FIBRINOGEN--MECHANISM OF ASSEMBLY

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DESCRIPTION: The objectives of this renewal proposal are 1) to determine the intracellular mechanisms by which the component chains of fibrinogen are assembled into a functional biologically active protein and 2) to map domains which play important roles in fibrin polymerization and crosslinking using recombinant engineered fibrinogen. To accomplish these objectives, the Applicant will 1) use a reconstituted ER vesicle system to characterize ER factors involved in fibrinogen assembly and to determine the association of fibrinogen precursors with such factors, 2) express deletion and substitution mutants of each of the fibrinogen chains in COS cells to map the critical domains necessary for assembly and secretion, 3) perform pulse-chase studies in HepG2 cells to define the sequential steps in fibrinogen assembly, 4) mutagenize secretion factors in a yeast expression system to study fibrinogen assembly and secretion, and 5) mutagenize recombinant fibrinogen in putative crosslinking and polymerization sites to define structure-function relationships.