute to the development of their therapies.
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