The broad goal of the proposed studies is to define the molecular mechanism of the activation of human blood coagulation by the S. aureus protein, staphylocoagulase (SC), and the role of the mechanism in the pathogenesis of endocarditis. SC binds tightly to human prothrombin (Pro) and induces formation of a functional catalytic site in the zymogen without the usual strictly required peptide bond cleavages. This unique conformational activation mechanism is hypothesized to involve initial encounter of SC and Pro, followed by activation of the catalytic site and occupation of regulatory proexosite I in two or more discrete conformational changes. The mechanism may involve conformational linkage between proexosite I occupation and catalytic site activation, stabilization by high affinity binding of SC to the active conformation, and is unlikely to require insertion of the SC amino-terminus into a binding pocket in the Pro catalytic domain. The mechanism underlying the unique specificity of SC-Pro to convert fibrinogen (Fbg) to fibrin (Fbn) is hypothesized to bypass and inhibit the normal reactions of Pro activation. This mechanism is central to the propagation of platelet- Fbn-bacteria vegetations on heart valves in endocarditis. Fbg clofting activity of the SC-Pro/T complexes is hypothesized to involve specific recognition of Fbg as a substrate through expression of a Fbg-binding exosite on the SC-Pro/T complexes, in addition to changes in catalytic site specificity. Biochemical, biophysical, and structural approaches employing novel active site-labeled fluorescent derivatives of Pro are proposed to test hypotheses for conformational activation of Pro by SC and the basis of its specificity for conversion of Fbg to Fbn.
Specific Aims are: (1) To determine the thermodynamic mechanism of conformational activation of Pro by SC; (2) To define the kinetic pathway of individual molecular events in conformational activation; (3) To elucidate the mechanism of specific recognition of Fbg as a substrate of SC-Pro/T complexes; and (4) To determine the three dimensional structures of SCI-327 bound to Pro/T species. The proposed studies are of fundamental significance in understanding how SC can circumvent the otherwise strict requirement for peptide bond cleavage in serine proteinase zymogen activation. The studies will provide new insight into the role of activation of Pro by SC in the pathogenesis of endocarditis and may ultimately allow therapy adjunctive to antibiotics to be developed based on inhibition of SC-activated blood coagulation.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Sarkar, Rita
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Vanderbilt University Medical Center
Schools of Medicine
United States
Zip Code
Kroh, Heather K; Bock, Paul E (2012) Effect of zymogen domains and active site occupation on activation of prothrombin by von Willebrand factor-binding protein. J Biol Chem 287:39149-57
Kroh, Heather K; Panizzi, Peter; Tchaikovski, Svetlana et al. (2011) Active site-labeled prothrombin inhibits prothrombinase in vitro and thrombosis in vivo. J Biol Chem 286:23345-56
Berny-Lang, M A; Aslan, J E; Tormoen, G W et al. (2011) Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells. Phys Biol 8:015014
Panizzi, Peter; Nahrendorf, Matthias; Figueiredo, Jose-Luiz et al. (2011) In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation. Nat Med 17:1142-6
Berny, Michelle A; Munnix, Imke C A; Auger, Jocelyn M et al. (2010) Spatial distribution of factor Xa, thrombin, and fibrin(ogen) on thrombi at venous shear. PLoS One 5:e10415
Kroh, Heather K; Panizzi, Peter; Bock, Paul E (2009) Von Willebrand factor-binding protein is a hysteretic conformational activator of prothrombin. Proc Natl Acad Sci U S A 106:7786-91
Munnix, Imke C A; Kuijpers, Marijke J E; Auger, Jocelyn et al. (2007) Segregation of platelet aggregatory and procoagulant microdomains in thrombus formation: regulation by transient integrin activation. Arterioscler Thromb Vasc Biol 27:2484-90
Friedrich, Rainer; Panizzi, Peter; Kawabata, Shun-Ichiro et al. (2006) Structural basis for reduced staphylocoagulase-mediated bovine prothrombin activation. J Biol Chem 281:1188-95
Panizzi, Peter; Friedrich, Rainer; Fuentes-Prior, Pablo et al. (2006) Fibrinogen substrate recognition by staphylocoagulase.(pro)thrombin complexes. J Biol Chem 281:1179-87
Panizzi, Peter; Friedrich, Rainer; Fuentes-Prior, Pablo et al. (2006) Novel fluorescent prothrombin analogs as probes of staphylocoagulase-prothrombin interactions. J Biol Chem 281:1169-78

Showing the most recent 10 out of 12 publications