title: Regulation of fibrinolysis by C-terminal lysines operates through plasminogen and plasmin but not tissue-type plasminogen activator.
abstract: BACKGROUND
  Binding of tissue-type plasminogen (Pgn) activator (t-PA) and Pgn to fibrin regulates plasmin generation, but there is no consistent, quantitative understanding of the individual contribution of t-PA finger and kringle 2 domains to the regulation of fibrinolysis. Kringle domains bind to lysines in fibrin, and this interaction can be studied by competition with lysine analogs and removal of C-terminal lysines by carboxypeptidase B (CPB).
  
  
  METHODS
  High-throughput, precise clot lysis assays incorporating the lysine analog tranexamic acid (TA) or CPB and genetically engineered variants of t-PA were performed. In particular, wild-type (WT) t-PA (F-G-K1-K2-P) and a domain-switched variant K1K1t-PA (F-G-K1-K1-P) that lacks kringle 2 but retains normal t-PA structure were compared to probe the importance of fibrin lysine binding by t-PA kringle 2.
  
  
  RESULTS
  WT t-PA showed higher rates of fibrinolysis than K1K1t-PA, but the inhibitory effects of TA or CPB were very similar for WT t-PA and the variant t-PA (< 10% difference). Urokinase plasminogen activator (u-PA)-catalyzed fibrinolysis was also inhibited by TA, even though Pgn activation could be stimulated. Fibrin treated with factor XIIIa (FXIIIa) generates crosslinked degradation products, but these did not affect the results obtained with WT t-PA and K1K1t-PA.
  
  
  CONCLUSIONS
  t-PA kringle 2 has a minor role in the initial interaction of t-PA and fibrin, but stimulation of fibrinolysis by C-terminal lysines (or inhibition by carboxypeptidases or TA) operates through Pgn and plasmin binding, not through t-PA. This is also true when fibrin is crosslinked by treatment with FXIIIa.
authors.affiliation: Biotherapeutics Group, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, UK.
authors.forename: M M C G
authors.initials: MM
authors.lastname: Silva
authors.forename: C
authors.initials: C
authors.lastname: Thelwell
authors.forename: S C
authors.initials: SC
authors.lastname: Williams
authors.forename: C
authors.initials: C
authors.lastname: Longstaff
mesh_terms: D001665:Binding Sites; D043424:Carboxypeptidase B / Q000737:chemistry; D005337:Fibrin / Q000737:chemistry; D005341:Fibrinolysin / Q000737:chemistry*; D005342:Fibrinolysis; D006801:Humans; D007700:Kinetics; D018082:Kringles; D008239:Lysine / Q000737:chemistry*; D010958:Plasminogen / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D014148:Tranexamic Acid / Q000737:chemistry; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry
chemical_list: D014148:Tranexamic Acid; D005337:Fibrin; D010958:Plasminogen; D043424:Carboxypeptidase B; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine