Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome

Background. The pathogenesis of Shiga toxin (Stx)mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. Muierials and methods: We measured cell Surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2 144 h), Stx- 1 (10(-11) mol L-1, 4-144 h), or their combination (TNF-alpha, 22 h and Stx-1 for the last 0.5-4 It of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). Results and conclusion: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha( and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx- I binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either- cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-a markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha. and Stx-l did not increase further the amount of either TF protein or TF mRNA . We conclude that cytokine-activated HGECs, but not