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Venous thromboembolism (VTE) is a common, deadly disease with an increasing incidence despite preventive efforts. Clinical observations have associated elevated antibody concentrations or antibody-based therapies with thrombotic events. However, how antibodies contribute to thrombosis is unknown. Here, we show that reduced blood flow enabled immunoglobulin M (IgM) to bind to FcµR and the polymeric immunoglobulin receptor (pIgR), initiating endothelial activation and platelet recruitment. Subsequently, the procoagulant surface of activated platelets accommodated antigen- and FcγR-independent IgG deposition. This leads to classical complement activation, setting in motion a prothrombotic vicious circle. Key elements of this mechanism were present in humans in the setting of venous stasis as well as in the dysregulated immunothrombosis of COVID-19. This antibody-driven thrombosis can be prevented by pharmacologically targeting complement. Hence, our results uncover antibodies as previously unrecognized central regulators of thrombosis. These findings carry relevance for therapeutic application of antibodies and open innovative avenues to target thrombosis without compromising hemostasis.
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The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.
Assuntos
Fator H do Complemento , Receptores de Complemento 3b , Proteínas Recombinantes de Fusão , Humanos , Fator H do Complemento/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/química , Fator H do Complemento/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Ativação do Complemento/efeitos dos fármacos , Antígenos CD55/genética , Antígenos CD55/metabolismo , Hemólise/efeitos dos fármacos , Via Alternativa do Complemento/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Eritrócitos/metabolismoRESUMO
Background: Haemostasis is a crucial process by which the body stops bleeding. It is achieved by the formation of a platelet plug, which is strengthened by formation of a fibrin mesh mediated by the coagulation cascade. In proinflammatory and prothrombotic conditions, multiple interactions of the complement system and the coagulation cascade are known to aggravate thromboinflammatory processes and increase the risk of arterial and venous thrombosis. Whether those interactions also play a relevant role during the physiological process of haemostasis is not yet completely understood. The aim of this study was to investigate the potential role of complement components and activation during the haemostatic response to mechanical vessel injury. Methods: We used a microvascular bleeding model that simulates a blood vessel, featuring human endothelial cells, perfusion with fresh human whole blood, and an inducible mechanical injury to the vessel. We studied the effects of complement inhibitors against components of the lectin (MASP-1, MASP-2), classical (C1s), alternative (FD) and common pathways (C3, C5), as well as a novel triple fusion inhibitor of all three complement pathways (TriFu). Effects on clot formation were analysed by recording of fibrin deposition and the platelet activation marker CD62P at the injury site in real time using a confocal microscope. Results: With the inhibitors targeting MASP-2 or C1s, no significant reduction of fibrin formation was observed, while platelet activation was significantly reduced in the presence of the FD inhibitor. Both common pathway inhibitors targeting C3 or C5, respectively, were associated with a substantial reduction of fibrin formation, and platelet activation was also reduced in the presence of the C3 inhibitor. Triple inhibition of all three activation pathways at the C3-convertase level by TriFu reduced both fibrin formation and platelet activation. When several complement inhibitors were directly compared in two individual donors, TriFu and the inhibitors of MASP-1 and C3 had the strongest effects on clot formation. Conclusion: The observed impact of complement inhibition on reducing fibrin clot formation and platelet activation suggests a role of the complement system in haemostasis, with modulators of complement initiation, amplification or effector functions showing distinct profiles. While the interactions between complement and coagulation might have evolved to support haemostasis and protect against bleeding in case of vessel injury, they can turn harmful in pathological conditions when aggravating thromboinflammation and promoting thrombosis.
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Complement activation in the diseases paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS) results in cytolysis and fatal thrombotic events, which are largely refractory to anticoagulation and/or antiplatelet therapy. Anticomplement therapy, however, efficiently prevents thrombotic events in PNH and aHUS, but the underlying mechanisms remain unresolved. We show that complement-mediated hemolysis in whole blood induces platelet activation similarly to activation by adenosine 5'-diphosphate (ADP). Blockage of C3 or C5 abolished platelet activation. We found that human platelets failed to respond functionally to the anaphylatoxins C3a and C5a. Instead, complement activation did lead to prothrombotic cell activation in the whole blood when membrane attack complex (MAC)-mediated cytolysis occurred. Consequently, we demonstrate that ADP receptor antagonists efficiently inhibited platelet activation, although full complement activation, which causes hemolysis, occurred. By using an established model of mismatched erythrocyte transfusions in rats, we crossvalidated these findings in vivo using the complement inhibitor OmCI and cobra venom factor. Consumptive complement activation in this animal model only led to a thrombotic phenotype when MAC-mediated cytolysis occurred. In conclusion, complement activation only induces substantial prothrombotic cell activation if terminal pathway activation culminates in MAC-mediated release of intracellular ADP. These results explain why anticomplement therapy efficiently prevents thromboembolisms without interfering negatively with hemostasis.
Assuntos
Síndrome Hemolítico-Urêmica Atípica , Hemoglobinúria Paroxística , Humanos , Ratos , Animais , Complexo de Ataque à Membrana do Sistema Complemento , Hemólise , Eritrócitos/metabolismo , Ativação do Complemento , Plaquetas/metabolismo , Hemoglobinúria Paroxística/genéticaRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.
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Antígeno B7-H1/metabolismo , Ativação do Complemento/imunologia , Complemento C3/antagonistas & inibidores , Complemento C5/antagonistas & inibidores , Hemoglobinúria Paroxística/patologia , Antígeno B7-H1/sangue , Antígenos CD55/genética , Antígenos CD59/genética , Complemento C3/imunologia , Complemento C5/imunologia , Granulócitos/metabolismo , Células-Tronco Hematopoéticas/citologia , Hemoglobinúria Paroxística/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Proteínas de Membrana/genética , Monócitos/metabolismoRESUMO
To discriminate between self and non-self surfaces and facilitate immune surveillance, the complement system relies on the interplay between surface-directed activators and regulators. The dimeric modulator FHR-1 is hypothesized to competitively remove the complement regulator FH from surfaces that strongly fix opsonic C3b molecules-a process known as "deregulation." The C-terminal regions of FH and FHR-1 provide the basis of this competition. They contain binding sites for C3b and host surface markers and are identical except for two substitutions: S1191L and V1197A (i.e., FH "SV"; FHR-1 "LA"). Intriguingly, an FHR-1 variant featuring the "SV" combination of FH predisposes to atypical hemolytic uremic syndrome (aHUS). The functional impact of these mutations on complement (de)regulation, and their pathophysiological consequences, have largely remained elusive. We have addressed these questions using recombinantly expressed wildtype, mutated, and truncated versions of FHR-1 and FH. The "SV" to "LA" substitutions did not affect glycosaminoglycan recognition and had only a small effect on C3b binding. In contrast, the two amino acids substantially affected the binding of FH and FHR-1 to α2,3-linked sialic acids as host surfaces markers, with the S-to-L substitution causing an almost complete loss of recognition. Even with sialic acid-binding constructs, notable deregulation was only detected on host and not foreign cells. The aHUS-associated "SV" mutation converts FHR-1 into a sialic acid binder which, supported by its dimeric nature, enables excessive FH deregulation and, thus, complement activation on host surfaces. While we also observed inhibitory activities of FHR-1 on C3 and C5 convertases, the high concentrations required render the physiological impact uncertain. In conclusion, the SV-to-LA substitution in the C-terminal regions of FH and FHR-1 diminishes its sialic acid-binding ability and results in an FHR-1 molecule that only moderately deregulates FH. Such FH deregulation by FHR-1 only occurs on host/host-like surfaces that recruit FH. Conversion of FHR-1 into a sialic acid binder potentiates the deregulatory capacity of FHR-1 and thus explains the pathophysiology of the aHUS-associated FHR-1 "SV" variant.
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Proteínas Sanguíneas/metabolismo , Fator H do Complemento/genética , Regulação da Expressão Gênica , Animais , Complemento C3/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Células Endoteliais/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Hemólise , Humanos , Mutação , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Coelhos , OvinosRESUMO
Blocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a "C3 bypass" activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.
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C3 Convertase da Via Alternativa do Complemento/fisiologia , Complemento C3/antagonistas & inibidores , Complemento C4b/fisiologia , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Modelos Imunológicos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Membrana Celular/imunologia , Complemento C5/química , Inativadores do Complemento/uso terapêutico , Complexo de Ataque à Membrana do Sistema Complemento/fisiologia , Resistência a Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Conformação ProteicaRESUMO
The alternative pathway regulator Factor H-like protein 1 (FHL-1) is composed of the first 7 N-terminal complement control protein domains of Factor H (FH) and protects host surfaces from uncontrolled complement attack. Although FHL-1 shares the N-terminal regulatory domains with FH, it was thought to be a weaker regulator. Recently, the regulatory activity of FHL-1 was shown to be comparable to FH. Nonetheless, the question remained whether FHL-1 is an indispensable, unique regulator. The discovery that FHL-1 is the predominant regulator on Bruch's membrane, a critical site for the onset and progression of age-related-macular degeneration (AMD), showed that FHL-1 is essential for complement regulation. A common single nucleotide polymorphism in FH/FHL-1 that predisposes for AMD underlines the important role of FHL-1 in this context. Reports that some cancer tissues specifically upregulate FHL-1 expression, thereby evading immune surveillance, suggests a pronounced regulatory activity of the splice variant. Several microorganisms specifically recruit FHL-1 to evade complement attack. From a phylogenetic point of view, FHL-1 appears much later than other complement regulators, which could imply a specific role that is possibly not systemic but rather tissue specific. This review focuses on the current knowledge of FHL-1 and its physiological and pathophysiological roles.
