Fibrinogen Assembly and Secretion

Human fibrinogen is a homodimer composed of three different (Aα, Bβ, γ) polypeptide chains. The chains are linked by 29 inter- and intrachain disulfide bonds. Each half-molecule has 6 intrachain disulfide bonds, which form loops in the carboxyl-terminal region of each of the chains. Aα chain has one disufide loop (Cys⁴⁴²-Cys⁴⁷²), Bβ has three (Cys²⁰¹-Cys²⁸⁶, Cys²¹¹-Cys²⁴⁰, and Cys³⁹⁴-Cys⁴⁰⁷), and γ has two loops (Cys¹⁵³-Cys¹⁸² and Cys³²⁶-Cys³³⁹). The intrachain loops are conserved in fibrinogens of different species. We changed, by site-directed mutagenesis, the cysteines, which form the intrachain loops, to serine or alanine. Fibrinogen chain assembly and secretion was determined in transiently transfected COS cells expressing two normal and a mutant fibrinogen chain. In the Bβ and γ chains, disruption of the disulfide loops closest to the “coiled-coil” region (Cys^Bβ211-Cys²⁴⁰, Cys^Bβ201-Cys²⁸⁶, and Cys^γ153-Cys¹⁸²) abolished chain assembly and secretion, indicating that the disulfide loops closest to the coiled-coil region are essential for chain assembly. By contrast, preventing formation of the disulfide loops, which are toward the carboxyl termini of each of the chains, had different effects. Disruption of the single Aα disulfide loop had no effect, as did disruption of BβCys³⁹⁴-Cys⁴⁰⁷. However, disruption of Cys^γ326-Cys³³⁹, which is similar in size and location to Cys^Bβ394-Cys⁴⁰⁷, allowed chain assembly to occur, but the assembled chains were not secreted.