Key Points• Heme activates complement alternative pathway in serum and on endothelial cell surfaces.• Heme-induced complement activation in the presence of complement mutations contributes as a secondary hit to the development of aHUS.Atypical hemolytic uremic syndrome (aHUS) is characterized by genetic and acquired abnormalities of the complement system leading to alternative pathway (AP) overactivation and by glomerular endothelial damage, thrombosis, and mechanical hemolysis. Mutations per se are not sufficient to induce aHUS, and nonspecific primary triggers are required for disease manifestation. We investigated whether hemolysis-derived heme contributes to aHUS pathogenesis. We confirmed that heme activates complement AP in normal human serum, releasing C3a, C5a, and sC5b9. We demonstrated that hemeexposed endothelial cells also activate the AP, resulting in cell-bound C3 and C5b9. This was exacerbated in aHUS by genetic abnormalities associated with AP overactivation. Heme interacted with C3 close to the thioester bond, induced homophilic C3 complexes, and promoted formation of an overactive C3/C5 convertase. Heme induced decreased membrane cofactor protein (MCP) and decay-accelerating factor (DAF) expression on endothelial cells, giving Factor H (FH) a major role in complement regulation. Finally, heme promoted a rapid exocytosis of Weibel-Palade bodies, with membrane expression of P-selectin known to bind C3b and trigger the AP, and the release of the prothrombotic von Willebrand factor. These results strongly suggest that hemolysis-derived heme represents a common secondary hit amplifying endothelial damage and thrombosis in aHUS. (Blood. 2013;122(2):282-292) IntroductionAtypical hemolytic uremic syndrome (aHUS) is a rare kidneypredominant thrombotic microangiopathy (TMA) associated with formation of fibrin-platelet clots in the glomerular microvasculature leading to mechanical hemolysis. 1 This disease is related to a dysregulation of the complement alternative pathway (AP), as shown by the identification of genetic or acquired abnormalities in AP regulators or activators in more than 60% of patients. 2 aHUS has an incomplete penetrance among mutation carriers, and a triggering event is presumably required for the disease manifestation. This primary hit can be an infection, pregnancy, drug, or other cell-activating event. Sera from aHUS patients carrying mutations deposit complement on endothelial cells activated by inflammatory cytokines, 3 suggesting continuous aggression on the endothelium. However, not all infections trigger aHUS in patients. It is frequently the second pregnancy postpartum period that is associated with the disease.4 Therefore, other factors appear to be necessary to exceed the threshold of tolerable endothelial stress leading to severe TMA lesions.In acute phase TMA, mechanical hemolysis induces the release of hemoglobin into the bloodstream. 5 This hemoglobin is readily oxidized to ferric hemoglobin (methemoglobin) which, in turn, liberates heme. In physiological conditions...