Investigation of residues in the fibrin(ogen) gamma chain involved in tissue plasminogen activator binding and plasminogen activation.
In order to characterize tissue plasminogen activator (t-PA) binding to gamma-chain residues in fibrinogen, we generated variant fibrinogens substituting alanine for gamma D316, gamma D318, gamma D320, and gamma K321. We measured thrombin-catalyzed polymerization and found normal polymerization with gamma K321A, no polymerization with gamma D316A, and, as reported by Lounes et al. in 2002, impaired polymerization with gamma D318A and gamma D320A. We measured t-PA binding in a solid-phase assay, and t-PA activity by the generation of plasmin. Comparing normal fibrin with fibrinogen, we found a seven-fold increase in binding and a two-fold increase in activity. Binding to all variant fibrinogens was the same as normal. In contrast, t-PA binding to all variant fibrins was weaker than binding to normal fibrin, 2.5-fold for gamma K321A, seven-fold for gamma D320A and 10-fold for gamma D316A and gamma D318A. Plasmin generation in the presence of variant fibrinogens was similar, although not identical, to normal, and plasmin generation in the presence of variant fibrins was impaired for the Asp to Ala variants. As the three variants with the weakest t-PA binding and least activity also showed impaired polymerization, our results support previous findings demonstrating the DD:E complex, found in the normal fibrin polymer, is necessary for the fibrin enhanced binding of t-PA and activation of plasminogen.
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Susan E
SE
Wilhelm
Karim C
KC
Lounes
Susan T
ST
Lord
D019943:Amino Acid Substitution; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D005341:Fibrinolysin / Q000096:biosynthesis; D005344:Fibrinopeptide A / Q000032:analysis; D005345:Fibrinopeptide B / Q000032:analysis; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
C027539:fibrinopeptides gamma; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin