Human fibrinogen γ-module comprising residues γ148-411 was expressed in Escherichia coli and refolded in vitro. Differential scanning calorimetry revealed that in addition to the two previously identified independently folded thermolabile domains, one in each half of the module, the γ-module also contains one or two thermostable domains that melt above 65 °C. To localize the latter, an NH2-terminal 6-kDa fragment was prepared by limited proteolysis of the recombinant γ-module. It melted at high temperature, indicating that this portion is folded into a compact structure that represents a thermostable domain, also identified in the proteolytic fibrinogen fragment D1 which contains the natural γ-module. Thus the NH2- terminal half of the γ-module forms two domains, a thermostable one and a thermolabile one, leaving the rest of the module to be responsible for the formation of the other one or two domains. The thermal stability of some domains was lower in the recombinant γ-module than in its natural counterpart in D1, reflecting most probably the loss of interactions with neighboring domains; however, the major functional sites were essentially preserved. The module bound Ca2+ and was stabilized by it against denaturation and proteolysis. It inhibited fibrin polymerization and was efficiently cross-linked by factor XIIIa. The γ-module supported adhesion of platelets via their GP IIbIIIa (α(IIb)β3) receptor in the same manner as D1 fragment. It also supported the adhesion of α(M)β2- (Mac-1-) transfected cells and in the fluid phase was more effective than D1 as an inhibitor of that adhesion, suggesting that the Mac-1 binding site is better exposed.
ASJC Scopus subject areas