Granulocyte proteases and hydrogen peroxide synergistically inactivate thrombomodulin of endothelial cells in vitro

Abstract

Leukocytes release lysosomal proteases and reactive oxygen species in response to various stimuli that damage the adjacent endothelial cells. We investigated the effects of granulocyte lysosomal proteases (granulocyte elastase [GE] and cathepsin G [CG] and/or hydrogen peroxide (H2O2) on thrombomodulin (TM) activity of endothelial cells. We wished to determine whether the activated leukocytes damage the nonthrombogenic systems of endothelial cells. When cultured human umbilical vein endothelial cells (HUVECs) were incubated with GE, TM activity of the cells (as judged by the protein C activation capacity) decreased to about 10% of control with the concomitant increase of immunoreactive TM concentration in the conditioned media. CG also decreased TM activity to about 20% of control with the concomitant increase in immunoreactive TM concentration in the conditioned media. The GE- or CG-induced inactivation of TM was not observed in the presence of alpha 1-proteinase inhibitor. Immunoblot analysis showed that CG cleaved purified TM to yield one major fragment with an M(r) of 43,000; TM activity of this fragment was about 10% of the control activity. When purified TM was incubated with GE, TM activity decreased to 10% of control, and no detectable band was found on immunoblotting, suggesting that CG and GE cleave TM into inactive fragments and that GE degrades the epitope structure of TM. Although H2O2 (1.0 mmol/L) enhanced chromium 51 release from prelabeled HUVECs after 30 minutes of incubation, it decreased TM activity only slightly.(ABSTRACT TRUNCATED AT 250 WORDS)