Severe Congenital Thrombocytopenia Characterized by Decreased Platelet Sialylation and Moderate Complement Activation Caused by Novel Compound Heterozygous Variants in GNE

BackgroundHereditary thrombocytopenias constitute a genetically heterogeneous cause of increased bleeding. We report a case of a 17-year-old boy suffering from severe macrothrombocytopenia throughout his life. Whole genome sequencing revealed the presence of two compound heterozygous variants in GNE encoding the enzyme UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, crucial for sialic acid biosynthesis. Sialic acid is required for normal platelet life span, and biallelic variants in GNE have previously been associated with isolated macrothrombocytopenia. Furthermore, sialic acid constitutes a key ligand for complement factor H (FH), an important inhibitor of the complement system, protecting host cells from indiscriminate attack.MethodsSialic acid expression and FH binding to platelets and leukocytes was evaluated by flow cytometry. The binding of FH to erythrocytes was assessed indirectly by measuring the rate of complement mediated hemolysis. Complement activation was determined by measuring levels of C3bBbP (alternative pathway), C4d (classical/lectin pathway) and soluble terminal complement complex assays.ResultsThe proband exhibited markedly decreased expression of sialic acid on platelets and leukocytes. Consequently, the binding of FH was strongly reduced and moderate activation of the alternative and classical/lectin complement pathways was observed, together with an increased rate of erythrocyte lysis.ConclusionWe report two previously undescribed variants in GNE causing severe congenital macrothrombocytopenia in a compound heterozygous state, as a consequence of decreased platelet sialylation. The decreased sialylation of platelets, leukocytes and erythrocytes affects the binding of FH, leading to moderate complement activation and increased hemolysis.

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Enterococcus faecalis Escapes Complement-Mediated Killing via Recruitment of Complement Factor H

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz226 ◽

2019 ◽

Vol 220 (6) ◽

pp. 1061-1070 ◽

Cited By ~ 2

Author(s):

Youssif M Ali ◽

Robert B Sim ◽

Wilhelm Schwaeble ◽

Mona I Shaaban

Keyword(s):

Enterococcus Faecalis ◽

Alternative Pathway ◽

Critical Role ◽

Intraperitoneal Administration ◽

Factor H ◽

Lectin Pathway ◽

High Dose ◽

Classical Pathway ◽

Complement Factor ◽

Important Species

AbstractBackgroundEnterococcus faecalis is considered to be the most important species of enterococci responsible for blood stream infections in critically ill patients. In blood, the complement system is activated via the classical pathway (CP), the lectin pathway (LP), or the alternative pathway (AP), and it plays a critical role in opsonophagocytosis of bacteria including E faecalis.MethodsIn a mouse model of enterococcus peritonitis, BALB-C mice were challenged with a high dose of E faecalis 12 hours after intraperitoneal administration of anti-Factor H (FH) antibodies or isotype control. Four hours later, control mice developed higher bacterial burden in blood and organs compared with mice treated with anti-FH antibodies.ResultsWe demonstrate that complement recognition molecules C1q, CL-11, and murine ficolin-A bind the enterococcus and drive the CP and the LP in human and mouse. We further describe that E faecalis evades the AP by recruitment of FH on its surface. Our results show a strong C3b deposition on E faecalis via both the CP and the LP but not through the AP.ConclusionsThese findings indicate that E faecalis avoids the complement phagocytosis by the AP via sequestering complement FH from the host blood.

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Limited tryptic cleavage of complement factor H abrogates recognition of sialic acid-containing surfaces by the alternative pathway of complement

Biochemical Journal ◽

10.1042/bj2830317 ◽

1992 ◽

Vol 283 (2) ◽

pp. 317-319 ◽

Cited By ~ 18

Author(s):

V Koistinen

Keyword(s):

Sialic Acid ◽

Cleavage Site ◽

Alternative Pathway ◽

Factor H ◽

Complement Factor H ◽

Complement Factor ◽

Complement Protein ◽

Sheep Erythrocytes ◽

Tryptic Cleavage ◽

N Terminus

The potency of complement factor H (H) in accelerating the decay of the alternative pathway C3 convertase, C3b,Bb (decay-accelerating activity), was used as a measure of the affinity of native versus trypsin-treated H for the complement protein C3b bound to surfaces. When about 99% of H was cleaved at the primary tryptic cleavage site 34 kDa from the N-terminus, its decay-accelerating activity on C3b,Bb on sheep erythrocytes fell about 60-fold, whereas the trypsin-treated H was only 3-4 times less potent than native H in dissociating C3b,Bb on Sepharose 4B. The residual decay-accelerating activity, remaining after the primary cleavage, was not affected by secondary cleavage at a site 120 kDa from the N-terminus, as shown with H preparations cleaved to different degrees. Because cell surface sialic acid is known to be responsible for the high affinity of H for C3b bound on sheep erythrocytes, the results strongly suggest that the integrity of the primary tryptic cleavage site of H is essential for the recognition of sialic acid-containing surfaces by the C3b-H complex.

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Control of Complement Activation by the Long Pentraxin PTX3: Implications in Age-Related Macular Degeneration

Frontiers in Pharmacology ◽

10.3389/fphar.2020.591908 ◽

2020 ◽

Vol 11 ◽

Author(s):

Matteo Stravalaci ◽

Francesca Davi ◽

Raffaella Parente ◽

Marco Gobbi ◽

Barbara Bottazzi ◽

...

Keyword(s):

Macular Degeneration ◽

Complement Activation ◽

Hot Spot ◽

Alternative Pathway ◽

Retinal Pigment ◽

Factor H ◽

Age Related Macular Degeneration ◽

Complement Factor ◽

Rpe Cells ◽

Age Related

Dysregulation of the complement system is central to age-related macular degeneration (AMD), the leading cause of blindness in the developed world. Most of the genetic variation associated with AMD resides in complement genes, with the greatest risk associated with polymorphisms in the complement factor H (CFH) gene; factor H (FH) is the major inhibitor of the alternative pathway (AP) of complement that specifically targets C3b and the AP C3 convertase. Long pentraxin 3 (PTX3) is a soluble pattern recognition molecule that has been proposed to inhibit AP activation via recruitment of FH. Although present in the human retina, if and how PTX3 plays a role in AMD is still unclear. In this work we demonstrated the presence of PTX3 in the human vitreous and studied the PTX3-FH-C3b crosstalk and its effects on complement activation in a model of retinal pigment epithelium (RPE). RPE cells cultured in inflammatory AMD-like conditions overexpressed the PTX3 protein, and up-regulated AP activating genes. PTX3 bound RPE cells in a physiological setting, however this interaction was reduced in inflammatory conditions, whereby PTX3 had no complement-inhibiting activity on inflamed RPE. However, on non-cellular surfaces, PTX3 formed a stable ternary complex with FH and C3b that acted as a “hot spot” for complement inhibition. Our findings suggest a protective role for PTX3 in response to complement dysregulation in AMD and point to a novel mechanism of complement regulation by this pentraxin with potential implications in pathology and pharmacology of AMD.

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Amnion epithelial cells are an effective source of factor H and prevent kidney complement deposition in factor H-deficient mice

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02386-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Federica Casiraghi ◽

Pamela Yossenaidy Rodriguez Ordonez ◽

Nadia Azzollini ◽

Marta Todeschini ◽

Daniela Rottoli ◽

...

Keyword(s):

Kidney Transplantation ◽

Epithelial Cells ◽

Complement Activation ◽

Therapeutic Option ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Factor H ◽

Complement Factor ◽

Complement Deposition ◽

Amnion Epithelial Cells

AbstractComplement factor H (FH) is the main plasma regulator of the alternative pathway of complement. Genetic and acquired abnormalities in FH cause uncontrolled complement activation amplifying, with the consequent accumulation of complement components on the renal glomeruli. This leads to conditions such as C3 glomerulopathy (C3G) and atypical hemolytic uremic syndrome (aHUS). There is no effective therapy for these diseases. Half of the patients progress to end-stage renal disease and the condition recurs frequently in transplanted kidneys. Combined liver/kidney transplantation is a valid option for these patients, but the risks of the procedure and donor organ shortages hamper its clinical application. Therefore, there is an urgent need for alternative strategies for providing a normal FH supply. Human amnion epithelial cells (hAEC) have stem cell characteristics, including the capability to differentiate into hepatocyte-like cells in vivo.Here, we administered hAEC into the livers of newborn Cfh−/− mice, which spontaneously developed glomerular complement deposition and renal lesions resembling human C3G. hAEC engrafted at low levels in the livers of Cfh−/− mice and produced sufficient human FH to prevent complement activation and glomerular C3 and C9 deposition. However, long-term engraftment was not achieved, and eventually hAEC elicited a humoral immune response in immunocompetent Cfh−/− mice.hAEC cell therapy could be a valuable therapeutic option for patients undergoing kidney transplantation in whom post-transplant immunosuppression may protect allogeneic hAEC from rejection, while allogeneic cells provide normal FH to prevent disease recurrence.

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Complement Factor H Family Proteins Modulate Monocyte and Neutrophil Granulocyte Functions

Frontiers in Immunology ◽

10.3389/fimmu.2021.660852 ◽

2021 ◽

Vol 12 ◽

Author(s):

Éva Kárpáti ◽

Mariann Kremlitzka ◽

Noémi Sándor ◽

Dávid Hajnal ◽

Andrea E. Schneider ◽

...

Keyword(s):

Immune Cells ◽

Complement Activation ◽

Vaccine Development ◽

Alternative Pathway ◽

Factor H ◽

Neutrophil Granulocyte ◽

Complement Factor ◽

Heterogeneous Effects ◽

Enhanced Secretion ◽

Main Regulator

Besides being a key effector arm of innate immunity, a plethora of non-canonical functions of complement has recently been emerging. Factor H (FH), the main regulator of the alternative pathway of complement activation, has been reported to bind to various immune cells and regulate their functions, beyond its role in modulating complement activation. In this study we investigated the effect of FH, its alternative splice product FH-like protein 1 (FHL-1), the FH-related (FHR) proteins FHR-1 and FHR-5, and the recently developed artificial complement inhibitor mini-FH, on two key innate immune cells, monocytes and neutrophilic granulocytes. We found that, similar to FH, the other factor H family proteins FHL-1, FHR-1 and FHR-5, as well as the recombinant mini-FH, are able to bind to both monocytes and neutrophils. As a functional outcome, immobilized FH and FHR-1 inhibited PMA-induced NET formation, but increased the adherence and IL-8 production of neutrophils. FHL-1 increased only the adherence of the cells, while FHR-5 was ineffective in altering these functions. The adherence of monocytes was increased on FH, recombinant mini-FH and FHL-1 covered surfaces and, except for FHL-1, the same molecules also enhanced secretion of the inflammatory cytokines IL-1β and TNFα. When monocytes were stimulated with LPS in the presence of immobilized FH family proteins, FH, FHL-1 and mini-FH enhanced whereas FHR-1 and FHR-5 decreased the secretion of TNFα; FHL-1 and mini-FH also enhanced IL-10 release compared to the effect of LPS alone. Our results reveal heterogeneous effects of FH and FH family members on monocytes and neutrophils, altering key features involved in pathogen killing, and also demonstrate that FH-based complement inhibitors, such as mini-FH, may have effects beyond their function of inhibiting complement activation. Thus, our data provide new insight into the non-canonical functions of FH, FHL-1, FHR-1 and FHR-5 that might be exploited during protection against infections and in vaccine development.

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Discovery and Development of the Oral Complement Factor D Inhibitor Danicopan (ACH-4471)

Current Medicinal Chemistry ◽

10.2174/0929867326666191001130342 ◽

2020 ◽

Vol 27 (25) ◽

pp. 4165-4180

Author(s):

Jason A. Wiles ◽

Manuel D. Galvan ◽

Steven D. Podos ◽

Michael Geffner ◽

Mingjun Huang

Keyword(s):

