title: The polymerization pocket "a" within the carboxyl-terminal region of the gamma chain of human fibrinogen is adjacent to but independent from the calcium-binding site.
abstract: The carboxyl-terminal region of the gamma 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 amino acids Val143 to Val411 (rFbggammaC30) or Val143 to Leu427 (gamma'C30) from the carboxyl end of the gamma or gamma' 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. rFbggammaC30 and gamma'C30 were both readily cross-linked by factor XIIIa, but only rFbggammaC30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, gammaC30-Q329R and gammaC30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbggammaC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbggammaC30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbggammaC30 and gamma'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 amino acids Gln329 and Asp364 play a crucial role in fibrin polymerization.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350, USA. cote@u.washington.edu
authors.forename: H C
authors.initials: HC
authors.lastname: Côté
authors.forename: K P
authors.initials: KP
authors.lastname: Pratt
authors.forename: E W
authors.initials: EW
authors.lastname: Davie
authors.forename: D W
authors.initials: DW
authors.lastname: Chung
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001704:Biopolymers; D002118:Calcium / Q000378:metabolism*; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008297:Male; D010974:Platelet Aggregation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: D001704:Biopolymers; D011994:Recombinant Proteins; D005340:Fibrinogen; D013917:Thrombin; D005341:Fibrinolysin; D002118:Calcium