title: Stability of a therapeutic layer of immobilized recombinant human tropoelastin on a plasma-activated coated surface.
abstract: OBJECTIVE
  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 (125)Iodine 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.
authors.affiliation: School of Molecular Bioscience,, University of Sydney, Sydney, NSW 2006, Australia.
authors.forename: Anna
authors.initials: A
authors.lastname: Waterhouse
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Bax
authors.forename: Steven G
authors.initials: SG
authors.lastname: Wise
authors.forename: Yongbai
authors.initials: Y
authors.lastname: Yin
authors.forename: Louise L
authors.initials: LL
authors.lastname: Dunn
authors.forename: Giselle C
authors.initials: GC
authors.lastname: Yeo
authors.forename: Martin K C
authors.initials: MK
authors.lastname: Ng
authors.forename: Marcela M M
authors.initials: MM
authors.lastname: Bilek
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Weiss
mesh_terms: D002448:Cell Adhesion; D020099:Coated Materials, Biocompatible / Q000737:chemistry* / Q000378:metabolism*; D004355:Drug Stability; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism*; D007457:Iodine Radioisotopes / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D007610:Kallikreins / Q000378:metabolism; D010447:Peptide Hydrolases / Q000097:blood; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013193:Stainless Steel / Q000737:chemistry; D013499:Surface Properties; D013917:Thrombin / Q000378:metabolism; D014333:Tropoelastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D020099:Coated Materials, Biocompatible; D055767:Immobilized Proteins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D014333:Tropoelastin; D013193:Stainless Steel; D010447:Peptide Hydrolases; D007610:Kallikreins; D013917:Thrombin