RESUMO
Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.
Assuntos
Complemento C5 , Ácidos Graxos , Ornithodoros , Animais , Ácidos Graxos/química , Camundongos , Complemento C5/antagonistas & inibidores , Desenho de Fármacos , Meia-Vida , Proteínas Inativadoras do Complemento/farmacologia , Proteínas Inativadoras do Complemento/química , Inativadores do Complemento/farmacologia , Inativadores do Complemento/farmacocinética , Inativadores do Complemento/química , HumanosRESUMO
Antimicrobial peptides (AMPs) are not only cytotoxic towards host pathogens or cancer cells but also are able to act as immunomodulators. It was shown that some human and non-human AMPs can interact with complement proteins and thereby modulate complement activity. Thus, AMPs could be considered as the base for complement-targeted therapeutics development. Arenicins from the sea polychaete Arenicola marina, the classical example of peptides with a ß-hairpin structure stabilized by a disulfide bond, were shown earlier to be among the most prospective regulators. Here, we investigate the link between arenicins' structure and their antimicrobial, hemolytic and complement-modulating activities using the derivative Ar-1-(C/A) without a disulfide bond. Despite the absence of this bond, the peptide retains all important functional activities and also appears less hemolytic in comparison with the natural forms. These findings could help to investigate new complement drugs for regulation using arenicin derivatives.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Proteínas de Helminto/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Inativadores do Complemento/química , Inativadores do Complemento/toxicidade , Eritrócitos/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Helminto/química , Proteínas de Helminto/toxicidade , Hemólise/efeitos dos fármacos , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Conformação Proteica , Coelhos , Carneiro Doméstico , Relação Estrutura-AtividadeRESUMO
BACKGROUND: A product of rational molecular design, PA-dPEG24 is the lead derivative of the PIC1 family of peptides with multiple functional abilities including classical complement pathway inhibition, myeloperoxidase inhibition, NET inhibition and antioxidant activity. PA-dPEG24 is composed of a sequence of 15 amino acid, IALILEPICCQERAA, and contains a monodisperse 24-mer PEGylated moiety at its C terminus to increase aqueous solubility. Here we explore a sarcosine substitution scan of the PA peptide to evaluate impacts on solubility in the absence of PEGylation and functional characteristics. METHODS: Sixteen sarcosine substitution variants were synthesized and evaluated for solubility in water. Aqueous soluble variants were then tested in standard complement, myeloperoxidase, NET formation and antioxidant capacity assays. RESULTS: Six sarcosine substitution variants were aqueous soluble without requiring PEGylation. Substitution with sarcosine of the isoleucine at position eight yielded a soluble peptide that surpassed the parent molecule for complement inhibition and myeloperoxidase inhibition. Substitution with sarcosine of the cysteine at position nine improved solubility, but did not otherwise change the functional characteristics compared with the parent compound. However, replacement of both vicinal cysteine residues at positions 9 and 10 with a single sarcosine residue reduced functional activity in most of the assays tested. CONCLUSIONS: Several of the sarcosine PIC1 variant substitutions synthesized yielded improved solubility as well as a number of unanticipated structure-function findings that provide new insights. Several sarcosine substitution variants demonstrate increased potency over the parent peptide suggesting enhanced therapeutic potential for inflammatory disease processes involving complement, myeloperoxidase, NETs or oxidant stress.
Assuntos
Antioxidantes/farmacologia , Ativação do Complemento/efeitos dos fármacos , Inativadores do Complemento/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Peptídeos/farmacologia , Peroxidase/antagonistas & inibidores , Sarcosina/farmacologia , Sequência de Aminoácidos , Antioxidantes/química , Inativadores do Complemento/química , Humanos , Oxirredução/efeitos dos fármacos , Peptídeos/química , Sarcosina/química , Solubilidade , Água/químicaRESUMO
Complement is crucial to the immune response, but dysregulation of the system causes inflammatory disease. Complement is activated by three pathways: classical, lectin, and alternative. The classical and lectin pathways are initiated by the C1r/C1s (classical) and MASP-1/MASP-2 (lectin) proteases. Given the role of complement in disease, there is a requirement for inhibitors to control the initiating proteases. In this article, we show that a novel inhibitor, gigastasin, from the giant Amazon leech, potently inhibits C1s and MASP-2, whereas it is also a good inhibitor of MASP-1. Gigastasin is a poor inhibitor of C1r. The inhibitor blocks the active sites of C1s and MASP-2, as well as the anion-binding exosites of the enzymes via sulfotyrosine residues. Complement deposition assays revealed that gigastasin is an effective inhibitor of complement activation in vivo, especially for activation via the lectin pathway. These data suggest that the cumulative effects of inhibiting both MASP-2 and MASP-1 have a greater effect on the lectin pathway than the more potent inhibition of only C1s of the classical pathway.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Complemento C1/antagonistas & inibidores , Inativadores do Complemento/química , Via Clássica do Complemento/efeitos dos fármacos , Lectina de Ligação a Manose da Via do Complemento/efeitos dos fármacos , Sanguessugas/química , Serina Proteases Associadas a Proteína de Ligação a Manose/antagonistas & inibidores , Peptídeos/química , Inibidores de Serina Proteinase/química , Animais , Domínio Catalítico/efeitos dos fármacos , Células Cultivadas , Inativadores do Complemento/farmacologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Peptídeos/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Inibidores de Serina Proteinase/farmacologiaRESUMO
The Ornithodoros moubata Complement Inhibitor (OmCI) binds complement component 5 (C5) with high affinity and, thus, selectively prevents proteolytic activation of the terminal lytic complement pathway. A recombinant version of OmCI (also known as Coversin and rEV576) has proven efficacious in several animal models of complement-mediated diseases and successfully completed a phase Ia clinical trial. Coversin is a small 17 kDa lipocalin protein which has a very short plasma half-life if not bound to C5; therefore, the drug requires frequent dosing. We have improved the pharmacokinetics of Coversin by N-terminal translational conjugation with a 600 residue polypeptide composed of Pro, Ala, and Ser (PAS) residues. To this end, PAS-Coversin as well as the unmodified Coversin were functionally expressed in the cytoplasm of E. coli and purified to homogeneity. Both versions showed identical affinity to human C5, as determined by surface plasmon resonance measurements, and revealed similar complement inhibitory activity, as measured in ELISAs with human serum. In line with the PEG-like biophysical properties, PASylation dramatically prolonged the plasma half-life of uncomplexed Coversin by a factor ≥50 in mice. In a clinically relevant in vitro model of the complement-mediated disease paroxysmal nocturnal hemoglobinuria (PNH) both versions of Coversin effectively reduced erythrocyte lysis. Unexpectedly, while the IC50 values were comparable, PAS-Coversin reached a substantially lower plateau of residual lysis at saturating inhibitor concentrations. Taken together, our data demonstrate two clinically relevant improvements of PASylated Coversin: markedly increased plasma half-life and considerably reduced background hemolysis of erythrocytes with PNH-induced phenotype.
Assuntos
Inativadores do Complemento/química , Inativadores do Complemento/farmacologia , Animais , Dicroísmo Circular , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/farmacocinética , Eritrócitos/efeitos dos fármacos , Feminino , Hemoglobinúria Paroxística/tratamento farmacológico , Hemólise/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Ressonância de Plasmônio de SuperfícieRESUMO
Complement control protein modules (CCPs) occur in numerous functionally diverse extracellular proteins. Also known as short consensus repeats (SCRs) or sushi domains each CCP contains approximately 60 amino acid residues, including four consensus cysteines participating in two disulfide bonds. Varying in length and sequence, CCPs adopt a ß-sandwich type fold and have an overall prolate spheroidal shape with N- and C-termini lying close to opposite poles of the long axis. CCP-containing proteins are important as cytokine receptors and in neurotransmission, cell adhesion, blood clotting, extracellular matrix formation, haemoglobin metabolism and development, but CCPs are particularly well represented in the vertebrate complement system. For example, factor H (FH), a key soluble regulator of the alternative pathway of complement activation, is made up entirely from a chain of 20 CCPs joined by short linkers. Collectively, therefore, the 20 CCPs of FH must mediate all its functional capabilities. This is achieved via collaboration and division of labour among these modules. Structural studies have illuminated the dynamic architectures that allow FH and other CCP-rich proteins to perform their biological functions. These are largely the products of a highly varied set of intramolecular interactions between CCPs. The CCP can act as building block, spacer, highly versatile recognition site or dimerization mediator. Tandem CCPs may form composite binding sites or contribute to flexible, rigid or conformationally 'switchable' segments of the parent proteins.
Assuntos
Enzimas Ativadoras do Complemento/química , Ativação do Complemento , Proteínas Inativadoras do Complemento/química , Desenho de Fármacos , Modelos Moleculares , Engenharia de Proteínas , Animais , Sítios de Ligação , Enzimas Ativadoras do Complemento/genética , Enzimas Ativadoras do Complemento/metabolismo , Fator H do Complemento/química , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Inativadores do Complemento/química , Inativadores do Complemento/metabolismo , Inativadores do Complemento/farmacologia , Proteínas Inativadoras do Complemento/genética , Proteínas Inativadoras do Complemento/metabolismo , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
BACKGROUND: A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. In the present study, we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. METHODS: Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC), was measured. RESULTS: SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59, respectively, in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7%, respectively. CONCLUSIONS: When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach.
Assuntos
Antígenos CD55/genética , Antígenos CD59/genética , Ativação do Complemento/genética , Inativadores do Complemento , Proteína Cofatora de Membrana/genética , Transgenes , Animais , Antígenos CD55/química , Antígenos CD59/química , Inativadores do Complemento/administração & dosagem , Inativadores do Complemento/química , Dependovirus , Vetores Genéticos , Humanos , Proteína Cofatora de Membrana/química , Camundongos , Estrutura Terciária de Proteína/genéticaRESUMO
HSCT-associated thrombotic microangiopathy (TA-TMA) is a severe complication with a poor prognosis. Recently, it has been reported that complement system dysregulation, such as CFH autoantibodies and deletions CFH-related genes 3 and 1, induced TA-TMA. In addition, C4d-positive renal arterioles are both a good marker of complement system activation and a useful diagnostic tool for TA-TMA. Because dysregulation of the complement system is associated with TA-TMA, the complement system might be a therapeutic target, such as eculizumab, a terminal complement inhibitor. Herein, we describe an eight-yr-old boy who developed TA-TMA accompanied by severe renal dysfunction. His renal specimen showed diffuse C4d deposition in the renal arterioles, which is consistent with TA-TMA. Although the patient gradually improved without eculizumab, renal arteriolar C4d staining in sample with TA-TMA shows the complement system activation and may guide the target therapy using the eculizumab.
Assuntos
Complemento C4b/química , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Fragmentos de Peptídeos/química , Artéria Renal/metabolismo , Microangiopatias Trombóticas/etiologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores , Biópsia , Criança , Inativadores do Complemento/química , Humanos , Rim/imunologia , Leucemia Mieloide Aguda/etiologia , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Prognóstico , Indução de Remissão , Microangiopatias Trombóticas/imunologia , Resultado do TratamentoRESUMO
Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (ß chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and ß chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure-function relationship between CVF and complement system.
Assuntos
Inativadores do Complemento/química , Inativadores do Complemento/farmacologia , Venenos Elapídicos/química , Elapidae/metabolismo , Lectinas Tipo C/química , Proteínas de Répteis/química , Proteínas de Répteis/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , China , Inativadores do Complemento/isolamento & purificação , Inativadores do Complemento/metabolismo , Cisteína/análise , Venenos Elapídicos/genética , Venenos Elapídicos/metabolismo , Venenos Elapídicos/farmacologia , Eritrócitos/efeitos dos fármacos , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Dados de Sequência Molecular , Peso Molecular , Oxirredução , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Carneiro DomésticoRESUMO
Complement inhibitors expressed on tumor cells provide an evasion mechanism against mAb therapy and may modulate the development of an acquired antitumor immune response. Here we investigate a strategy to amplify mAb-targeted complement activation on a tumor cell, independent of a requirement to target and block complement inhibitor expression or function, which is difficult to achieve in vivo. We constructed a murine fusion protein, CR2Fc, and demonstrated that the protein targets to C3 activation products deposited on a tumor cell by a specific mAb, and amplifies mAb-dependent complement activation and tumor cell lysis in vitro. In syngeneic models of metastatic lymphoma (EL4) and melanoma (B16), CR2Fc significantly enhanced the outcome of mAb therapy. Subsequent studies using the EL4 model with various genetically modified mice and macrophage-depleted mice revealed that CR2Fc enhanced the therapeutic effect of mAb therapy via both macrophage-dependent FcγR-mediated antibody-dependent cellular cytotoxicity, and by direct complement-mediated lysis. Complement activation products can also modulate adaptive immunity, but we found no evidence that either mAb or CR2Fc treatment had any effect on an antitumor humoral or cellular immune response. CR2Fc represents a potential adjuvant treatment to increase the effectiveness of mAb therapy of cancer.