. The overall goal of the proposed studies is to define the molecular mechanism by which the Staphylococcus aureus protein, staphylocoagulase (SC), activates blood coagulation, and its role in the pathology of acute bacterial endocarditis. Thermodynamic, kinetic, and structural approaches are proposed to address major gaps in the understanding of the unique mechanism by which SC activates prothrombin (ProT) non-proteolytically and forms an SOProT complex that specifically converts fibrinogen (Fbg) into fibrin. The studies focus on 6 areas: (1) Structure-function relationships governing the critical SC N-terminal insertion that induces conformational ProT activation will be investigated to characterize the specificity of the N-terminal binding cleft, and to evaluate the hypothesis that insertion is a reversible equilibrium process that can induce different levels of activation. (2) The pathway of individual binding and conformational change events in the molecular mechanism of conformational activation of ProT by SC will be defined in fluorescence- and activity-monitored rapid-reaction kinetic studies. (3) The structure-function relationships between the two modes of Fbg interactions, as a substrate of the active SC?ProT complex bound via N-terminal SC domains, and as a Fbg adhesion protein through C-terminal SC repeat sequences will be investigated in binding, kinetic, and crystallographic studies. (4) Mutagenesis-based structure-function studies will test the hypothesis that autolysis loop residues of human and bovine ProT control the species specificity of SC-ProT activation. (5) The mechanisms through which proteins of the newly discovered zymogen activator and adhesion p/otein (ZAAP) family of SC homologs activate zymogens, cleave protein substrates, and bind to plasma and extracellular matrix proteins will be investigated. (6) The mechanism of inhibition of SC-ProT activation by antibodies specific for the N-terminal sequence will be investigated as a potential therapeutic approach for treating endocarditis. The studies proposed will provide a detailed understanding of the molecular mechanisms of SC-activated blood coagulation. This may enable the design of new mechanism-based therapeutic agents for treating endocarditis. Extension of the studies to other ZAAP family members will provide new insight into the roles of these novel proteins that target the human blood coagulation system in the pathogenesis of life-threatening staphylococcal and streptococcal diseases.

Public Health Relevance

. Minor injury to the surface of a heart valve in combination with bacteria in the blood stream can cause acute bacterial endocarditis, an infection that is alarmingly, 25-40% fatal despite antibiotic therapy. The common bacterium, S. aureus, secretes staphylocoagulase, a protein that activates human blood clotting and thereby propagates growths on heart valves that contain bacteria enmeshed by blood clots. Biochemical research is proposed to define the molecular mechanisms of the pathogenic activity of staphylocoagulase, and ultimately to design new drugs to inhibit its role in endocarditis.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HL071544-10
Application #
8212433
Study Section
Special Emphasis Panel (NSS)
Program Officer
Sarkar, Rita
Project Start
2003-01-01
Project End
2013-06-30
Budget Start
2012-02-01
Budget End
2013-06-30
Support Year
10
Fiscal Year
2012
Total Cost
$341,921
Indirect Cost
$119,171
Name
Vanderbilt University Medical Center
Department
Pathology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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