Inhibition of Coagulation Activation and Inflammation by a Novel Factor Xa Inhibitor Synthesized from the Earthworm Eisenia andrei

Abstract

We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (pϽ0.0001), indicating that protease-activated receptor-2 (PAR-2) was effectively inactivated. We also found that ESP reduced the expressions of pro-inflammatory cytokines (IL-1a a, IL-1b b, IL-8, IL-16, MCP-1, MIP-1a a and MIP-1b b) by cultured cells treated with both ESP and FXa. Our results provide the first evidence that ESP might interrupt coagulation cascades by inhibiting FXa, and thereby may effectively control the bidirectional alternation between coagulation and inflammation.

Tissue Factor-Expressing Tumor-Derived Extracellular Vesicles Activate Quiescent Endothelial Cells via Protease-Activated Receptor-1

“…Our results suggest that PAR-1 is the dominant receptor on unstimulated endothelial cells for the TF–FVIIa–FX complex on tumor-derived EV. Endothelial responses induced by FXa have largely been ascribed to PAR-2 ( 21 , 22 , 26 , 27 ); however, this was not the case in our study, likely due to the weak PAR-2 expression on the endothelial cells. Our study focused on the 6-h acute response of unstimulated ECs upon exposure to tumor-derived EV.…”

“…Purified FXa has been shown to activate endothelial cells in vitro , inducing the secretion of pro-inflammatory cytokines and upregulating adhesion molecules ( 24 , 25 ). This effect could be mediated by both PAR-1 and PAR-2; however, FXa is presumed to signal preferentially through PAR-2 ( 21 , 22 , 26 , 27 ).…”

Pyrosequencing the salivary transcriptome of Haemadipsa interrupta (Annelida: Clitellata: Haemadipsidae): anticoagulant diversity and insight into the evolution of anticoagulation capabilities in leeches

“…(), Joo et al. (), and Lee et al. () each isolated and characterized anticoagulation‐related proteins from lumbricid clitellates (galactose‐binding lectin from Lumbricus terrestris Linnaeus 1758 and factor Xa inhibiting eisenstasin from Eisenia andrei Bouché 1972).…”

7‐difluoromethyl‐5,4′‐dimethoxygenistein, a novel agent protecting against vascular endothelial injury caused by oxidative stress

“…22,23 The HUVE-12 cell line, originally from the American Type Culture Collection (Bethesda, MD, USA), is a spontaneously transformed cell line derived from the human umbilical vein endothelium that is commonly used in the cardiovascular pharmacological or physiological studies. 24,25 Thus, we chose to use this cell line in the present study.…”