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Pharmaceutical Research

, Volume 28, Issue 6, pp 1415–1421 | Cite as

Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface

  • Anna Waterhouse
  • Daniel V. Bax
  • Steven G. Wise
  • Yongbai Yin
  • Louise L. Dunn
  • Giselle C. Yeo
  • Martin K. C. Ng
  • Marcela M. M. Bilek
  • Anthony S. WeissEmail author
Research Paper

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.

KEY WORDS

kallikrein plasma-activated thrombin tropoelastin 

ABBREVIATIONS

125I

iodine-125

316L SS

316L stainless steel

ANOVA

analysis of variance

PAC

plasma-activated coating

PEO

polyethylene oxide

R515A

point-mutant tropoelastin SHEL∆26A(R515A)

SDS

sodium dodecyl sulfate

SEM

scanning electron microscopy

TE

tropoelastin (SHEL∆26A)

Notes

ACKNOWLEDGMENTS

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.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Anna Waterhouse
    • 1
  • Daniel V. Bax
    • 2
  • Steven G. Wise
    • 3
  • Yongbai Yin
    • 2
  • Louise L. Dunn
    • 3
  • Giselle C. Yeo
    • 1
  • Martin K. C. Ng
    • 4
  • Marcela M. M. Bilek
    • 2
  • Anthony S. Weiss
    • 1
    Email author
  1. 1.School of Molecular Bioscience,University of SydneySydneyAustralia
  2. 2.Applied & Plasma Physics Group, Physics SchoolUniversity of SydneySydneyAustralia
  3. 3.The Heart Research InstituteSydneyAustralia
  4. 4.Department of Cardiology,Royal Prince Alfred HospitalSydneyAustralia

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