Analysis of engineered fibrinogen variants suggests that an additional site mediates platelet aggregation and that "B-b" interactions have a role in protofibril formation.
The C-terminal domain of the fibrinogen gamma-chain includes multiple functional sites that have been defined in high-resolution structures and biochemical assays. Calcium binds to this domain through the side chains of gammaD318 and gammaD320 and the backbone carbonyls of gammaF322 and gammaG324. We have examined variant fibrinogens with alanine at position gamma318 and/or gamma320 and found that calcium binding, fibrin polymerization, and fibrinogen-mediated platelet aggregation, but not FXIIIa-catalyzed cross-linking, were abnormal. When measured by turbidity, thrombin-catalyzed polymerization was severely reduced, and batroxobin-catalyzed polymerization was completely obliterated. Moreover, thrombin-catalyzed polymerization was abolished by the peptide GHRP, which binds to the polymerization site in the beta-chain but does not inhibit polymerization of normal fibrinogen. ADP-induced platelet aggregation was also severely impaired. In contrast, as measured by SDS-PAGE, FXIIIa introduced cross-links between gamma-chains for all three variants, as expected if the gamma-chain C-terminal sites were normal. In addition, binding of the monoclonal antibody 4A5, which recognizes the C-terminal residues, was not different from normal. These data suggest two specific conclusions: (1) a site in the gamma-module other than the C-terminus is critical for platelet aggregation and (2) "B-b" interactions have a role in protofibril formation.
Lounes
Karim C
KC
Department of Pathology and Laboratory Medicine and Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-7525.
Ping
Lifang
L
Gorkun
Oleg V
OV
Lord
Susan T
ST
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics; D001666:Binding Sites, Antibody / Q000235:genetics; D016466:CHO Cells; D002850:Chromatography, Gel; D006224:Cricetinae; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D005341:Fibrinolysin / Q000378:metabolism; D005344:Fibrinopeptide A / Q000235:genetics / Q000378:metabolism; D005345:Fibrinopeptide B / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009391:Nephelometry and Turbidimetry; D010974:Platelet Aggregation / Q000235:genetics / Q000502:physiology; D011108:Polymers / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D011108:Polymers; D011994:Recombinant Proteins; C027539:fibrinopeptides gamma; D005344:Fibrinopeptide A; D001224:Aspartic Acid; D005345:Fibrinopeptide B; D005340:Fibrinogen; D005341:Fibrinolysin; D000409:Alanine