Alexa Fluor 680-NH-CO-CH2-S-CH2-Phe-Pro-Arg-CH2-prothrombin

Excerpt

The fluorescently labeled prothrombin analog, Alexa Fluor 680-NH-CO-CH₂-S-CH₂-Phe-Pro-Arg-CH₂-prothrombin, abbreviated as AF680-ProT, is a probe developed by Panizzi et al. for detecting Staphylococcus aureus (S. aureus) endocarditis by targeting staphylocoagulase (1).

Infective endocarditis is a life-threatening condition that is commonly caused by S. aureus, and the hallmark of S. aureus endocarditis is the development of vegetations on heart valves (2). After entering into the bloodstream, S. aureus proliferates quickly, colonizes on either damaged or normal heart valves, and leads to a rapid progressive endocarditis with destruction of the heart valves (3-5). In this pathological process, adherence of S. aureus to heart valves is the key to initiate the vegetation formation, and SC is one of the most important stimulators of the initiation. SC is an extracellular protein secreted by coagulase-positive S. aureus, and it can bind prothrombin with a high affinity (K_d = ~17–72 pM) in a 1:1 stoichiometry (6-8). This binding forms an active SC-prothrombin complex, which induces plasma coagulation via specific cleavage of fibrinogen to fibrin (7, 9). The N-terminal D1 and D2 regions of SC are involved in the binding with prothrombin and its activation, and the C-terminal repeat region, comprising 27-amino-acid tandem repeats, is associated with the adherence of SC to fibrinogen (10, 11).

Panizzi et al. synthesized an engineered analog of human prothrombin in which the active site was modified by a thrombin inhibitor that possesses a protected thiol group (1, 6). Thus, the prothrombin analog could react with a thiol-specific Alexa Fluor 680 dye (AF680-ProT) for fluorescence imaging or with diethylenetriamine pentaacetic acid (DTPA) for ⁶⁴Cu-labeling (⁶⁴Cu-DTPA-ProT). The rationale underlying this design is the SC-dependent prothrombin recruitment, which simultaneously tethers the active SC-prothrombin analog complex into the fibrin(ogen)-rich S. aureus vegetations. Imaging studies by Panizzi et al. showed that the two probes could bind SC and intercalate into growing bacterial vegetations on the heart valves, with the potential to detect S. aureus endocarditis and monitor antibiotic therapy via noninvasive optical imaging and positron emission tomography (1).

This chapter summarizes the data obtained with AF680-ProT, and another chapter summarizes the data obtained with ⁶⁴Cu-DTPA-ProT.