Small Molecules ◽

Clinical Studies ◽

Alternative Pathway ◽

Phase 1 ◽

Immune Defense ◽

Host Cells ◽

Lectin Pathway ◽

Central Component ◽

Classical Pathway ◽

Complement Factor

Complement plays a vital role in our innate immune defense against invasive microorganisms. Excessive complement activation or insufficient control of activation on host cells, however, is associated with several chronic disorders. Essential to the activation and amplification of the Alternative Pathway (AP) of complement, Complement Factor D (CFD) is a specific serine protease that cleaves its unique substrate, Complement Factor B (CFB) in complex with an activated form of complement component 3 (C3), to generate the AP C3 convertases C3(H2O)Bb and C3bBb. These convertases comprise a central component in eliciting effector responses following AP activation, and they also enable a powerful amplification loop for both the Classical Pathway (CP) and Lectin Pathway (LP) of complement. Because CFD is not required for the activation of either the CP or LP, selective CFD inhibition presents a favorable therapeutic approach to modulating complement activity that leaves intact the effector functions following CP and LP activation and thus poses a lower risk of bacterial infection than other complement-directed approaches. This review provides an update on inhibitors of CFD, which have evolved from irreversible small molecules that demonstrate poor selectivity to reversible small molecules and monoclonal antibodies that demonstrate exceptional selectivity and potency. The reversible small-molecule inhibitor danicopan (ACH-4471) has emerged recently as a promising therapeutic candidate. An overview of its discovery, preclinical pharmacology, Phase 1 clinical studies in healthy volunteers, and Phase 2 clinical studies in Paroxysmal Nocturnal Hemoglobinuria (PNH) patients is presented.

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Hiding in plain sight: immune evasion by the staphylococcal protein SdrE

Biochemical Journal ◽

10.1042/bcj20170132 ◽

2017 ◽

Vol 474 (11) ◽

pp. 1803-1806

Author(s):

Andrew B. Herr ◽

Alexander W. Thorman

Keyword(s):

Host Cell ◽

Immune Evasion ◽

Complement Activation ◽

Antimicrobial Agents ◽

Factor H ◽

Surface Proteins ◽

Host Cells ◽

Complement Factor ◽

Bacterial Killing ◽

Extracellular Matrix Components

The human immune system is responsible for identification and destruction of invader cells, such as the bacterial pathogen Staphylococcus aureus. In response, S. aureus brings to the fight a large number of virulence factors, including several that allow it to evade the host immune response. The staphylococcal surface protein SdrE was recently reported to bind to complement Factor H, an important regulator of complement activation. Factor H attaches to the surface of host cells to inhibit complement activation and amplification, preventing the destruction of the host cell. SdrE binding to Factor H allows S. aureus to mimic a host cell and reduces bacterial killing by granulocytes. In a new study published in Biochemical Journal, Zhang et al. describe crystal structures of SdrE and its complex with the C-terminal portion of Factor H. The structure of SdrE and its interaction with the Factor H peptide closely resemble a family of surface proteins that recognize extracellular matrix components such as fibrinogen. However, unbound SdrE forms a novel ‘Closed’ conformation with an occluded peptide-binding groove. These structures reveal a fascinating mechanism for immune evasion and provide a potential avenue for the development of novel antimicrobial agents to target SdrE.

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Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

Blood ◽

10.1182/blood.2020008248 ◽

2020 ◽

Vol 136 (18) ◽

pp. 2080-2089 ◽

Cited By ~ 13

Author(s):

Jia Yu ◽

Xuan Yuan ◽

Hang Chen ◽

Shruti Chaturvedi ◽

Evan M. Braunstein ◽

...

Keyword(s):

Complement Activation ◽

Alternative Pathway ◽

Atypical Hemolytic Uremic Syndrome ◽

Factor H ◽

Spike Protein ◽

Target Cells ◽

Antiphospholipid Antibody ◽

Antiphospholipid Antibody Syndrome ◽

Complement Factor ◽

Spike Proteins

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly contagious respiratory virus that can lead to venous/arterial thrombosis, stroke, renal failure, myocardial infarction, thrombocytopenia, and other end-organ damage. Animal models demonstrating end-organ protection in C3-deficient mice and evidence of complement activation in humans have led to the hypothesis that SARS-CoV-2 triggers complement-mediated endothelial damage, but the mechanism is unclear. Here, we demonstrate that the SARS-CoV-2 spike protein (subunit 1 and 2), but not the N protein, directly activates the alternative pathway of complement (APC). Complement-dependent killing using the modified Ham test is blocked by either C5 or factor D inhibition. C3 fragments and C5b-9 are deposited on TF1PIGAnull target cells, and complement factor Bb is increased in the supernatant from spike protein–treated cells. C5 inhibition prevents the accumulation of C5b-9 on cells, but not C3c; however, factor D inhibition prevents both C3c and C5b-9 accumulation. Addition of factor H mitigates the complement attack. In conclusion, SARS-CoV-2 spike proteins convert nonactivator surfaces to activator surfaces by preventing the inactivation of the cell-surface APC convertase. APC activation may explain many of the clinical manifestations (microangiopathy, thrombocytopenia, renal injury, and thrombophilia) of COVID-19 that are also observed in other complement-driven diseases such as atypical hemolytic uremic syndrome and catastrophic antiphospholipid antibody syndrome. C5 inhibition prevents accumulation of C5b-9 in vitro but does not prevent upstream complement activation in response to SARS-CoV-2 spike proteins.

