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Title page for ETD etd-12162009-101657

Type of Document Dissertation
Author Kroh, Heather K.
URN etd-12162009-101657
Title Molecular Mechanism of Prothrombin Activation by von Willebrand Factor-Binding Protein
Degree PhD
Department Pathology
Advisory Committee
Advisor Name Title
Richard L. Hoover Committee Chair
Paul E. Bock Committee Member
  • zymogens
  • coagulation
  • serine proteinases
  • enzymology
Date of Defense 2009-12-07
Availability unrestricted
The studies in this dissertation defined the molecular mechanism of prothrombin (ProT) activation by a recently-identified Staphylococcus aureus exoprotein, von Willebrand factor-binding protein (VWbp). We demonstrated that VWbp activates ProT conformationally in a mechanism requiring insertion of its NH2-terminal Val1-Val2 residues into the NH2-terminal binding cleft on ProT. Such non-proteolytic activation of ProT has been previously identified for only one other protein, staphylocoagulase (SC), which shares structural homology with VWbp. Kinetic studies of ProT activation by VWbp showed that it activates ProT by a unique substrate-dependent, hysteretic mechanism. The NH2-terminal domains of VWbp (VWbp(1-263)) bind weakly to ProT, forming an inactive complex that is fully activated through a slow conformational change mediated by binding of tripeptide substrates or the physiological substrate, fibrinogen (Fbg). The activity of VWbp(1-474) is increased compared to VWbp(1-263), supporting a contribution from the COOH-terminal region in ProT activation. The role of allostery in regulating binding and activation of ProT by VWbp was also demonstrated from the effect of the fragment 1 and 2 domains of ProT on VWbp function, as well as the cofactor effect of substrate on complex formation. Specifically, loss of fragment 1 of ProT or active-site occupation enhances affinity for both VWbp(1-263) and VWbp(1-474), suggesting a regulatory role for fragment 1 and verifying the substrate dependence of the activating conformational change. Further, the activation state of anion-binding (pro)exosite I influences ProT-VWbp binding. This pattern is consistent with that seen during the physiological pathway of ProT activation, where high-affinity exosite expression is linked to formation of a competent active site. Slow, progressive loss of the fragment domains of ProT*•VWbp occurs through autocatalysis, reinforcing the potential role of these domains in ProT activation during S. aureus infections. Together, these findings reveal a pattern of regulation of ligand recognition similar to that of normal thrombin, but with strictly defined recognition of Fbg by the ProT*•VWbp complex. The restricted substrate specificity provided by the hysteretic activation mechanism may allow VWbp to serve as a virulence factor in propagating the growth of fibrin-rich vegetations during acute infective endocarditis.
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