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. 2017 Aug 15;7:8126. doi: 10.1038/s41598-017-05326-9

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Functional studies of tCoa-RGD fusion proteins. (A) Modeling, docking, and MD simulation. (a) Amino acid sequence of tCoa-RGD, its domains, and 3D structure. (b) tCoa-RGD has a helical structure within its two domains. (c) The 3D structure of tCoa-RGD in complex with prothrombin after equilibration in MD simulation. Insertion of tCoa-N-terminal into prothrombin is highlighted by a circle. IVTKDY hexapeptide at the N-terminus of protein interacts strongly with zymogen activation domain of prothrombin in a “molecular sexuality mechanism.” (d) The 3D structure of tCoa-RGD-prothrombin in complex with the extracellular domain of α_vβ₃ integrins after equilibration in MD simulation. The interaction of RGD domain with integrin residues is highlighted with a circle. (B) Functional studies to determine coagulase activity of the fusion proteins (a), and retention of antibody activity by tCoa-RGD to α_vβ₃ integrins by ELISA (b, c) and FACS (d). (a) Retention of coagulase activity was determined by the capability of tCoa and tCoa-RGD to convert fibrinogen into fibrin through the conformational activation of ProT. tCoa-RGD presented coagulase activity comparable to that of tCoa at each time point, whereas ProT alone did not potentiate coagulation and there was no detectable absorbance. (b) In vitro binding studies demonstrated that tCoa-RGD specifically bind to immobilized α_vβ₃ integrins in a concentration dependent manner, with the highest binding at 1.2 nM concentration. (c) In presence of His-tag removed tCoa-RGD but not tCoa, binding of tCoa-RGD to the α_vβ₃ integrins was significantly inhibited (>70%). (d) FACS analysis verified differential binding of tCoa (peck 1) and tCoa-RGD (peck 2, 3) on endothelial cells in suspension. In this histogram, M1 marker covers the negative cells presenting no/none-specific binding while M2 marker comprises the positive cells. Accordingly, specific binding of tCoa-RGD to the α_vβ₃ integrins was determined 64.24% (peck 3). None-specific binding for tCoa and tCoa-RGD was measured 98% and 36.05%, respectively. Correspondingly, the measured fluorescence intensity for tCoa-RGD was eight times higher than that of tCoa. (C) Tracing of fluorescently labeled drugs in vivo. tCoa-RGD labeled with FITC injected to (a) healthy mice with no tumor; while (b) tCoa and (c) tCoa-RGD injected to mice bearing SKOV3 ovarian carcinoma xenografts. tCoa-RGD fusion protein but not tCoa showed specific accumulation at the subcutaneously implanted tumor site in C57Bl/6 nude mice.