Properdin Is a Key Player in Lysis of Red Blood Cells and Complement Activation on Endothelial Cells in Hemolytic Anemias Caused by Complement Dysregulation

Abstract

The complement system alternative pathway (AP) can be activated excessively in inflammatory diseases, particularly when there is defective complement regulation. For instance, deficiency in complement regulators CD55 and CD59, leads to paroxysmal nocturnal hemoglobinuria (PNH), whereas Factor H mutations predispose to atypical hemolytic uremic syndrome (aHUS), both causing severe thrombohemolysis. Despite eculizumab being the treatment for these diseases, benefits vary considerably among patients. Understanding the molecular mechanisms involved in complement regulation is essential for developing new treatments. Properdin, the positive AP regulator, is essential for complement amplification by stabilizing enzymatic convertases. In this study, the role of properdin in red blood cell (RBC) lysis and endothelial cell opsonization in these AP-mediated diseases was addressed by developing in vitro assays using PNH patient RBCs and human primary endothelial cells, where the effects of inhibiting properdin, using novel monoclonal antibodies (MoAbs) that we generated and characterized, were compared to other complement inhibitors. In in vitro models of PNH, properdin inhibition prevented hemolysis of patient PNH type II and III RBCs more than inhibition of Factor B, C3, and C5 (>17-fold, or >81-fold, or >12-fold lower molar IC 90 values, respectively). When tested in an in vitro aHUS hemolysis model, the anti-properdin MoAbs had 11-fold, and 86-fold lower molar IC 90 values than inhibition of Factor B, or C3, respectively ( P < 0.0001). When comparing target/inhibitor ratios in all hemolysis assays, inhibiting properdin was at least as efficient as the other complement inhibitors in most cases. In addition, using in vitro endothelial cell assays, the data indicate a critical novel role for properdin in promoting complement activation on human endothelial cells exposed to heme (a hemolysis by-product) and rH19-20 (to inhibit Factor H cell-surface protection), as occurs in aHUS. Inhibition of properdin or C3 in this system significantly reduced C3 fragment deposition by 75%. Altogether, the data indicate properdin is key in promoting RBC lysis and complement activation on human endothelial cells, contributing to the understanding of PNH and aHUS pathogenesis. Further studies to determine therapeutic values of inhibiting properdin in complement-mediated diseases, in particular those that are characterized by AP dysregulation, are warranted.

Complement activation is a crucial driver of acute kidney injury in rhabdomyolysis

“…This process could be mediated also by properdin. 33,34 Moreover, the injection of heme-scavenger hemopexin, which can be filtered through the glomerular basement membrane, partially improved kidney function and vascular aggression, as described. 7 Hemopexin decreased C3-deposits, showing a direct role of heme in the intrarenal complement activation.…”

Properdin oligomers adopt rigid extended conformations supporting function

“…Our identification of FP oligomers as rigid molecules with a potential for bridging C3b-opsonized cells with other cells may assist future studies analyzing the role of FP in complement-driven pathogenesis and facilitate new strategies for therapeutic modulation of FP activity. Inhibition of FP by function-blocking anti-mouse FP mAbs or FP gene deletion has demonstrated beneficial effects in murine models of arthritis ( Kimura et al, 2010 ), renal ischemia-reperfusion injury ( Miwa et al, 2013 ), allergen-induced airway inflammation ( Wang et al, 2015 ), abdominal aortic aneurysm ( Bertram et al, 2015 ), and atypical hemolytic uremic syndrome ( Ueda et al, 2018 ; Chen et al, 2020 ). Finally, our results should promote experiments clarifying how FP oligomers may act as C3b-independent pattern recognition molecules capable of initiating the AP.…”

Mechanisms Driving Neutrophil-Induced T-cell Immunoparalysis in Ovarian Cancer

“…To inhibit the function of properdin, 100µg/mL of 6E11A4 antiproperdin monoclonal antibody was added to cocultures. 6E11A4 was generated as previously described (19). Briefly, the anti-properdin 6E11A4 hybridoma cell line was developed by Genscript USA Inc. using human properdin that was purified as described previously (20).…”

“…Briefly, the anti-properdin 6E11A4 hybridoma cell line was developed by Genscript USA Inc. using human properdin that was purified as described previously (20). The antibody was purified by Protein G chromatography and the isotype was determined to be IgG1 using a mouse MoAb isotyping test kit (Abd Serotec) (19). Inhibition of both the classical and alternative pathways involved pretreatment with SALO and addition of 6E11A4.…”