IN-VITRO ASSEMBLY OF THE COMPONENT CHAINS OF FIBRINOGEN REQUIRES ENDOPLASMIC-RETICULUM FACTORS

Human fibrinogen (340 kDa) is a dimer, with each identical half-molecu le composed of three different polypeptides (A alpha, 66 kDa; B beta, 55 kDa; and gamma, 48 kDa). To understand the mechanisms of chain asse mbly, a coupled in vitro transcription translation system capable of a ssembling fibrinogen chains was developed, Fibrinogen chain assembly w as assayed in an expression system coupled to rabbit reticulocyte lysa te in the presence or absence of dog pancreas microsomal membranes, Fi brinogen chain assembly required microsomal membranes and oxidized glu tathione. Co-expression of two of the chains, B beta and gamma or A al pha and gamma, yielded free chains and two-chain complexes, Unlike com binations of A alpha with gamma and B beta with gamma, co expression o f A alpha and B beta did not form a single two-chain complex but produ ced a mixture of two-chain complexes. Co-expression of all three chain s yielded free chains, two-chain complexes, and higher molecular weigh t complexes that corresponded to a half-molecule and to fully formed f ibrinogen. Upon treatment of this mixture with thrombin and factor XII Ia, a gamma .gamma dimer, similar to that obtained from crosslinked hu man fibrin, was produced, indicating that properly folded fibrinogen w as formed in vitro. Molecular chaperones may participate in fibrinogen assembly, since antibodies to resident proteins of the endoplasmic re ticulum (BiP, Hsp90, protein disulfide isomerase, and calnexin) co-pre cipitated the chaperones together with nascent fibrinogen chains and c omplexes.