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Dendrimer end-terminal motif-dependent evasion of human complement and complement activation through IgM hitchhiking

Nature Communications ◽

10.1038/s41467-021-24960-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lin-Ping Wu ◽

Mario Ficker ◽

Jørn B. Christensen ◽

Dmitri Simberg ◽

Panagiotis N. Trohopoulos ◽

...

Keyword(s):

Pattern Recognition ◽

Complement Activation ◽

Alternative Pathway ◽

Factor H ◽

The Body ◽

Lectin Pathway ◽

Blood Proteins ◽

Triggering Mechanisms ◽

Natural Igm ◽

Mannan Binding Lectin

AbstractComplement is an enzymatic humoral pattern-recognition defence system of the body. Non-specific deposition of blood biomolecules on nanomedicines triggers complement activation through the alternative pathway, but complement-triggering mechanisms of nanomaterials with dimensions comparable to or smaller than many globular blood proteins are unknown. Here we study this using a library of <6 nm poly(amido amine) dendrimers bearing different end-terminal functional groups. Dendrimers are not sensed by C1q and mannan-binding lectin, and hence do not trigger complement activation through these pattern-recognition molecules. While, pyrrolidone- and carboxylic acid-terminated dendrimers fully evade complement response, and independent of factor H modulation, binding of amine-terminated dendrimers to a subset of natural IgM glycoforms triggers complement activation through lectin pathway-IgM axis. These findings contribute to mechanistic understanding of complement surveillance of dendrimeric materials, and provide opportunities for dendrimer-driven engineering of complement-safe nanomedicines and medical devices.

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Complement factor H protects tumor cell-derived exosomes from complement-dependent lysis and phagocytosis

PLoS ONE ◽

10.1371/journal.pone.0252577 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252577

Author(s):

Ryan T. Bushey ◽

Elizabeth B. Gottlin ◽

Michael J. Campa ◽

Edward F. Patz

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Cell Lines ◽

Alternative Pathway ◽

Metastatic Potential ◽

Factor H ◽

Host Cells ◽

Complement Factor H ◽

Complement Factor ◽

Classical Complement Pathway

Exosomes are a class of extracellular vesicles (EVs) that are mediators of normal intercellular communication, but exosomes are also used by tumor cells to promote oncogenesis and metastasis. Complement factor H (CFH) protects host cells from attack and destruction by the alternative pathway of complement-dependent cytotoxicity (CDC). Here we show that CFH can protect exosomes from complement-mediated lysis and phagocytosis. CFH was found to be associated with EVs from a variety of tumor cell lines as well as EVs isolated from the plasma of patients with metastatic non-small cell lung cancer. Higher levels of CFH-containing EVs correlated with higher metastatic potential of cell lines. GT103, a previously described antibody to CFH that preferentially causes CDC of tumor cells, was used to probe the susceptibility of tumor cell-derived exosomes to destruction. Exosomes were purified from EVs using CD63 beads. Incubation of GT103 with tumor cell-derived exosomes triggered exosome lysis primarily by the classical complement pathway as well as antibody-dependent exosome phagocytosis by macrophages. These results imply that GT103-mediated exosome destruction can be triggered by antibody Fc-C1q interaction (in the case of lysis), and antibody-Fc receptor interactions (in the case of phagocytosis). Thus, this work demonstrates CFH is expressed on tumor cell derived exosomes, can protect them from complement lysis and phagocytosis, and that an anti-CFH antibody can be used to target tumor-derived exosomes for exosome destruction via innate immune mechanisms. These findings suggest that a therapeutic CFH antibody has the potential to inhibit tumor progression and reduce metastasis promoted by exosomes.

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