The Roles of Complement Activation vs. Shiga Toxin Binding to Endothelium in eHUS.

Summary/AbstractEpidemic hemolytic uremic syndrome (eHUS) is a leading cause of acute renal injury and failure in children(65% of cases of eHUS), who may also suffer seizures, neurocognitive deficits from cerebral ischemic events,and other organ system failure. This project will examine the mechanisms whereby Shiga toxin (Stx)-inducedcomplement activation and Stx interaction with the endothelium generate procoagulant tissue factor (TF)activity and platelet adhesion molecules in this thrombotic microangiopathy. We hypothesize that Stx activatesthe mannose binding lectin-2 (MBL-2) pathway of complement, leading to the binding of the complementeffectors C3b, C5, C5b-9, and MBL2 on or near endothelial cell (EC) Gb3, the inducible receptor on ECs forStx, and on or near toll-like receptor 4 (TLR4). Activation of endothelium via cytokines and Stx follows, withsubsequent upregulation of platelet adhesion molecules and expression of EC pro-coagulant tissue factor (TF)activity, leading to the development of eHUS and further thrombin and factor Xa-mediated complementactivation. Specific Aims/Hypotheses:1: To demonstrate that complement activation by Stx leads to C3d, C5a, and MBL-2 binding to ECsand the appearance of membrane-bound C5b-9, with expression of procoagulant TF activity andplatelet adhesion molecules (P-selectin, high MW von Willebrand Factor (vWF) multimers). To showthat complement is activated via the MBL-2 lectin pathway, and not the alternative pathway.2: To demonstrate that Stx, complement activation, and upregulated procoagulant TF and plateletadhesion molecules contribute to platelet thrombus formation during flow using an in vitro flow modelof eHUS employing monolayers of HUVECs or HGECs. The studies incorporate the effects of shearrate (convection of complement components, clotting factors) characteristic of glomerular arterioles,and shear stress, which are in vivo conditions.3: To identify the molecular mechanisms of complement activation in a novel humanized MBL-2mouse model of Stx-2-induced renal injury.This proposal will demonstrate that Stx leads to MBL-2 lectin pathway complement activation, procoagulant ECTF activity, and platelet-fibrin thrombus formation in flowing blood, in vitro and in vivo, thereby providing noveltherapeutic targets (i.e., MBL-2 and/or TF) for a devastating disease that presently lacks a definitive therapy.1