Novel Fluorescent Prothrombin Analogs as Probes of Staphylocoagulase-Prothrombin Interactions

Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin

“…A more complex mechanism involving significant activity for the inactive complex failed to produce consistent fits or reasonable parameters. The possibility that autocatalytic cleavage of ProT to Pre 1, previously reported for SC (6) and SC(1-325) (20), accounted for the hysteresis was evaluated by Western blot analysis under conditions identical to those used for collecting the progress curves (see SI). At the highest VWbp(1-263) concentration (10 M) in the kinetic studies, 23 Ϯ 9% (mean and range) of ProT was converted into Pre 1 over the whole time course for both substrates.…”

Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes

“…This is substantiated by our solution studies demonstrating the protection of the Fbg clotting activity of SC-(1-325)⅐(pro)thrombin from inhibition by the exosite I-specific ligand, Hir-(54 -65)(SO 3 Ϫ ) and displacement of a fluorescein-labeled derivative of the peptide by SC-(1-325) and SC D2 binding (3,4). This puzzling observation implies that Fbg presentation to the thrombin active site is taken over by the bacterial cofactor mechanistically similar to interactions of substrate Pg with SK and SAK.…”

“…Fbg (fraction I; Sigma) was purified further by chromatography on lysine-Sepharose to remove plasminogen, gelatin-Sepharose to remove fibronectin, and Sephacryl HR 400 to remove aggregates. Protein concentrations were determined by absorbance at 280 nm with the following absorption coefficients ((mg/ml) Ϫ1 cm Ϫ1 ) and molecular weights: ProT, 1.47, 71,600; thrombin, 1.74, 36,600; Fbg, 1.54, 340,000; and Met-SC-(1-325) and SC-(1-325), 1.00, 38,000 (4,22,23). Fluorescent active site-labeled ProT analogs were prepared and characterized as described in the preceding paper (4).…”

“…We recently solved the crystal structure of the immediate human thrombin zymogen precursor, prethrombin 2 (Pre 2), bound to an SC fragment, SC-(1-325), which possesses full ProT activator and Fbg clotting activity (3,4). The structure demonstrates that SC activates ProT conformationally by a mechanism known as "molecular sexuality" (5).…”

Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism

“…Active site labeling of the conformationally activated zymogens plasminogen and prothrombin (27,57) provides the advantage of studying ligand binding uncoupled from catalytic activity. Introducing the fluorescent label 5-fluorescein ([5F]) and a tripeptide chloromethyl ketone in the Pm active site (FFR-Pm) does not affect the affinity for SK, whereas labeled [Glu]Pg and [Lys]Pg analogs bind SK with ϳ5-fold lower affinity than the native proteins (26,48,58).…”