The current proposal is based on discovery of a new fibrinogen gene exon (exon VI) that encodes an extension of the fibrinogen alpha chain. The extended alpha chain (alpha/E) is incorporated primarily into a larger than normal fibrinogen molecule that represents a long overlooked, naturally occurring fibrinogen species. Both the novel alpha/E-and the common alpha-fibrinogen are symmetrically structured molecules of the formula (alpha/E beta gamma)2 and (alpha beta gamma)2, respectively, the proposed studies will ascertain whether these two fibrinogen forms are functionally different and, if so, to elucidate the structural basis for the difference(s). Towards this end, the forms will be compared in terms of their: susceptibility to thrombin and plasmin; fibrinopeptide release; clotting time; gamma chain crosslinking; calcium binding; alpha polymer formation; degree of alpha/E-gamma crosslinking; clot permeation and turbidity; clot structure of confocal laser 3-D microscopy; and electron microscopy. Peptide analysis and site-directed mutagenesis will complement other techniques to define the unique exon VI-derived features of alpha/E sites of cleavage during fibrin formation; polymerization sites; disulfide bridges; and N-glycosylation sites and carbohydrate structure. These experiments will test the central hypothesis that some functions currently ascribed to alpha-fibrinogen actually derive from the presence of alpha/E-fibrinogen and may reveal new aspects of fibrinogen metabolism. They will provide evidence against which to evaluate the variant's postulated tetranodular structure and the role of disulfide bridges involving its extra domain in driving selective assembly of symmetrical hexamers. The structure/function studies will inform and be informed by exploration of the physiology of (alpha/E beta gamma)2. One focus will be development of model systems enriched for one type of fibrinogen of the other for comparison of parameters relating to wound healing. Other physio-logical studies will include: developing a ELISA for quantitation of alpha/E levels in human plasma samples from healthy and diseased individuals; immunohistochemical and immunoelectrophoretic evaluation of vascular and extravascular human tissues for presence of alpha/E-containing fibrinogen/fibrin; and examination of specific cell binding to the extra domain. The multifaceted approach exploits material, reagents, and concentrated fibrinogen expertise at the NY Blood Center and is geared to providing a broad foundation for understanding the relative roles of the common form and novel variant of this clotting protein in health and disease.
