Richard B. Pouw

TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)‐injury. MBL‐associated serine protease (MASP)‐2 appears to be crucial in this process. The serpin C1‐inhibitor is the major inhibitor of MASP‐2. In addition, aprotinin, a Kunitz‐type inhibitor, was shown to inhibit MASP‐2 activity in vitro. In this study we investigated whether the Kunitz‐type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP‐2. Ex vivo LP was induced and detected by C4‐deposition on mannan‐coated plates. The MASP‐2 activity was measured in a fluid‐phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI‐domains contribute to MASP‐2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP‐2, without affecting MASP‐1 or the classical pathway proteases C1s and C1r. Kunitz‐2 domain of TFPI is required for the inhibition of MASP‐2. Considering the role of MASP‐2 in complement‐mediated I/R‐injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

Complement Regulator FHR-3 Is Elevated either Locally or Systemically in a Selection of Autoimmune Diseases

The human complement factor H-related protein-3 (FHR-3) is a soluble regulator of the complement system. Homozygous cfhr3/1 deletion is a genetic risk factor for the autoimmune form of atypical hemolytic-uremic syndrome (aHUS), while also found to be protective in age-related macular degeneration (AMD). The precise function of FHR-3 remains to be fully characterized. We generated four mouse monoclonal antibodies (mAbs) for FHR-3 (RETC) without cross-reactivity to the complement factor H (FH)-family. These antibodies detected FHR-3 from human serum with a mean concentration of 1 μg/mL. FHR-3 levels in patients were significantly increased in sera from systemic lupus erythematosus, rheumatoid arthritis, and polymyalgia rheumatica but remained almost unchanged in samples from AMD or aHUS patients. Moreover, by immunostaining of an aged human donor retina, we discovered a local FHR-3 production by microglia/macrophages. The mAb RETC-2 modulated FHR-3 binding to C3b but not the binding of FHR-3 to heparin. Interestingly, FHR-3 competed with FH for binding C3b and the mAb RETC-2 reduced the interaction of FHR-3 and C3b, resulting in increased FH binding. Our results unveil a previously unknown systemic involvement of FHR-3 in rheumatoid diseases and a putative local role of FHR-3 mediated by microglia/macrophages in the damaged retina. We conclude that the local FHR-3/FH equilibrium in AMD is a potential therapeutic target, which can be modulated by our specific mAb RETC-2.

Complement Factor H-Related Protein 4A Is the Dominant Circulating Splice Variant of CFHR4

Recent research has elucidated circulating levels of almost all factor H-related (FHR) proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH), fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs) that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 ± 1.46 µg/mL) were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

Self-damage caused by dysregulation of the complement alternative pathway: Relevance of the factor H protein family

The alternative pathway is a continuously active surveillance arm of the complement system, and it can also enhance complement activation initiated by the classical and the lectin pathways. Various membrane-bound and plasma regulatory proteins control the activation of the potentially deleterious complement system. Among the regulators, the plasma glycoprotein factor H (FH) is the main inhibitor of the alternative pathway and its powerful amplification loop. FH belongs to a protein family that also includes FH-like protein 1 and five factor H-related (FHR-1 to FHR-5) proteins. Genetic variants and abnormal rearrangements involving the FH protein family have been linked to numerous systemic and organ-specific diseases, including age-related macular degeneration, and the renal pathologies atypical hemolytic uremic syndrome, C3 glomerulopathies, and IgA nephropathy. This review covers the known and recently emerged ligands and interactions of the human FH family proteins associated with disease and discuss the very recent experimental data that suggest FH-antagonistic and complement-activating functions for the FHR proteins.