Immobilization of human thrombomodulin to expanded polytetrafluoroethylene.
BACKGROUND
The success of synthetic grafts for vascular reconstruction remains limited by thrombosis and intimal hyperplasia. In addition to the well-described antithrombotic effects of thrombomodulin, we have demonstrated that recombinant human thrombomodulin (rTM) inhibits arterial smooth muscle cell proliferation induced by thrombin. This study investigated the binding of functional rTM to expanded polytetrafluoroethylene (ePTFE).


METHODS
Immobilization of rTM was achieved by either (1) a direct coating or (2) a two-step binding process using a water-soluble condensing cross-reaction agent EDAC to modify the ePTFE surface followed by binding of rTM. The samples were then subjected to a tangential shaken wash. The evidence of bound rTM was evaluated by both morphologic and functional studies.


RESULTS
SEM, BSI, and X-ray microanalysis identified that the two-step binding method resulted in significantly greater binding of rTM molecules to ePTFE pre- and post a 7-h wash than the direct coating method. With the two-step binding method rTM ranging from 0.25 to 12.5 microg immobilized to ePTFE-activated protein C (APC) in a concentration-dependent manner by more than 6000-fold compared to the buffer control (P < 0.04) and 50-85% more than direct coating (P < 0.004). With direct coating, the level of APC dropped significantly to near 40% of the preshaken level at 2 h and diminished to 26% at 7 h. Whereas, the level of APC with the two-step binding stabilized at 51 and 49% after being shaken 2 and 7 h, respectively.


CONCLUSIONS
Functional rTM binding to ePTFE was significantly improved with a new two-step binding method.
Department of Surgery, Division of Vascular Surgery, University of Massachusetts Medical School, Worcester, 01655, USA. jianming.li@umassmed.edu
Jian-ming
JM
Li
Michael J
MJ
Singh
Peter R
PR
Nelson
Gregory M
GM
Hendricks
Mazen
M
Itani
Michael J
MJ
Rohrer
Bruce S
BS
Cutler
D020099:Coated Materials, Biocompatible; D004577:Electron Probe Microanalysis; D006801:Humans; D007158:Immunologic Techniques; D008855:Microscopy, Electron, Scanning; D010812:Physical Stimulation; D011138:Polytetrafluoroethylene / Q000378:metabolism*; D011486:Protein C / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013194:Staining and Labeling; D018180:Thrombomodulin / Q000378:metabolism* / Q000502:physiology
D020099:Coated Materials, Biocompatible; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D011138:Polytetrafluoroethylene