The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies.
Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.
Longstaff
Colin
C
National Institute for Biological Standards and Control, South Mimms, UK. colin.longstaff@nibsc.hpa.org.uk
Thelwell
Craig
C
Williams
Stella C
SC
Silva
Marta M C G
MM
Szabó
László
L
Kolev
Krasimir
K
D005337:Fibrin / Q000378:metabolism* / Q000648:ultrastructure*; D005342:Fibrinolysis / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D010958:Plasminogen / Q000378:metabolism; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D005337:Fibrin; D010958:Plasminogen; D013917:Thrombin; D010959:Tissue Plasminogen Activator