The dual diverse dynamic reversible effects of ankaferd blood stopper on EPCR and PAI-1 inside vascular endothelial cells with and without LPS challenge

Objective: Ankaferd blood stopper (ABS) is comprised of a mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS is used as a topical hemostatic agent due to its antihemorrhagic effect, yet its hemostatic mechanism of action remains to be investigated. ABS does not affect the levels of coagulation factors II, V, VII, VIII, IX, X, XI and XII. The aim of this study was to investigate the effects of ABS on endothelium and immune response. As such, we evaluated changes in endothelial cell protein C receptor (EPCR) and plasminogen activator inhibitor type-1 (PAI-1) expression inside human umbilical vein endothelial cells (HUVECs) in the presence and absence of lipopolysaccharides (LPSs). Material and Methods: We exposed HUVECs to 10 μL and 100 μL of ABS for 5 min, 25 min, 50 min, 6 h, and 24 h. Additionally, 10 μg mL^-1 of LPS was administered for 1 h to observe the effects of LPS challenge on HUVECs, and then the cells were treated with ABS for 5 min, 25 min, 50 min, and 6 h to observe the effects of ABS on HUVECs. Total RNA was isolated from HUVECs and then the level of expression of EPCR and PAI-1 mRNA was measured. Results: Cells were microscopically observed to arise from the surface and adhere to each other following the administration of ABS to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. ABS had a negative effect on EPCR and PAI-1 expression; the effect in response to 100 μL was greater than that to 10 μL. EPCR and PAI-1 expression increased over time in response to LPS and 10 μL of ABS. EPCR and PAI-1 expression was very low during the first hour of exposure to LPS and 100 μL of ABS, but after 6 h increased to levels similar to those observed in response to LPS and 10 μL of ABS. Conclusion: It was observed that ABS had dual diverse dynamic reversible effects on EPCR and PAI-1 expression in HUVECs, which were dependent on dose and concentration. ABS might play a role in numerous cellular mechanisms, in addition to having hemostatic effects.