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| Chemical name: | Alexa Fluor 680-NH-CO-CH2-S-CH2-Phe-Pro-Arg-CH2-prothrombin | |
| Abbreviated name: | AF680-ProT | |
| Synonym: | ||
| Agent Category: | Proteins | |
| Target: | Staphylocoagulase | |
| Target Category: | Bacterial enzyme | |
| Method of detection: | Optical imaging | |
| Source of signal / contrast: | Alexa Fluor 680 | |
| Activation: | No | |
| Studies: |
| No structure is available. |
In vitro
Background
[PubMed]
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 (Kd = ~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.
Related Resource Links:
The nucleotide and protein sequences of staphylocoagulase in GenBank
Synthesis
[PubMed]
Panizzi et al. described the synthesis of AF680-ProT in detail (1, 6). Prothrombin was purified from human plasma (absorption coefficient, 1.47 (mg/ml)−1cm−1; molecular weight, 71.6 kDa). Met-SC-(1–325)-His6 fragment was expressed in E. coli strain BL21(DE3) plysS and purified from the soluble fraction after centrifugation. An active site was first induced through the binding of Met-SC-(1–325)-His6 with prothrombin. This site was then inactivated via Ser195 and His57 alkylation with the thiol group-possessing thrombin inhibitor Nα-[(acetylthio)acetyl]-d-Phe-Pro-Arg-CH2Cl (ATA-FPR-CH2Cl). The resulting Met-SC-(1–325)-His6·ATA-FPR-ProT complex was fluorescently labeled through the reaction with a 10-fold molar excess of thiol-reactive Alexa Fluor 680 C2 maleimide. Finally, the Met-SC-(1–325)-His6 was dissociated with NaSCN from the fluorescently labeled complex, and the desired product, AF680-ProT, was separated with gel filtration chromatography. The chemical purity of AF680-ProT and the number of dye moieties per probe were not described in detail.
In Vitro Studies: Testing in Cells and Tissues
[PubMed]
Panizzi et al. first determined the activity of the residual active prothrombin in the final product because the residual active prothrombin could trigger downstream activation of the clotting cascade in vivo (1). The results showed a residual activity of 0.3% for AF680-ProT, indicating less potential to trigger clotting cascade when used in vivo.
Panizzi et al. then verified whether the recombinant N-terminal fragment SC (1–325) was able to activate mouse prothrombin. The investigators found that the SC (1–325)-prothrombin complex hydrolyzed the chromogenic substrate of H-d-Phe-Pip-Arg-pNA (S2238) at a rate that was equivalent to that of mouse thrombin (kcat = 26 ± 1 s−1) (1, 6). The supernatants of various S. aureus strains were able to clot mouse plasma. These data indicate that the mouse is an appropriate model organism to study SC function and regulation in vivo.
To determine the molecular mechanism underlying prothrombin recruitment to vegetations, Panizzi et al. performed in vitro native gel binding experiments using either the N-terminal active fragment SC (1–325) or the full-length SC (1–660) (1, 6). After incubation of these SC forms with prothrombin and fibrinogen fragment D (FragD), SC (1–325) and prothrombin bound together but lacked the ability to interact with FragD. However, SC (1–660) formed an SC-prothrombin-FragD ternary complex, and multiple FragD subunits interacted with a single SC molecule. The SC recombinant fragment that contained only the pseudorepeat and the first SC repeat could bind FragD with a Kd of 36 ± 8 nM. These results suggest that SC could interact with at least four fibrinogen or fibrin molecules per SC molecule to form a megaprotein complex, and this complex anchored the active SC-prothrombin complex to the growing vegetation.
Animal Studies
Rodents
[PubMed]
To test the feasibility of AF680-ProT to image vegetations, Panizzi et al. established several mouse models of infective endocarditis by inserting a segment of suture material down the right carotid artery into the heart to induce damage of the aortic valve and by injecting bacteria (1 × 106 colony-forming units per 100 ìl phosphate-buffered saline (PBS)) via the tail vein 24 h later (1). Bacterial strains used to establish disease models included SC-positive S. aureus strains (Newman D2 Tager 104, Xen29, and Xen8.1), SC-negative S. epidermidis FDA strain PCI 1200, and Newman SC and von Willebrand factor–binding protein (vWbp) double-knockout S. aureus (n = 6 mice/group). Newman D2 Tager 104, Xen29, and Xen8.1 are vancomycin-susceptible. Endocarditis was observed in >85% of the mice, but the size of formed vegetations and the extent of occlusion of the aortic valve varied, potentially correlating with the extent of denuded endothelium caused by the mechanical injury. On day 2 after suture insertion and 24 h after induction of bacteremia, 30–45 ìg AF680-ProT was injected via the tail vein, and imaging was performed 24 h later with fluorescence molecular tomography fused to X-ray computed tomography (FMT-CT). The isotropic spatial resolution was 110 ìm for CT and 1 mm for FMT.
FMT-CT imaging showed that AF680-ProT accumulated in the vegetations, which was consistent in mice induced with Tager 104, Xen29, and Xen8.1 strains, but not in mice infected with S. epidermidis or in mice without bacteremia. The fluorescence intensity in the aortic outflow tract in mice with S. aureus–induced vegetations was 20- to 28-fold higher than that in the mice without bacteremia and the mice with S. epidermidis challenge (1). AF680-ProT had a blood half-life of 79 ± 14 min.
The expression pattern of SC within vegetations was investigated in the excised aortas with vegetations (1). AF680-ProT was co-localized with SC at the interface of vegetations with the host's circulation, although bacteria were present throughout the vegetations. This result was consistent with the findings from immunoreactive staining for SC and vWbp and from in situ hybridization for SC RNA, which showed that the proteins and RNA were all limited to the periphery of vegetations. This SC expression pattern was considered to be the result of the differential expression of SC during vegetation development; as in younger lesions with lower bacterial burden, the entire S. aureus population stained positive for SC.
