Investigation of residues in the fibrin(ogen) γ chain involved in tissue plasminogen activator binding and plasminogen activation

In order to characterize tissue plasminogen activator (t-PA) binding to γ-chain residues in fibrinogen, we generated variant fibrinogens substituting alanine for γD316, γD318, γD320, and γK321. We measured thrombin-catalyzed polymerization and found normal polymerization with γK321A, no polymerization with γD316A, and, as reported by Lounes et al. in 2002, impaired polymerization with γD318A and γD320A. 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 γK321A, seven-fold for γD320A and 10-fold for γD316A and γD318A. 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.