Figure 5.

Cytoprotective signaling activity of APC/PC derivatives in established cellular assays. (A) The endothelial cell permeability in response to LPS (10 ng/mL for 4 hours) following their treatment with APC derivatives (5-100 nM for 3 hours) was quantitated by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional cell monolayers as described in “Materials and methods.” (B) The same as panel A except that the activation of PC derivatives by the thrombin-W215A/E217A mutant (WE-Thr) was coupled to its barrier protective function. Endothelial cells were initially incubated with increasing concentrations of PC derivatives (0-100 nM) plus the thrombin mutant (5 nM) for 3 hours before inducing cell permeability with LPS (10 ng/mL for 4 hours). (C) The same as panel B except that the effect of cell surface WE-Thr–activated PC derivatives on NF-κB activation in response to LPS was measured by an ELISA. (D) The same as panel C except that the effect WE-Thr–activated PC derivatives on cell surface expression of intercellular adhesion molecule (ICAM)-1 (gray bars) and vascular cell adhesion molecule (VCAM)-1 (black bars) was monitored by a cell-based ELISA. (E) The same as panel C except that the adherence of THP-1 cells to LPS-stimulated endothelial cells was monitored. *P < .05 and **P < .01 in both panels. OD, optical density.