Generation Of C5a Research Articles

Complement C5a Functions as a Master Switch for the pH Balance in Neutrophils Exerting Fundamental Immunometabolic Effects.

During sepsis, excessive activation of the complement system with generation of the anaphylatoxin C5a results in profound disturbances in crucial neutrophil functions. Moreover, because neutrophil activity is highly dependent on intracellular pH (pHi), we propose a direct mechanistic link between complement activation and neutrophil pHi In this article, we demonstrate that in vitro exposure of human neutrophils to C5a significantly increased pHi by selective activation of the sodium/hydrogen exchanger. Upstream signaling of C5a-mediated intracellular alkalinization was dependent on C5aR1, intracellular calcium, protein kinase C, and calmodulin, and downstream signaling regulated the release of antibacterial myeloperoxidase and lactoferrin. Notably, the pH shift caused by C5a increased the glucose uptake and activated glycolytic flux in neutrophils, resulting in a significant release of lactate. Furthermore, C5a induced acidification of the extracellular micromilieu. In experimental murine sepsis, pHi of blood neutrophils was analogously alkalinized, which could be normalized by C5aR1 inhibition. In the clinical setting of sepsis, neutrophils from patients with septic shock likewise exhibited a significantly increased pHi These data suggest a novel role for the anaphylatoxin C5a as a master switch of the delicate pHi balance in neutrophils resulting in profound inflammatory and metabolic changes that contribute to hyperlactatemia during sepsis.

TLR7/8 activation in neutrophils impairs immune complex phagocytosis through shedding of FcgRIIA.

Neutrophils play a crucial role in host defense. However, neutrophil activation is also linked to autoimmune diseases such as systemic lupus erythematosus (SLE), where nucleic acid-containing immune complexes (IC) drive inflammation. The role of Toll-like receptor (TLR) signaling in processing of SLE ICs and downstream inflammatory neutrophil effector functions is not known. We observed that TLR7/8 activation leads to a furin-dependent proteolytic cleavage of the N-terminal part of FcgRIIA, shifting neutrophils away from phagocytosis of ICs toward the programmed form of necrosis, NETosis. TLR7/8-activated neutrophils promoted cleavage of FcgRIIA on plasmacytoid dendritic cells and monocytes, resulting in impaired overall clearance of ICs and increased complement C5a generation. Importantly, ex vivo derived activated neutrophils from SLE patients demonstrated a similar cleavage of FcgRIIA that was correlated with markers of disease activity, as well as complement activation. Therapeutic approaches aimed at blocking TLR7/8 activation would be predicted to increase phagocytosis of circulating ICs, while disarming their inflammatory potential.

Complement C5a-C5aR1 signalling drives skeletal muscle macrophage recruitment in the hSOD1G93A mouse model of amyotrophic lateral sclerosis

BackgroundThe terminal pathway of the innate immune complement system is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Terminal complement activation leads to generation of C5a, which through its receptor, C5aR1, drives immune cell recruitment and activation. Importantly, genetic or pharmacological blockage of C5aR1 improves motor performance and reduces disease pathology in hSOD1G93A rodent models of ALS. In this study, we aimed to explore the potential mechanisms of C5aR1-mediated pathology in hSOD1G93A mice by examining their skeletal muscles.ResultsWe found elevated levels of C1qB, C4, fB, C3, C5a, and C5aR1 in tibialis anterior muscles of hSOD1G93A mice, which increased with disease progression. Macrophage cell numbers also progressively increased in hSOD1G93A muscles in line with disease progression. Immuno-localisation demonstrated that C5aR1 was expressed predominantly on macrophages within hSOD1G93A skeletal muscles. Notably, hSOD1G93A × C5aR1-/- mice showed markedly decreased numbers of infiltrating macrophages, along with reduced neuromuscular denervation and improved grip strength in hind limb skeletal muscles, when compared to hSOD1G93A mice.ConclusionThese results indicate that terminal complement activation and C5a production occur in skeletal muscle tissue of hSOD1G93A mice, and that C5a-C5aR1 signalling contributes to the recruitment of macrophages that may accelerate muscle denervation in these ALS mice.