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Processamento Alternativo , Proteínas Inativadoras do Complemento C3b/genética , Regulação da Expressão Gênica , Animais , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Humanos , Ligação ProteicaRESUMO
Factor H (FH), a member of the regulators-of-complement-activation (RCA) family of proteins, circulates in human plasma at concentrations of 180-420 mg/L where it controls the alternative pathway (AP) of complement in the fluid phase and on cell surfaces. When the regulatory function of FH is impaired, complement-mediated tissue injury and inflammation occur, leading to diseases such as atypical hemolytic uremic syndrome (a thrombotic microangiopathy or TMA), C3 glomerulopathy (C3G) and monoclonal gammopathy of renal significance (MGRS). A pathophysiological cause of compromised FH function is the development of autoantibodies to various domains of the FH protein. FH autoantibodies (FHAAs) are identified in 10.9% of patients with aHUS, 3.2% of patients with C3G, and rarely in patients with MGRS. The phenotypic variability of FHAA-mediated disease reflects both the complexity of FH and the epitope specificity of FHAA for select regions of the native protein. In this paper, we have characterized FHAA epitopes in a large cohort of patients diagnosed with TMA, C3G or MGRS. We explore the epitopes recognized by FHAAs in these diseases and the association of FHAAs with the genetic deletion of both copies of the CFHR1 gene to show how these disease phenotypes are associated with this diverse spectrum of autoantibodies.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/imunologia , Autoanticorpos/sangue , Glomerulonefrite/imunologia , Paraproteinemias/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndrome Hemolítico-Urêmica Atípica/sangue , Síndrome Hemolítico-Urêmica Atípica/epidemiologia , Síndrome Hemolítico-Urêmica Atípica/genética , Biomarcadores/sangue , Criança , Pré-Escolar , Proteínas Inativadoras do Complemento C3b/genética , Fator H do Complemento/imunologia , Epitopos , Feminino , Deleção de Genes , Predisposição Genética para Doença , Glomerulonefrite/sangue , Glomerulonefrite/epidemiologia , Glomerulonefrite/genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Paraproteinemias/sangue , Paraproteinemias/epidemiologia , Paraproteinemias/genética , Fenótipo , Prevalência , Estudos Retrospectivos , Estados Unidos/epidemiologia , Adulto JovemRESUMO
Background: Eculizumab blocks the lytic complement pathway by inhibiting C5 and has become the standard of care for certain complement-mediated diseases. Previously, we have shown that strong complement activation in vitro overrides the C5 inhibition by Eculizumab, which accounts for residual terminal pathway activity. Results: Here we show that the levels of residual hemolysis in ex vivo assays differ markedly (up to 3.4-fold) across sera collected from different paroxysmal nocturnal hemoglobinuria (PNH) patients on Eculizumab treatment. This large variability of residual activity was also found in sera of healthy donors, thus cross-validating the findings in patients. While PNH patients with residual lytic activities of 11-30% exhibited hemolysis levels around the upper limit of normal (i.e., plasma LDH of ~250 u/L), as expected for PNH patients on Eculizumab therapy, we found sustained and markedly increased LDH levels of around 400 u/L for the patient with the highest residual activity of 37%. Furthermore, the clinical history of nine out of 14 PNH patients showed intravascular breakthrough hemolysis at the time of documented infections despite ample amounts of administered Eculizumab and/or experimentally determined excess over C5. Conclusion: The occurrence of extraordinary high levels of residual terminal pathway activity in PNH patients receiving Eculizumab is rare, but can impair the suppression of hemolysis. The commonly observed low levels of residual terminal pathway activity seen for most PNH patients can exacerbate during severe infections and, thus, can cause pharmacodynamic breakthrough hemolysis in PNH patients treated with Eculizumab.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/uso terapêutico , Hemoglobinúria Paroxística/sangue , Hemoglobinúria Paroxística/tratamento farmacológico , HumanosRESUMO
The plasma proteins Factor H (FH) and its alternate splice variant FH-like protein 1 (FHL-1) are the major regulators of the complement alternative pathway. The indiscriminate nature of alternative pathway activation necessitates the regulators to be host selective, but the underlying principles of selectivity remained largely elusive. By analyzing human FH and FHL-1 for protection of different host and foreign cells (rabbit and yeast), we uncovered a 2-fold discriminatory mechanism of FH in favor of self: relative to FHL-1, FH exhibits a regulatory benefit on self but importantly, also, a regulatory penalty on nonself surfaces, yielding a selectivity factor of â¼2.4 for sialylated host surfaces. We further show that FHL-1 possesses higher regulatory activity than known but is relatively unselective. The reason for this unexpected high activity of FHL-1 is the observation that the complement regulatory site in FH exceeds the established first four domains. Affinity for C3b, cofactor and decay-accelerating activities, and serum assays demonstrate that the regulatory site extends domains 1-4 and includes domains 5-7. But unlike FH, FHL-1 exhibits a fast plasma clearance in mice, occurs sparsely in human plasma (at one fortieth of the FH concentration), and resists deregulation by FH-related proteins. These physiological differences and its late phylogenetic occurrence argue that FHL-1 is crucial for local rather than systemic compartments. In conclusion, we demonstrate a 2-fold discriminatory power of FH to promote selectivity for self over foreign and show that FHL-1 is more active than known but specialized for regulation on local tissues.