title: A high affinity interaction of plasminogen with fibrin is not essential for efficient activation by tissue-type plasminogen activator.
abstract: Fibrin (Fn) enhances plasminogen (Pg) activation by tissue-type plasminogen activator (tPA) by serving as a template onto which Pg and tPA assemble. To explore the contribution of the Pg/Fn interaction to Fn cofactor activity, Pg variants were generated and their affinities for Fn were determined using surface plasmon resonance (SPR). Glu-Pg, Lys-Pg (des(1-77)), and Mini-Pg (lacking kringles 1-4) bound Fn with K(d) values of 3.1, 0.21, and 24.5 μm, respectively, whereas Micro-Pg (lacking all kringles) did not bind. The kinetics of activation of the Pg variants by tPA were then examined in the absence or presence of Fn. Whereas Fn had no effect on Micro-Pg activation, the catalytic efficiencies of Glu-Pg, Lys-Pg, and Mini-Pg activation in the presence of Fn were 300- to 600-fold higher than in its absence. The retention of Fn cofactor activity with Mini-Pg, which has low affinity for Fn, suggests that Mini-Pg binds the tPA-Fn complex more tightly than tPA alone. To explore this possibility, SPR was used to examine the interaction of Mini-Pg with Fn in the absence or presence of tPA. There was 50% more Mini-Pg binding to Fn in the presence of tPA than in its absence, suggesting that formation of the tPA-Fn complex exposes a cryptic site that binds Mini-Pg. Thus, our data (a) indicate that high affinity binding of Pg to Fn is not essential for Fn cofactor activity, and (b) suggest that kringle 5 localizes and stabilizes Pg within the tPA-Fn complex and contributes to its efficient activation.
authors.affiliation: Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Thrombosis and Atherosclerosis Research Institute, Hamilton, Ontario L8L 2X2, Canada.
authors.forename: Paul Y
authors.initials: PY
authors.lastname: Kim
authors.forename: Long D
authors.initials: LD
authors.lastname: Tieu
authors.forename: Alan R
authors.initials: AR
authors.lastname: Stafford
authors.forename: James C
authors.initials: JC
authors.lastname: Fredenburgh
authors.forename: Jeffrey I
authors.initials: JI
authors.lastname: Weitz
mesh_terms: D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D005337:Fibrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018082:Kringles; D010958:Plasminogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D020349:Surface Plasmon Resonance; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator