Molecular basis of heme-driven pathway activation in hemolytic disorders
Marie-T. Hopp1, Diana Imhof1
1University of Bonn, Pharmaceutical Biochemistry and Bioanalyitcs, Bonn, Germany
Hemolysis results in an accumulation of labile heme, which leads to proinflammatory and prothrombotic complications. As a regulatory molecule, heme can affect the function and/or stability of proteins through binding to short, surface-exposed amino acid stretches. As such, the stimulation of the complement and coagulation system through direct heme binding to participating proteins (e.g., C3, fibrinogen, and APC) was described. On the cellular level, heme-triggered proinflammatory and procoagulant effects under hemolytic conditions were attributed to the activation of the TLR4- signaling pathway, but direct binding of heme to the receptor has not been analyzed so far. Herein, the functional influence of heme binding to a selection of proinflammatory and procoagulant proteins is demonstrated, characterized by the combination of biochemical approaches. The heme-binding properties of these proteins were analyzed by using surface plasmon resonance spectroscopy. In order to characterize the respective heme-binding sites, the proteins were screened for potential heme-binding motifs by using the webserver HeMoQuest. Subsequently, these motifs were synthesized as nonapeptides and analyzed for heme binding via UV/vis spectroscopy. Promising sites were further evaluated by molecular docking simulations of the heme- protein complexes. Taken together, these results extend the understanding of hemolysis-derived heme as a regulator within the triad of inflammation, the complement, and the blood coagulation system on the molecular level, which will support an improved understanding of the progression of thrombosis and inflammation under hemolytic conditions.