Complement Component C3 Binds to the A3 Domain of von Willebrand Factor.

von Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (~280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.

Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers.

Shiga toxin 1 (Stx-1) and Stx-2 produced by enterohemorrhagic Escherichia coli cause the diarrhea-associated hemolytic uremic syndrome (HUS). This type of HUS is characterized by obstruction of the glomeruli and renal microvasculature by platelet-fibrin thrombi, acute renal failure, thrombocytopenia, microvascular hemolytic anemia, and plasma levels of von Willebrand factor (VWF)-cleaving protease (ADAMTS13) activity that are within a broad normal range. We investigated the mechanism of initial platelet accumulation on Stx-stimulated endothelial cells. Stx-1 or Stx-2 (1-10 nM) stimulated the rapid secretion of unusually large (UL) VWF multimeric strings from human umbilical vein endothelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs). Perfused normal human platelets immediately adhered to the secreted ULVWF multimeric strings. Nanomolar concentrations (1-10 nM) of the Shiga toxins were as effective in inducing the formation of ULVWF-platelet strings as millimolar concentrations (0.1-20 mM) of histamine. The rate of ULVWF-platelet string cleavage by plasma or recombinant ADAMTS13 was delayed by 3 to 10 minutes (or longer) in the presence of 10 nM Stx-1 or Stx-2 compared with 20 mM histamine. Stx-induced formation of ULVWF strings, and impairment of ULVWF-platelet string cleavage by ADAMTS13, may promote initial platelet adhesion above glomerular endothelial cells. These processes may contribute to the evolution of glomerular occlusion by platelet and fibrin thrombi in diarrhea-associated HUS.