• C5 inhibitors
• aHUS
• atypical hemolytic uremic syndrome
• complement
• eculizumab
• ravulizumab
• thrombotic microangiopathy

• Victor Babeș University of Medicine and Pharmacy Timișoara

• alternative pathway
• complement
• factor B
• inhibitors
• pharmacological treatment
• therapeutic target

• Humans
• Complement Factor B/metabolism
• Complement Factor B/antagonists & inhibitors
• Animals
• Complement Activation/drug effects
• Complement Inactivating Agents/therapeutic use
• Complement Pathway, Alternative/drug effects
• Molecular Targeted Therapy
• Autoimmune Diseases/immunology
• Autoimmune Diseases/drug therapy
• Autoimmune Diseases/therapy

• Novartis Pharma

• complement
• alternative pathway
• factor b
• aptamer
• inhibitor

• Aptamers, Nucleotide
• Humans
• Atypical Hemolytic Uremic Syndrome/genetics
• Atypical Hemolytic Uremic Syndrome/immunology
• Complement Factor B/genetics
• Complement Pathway, Alternative/immunology
• Gain of Function Mutation
• Complement C3b/metabolism
• Protein Binding

• R35 GM140852 NIGMS NIH HHS
• R35GM140852 HHS | NIH | National Institute of General Medical Sciences (NIGMS)

• autoinflammation
• cytokines
• inflammasome
• interferons
• trained immunity

• Immunity, Innate/genetics
• Humans
• Epigenesis, Genetic/immunology
• Inflammation/immunology
• Inflammation/genetics
• Hereditary Autoinflammatory Diseases/immunology
• Hereditary Autoinflammatory Diseases/genetics
• Autoimmune Diseases/immunology
• Autoimmune Diseases/genetics
• Animals

• 833247 European Research Council
• Spinoza Grant of the Netherlands Organization for Scientific Research

• Autoantibodies
• C3 glomerulopathy
• Complement pathway
• Thrombotic microangiopathy

• Child
• Humans
• Adolescent
• Atypical Hemolytic Uremic Syndrome/genetics
• Glomerulonephritis, Membranoproliferative/drug therapy
• Glomerulonephritis
• Autoantibodies
• Immunoglobulin G
• Antilymphocyte Serum/therapeutic use
• Complement Factor H/genetics

• 5/7/1090/2013-RHN Indian Council of Medical Research
• 5/4/7-18/Nephro/2020-NCDII Indian Council of Medical Research
• BT/PR11030/MED/30/1644/2016 Department of Biotechnology, Government of India (IN)

• Alternative pathway of complement activation
• atypical hemolytic uremic syndrome (aHUS)
• cofactor activity (CA)
• haploinsufficiency
• thrombotic microangiopathy (TMA)

• R01 EY028602 NEI NIH HHS
• R21 AR076534 NIAMS NIH HHS
• R35 GM136352 NIGMS NIH HHS

• C3 glomerulopathy
• atypical hemolytic uremic syndrome
• complement
• genes
• membranoproliferative glomerulonephritis

• Humans
• Complement System Proteins/genetics
• Complement Activation/genetics
• Atypical Hemolytic Uremic Syndrome/diagnosis
• Atypical Hemolytic Uremic Syndrome/genetics
• Gene Frequency
• Kidney Diseases

• Alternative pathway
• C3
• CFB
• COVID-19
• Coding SNPs
• Complement
• Host genomics
• SARS-CoV-2

• Male
• Female
• Humans
• Complement Factor B/genetics
• Complement C3/genetics
• Polymorphism, Single Nucleotide
• Genotype
• Macular Degeneration/genetics
• Complement Factor H/genetics
• COVID-19
• SARS-CoV-2
• Complement C2/genetics

• aHUS
• complement
• endothelial C5b-9 formation
• endothelial cells
• rare variants

• C3 convertase
• C3 glomerulopathies
• C3 nephritic factor (C3Nef)
• complement regulation
• extracellar matrix
• factor B (FB)
• factor H (FH)

• Animals
• Complement C3/genetics
• Complement C3/metabolism
• Complement Pathway, Alternative/genetics
• Properdin/genetics
• Properdin/metabolism
• Complement C3-C5 Convertases/metabolism
• Complement C3 Nephritic Factor/metabolism
• Kidney Diseases
• Extracellular Matrix/metabolism

• R01 DK110023 NIDDK NIH HHS
• National Institutes of Health

• alternative pathway
• clinical
• diagnostic
• laboratory

• Humans
• Complement System Proteins/genetics
• Complement Activation
• Prognosis
• Complement Pathway, Alternative/genetics

• R01 DK125823 NIDDK NIH HHS
• R01 DK130255 NIDDK NIH HHS
• R01 DK113586 NIDDK NIH HHS
• R01 DK076690 NIDDK NIH HHS

• alternative pathway of complement
• complement inactivating agents
• complement system
• glomerular diseases
• rare kidney diseases

• anaphylatoxin
• autoimmunity
• cancer
• complement C3
• inflammation
• opsonin

• C3 glomerulopathies
• MIDAS
• complement system
• convertase
• molecular modeling

Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non‐globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross‐linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.

PDB Code(s): 7NOZ;

• complement
• convertase
• innate immunity
• macromolecular complexes
• nanobody
• structural biology

• Complement C3-C5 Convertases/chemistry
• Complement C3-C5 Convertases/metabolism
• Complement Factor B/chemistry
• Complement Factor B/metabolism
• Enzyme Precursors
• Macromolecular Substances
• Properdin/chemistry
• Properdin/metabolism
• Single-Domain Antibodies
• Thrombospondins

• Lundbeckfonden

• COVID-19
• MIS-C
• SNPs
• children
• complement

• Covid-19 Prefecture of Central Macedonia
• IDG Pfizer Greece

As part of the innate immune system, the complement system plays a key role in defense against pathogens and in host cell homeostasis. This enzymatic cascade is rapidly triggered in the presence of activating surfaces. Physiologically, it is tightly regulated on host cells to avoid uncontrolled activation and self-damage. In cases of abnormal complement dysregulation/overactivation, the endothelium is one of the primary targets. Complement has gained momentum as a research interest in the last decade because its dysregulation has been implicated in the pathophysiology of many human diseases. Thus, it appears to be a promising candidate for therapeutic intervention. However, detecting abnormal complement activation is challenging. In many pathological conditions, complement activation occurs locally in tissues. Standard routine exploration of the plasma concentration of the complement components shows values in the normal range. The available tests to demonstrate such dysregulation with diagnostic, prognostic, and therapeutic implications are limited. There is a real need to develop tools to demonstrate the implications of complement in diseases and to explore the complex interplay between complement activation and regulation on human cells. The analysis of complement deposits on cultured endothelial cells incubated with pathologic human serum holds promise as a reference assay. This ex vivo assay most closely resembles the physiological context. It has been used to explore complement activation from sera of patients with atypical hemolytic uremic syndrome, malignant hypertension, elevated liver enzymes low platelet syndrome, sickle cell disease, pre-eclampsia, and others. In some cases, it is used to adjust the therapeutic regimen with a complement-blocking drug. Nevertheless, an international standard is lacking, and the mechanism by which complement is activated in this assay is not fully understood. Moreover, primary cell culture remains difficult to perform, which probably explains why no standardized or commercialized assay has been proposed. Here, we review the diseases for which endothelial assays have been applied. We also compare this test with others currently available to explore complement overactivation. Finally, we discuss the unanswered questions and challenges to overcome for validating the assays as a tool in routine clinical practice.

• complement
• diagnostics
• endothelial cells
• explorations
• kidney injury
• nephrology
• prognostics
• therapeutics

• CLL
• NHL
• complement system
• obinutuzumab
• rituximab

Thrombotic microangiopathy (TMA) gathers consumptive thrombocytopenia, mechanical haemolytic anemia, and organ damage. Hemolytic uremic syndromes (HUS) are historically classified as primary or secondary to another disease once thrombotic thrombocytopenic purpura (TTP), Shiga-toxin HUS, and cobalamin C-related HUS have been ruled out. Complement genetics studies reinforced the link between complement dysregulation and primary HUS, contributing to reclassifying some pregnancy- and/or post-partum-associated HUS and to revealing complement involvement in severe and/or refractory hypertensive emergencies. By contrast, no firm evidence allows a plausible association to be drawn between complement dysregulation and Shiga-toxin HUS or other secondary HUS. Nevertheless, rare complement gene variants are prevalent in healthy individuals, thus providing an indication that an investigation into complement dysregulation should be carefully balanced and that the results should be cautiously interpreted with the help of a trained geneticist. Several authors have suggested reclassifying HUS in two entities, regardless of they are complement-mediated or not, since the use of eculizumab, an anti-C5 antibody, dramatically lowers the proportion of patients who die or suffer from end-stage renal disease within the year following diagnosis. Safety and the ideal timing of eculizumab discontinuation is currently under investigation, and the long-term consequences of HUS should be closely monitored over time once patients exit emergency departments.

• complement
• critical care
• eculizumab
• haemolytic uremic syndrome
• thrombotic microangiopathies

Pathogenic gain-of-function variants in complement Factor B were identified as causative of atypical Hemolytic Uremic syndrome (aHUS) in 2007. These mutations generate a reduction on the plasma levels of complement C3. A four-month-old boy was diagnosed with hypocomplementemic aHUS in May 2000, and he suffered seven recurrences during the following three years. He developed a severe hypertension which required 6 anti-hypertensive drugs and presented acrocyanosis and several confusional episodes. Plasma infusion or exchange, and immunosuppressive treatments did not improve the clinical evolution, and the patient developed end-stage renal disease at the age of 3 years. Hypertension and vascular symptoms persisted while he was on peritoneal dialysis or hemodialysis, as well as after bilateral nephrectomy. C3 levels remained low, while C4 levels were normal. In 2005, a heterozygous gain-of-function mutation in Factor B (K323E) was found. A combined liver and kidney transplantation (CLKT) was performed in March 2009, since there was not any therapy for complement inhibition in these patients. Kidney and liver functions normalized in the first two weeks, and the C3/C4 ratio immediately after transplantation, indicating that the C3 activation has been corrected. After remaining stable for 4 years, the patient suffered a B-cell non-Hodgkin lymphoma that was cured by chemotherapy and reduction of immunosuppressive drugs. Signs of liver rejection with cholangitis were observed a few months later, and a second liver graft was done 11 years after the CLKT. One year later, the patient maintains normal kidney and liver functions, also C3 and C4 levels are within the normal range. The 12-year follow-up of the patient reveals that, in spite of severe complications, CLKT was an acceptable therapeutic option for this aHUS patient.

• atypical hemolytic uremic syndrome
• combined liver-kidney transplantation
• complement
• factor B
• rare diseases

• Complement activation
• Complement biomarkers
• Complement mediated diseases
• Complement system
• Complement therapeutics

Complement factor B (FB) mutant variants are associated with excessive complement activation in kidney diseases such as atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy and membranoproliferative glomerulonephritis (MPGN). Patients with aHUS are currently treated with eculizumab while there is no specific treatment for other complement-mediated renal diseases. In this study the phenotype of three FB missense variants, detected in patients with aHUS (D371G and E601K) and MPGN (I242L), was investigated. Patient sera with the D371G and I242L mutations induced hemolysis of sheep erythrocytes. Mutagenesis was performed to study the effect of factor D (FD) inhibition on C3 convertase-induced FB cleavage, complement-mediated hemolysis, and the release of soluble C5b-9 from glomerular endothelial cells. The FD inhibitor danicopan abrogated C3 convertase-associated FB cleavage to the Bb fragment in patient serum, and of the FB constructs, D371G, E601K, I242L, the gain-of-function mutation D279G, and the wild-type construct, in FB-depleted serum. Furthermore, the FD-inhibitor blocked hemolysis induced by the D371G and D279G gain-of-function mutants. In FB-depleted serum the D371G and D279G mutants induced release of C5b-9 from glomerular endothelial cells that was reduced by the FD-inhibitor. These results suggest that FD inhibition can effectively block complement overactivation induced by FB gain-of-function mutations.

