The polymerization pocket 'a' within the carboxyl-terminal region of the γ chain of human fibrinogen is adjacent to but independent from the calcium- binding site

The carboxyl-terminal region of the γ chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross- linking, and binding to the platelet fibrin(ogen) receptor. Protein fragments encoding amine acids Val¹⁴³ to Val⁴¹¹ (rFbgγC30) or Val¹⁴³ to Leu⁴²⁷ (γ'C30) from the carboxyl end of the γ or γ' chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbgγC30 and γ'C30 were both readily cross-linked by factor XIIIa, but only rFbgγC30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, γC30-Q329R and γC30- D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbgγC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbgγC30 inhibited (desAA)fibrin polymerization in a dose- dependent manner, while the two mutant forms did not. Similarly, rFbgγC30 and γ'C30 were protected from plasmin degradation by the presence of Ca²⁺ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca²⁺-binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket 'a', which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amine acids Gln³²⁹ and Asp³⁶⁴ play a crucial role in fibrin polymerization.