Objective: To explore whether the improvement in organ function and the vasoactive effect observed in the clinical studies of drotrecogin alfa (activated) (recombinant human activated protein C, rhAPC) in sepsis are a result of rhAPC's effect on endothelial cell (EC) permeability and modulation of the intracellular cytoskeleton via the Rho kinase signaling pathway.
Design: Findings regarding dose and duration of exposure to the drug with sequential addition of rhAPC and mediators (thrombin, histamine, interleukin-1β).
Setting: Research laboratory in a pharmaceutical company.
Subjects: Cultured primary human EC from different tissues and vascular beds.
Interventions: A monolayer of EC was incubated with either rhAPC, thrombin, histamine, or interleukin-1β alone or with rhAPC in combination with thrombin or interleukin-β. The effect of rhAPC and mediators on EC permeability was monitored with measurement of electrical resistance. The effect on Rho kinase pathway signaling was monitored by the levels of phosphorylated myosin light chain and blockage with the Rho kinase specific inhibitor, Y27632.
Measurements and Main Results: Thrombin alone induced an early, concentration-dependent, and transient leakiness of EC. Interleukin-1β (0.5 ng/mL) induced an early, irreversible leakiness of EC. rhAPC (0.05-0.2 μg/mL, approximate median therapeutic blood levels) alone had no effect on EC permeability. rhAPC at ≥1 μg/mL induced an early EC leakage. rhAPC (0.19 μg/mL) attenuated the leakage induced by 0.5 ng/mL interleukin-1β on microvascular EC derived from lung and skin and partially attenuated the leakage induced by 0.25 nM thrombin on human coronary arterial ECs. Levels of phosphorylated myosin light chain increased rapidly in human coronary arterial ECs when stimulated with thrombin or rhAPC (about 100-fold less potent) in a concentration-dependent manner via the Rho kinase signaling pathway. Short (5 mins) preconditioning of human coronary arterial ECs with 0.19 μg/mL rhAPC partially blocked the increase in phosphorylated myosin light chain levels induced by thrombin (0.06-0.2 nM).
Conclusions: At concentrations exceeding physiologic and therapeutic levels, rhAPC increases EC permeability, an effect not seen at lower concentrations. The data suggest that interpretation of published in vitro and in vivo data of rhAPC and EC permeability should take into consideration the concentrations of rhAPC used or achieved. Other preliminary novel observations suggest that studying the effects of rhAPC on EC permeability and intracellular cytoskeletal organization may provide understanding of the effect of rhAPC on EC function.