Inherited CD55 Deficiency in Patients with Early Onset Protein-Losing Enteropathy and Thrombosis

Abstract Studies of monogenic gastrointestinal diseases have revealed molecular pathways crucial for gut homeostasis and enabled the development of targeted therapies. We studied 11 patients with abdominal pain and diarrhea caused by protein-losing enteropathy with primary intestinal lymphangiectasia, hypoproteinemia, malabsorption, bowel inflammation, recurrent infections, and angiopathic thromboembolic disease. DNA sequencing identified autosomal recessive mutations in the gene encoding CD55/Decay accelerating factor, leading to loss of protein expression. CD55 regulatory function including complement deposition, costimulation, and anaphylatoxin responses were assessed by shRNA and CRISPR-mediated gene suppression and rescue of CD55 expression using lentiviruses. In all patients, Patient T lymphocytes and CD55-deficient cell lines displayed uncontrolled complement activation causing C3d deposition and generation of C5a. Genetic reconstitution of CD55 or treatment with a complement-inhibitory therapeutic antibody normalized complement activation. Patient T lymphocytes produced increased tumor necrosis factor, which was associated with a decreased ratio of the anti-coagulatory protein thrombomodulin to the pro-coagulatory protein tissue factor on endothelial cells. Additionally, CD55 costimulation by CD97 and contingent production of interleukin-10 were defective in patient T lymphocytes. Altogether, CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and PLE (CHAPLE) disease is caused by abnormal complement activation due to autosomal recessive loss-of-function mutations in CD55, and may be effectively treated with complement-inhibitory therapeutics.

Complement drives neurodegeneration in Gaucher disease

Abstract GBA1 mutations result in excess storage of glucosylceramide (GC) and the induction of Gaucher disease (GD). GD is frequently associated with elevated levels of pro-inflammatory cytokines and the development of brain inflammation. The mechanisms underlying GC-driven brain inflammation in GD are ill-defined. Recently, we described immune complexes of GC-specific IgG autoantibodies in experimental and clinical GD, which induced massive complement activation and C5a generation. Further, we found that C5a-mediated activation of its cognate C5a receptor 1 (C5aR1) tips the balance between GC formation and degradation, thereby fueling excess GC accumulation and inflammation in visceral tissues in experimental and clinical GD. Previously, the C5a/C5aR1 axis was found to regulate the blood brain barrier integrity in systemic lupus and promote neurodegeneration in Alzheimer’s disease. Here, we determined the production of C5a in the brain of Gba1 D409V/knockout (9V/null) GD-prone mice. C5a production in the brain of 9V/null mice was markedly elevated, when compared to WT control mice. Also, 9V/null mice suffered from massive accumulation of GC in the brain and loss of neurons. To assess the relevance of C5a/C5aR1 axis activation for brain inflammation in GD, we targeted glucocerebrosidase (GCase) with conduritol B epoxide (CBE) in WT and C5aR1−/− mice. Strikingly, CBE-injected WT mice died within 30 days. In contrast, all C5aR1−/− mice survived the 60 days observation window, were protected from CBE-induced accumulation of GC in the brain, showed a marked reduction of microglial cell activation and only a minor loss of neurons. Our data suggest that the C5a/C5aR1 axis is a critical driver of neurodegeneration in experimental GD.

Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition

The terminal complement split product C5a has been described as an important mediator in inflammatory diseases. C5a is generated upon cleavage of C5 and earlier research suggests that, besides the known C5 convertases formed upon activation of the complement pathways, various enzymes could activate C5 directly. We demonstrate that eculizumab effectively blocks C5 activation when mediated by C5-convertase formation, but fails to block C5a generation resulting from direct enzymatic cleavage by trypsin and thrombin. C5a generated by these enzymes is shown to be fully biologically functional and can be blocked by IFX-1, a specific monoclonal anti-human C5a antibody. We further report clinical cases of atypical hemolytic uremic syndrome (aHUS) and C3 Glomerulonephritis (C3GN) patients under treatment with eculizumab presenting substantially elevated C5a levels. Thus, blocking the C5 convertase mediated activation of C5 may not be efficient to control C5a-mediated effects in human disease and that a targeted approach is warranted.

Synergy between the classical and alternative pathways of complement is essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus infection.

The pandemic influenza A(H1N1) 2009 virus caused significant morbidity and mortality worldwide thus necessitating the need to understand the host factors that influence its control. Previously, the complement system has been shown to provide protection during the seasonal influenza virus infection, however, the role of individual complement pathways is not yet clear. Here, we have dissected the role of intact complement as well as of its individual activation pathways during the pandemic influenza virus infection using mouse strains deficient in various complement components. We show that the virus infection in C3-/- mice results in increased viral load and 100% mortality, which can be reversed by adoptive transfer of naïve wild-type (WT) splenocytes, purified splenic B cells, or passive transfer of immune sera from WT, but not C3-/- mice. Blocking of C3a and/or C5a receptor signaling in WT mice using receptor antagonists and use of C3aR-/- and C5aR-/- mice showed significant mortality after blocking/ablation of C3aR, with little or no effect after blocking/ablation of C5aR. Intriguingly, deficiency of C4 and FB in mice resulted in only partial mortality (24%-32%) suggesting a necessary cross-talk between the classical/lectin and alternative pathways for providing effective protection. In vitro virus neutralization experiments performed to probe the cross-talk between the various pathways indicated that activation of the classical and alternative pathways in concert, owing to coating of viral surface by antibodies, is needed for its efficient neutralization. Examination of the virus-specific complement-binding antibodies in virus positive subjects showed that their levels vary among individuals. Together these results indicate that cooperation between the classical and alternative pathways not only result in efficient direct neutralization of the pandemic influenza virus, but also lead to the optimum generation of C3a, which when sensed by the immune cells along with the antigen culminates in generation of effective protective immune responses.

Complement drives glucosylceramide accumulation and tissue inflammation in Gaucher disease.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

Cellular reactions and compensatory tissue re-organization during spontaneous recovery after spinal cord injury in neonatal mice.

Following incomplete spinal cord injuries, neonatal mammals display a remarkable degree of behavioral recovery. Previously, we have demonstrated in neonatal mice a wholesale re-establishment and reorganization of synaptic connections from some descending axon tracts (Boulland et al.: PLoS One 8 (2013)). To assess the potential cellular mechanisms contributing to this recovery, we have here characterized a variety of cellular sequelae following thoracic compression injuries, focusing particularly on cell loss and proliferation, inflammation and reactive gliosis, and the dynamics of specific types of synaptic terminals. Early during the period of recovery, regressive events dominated. Tissue loss near the injury was severe, with about 80% loss of neurons and a similar loss of axons that later make up the white matter. There was no sign of neurogenesis, no substantial astroglial or microglial proliferation, no change in the ratio of M1 and M2 microglia and no appreciable generation of the terminal complement peptide C5a. One day after injury the number of synaptic terminals on lumbar motoneurons had dropped by a factor of 2, but normalized by 6 days. The ratio of VGLUT1/2+ to VGAT+ terminals remained similar in injured and uninjured spinal cords during this period. By 24 days after injury, when functional recovery is nearly complete, the density of 5-HT+ fibers below the injury site had increased by a factor of 2.5. Altogether this study shows that cellular reactions are diverse and dynamic. Pronounced recovery of both excitatory and inhibitory terminals and an increase in serotonergic innervation below the injury, coupled with a general lack of inflammation and reactive gliosis, are likely to contribute to the recovery. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 928-946, 2017.

Complement and sepsis-induced heart dysfunction.

It is well known that cardiac dysfunction develops during sepsis in both humans and in rodents (rats, mice). These defects appear to be reversible, since after "recovery" from sepsis, cardiac dysfunction disappears and the heart returns to its function that was present before the onset of sepsis. Our studies, using in vivo and in vitro models, have demonstrated that C5a and its receptors (C5aR1 and C5aR2) play key roles in cardiac dysfunction developing during sepsis. Use of a neutralizing antibody to C5a largely attenuates cardiac dysfunction and other adverse events developing during sepsis. The molecular basis for cardiac dysfunctions is linked to generation of C5a and its interaction with C5a receptors present on surfaces of cardiomyocytes (CMs). It is established that C5a interactions with C5a receptors leads to significant reductions involving faulty contractility and relaxation in CMs. In addition, C5a interactions with C5a receptors on CMs results in reductions in Na+/K+-ATPase in CMs. This ATPase is essential for intact action potentials in CMs. The enzymatic activity and protein for this ATPase were strikingly reduced in CMs during sepsis by unknown mechanisms. In addition, C5a interactions with C5aRs also caused reductions in CM homeostatic proteins that regulate cytosolic [Ca2+]i in CMs: sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and Na+/Ca2+ exchanger (NCX). In the absence of C5a receptors, defects in SERCA2 and NCX in CMs after sepsis are strikingly attenuated. These observations suggest new strategies to protect the heart from dysfunction developing during sepsis.

Complement Activation in a Murine Model of Sickle Cell Disease: Inhibition of Vaso-Occlusion By Blocking C5 Activation

▪IntroductionInnate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. The alternative complement pathway is abnormally activated in SCD and is additionally activated by phosphatidylserine (PS) on the outer leaflet of SS-red blood cells (SS-RBC). PS on the surface of SS-RBC and activated platelets accelerates the assembly of prothrombinase complexes leading to generation of thrombin, which can cleave circulating C5 protein into two biologically active fragments, C5a and C5b. C5a is an anaphylatoxin, a potent pro-inflammatory mediator, that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion (VO). Active C5b fragments stimulate the formation of membrane attack complexes (MAC) on SS-RBC that increases their susceptibility to lysis. We hypothesize that complement activation on the surface of SS-RBC may stimulate VO and RBC turnover in SCD.Methods and ResultsWhole blood from Townes-AA, -AS, and -SS mice (n=4) was collected in EDTA and RBC were immunostained with erythroid specific anti-Ter119 (Ly-76) IgG conjugated to PE-Cy7, anti-C5b-9 (MAC) IgG conjugated to Alexa Fluor 647, and anti-C3 (also specific for activation fragments C3b, iC3b, C3d and C3dg) IgG conjugated to PE. The percentages of Ter119-positive RBC that were positive for MAC were 6.3%, 6.5%, and 26.0% for AA-, AS-, and SS-RBCs, respectively (p<0.01 SS vs. AA and AS) suggesting enhanced C5 activation in Townes-SS mice. However, EDTA plasma C5a levels measured by ELISA were not significantly different between Townes-AA, -AS and -SS mice. C3 activation fragments were also found on a subset of MAC positive RBC. We used a mouse model of hypoxia/reoxygenation (H/R)-induced stasis to investigate the role of complement in VO. Townes-SS mice with implanted dorsal skin-fold chambers were infused with 30 µg of either anti-C5 IgG mAb BB5.1 (which blocks murine C5 cleavage ) (n=4), annexin V (n=2), or with isotype control IgG (n=4) 30 minutes prior to H/R (1 hour of hypoxia at 7% O2, followed by reoxygenation for 4 hours in room air). Percent microvascular stasis (% non-flowing venules) in the subcutaneous venules in the dorsal skin fold chamber window was measured using intravital microscopy at 1 and 4 hours post-hypoxia. Percent stasis was significantly lower in the Townes-SS mice receiving anti-C5 IgG or annexin V than the mice receiving the isotype control IgG at 1 and 4 hours post-hypoxia (Figure 1, means ± SD, *p<0.01 anti-C5 IgG or annexin V vs. control IgG). This finding implies that H/R may acutely activate C5 , thus leading to stasis. Initial studies suggest increased plasma C5a levels in Towne-SS mice treated with isotype control IgG compared to mice treated with anti-C5 IgG in response to H/R.ConclusionsThese results demonstrate an increased percentage of SS-RBC expressing MAC on their surface and inhibition of H/R-induced stasis with anti-C5 IgG. However, circulating C5a levels were not different between untreated Townes-AA, -AS, or -SS mice suggesting that C5a is not chronically elevated in SCD. Rather, the generation of C5a may occur locally and in the acute setting. Current studies underway are examining the effects of anti-C5 IgG on MAC deposition and RBC half-lives; the mechanism(s) of complement activation in SCD mice (classical, alternative, tic-over, etc); the ability of recombinant murine C5a to induce stasis; and complement activation markers on RBC and in plasma from SCD patients and controls. [Display omitted] DisclosuresChen:Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

High-Fat Diet-Induced Complement Activation Mediates Intestinal Inflammation and Neoplasia, Independent of Obesity.

