The mixtures were injected separately into the control flow cell (blank channel) and into a flow cell coupled with heparin-albumin (A) or C3d (B, C)
The mixtures were injected separately into the control flow cell (blank channel) and into a flow cell coupled with heparin-albumin (A) or C3d (B, C). FH most probably mediates complement-induced endothelial cell damage in aHUS. The match (C) system consists Presatovir (GS-5806) of a set of plasma and membrane-bound proteins and protects the organism against invading microbes, removes debris from plasma and tissues, and enhances cell-mediated immune responses. Complement can be activated through three pathways, of which the alternative pathway (AP) acts independently of antibodies. This pathway is initiated spontaneously in plasma leading to an attack against all particles, membranes, and cells that are plasma-exposed and not specifically protected.1 The key molecule in AP activation is C3 and this protein is spontaneously hydrolyzed generating C3(H2O), which is responsible for the formation of the fluid-phase C3 convertase of AP, and eventually leading to deposition of some C3b onto practically all surfaces in contact with plasma. In the absence of regulators the activation proceeds via an amplification cascade and leads to opsonization with C3b, generation of chemotactic and anaphylatoxic peptides, and formation of C membrane attack complexes resulting in cell lysis. Regulation of AP activation occurs at the level of C3b and the AP C3 convertase C3bBb by the plasma proteins factor H (FH)2 and FH-like protein 1 (FHL-1) and by three membrane-bound molecules (CD35, CD46, and CD55). FH is composed of 20 short consensus repeat Presatovir (GS-5806) domains (SCRs) each consisting of 60 amino acids. It regulates AP activation by competing with factor B for binding to Presatovir (GS-5806) C3b, by acting as a co-factor for factor I leading to proteolytic inactivation of C3b, and by enhancing dissociation of the C3bBb complex.3C5 FH is the Cd200 only known regulator involved in down-regulating AP activation on host structures that lack the membrane-bound regulators (eg, basement membranes in kidney glomeruli).6,7 Expression or binding of FH contributes to protection against complement of certain tumor cells8 and pathogenic microbes (eg, sp., and has been suggested within SCRs 16 to 20.11 It appears possible to convert an activator surface into a nonactivator surface by associating heparin onto the surface.25 The physiological role of heparin-binding by FH and the role of the individual heparin- and the C3b-binding sites have not been Presatovir (GS-5806) characterized so far. Also, the exact mechanism how AP discriminates between activators and nonactivators is not yet known. It has recently been shown that atypical hemolytic uremic Presatovir (GS-5806) syndrome (aHUS) is associated with mutations in the FH gene that cause either truncation of FH or point mutations.26C30 The hot spot for the point mutations is within SCRs 19 to 20 of FH. In addition, FH derived from plasma of some of these patients binds heparin with lower affinity than normal FH.29 The aHUS syndrome is characterized by endothelial cell damage, microthrombosis, renal failure, and AP activation.31 Therefore, it seems that protection of endothelial cells from the AP attack is impaired in this condition. FH is known to bind to endothelial cells via the most carboxy-terminal domain.29 So far the effect of the aHUS-associated mutations to binding of FH to both heparin and C3b/C3d in addition to endothelial cells has been measured in one study only29 and both the analyzed single amino acid mutants (R1210C, R1215G) showed decreased binding to heparin, C3b/C3d, and endothelial cells. The observed combination of loss-of-function could be due to altered overall conformation of the domain SCR20, for example secondary to mispairing of the cysteine bridges (R1210C) or introduction of glycine to a stretch that in the previously reported tertiary structures of SCR domains is in the middle of a -strand (R1215G). Therefore, the exact location of the endothelial cell-binding site and its relation to the previously described heparin- and C3b/C3d-binding sites within SCRs 19 to 20 have not been defined. The goal of this study was to characterize the relationship between the binding sites of FH for heparin, C3d, and endothelial cells on SCRs 19 to 20. By site-directed mutagenesis of positively charged amino acids in SCR20 and the use of an aHUS patient-derived FH mutant (E1172Stop) that lacks SCR20 we show that binding of FH to endothelial cells is mediated by the heparin-, but not the C3d-binding site on SCRs 19.