Kelly A. Hogan, Ph.D.

Dissertation research performed under the direction of Susan T. Lord

ABSTRACT
    Fibrinogen is the key structural protein involved in blood coagulation and consists of six polypeptide chains (Aa, Bb, g)2. The dysfibrinogen Vlissingen/Frankfurt IV (V/F IV) is characterized as a deletion of Asn319 and Asp320 from the C-terminus of the g-chain of fibrinogen.  This dysfibrinogen, which was identified in several family members who are all heterozygous for the in-frame 6 bp deletion, is associated with both venous and arterial thrombosis. Here, we describe the generation of both human recombinant fibrinogen and gene-targeted mice containing the V/F IV deletion.
    The recombinant fibrinogen did not polymerize; this loss of normal polymerization was due to the lack of “A-a” interactions.  Moreover, functions associated with the C-terminal end of the g chain, such as platelet aggregation and Factor XIII cross-linking, were also disrupted, suggesting that this deletion of two residues affected the overall structure of the C-terminal domain of the g-chain. We found that polymerization of a 1:1 mixture of variant and normal fibrinogen was the same as polymerization of a 1:1 mixture of buffer and normal fibrinogen, indicating that the variant molecules did not incorporate into a normal clot.  Because there are two copies of each polypeptide per molecule, we concluded that the heterozygous V/F IV plasma fibrinogen is a mix of homodimers and heterodimers, such that the incorporation of heterodimers into the fibrin clot impairs polymerization.  We suggest that incorporation of heterodimers can induce clinical symptoms.
    Analysis of fibrinogen from V/F IV gene-targeted mice demonstrated that the calcium binding site and the “a” are disrupted by the deletion, similar to the human protein. The heterozygous mice provide the first opportunity to examine the association of thrombophilia and dysfibrinogenemia on a controlled genetic background. To date, these mice do not have overt thrombotic disease, but further examination is required. In contrast, the homozygous mice have a bleeding phenotype, evident within the first few days of birth. Unexpectedly, the mutation affected the concentration of fibrinogen in plasma, making this the first hypodysfibrinogenemia modeled in mice.