Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

Factor XII (FXII) is the zymogen of a plasma protease (FXIIa) that contributes to bradykinin generation by converting prekallikrein to the protease plasma kallikrein (PKa). FXII conversion to FXIIa by autocatalysis or PKa-mediated cleavage is enhanced when the protein binds to negatively charged "surfaces" such as polymeric orthophosphate. FXII is comprised of non-catalytic (heavy chain) and catalytic (light chain) regions. The heavy chain promotes FXII surface-binding and surface-dependent activation, but restricts activation when FXII is not surface-bound. From the N-terminus, the heavy chain contains fibronectin type II (FN2), epidermal growth factor-1 (EGF1), fibronectin type I (FN1), EGF2, and kringle (KNG) domains, and a proline-rich region (PRR). It shares this organization with its homolog, pro-hepatocyte growth factor activator (Pro-HGFA). To study the importance of heavy chain domains to FXII function, we prepared FXII with replacements of each domain with corresponding Pro-HGFA domains, and tested them in activation and activity assays. EGF1 is required for surface-dependent FXII autoactivation and surface-dependent prekallikrein activation by FXIIa. KNG and FN2 are important for limiting FXII activation in the absence of a surface by a process that may require interactions between a lysine/arginine binding site on KNG and basic residues elsewhere on FXII. This interaction is disrupted by the lysine analog Ɛ-aminocaproic acid. A model is proposed in which an Ɛ-aminocaproic acid-sensitive interaction between the KNG and FN2 domains maintains FXII in a conformation that restricts activation. Upon binding to a surface through EGF1, the KNG/FN2-dependent mechanism is inactivated, exposing the FXII activation cleavage site.

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Background: Snake venom of Notechis scutatus contains notecarin D, a coagulation factor Xa homolog. Results: Notecarin D binds with extraordinarily high affinity to factor V/Va in the absence of membranes. Conclusion: Notecarin D lacks the restricted membrane dependence of factor Xa for binding factor V/Va. Significance: Notecarin D likely bypasses the membrane requirement for high affinity factor Va binding, gaining control of blood coagulation after envenomation of mammalian prey.

Background: X-ray structures indicate remarkable conformational changes are involved in trapping serpin-protease acylintermediate complexes.Results: Rapid kinetics showed two kinetically resolvable complexes with distinct structural attributes involved in conformational trapping. Conclusion:The intermediate complex properties support a role for helix F in the trapping mechanism. Significance: New insights into the mechanism of conformational trapping of serpin-protease complexes are provided.