To image the efficiency of AF680-ProT for monitoring vancomycin therapy, mice were given daily intraperitoneal injections of 10 ìg vancomycin or PBS for 6 days (n = 8–10 mice/group) (1). FMT-CT imaging at 48 h after infection indicated that AF680-ProT was able to quantify the effect of vancomycin, showing that vancomycin treatment eliminated bacteria in vegetations and that termination of the therapy resulted in recurrence of the infection and a high risk of mortality, similar to the relapse observed in some patients with infective endocarditis.
To determine the specificity of AF680-ProT for endocarditic vegetations that contain SC, femoral artery thrombosis was first induced via topical application of FeCl3 (1). No accumulation of AF680-ProT was observed in the thrombus after injection of 25 ìg AF680-ProT. Endocarditis was then induced with the S. aureus strain that is deficient of both SC and vWbp (n = 10–13 mice). Only background levels of AF680-ProT accumulation were detected in the vegetations. Improved survival was also observed in these mice, suggesting that SC increases the virulence of S. aureus. Histology revealed leukocyte infiltration and the absence of the protective fibrin barrier in the vegetations induced by the SC and vWbp double-knockout S. aureus, indicating an impaired ability of the bacteria to evade the host defense. In the mice infected with an S. aureus strain that lacks only SC (n = 5 mice), AF680-ProT accumulation in the vegetations was reduced to 14% compared to mice that were infected with isogenic wild-type Newman strain bacteria (P < 0.0001).
Toxicity of the AF680-ProT was studied after three injections of 10 times (250 μg) the imaging dose over one week (n = 5 mice) (1). Pathological examination of the normal tissue sections revealed no abnormalities such as inflammatory foci, necrosis, clots, or bleeding. No differences were observed for the prothrombin time for either human or mouse plasma after incubation with the prothrombin analog (250 μg) at 37ºC. Tail tip bleeding assay also showed no significant changes in clotting parameters in mice injected with the probe (n = 5 mice).
References
- 1.
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- 2.
- Chorianopoulos E. et al. The role of endothelial cell biology in endocarditis. Cell Tissue Res. 2009;335(1):153–63. [PubMed: 19015889]
- 3.
- Friedrich R. et al. Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation. Nature. 2003;425(6957):535–9. [PubMed: 14523451]
- 4.
- Panizzi P. et al. The staphylocoagulase family of zymogen activator and adhesion proteins. Cell Mol Life Sci. 2004;61(22):2793–8. [PMC free article: PMC2291352] [PubMed: 15558209]
- 5.
- Hacisalihoglu A. et al. Restricted active site docking by enzyme-bound substrate enforces the ordered cleavage of prothrombin by prothrombinase. J Biol Chem. 2007;282(45):32974–82. [PMC free article: PMC2292459] [PubMed: 17848548]
- 6.
- Panizzi P. et al. Novel fluorescent prothrombin analogs as probes of staphylocoagulase-prothrombin interactions. J Biol Chem. 2006;281(2):1169–78. [PMC free article: PMC2292460] [PubMed: 16230340]
- 7.
- Panizzi P. et al. Fibrinogen substrate recognition by staphylocoagulase.(pro)thrombin complexes. J Biol Chem. 2006;281(2):1179–87. [PMC free article: PMC2291351] [PubMed: 16230339]
- 8.
- Friedrich R. et al. Structural basis for reduced staphylocoagulase-mediated bovine prothrombin activation. J Biol Chem. 2006;281(2):1188–95. [PMC free article: PMC2292465] [PubMed: 16230338]
- 9.
- Munnix I.C. et al. Segregation of platelet aggregatory and procoagulant microdomains in thrombus formation: regulation by transient integrin activation. Arterioscler Thromb Vasc Biol. 2007;27(11):2484–90. [PMC free article: PMC2376762] [PubMed: 17761939]
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- Watanabe S. et al. Structural comparison of ten serotypes of staphylocoagulases in Staphylococcus aureus. J Bacteriol. 2005;187(11):3698–707. [PMC free article: PMC1112059] [PubMed: 15901693]
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- Hirose M. et al. Identification of staphylocoagulase genotypes I-X and discrimination of type IV and V subtypes by multiplex PCR assay for clinical isolates of Staphylococcus aureus. Jpn J Infect Dis. 2010;63(4):257–63. [PubMed: 20657065]
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- Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.[J Biol Chem. 2020]Specificity and affinity of the N-terminal residues in staphylocoagulase in binding to prothrombin.Maddur AA, Kroh HK, Aschenbrenner ME, Gibson BHY, Panizzi P, Sheehan JH, Meiler J, Bock PE, Verhamme IM. J Biol Chem. 2020 Apr 24; 295(17):5614-5625. Epub 2020 Mar 10.
- Fibrinogen substrate recognition by staphylocoagulase.(pro)thrombin complexes.[J Biol Chem. 2006]Fibrinogen substrate recognition by staphylocoagulase.(pro)thrombin complexes.Panizzi P, Friedrich R, Fuentes-Prior P, Richter K, Bock PE, Bode W. J Biol Chem. 2006 Jan 13; 281(2):1179-87. Epub 2005 Oct 17.
- In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.[Nat Med. 2011]In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.Panizzi P, Nahrendorf M, Figueiredo JL, Panizzi J, Marinelli B, Iwamoto Y, Keliher E, Maddur AA, Waterman P, Kroh HK, et al. Nat Med. 2011 Aug 21; 17(9):1142-6. Epub 2011 Aug 21.
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