caa a22 4500 445311819 CHVBK 20180317142632.0 cr unu---uuuuu 170323e20110601xx s 000 0 eng 10.1007/s11095-010-0327-z doi (NATIONALLICENCE)springer-10.1007/s11095-010-0327-z Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface [Elektronische Daten] [Anna Waterhouse, Daniel Bax, Steven Wise, Yongbai Yin, Louise Dunn, Giselle Yeo, Martin Ng, Marcela Bilek, Anthony Weiss] ABSTRACT: Purpose: To modify blood-contacting stainless surfaces by covalently coating them with a serum-protease resistant form of tropoelastin (TE). To demonstrate that the modified TE retains an exposed, cell-adhesive C-terminus that persists in the presence of blood plasma proteases. Methods: Recombinant human TE and a point mutant variant (R515A) of TE were labeled with 125Iodine and immobilized on plasma-activated stainless steel (PAC) surfaces. Covalent attachment was confirmed using rigorous detergent washing. As kallikrein and thrombin dominate the serum degradation of tropoelastin, supraphysiological levels of these proteases were incubated with covalently bound TE and R515A, then assayed for protein levels by radioactivity detection. Persistence of the C-terminus was assessed by ELISA. Results: TE was significantly retained covalently on PAC surfaces at 88 ± 5% and 71 ± 5% after treatment with kallikrein and thrombin, respectively. Retention of R515A was 100 ± 1.3% and 87 ± 2.3% after treatment with kallikrein and thrombin, respectively, representing significant improvements over TE. The functionally important C-terminus was cleaved in wild-type TE but retained by R515A. Conclusions: Protein persists in the presence of human kallikrein and thrombin when covalently immobilized on metal substrata. R515A displays enhanced protease resistance and retains the C-terminus presenting a protein interface that is viable for blood-contacting applications. Springer Science+Business Media, LLC, 2010 kallikrein nationallicence plasma-activated nationallicence thrombin nationallicence tropoelastin nationallicence 125I : iodine-125 nationallicence 316L SS : 316L stainless steel nationallicence ANOVA : analysis of variance nationallicence PAC : plasma-activated coating nationallicence PEO : polyethylene oxide nationallicence R515A : point-mutant tropoelastin SHEL∆26A(R515A) nationallicence SDS : sodium dodecyl sulfate nationallicence SEM : scanning electron microscopy nationallicence TE : tropoelastin (SHEL∆26A) nationallicence Waterhouse Anna School of Molecular Bioscience,, University of Sydney, 2006, Sydney, NSW, Australia aut Bax Daniel Applied & Plasma Physics Group, Physics School, University of Sydney, 2006, Sydney, NSW, Australia aut Wise Steven The Heart Research Institute, 2042, Sydney, NSW, Australia aut Yin Yongbai Applied & Plasma Physics Group, Physics School, University of Sydney, 2006, Sydney, NSW, Australia aut Dunn Louise The Heart Research Institute, 2042, Sydney, NSW, Australia aut Yeo Giselle School of Molecular Bioscience,, University of Sydney, 2006, Sydney, NSW, Australia aut Ng Martin Department of Cardiology,, Royal Prince Alfred Hospital, 2050, Sydney, NSW, Australia aut Bilek Marcela Applied & Plasma Physics Group, Physics School, University of Sydney, 2006, Sydney, NSW, Australia aut Weiss Anthony School of Molecular Bioscience,, University of Sydney, 2006, Sydney, NSW, Australia aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/6(2011-06-01), 1415-1421 0724-8741 28:6<1415 2011 28 11095 https://doi.org/10.1007/s11095-010-0327-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-010-0327-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Waterhouse Anna School of Molecular Bioscience,, University of Sydney, 2006, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Bax Daniel Applied & Plasma Physics Group, Physics School, University of Sydney, 2006, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Wise Steven The Heart Research Institute, 2042, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Yin Yongbai Applied & Plasma Physics Group, Physics School, University of Sydney, 2006, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Dunn Louise The Heart Research Institute, 2042, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Yeo Giselle School of Molecular Bioscience,, University of Sydney, 2006, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Ng Martin Department of Cardiology,, Royal Prince Alfred Hospital, 2050, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Bilek Marcela Applied & Plasma Physics Group, Physics School, University of Sydney, 2006, Sydney, NSW, Australia aut NATIONALLICENCE 700 1- Weiss Anthony School of Molecular Bioscience,, University of Sydney, 2006, Sydney, NSW, Australia aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/6(2011-06-01), 1415-1421 0724-8741 28:6<1415 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer