Shiga toxin downregulates tissue factor pathway inhibitor, modulating an increase in the expression of functional tissue factor on endothelium

Introduction: Endothelial expression of tissue factor (TF) may play a major role in (Stx)-related hemolytic uremic syndrome. We examined human umbilical vein endothelial cell (HUVEC) monolayers to determine the interaction between TF and TF pathway inhibitor (TFPI), hypothesizing that changes in TFPI modulate TF expression. Materials and Methods: We studied 1) cell surface expression of globotriasylceramide (Gb3, the receptor for Stx) with Stx-1 (10 pM), TNF alpha (20 Ng/ml), or Stx-1 plus TNFa compared to control, 2) gene expression of TF and TFPI, 3) total cellular and cell surface antigenic TF and TFPI, 4) TFPI secretion into supernatant, and 5) factor Xa production. Results and Conclusions: Gb3 expression, negligible with control and Stx-1 alone, increased significantly with TNFa and with Stx-1 plus TNF alpha. TF mRNA increased 1.25 +/- 0.32-fold (N = 9; p = 0.041) with Stx-1 alone vs. 2.82 +/- 0.92-fold (N = 13; p < 0.0005) with TNFa alone. However, Stx-1 plus TNFa yielded a 6.51 +/- 3.48-fold increase (N = 17; p < 0.0005). TFPI mRNA decreased with TNFa (p < 0.001) and Stx-1 plus TNF alpha (p < 0.0005). Total cellular and cell surface TF antigen increased significantly with TNF alpha, but no further with Stx-1 plus TNF alpha. Total TFPI cellular and cell surface antigen levels, and TFPI secretion decreased significantly with Stx-1 plus TNF alpha. Median factor Xa production for Stx-1 plus TNF alpha vs TNF alpha alone increased (p < 0.001) 3.24-fold. Our results indicate that a subinhibitory concentration of Stx-1 plus TNFa impairs TFPI gene expression, synthesis, cell-surface association, and secretion, leading to augmented functional TF. (C) 2013 Elsevier Ltd. All rights reserved.