Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface
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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.
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.
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.
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.
KEY WORDSkallikrein plasma-activated thrombin tropoelastin
- 316L SS
316L stainless steel
analysis of variance
point-mutant tropoelastin SHEL∆26A(R515A)
sodium dodecyl sulfate
scanning electron microscopy
This work was supported by grants from the Australian Research Council, the National Health and Medical Research Council, and the University of Sydney Medical Foundation.
- 4.Colman RW, Marder VJ, Clowes AW, George JN, Goldhaber SZ, editors. Hemostasis and Thrombosis. Basic principles and clinical practice. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. p. 437–41.Google Scholar
- 24.Yang Z, Wang J, Luo R, Maitz MF, Jing F, Sun H, Huang N. The covalent immobilization of heparin to pulsed-plasma polymeric allylamine films on 316L stainless steel and the resulting effects on hemocompatibility. Biomaterials. In Press, Corrected Proof: 2009.Google Scholar
- 26.Andersson J, Bexborn F, Klinth J, Nilsson B, Ekdahl KN. Surface-attached PEO in the form of activated pluronic with immobilized factor H reduces both coagulation and complement activation in a whole-blood model. J Biomed Mater Res A. 2005;76A:25–34.Google Scholar