Abstract
Post-diarrheal hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children, is most often caused by Shiga toxin (Stx)-producing Escherichia coli infection. The most widely recognized tissue damage in HUS occurs within the kidney, most prominently with local thrombosis and platelet-fibrin accumulation, suggesting activation of the coagulation cascade. We have shown that human proximal tubular epithelial cells (HK-2 cells) exposed to Stx-1 augment their constitutive surface tissue factor (TF) activity by 3.2-fold, and that Stx-1 treatment following TNF-alpha exposure enhances TF activity by 2.7-fold on human glomerular endothelial cells (HGECs). We investigated the possibility of modulating this cellular response to Stx-1 by blocking the TF pathway with active site-inactivated recombinant factor VIIa (irFVIIa), which retains the ability to bind TF, but is enzymatically inactive, thereby inhibiting thrombin formation. Moreover, its action is limited to exposed, functional TF and does not significantly prolong the bleeding time in vivo. Monolayers of both HK-2 cells and TNF-alpha-activated HGECs were incubated at 37°C with Stx-1. Functional TF was determined chromogenically as the production rate of factor Xa. The use of 60 nM irFVIIa completely abrogated the enhanced activity of TF caused by 4 hours incubation with 10 pM Stx-1 of TNF-alpha-activated HGECs (N = 4; Figure 1). On HK-2 cells, irFVIIa (60 – 200 nM) also effectively reversed the observed TF upregulation after exposure to 1 nM Stx-1 for 22 hours as well as blocked the constitutive TF expression which is seen with this cell type (N = 3; Figure 2). The TF pathway may prove to be of major importance in the pathogenesis of HUS, and direct targeting of this pathway with irFVIIa might provide a novel therapeutic approach to this disease.