• C3 glomerulopathy
• atypical hemolytic uremic syndrome
• complement
• danicopan
• factor B
• factor D

• complement
• genetic renal disease
• glomerulopathy
• hemolytic uremic syndrome
• human genetics
• immune complexes
• membranoproliferative glomerulonephritis (MPGN)

• complement
• genetic renal disease
• glomerulopathy
• hemolytic uremic syndrome
• human genetics
• immune complexes
• membranoproliferative glomerulonephritis (MPGN)

• atypical hemolytic uremic syndrome
• case report
• complement
• complement factor b
• gene mutation

• Atypical Hemolytic Uremic Syndrome/genetics
• Complement Factor B/genetics
• Humans
• Kidney Tubular Necrosis, Acute/genetics
• Male
• Middle Aged
• Mutation/genetics

• 81800633 National Natural Science Foundation of China
• JLSCZD2019-076 Grants from the Finance Department of Jilin Province

• Thrombotic microangiopathy (TMA)
• hyperhomocysteinemia
• methylene tetrahydrofolate reductase (MTHFR)
• methylmalonic aciduria and homocystinuria type C protein (MMACHC)

• C3
• Cell imaging
• Complement system
• Endothelial cells
• Flow cytometry

• Atypical Hemolytic Uremic Syndrome/blood
• Atypical Hemolytic Uremic Syndrome/diagnosis
• Atypical Hemolytic Uremic Syndrome/immunology
• Cells, Cultured
• Complement Activation/physiology
• Complement C3/metabolism
• Endothelial Cells/cytology
• Endothelial Cells/metabolism
• Endothelial Cells/pathology
• Flow Cytometry/methods
• Humans
• Protein Binding

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 IC₉₀ values, respectively). When tested in an in vitro aHUS hemolysis model, the anti-properdin MoAbs had 11-fold, and 86-fold lower molar IC₉₀ 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.

• PNH
• aHUS
• alternative pathway
• complement
• endothelial cell
• hemolysis
• monoclonal antibodies
• properdin

• acute kidney injury
• atypical hemolytic-uremic syndrome
• biosimular
• complement-mediated thrombotic microangiopathy
• eculizumab
• pathogenetic therapy

• Antithrombin
• CYP
• Factor B
• Factor VIII
• PolyPhen-2
• missense variants
• structural analysis
• structural bioinformatics

• Blood Coagulation Factors/chemistry
• Blood Coagulation Factors/genetics
• Cytochrome P-450 Enzyme System/chemistry
• Cytochrome P-450 Enzyme System/genetics
• Humans
• Molecular Dynamics Simulation/standards
• Mutation, Missense
• Sequence Analysis, Protein/methods
• Sequence Analysis, Protein/standards
• Software/standards

• R01 HL062565 NHLBI NIH HHS

• complement
• glomerulonephritis
• pediatrics

• Atypical hemolytic uremic syndrome
• Complement pathway, alternative
• Diagnosis, differential
• Eculizumab
• Thrombotic microangiopathies

• Atypical Hemolytic Uremic Syndrome/diagnosis
• Atypical Hemolytic Uremic Syndrome/therapy
• Complement Inactivating Agents/therapeutic use
• Consensus
• Diagnosis, Differential
• Female
• Humans
• Pregnancy
• Thrombotic Microangiopathies/diagnosis

Vascular diseases are multifactorial, often requiring multiple challenges, or ‘hits’, for their initiation. Intra-vascular hemolysis illustrates well the multiple-hit theory where a first event lyses red blood cells, releasing hemolysis-derived products, in particular cell-free heme which is highly toxic for the endothelium. Physiologically, hemolysis derived-products are rapidly neutralized by numerous defense systems, including haptoglobin and hemopexin which scavenge hemoglobin and heme, respectively. Likewise, cellular defense mechanisms are involved, including heme-oxygenase 1 upregulation which metabolizes heme. However, in cases of intra-vascular hemolysis, those systems are overwhelmed. Heme exerts toxic effects by acting as a damage-associated molecular pattern and promoting, together with hemoglobin, nitric oxide scavenging and ROS production. In addition, it activates the complement and the coagulation systems. Together, these processes lead to endothelial cell injury which triggers pro-thrombotic and pro-inflammatory phenotypes. Moreover, among endothelial cells, glomerular ones display a particular susceptibility explained by a weaker capacity to counteract hemolysis injury. In this review, we illustrate the ‘multiple-hit’ theory through the example of intra-vascular hemolysis, with a particular focus on cell-free heme, and we advance hypotheses explaining the glomerular susceptibility observed in hemolytic diseases. Finally, we describe therapeutic options for reducing endothelial injury in hemolytic diseases.

• damage-associated molecular patterns (DAMPs)
• endothelial cells
• glomerular endothelium
• heme
• hemolytic diseases
• two-stage model disease

• complement
• complement component C3
• convertase
• crystal structure
• factor B
• properdin
• regulation

• Binding Sites
• Complement C3-C5 Convertases/chemistry
• Complement C3-C5 Convertases/metabolism
• Complement System Proteins/chemistry
• HEK293 Cells
• Humans
• Models, Molecular
• Mutation
• Properdin/chemistry
• Properdin/metabolism
• Protein Binding
• Protein Conformation
• Protein Multimerization
• Protein Processing, Post-Translational
• Proteolysis
• Structure-Activity Relationship

Thrombotic microangiopathies (TMAs) are a heterogeneous group of syndromes presenting with a distinct clinical triad: microangiopathic hemolytic anemia, thrombocytopenia, and organ damage. We currently recognize two major entities with distinct pathophysiology: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). Beyond them, differential diagnosis also includes TMAs associated with underlying conditions, such as drugs, malignancy, infections, scleroderma-associated renal crisis, systemic lupus erythematosus (SLE), malignant hypertension, transplantation, HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), and disseminated intravascular coagulation (DIC). Since clinical presentation alone is not sufficient to differentiate between these entities, robust pathophysiological features need to be used for early diagnosis and appropriate treatment. Over the last decades, our understanding of the complement system has evolved rapidly leading to the characterization of diseases which are fueled by complement dysregulation. Among TMAs, complement-mediated HUS (CM-HUS) has long served as a disease model, in which mutations of complement-related genes represent the first hit of the disease and complement inhibition is an effective and safe strategy. Based on this knowledge, clinical conditions resembling CM-HUS in terms of phenotype and genotype have been recognized. As a result, the role of complement in TMAs is rapidly expanding in recent years based on genetic and functional studies. Herein we provide an updated overview of key pathophysiological processes underpinning complement activation and dysregulation in TMAs. We also discuss emerging clinical challenges in streamlining diagnostic algorithms and stratifying TMA patients that could benefit more from complement modulation. With the advent of next-generation complement therapeutics and suitable disease models, these translational perspectives could guide a more comprehensive, disease- and target-tailored complement intervention in these disorders.

• HELLP syndrome
• complement inhibitors
• hemolytic uremic syndrome
• thrombotic microangiopathy
• transplant-associated thrombotic microangiopathy

• Endothelial Cells/pathology
• Endothelial Cells/physiology
• Endothelium/anatomy & histology
• Endothelium/pathology
• Endothelium/physiology
• Endothelium/physiopathology
• Humans
• Kidney/anatomy & histology
• Kidney/pathology
• Kidney/physiology
• Kidney/physiopathology
• Kidney Diseases/pathology
• Kidney Diseases/physiopathology
• Kidney Diseases/therapy