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

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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.

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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)

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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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Authors and Affiliations

  1. School of Molecular Bioscience,, University of Sydney, Sydney, NSW, 2006, Australia

    Anna Waterhouse, Giselle C. Yeo & Anthony S. Weiss

  2. Applied & Plasma Physics Group, Physics School, University of Sydney, Sydney, NSW, 2006, Australia

    Daniel V. Bax, Yongbai Yin & Marcela M. M. Bilek

  3. The Heart Research Institute, Sydney, NSW, 2042, Australia

    Steven G. Wise & Louise L. Dunn

  4. Department of Cardiology,, Royal Prince Alfred Hospital, Sydney, NSW, 2050, Australia

    Martin K. C. Ng

Authors
  1. Anna Waterhouse
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  2. Daniel V. Bax
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  9. Anthony S. Weiss
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Correspondence to Anthony S. Weiss.

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Waterhouse, A., Bax, D.V., Wise, S.G. et al. Stability of a Therapeutic Layer of Immobilized Recombinant Human Tropoelastin on a Plasma-Activated Coated Surface. Pharm Res 28, 1415–1421 (2011). https://doi.org/10.1007/s11095-010-0327-z

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  • DOI: https://doi.org/10.1007/s11095-010-0327-z

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