Effect of zymogen domains and active site occupation on activation of prothrombin by von Willebrand factor-binding protein.

Kroh HK, Bock PE
J Biol Chem. 2012 287 (46): 39149-57

PMID: 23012355 · PMCID: PMC3493955 · DOI:10.1074/jbc.M112.415562

Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.

MeSH Terms (20)

Binding, Competitive Blood Coagulation Carrier Proteins Catalytic Domain Enzyme Precursors Fibrin HEK293 Cells Humans Kinetics Platelet Membrane Glycoproteins Protein Binding Protein Conformation Protein Structure, Tertiary Prothrombin Recombinant Proteins Staphylococcus aureus Thermodynamics Thrombin Virulence Factors von Willebrand Factor

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