Interaction of Shiga Toxin with the A-domains and Multimers of von Willebrand Factor

Nathan C Lo,Nancy A Turner,Miguel A Cruz,Joel Moake

doi:10.1074/jbc.m113.487413

Abstract

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr(1605)-Met(1606) peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.

Highlights

von Willebrand Factor (VWF) is a multimeric glycoprotein that causes platelet adherence and aggregation and is cleaved by ADAMTS-13
Quantification of these images demonstrated that anchored ultra-large VWF (ULVWF) strings had 8-fold more Shiga toxin (Stx)-1 attached than Ctx (Fig. 2A)
The experiments described in this study demonstrate that Stx binds to the A1 and A2 domains of VWF and that binding results in a decreased rate of ADAMTS-13-mediated cleavage of the 1605–1606 peptide bond in the A2 domain of VWF

Summary

Background

VWF is a multimeric glycoprotein that causes platelet adherence and aggregation and is cleaved by ADAMTS-13. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS13-mediated cleavage of the Tyr1605-Met1606 peptide bond in the A2 domain. Thrombocytopenia characteristic of DHUS and accumulation of platelets atop intact (i.e. non-desquamated) glomerular endothelial cells in the renal circulation have been shown in sophisticated and carefully timed morphological studies [13, 15] These thrombi may be the result of increased secretion or persistence of von Willebrand Factor (VWF), a multimeric glycoprotein secreted by endothelial cells that mediates platelet adherence and aggregation [3, 4, 16]. We have investigated the binding and functional interactions among Shiga-like toxin, VWF, and ADAMTS-13 to determine the molecular explanation for thrombus formation in DHUS

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION