Effect of Agkistrodon Acutus Venom PCA on HUVEC Activity

Abstract

To investigate the effects of Agkistrodon acutus venom protein C activator(PCA) on ultrastructure of human umbilical vein endothelial cells(HUVEC), and the levels of tissue factor(TF), vascular von Willebrand factor (vWF) and endothelin-1 secreted by HUVEC and to clarify the anti-thrombotic mechanism of PCA. The experiments were divided into control group(DMEM), LPS group (LPS 0.1 µg/ml), PCA group(PCA 1 µg/ml) and PCA+LPS group (1 µg/ml PCA+ 0.1 µg/ml LPS). The morphology of endoplasmic reticulum, mitochondria and the number of autophagosome in HUVEC were observed by transmission electron microscopy. The TF, vWF and ET-1 were measured in the medium of each group by ELISA; RT-PCR was used to detect mRNA expression level of vWF and ET-1 in cells; and the protein expression level of TF in cells was detected by Western blot. Compared with the control group, the ultrastructural changes of HUVEC in the LPS group included the cell membrane getting rough, swelling of mitochondria and endoplasmic reticulum, and autophagosome increase, however, the ultrastructure differences between PCA and control group were not significant. Compared with the ultrastructure of HUVECs in LPS group, the swelling of mitochondria and endoplasmic reticulum disappeared in the LPS+PCA group, and the number of autophagosome decreased obviously. Compared with the control group, the content of ET-1, vWF and TF in cell culture supernatant, and the protein expression level of vWF, ET-1 gene and TF protein were significantly increased in LPS group (P<0.05); the expression levels of the 3 factors in the cell culture supernatant and cells in PCA group were not significantly different from the control group (P>0.05). The expression levels of TF, vWF and ET-1 in LPS group were significantly lower than those in LPS+PCA group (P<0.05). PCA(1 µg/ml) can reduce the ultrastructural changes of HUVEC induced by LPS, and inhibit the increase of TF, vWF and ET-1 secretion from HUVEC induced by LPS.