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Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1α, IFN-γ, LTB-4, MMP-8 and -9, and IL-1Ra with more than 60% (p < 0.05 for all) reduced the upregulation of CD11b on granulocytes and monocytes by 59 and 40%, respectively (p < 0.05), and attenuated monocytic tissue factor expression by 33% (p < 0.001). Blocking CD14 attenuated IL-6 and TNF by more than 50% (p < 0.05). In contrast to single inhibition, combined C5 and CD14 was required for a significantly attenuated prothrombin cleavage (72%, p < 0.05). Markers of thromboinflammation were also quantified in two patients admitted to the hospital with sickle cell disease (SCD) crisis. Both SCD patients had pronounced hemolysis and depleted plasma hemopexin and haptoglobin. Plasma heme and complement activation was markedly increased in one patient, a coinciding observation as demonstrated ex vivo. In conclusion, heme-induced thromboinflammation was largely attenuated by C5 inhibition alone, with a beneficial effect of adding a CD14 inhibitor to attenuate prothrombin activation. Targeting C5 has the potential to reduce thromboinflammation in SCD crisis patients. Copyright 2019 by The American Association of Immunologists. This article is distributed under The American Association of Immunologists, Inc., Reuse Terms and Conditions for Author Choice articles.

The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab. © 2017 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).