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.
National Institute for Biological Standards and Control, South Mimms, UK. colin.longstaff@nibsc.hpa.org.uk
Colin
C
Longstaff
Craig
C
Thelwell
Stella C
SC
Williams
Marta M C G
MM
Silva
László
L
Szabó
Krasimir
K
Kolev
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