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Inativadores do Complemento/administração & dosagem , Neoplasias/terapia , Animais , Anticorpos Monoclonais/química , Inativadores do Complemento/química , Proteínas do Sistema Complemento/metabolismo , Proteínas do Sistema Complemento/fisiologia , Modelos Animais de Doenças , Imunoconjugados/química , Imunoconjugados/uso terapêutico , Imunoglobulina G/administração & dosagem , Imunoglobulina G/química , Imunoterapia/métodos , Linfoma/tratamento farmacológico , Linfoma/patologia , Linfoma/terapia , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Metástase Neoplásica , Neoplasias/patologia , Receptores de Complemento 3d/administração & dosagem , Receptores de Complemento 3d/química , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Seven flavonoids (1-7), two triterpenoids (8 and 9) and four steroids (10, 11, 12 and 13) were isolated from the whole plant of Tiarella polyphylla. Based on the physicochemical and spectroscopic analyses, their structures were identified as myricetin (1), astragalin (2), afzelin (3), quercitrin (4), myricitin (5), nicotiflorin (6), isoquercitrin (7), tiarellic acid (8), 3beta-hydroxy-20(29)lupen-27-oic acid (9), beta-sitosterol-3-O-beta-D-glucoside (10), stigmasterol-3-O-beta-D-glucoside (11), beta-sitosterol (12) and ergosterol endoperoxide (13). All 13 compounds, with the exception of tiarellic acid were isolated for the first time from T. polyphylla. In the anti-complementary assay, the steroids (12 and 13) exhibited potent activities; whereas, the flavonoids (1 to 7) showed weak or no activities, but even the triterpenoids (8 and 9) and steroidal saponins (10 and 11) evoked hemolysis.
Assuntos
Saxifragaceae/química , Fenômenos Químicos , Físico-Química , Inativadores do Complemento/química , Inativadores do Complemento/isolamento & purificação , Inativadores do Complemento/farmacologia , Via Clássica do Complemento/efeitos dos fármacos , Flavonoides/química , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Saponinas/química , Saponinas/isolamento & purificação , Saponinas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade , Triterpenos/química , Triterpenos/isolamento & purificação , Triterpenos/farmacologiaRESUMO
The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/química , Inativadores do Complemento/farmacologia , Desenho de Fármacos , Hemoglobinúria Paroxística/tratamento farmacológico , Animais , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Ensaios Clínicos como Assunto , Complemento C5/fisiologia , Inativadores do Complemento/uso terapêutico , Aprovação de Drogas , Avaliação Pré-Clínica de Medicamentos , Hemoglobinúria Paroxística/imunologia , Humanos , Camundongos , Engenharia de ProteínasRESUMO
Systemic activation of complement is a pathophysiological response common to severe disturbances such as hemorrhagic shock, major burn injury and sepsis. Intravenous infusion of cobra venom factor (CVF) has been used as an animal model of acute respiratory distress syndrome (ARDS), and reliably and selectively induces rapid intravascular activation of the complement system, leading to acute organ damage. In the present study, we have used different complement inhibitors to investigate the roles of complement products in CVF-induced responses in the rat. Rats were treated with either a C5a receptor antagonist (C5aRA, AcF-[OP(d-Cha)WR], 1 mg/kg i.v. or 10 mg/kg p.o.), a C3a receptor antagonist (C3aRA, N(2)-[(2,2-diphenylethoxy)acetyl]-l-arginine, 0.1 mg/kg i.v.) or a convertase inhibitor, rosmarinic acid (RMA, 10 mg/kg i.v.), prior to CVF-induced complement challenge. Intravenous CVF resulted in hallmark events evident in the development of ARDS, including systemic neutropenia followed by neutrophil migration to the lung and bronchoalveolar vascular leakage, blood pressure alterations, and an increase in TNFalpha levels in both serum and bronchoalveolar lavage fluid. These hemodynamic changes were differentially inhibited by antagonism of C5a receptors, C3a receptors or by inhibition of the entire complement cascade using RMA. This evidence strongly implicates complement factors in the development of lung injury associated with systemic complement activation and identifies complement inhibition as a potential therapeutic target for acute syndromes such as ARDS and other severe systemic shock states mediated by activation of complement.