Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress

Verotoxin-1 (VT-1)-producing Escherichia coli is the causative agent of pos tdiarrheal hemolytic uremic syndrome (D+HUS) of children, which leads to re nal and other organ microvascular thrombosis. Why thrombi form only on arte rioles and capillaries is not known. This study investigated whether VT-1 d irectly affected endothelial antithrombogenic properties promoting platelet deposition and thrombus formation on human microvascular endothelial cell line (HMEC-1) under high shear stress. Human umbilical vein endothelial cel ls (HUVECs) were used for comparison as a large-vessel endothelium. HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT -1 (2-50 pM) and then perfused at 60 dynes/cm(2) with heparinized human blo od prelabeled with mepacrine. Results showed that VT-1 significantly increa sed platelet adhesion and thrombus formation on HMEC-1 in comparison with u nstimulated control cells. An increase in thrombus formation was also obser ved on HUVECs exposed to VT-1, but to a remarkably lower extent. The greate r sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor (20-fold more) as docu mented by FACS analysis. The HMEC-1 line had a comparable susceptibility to the thrombogenic effect of VT-1 as primary human microvascular cells of th e same dermal origin (HDMECs). The adhesive molecules involved in VT-induce d thrombus formation were also studied. Blocking the binding of von Willebr and factor to platelet glycoprotein lb by aurintricarboxylic acid (ATA) or inhibition of platelet alpha (IIb)beta (3)-integrin by chimeric 7E3 Fab res ulted In a significant reduction of VT-1-induced thrombus formation, sugges ting the involvement of von Willebrand factor-platelet interaction at high shear stress in this phenomenon. Functional blockade of endothelial beta3-i ntegrin subunit, vitronectin receptor, P-selectin, and PECAM-1 with specifi c antibodies was associated with a significant decrease of the endothelial area covered by thrombi. Confocal microscopy studies revealed that VT-1 inc reased the expression of vitronectin receptor and P-selectin and redistribu ted PECAM-1 away from the cell-cell border of HMEC-1, as well as of HDMECs, thus indicating that the above endothelial adhesion molecules are directly involved and possibly determine the effect of VT-1 on enhancing platelet a dhesion and thrombus formation In microvascular endothelium. These results might help to explain why thrombi In HUS localize In microvessels rather th an in larger ones and provide insights on the molecular events involved in the process of microvascular thrombosis associated with D+HUS.