This study characterizes the relations between diet and metabolic conditions on risk for a common cancer and identifies complement activation as a novel target for cancer prevention. Mol Cancer Res; 14(10); 953-65. ©2016 AACR.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003.

BackgroundAntibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling.MethodsPrimary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003).ResultsTreatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement.ConclusionsDespite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.

Novel biomarker and easy to perform ELISA for monitoring complement inhibition in patients with atypical hemolytic uremic syndrome treated with eculizumab

Atypical hemolytic uremic syndrome (aHUS) is a devastating disease characterized by thrombus formation in the microvasculature and is associated with complement dysregulation. The recommended treatment is eculizumab, an humanized monoclonal antibody, which binds complement protein C5 and thereby preventing the assembly of the terminal complement complex (TCC, soluble C5b-9) and the generation of C5a, an anaphylatoxin.The objective of the study was to identify a reliable biomarker, which estimates the degree of complement inactivation under normal and pathophysiological conditions, such as an infection. Here we report on serial measurements of the soluble form of C5b-9, measured in plasma and the TCC capacity (soluble C5b9 after ex-vivo activation, measured in serum) in seven patients with aHUS who were treated with eculizumab. By measuring the latter we were able to assess the maximum possible soluble C5b-9 production under a potent complement trigger. Patients were followed up over a median duration of 3.8years either on biweekly (q2w) or a three-weekly (q3w) maintenance therapy interval.As expected, eculizumab treatment resulted in a profound decrease of TCC capacity (median 55, IQR 44–88AU/ml) compared to baseline (1249, 529–1806AU/ml, p<0.0001) and there was no significant difference in TCC capacity measurements on q2w vs. q3w maintenance dosing schedule. However, during q3w maintenance significantly more single measurements of TCC capacity above the lower cut-off level were detected (50% on q3w vs. 5% on q2w maintenance, p<0.045). Higher TCC capacity levels were associated with lower eculizumab levels.The TCC capacity may represent a novel and easy to perform parameter to determine level of complement inhibition in patients treated with eculizumab, especially because it identifies the residual capacity of inhibition at pathophysiological stages.

Mechanisms of Coagulation-Complement pathway interplay in ischemia/reperfusion induced tissue injury.

Abstract Impaired fibrinolysis in systemic lupus erythematosus (SLE) is a contributing factor in disease complications including hyper-coagulation, tissue ischemia/reperfusion (IR), and increased risk of thrombosis: including stroke and deep vein thrombosis. Aberrant complement activation has been identified as a major factor in tissue injury in SLE. Previous work in the laboratory focused on mechanisms of complement activation in IR induced tissue injury in B6.MRL/lpr mice, an autoimmune prone mouse strain with a propensity to develop lymphadenopathy, glomerulonephritis, and polyarthritis. This work identified a C3-independent mechanism of downstream complement C5a generation contributing to tissue injury. Evidence of interplay between the coagulation and complement pathways was identified. Current work elucidates the mechanism by which the two pathways interact. Using a superior mesenteric artery IR model, we utilized complement and specific extrinsic or intrinsic coagulation pathway inhibitors to evaluate and compare resulting tissue pathology, generation of secondary inflammatory mediators, extent of innate immune activation, and generation of breakdown products involved in regulating feedback regulatory loops after ischemic/reperfusion injury. Inhibition of the extrinsic pathway had a greater impact on downstream complement activation, tissue pathology, vascular integrity, and recruitment of innate immune components versus the inhibition of the extrinsic pathway. Understanding the mechanisms of interplay between the pathways in the autoimmune mouse has significant clinical implications – especially in autoimmune diseases in which inflammation and hypercoagulability are known manifestations.

Plasmin as a complement C5 convertase

Both the complement and coagulation/fibrinolytic systems are essential components of the host defense system that evolved early in the evolution of vertebrates (Krem and Di Cera, 2002). While the coagulation and fibrinolytic systems are necessary for maintenance of the integrity of the vasculature, these systems also respond to infections, and together with the complement system, play an important role in innate and adaptive immunity. There are two pathways in mammals that initiate coagulation and three that initiate complement while fibrinolysis commences in response to the presence of a fibrin clot (Bajic et al., 2015, Smith et al., 2015). The intrinsic or contact pathway for initiation of coagulation and the alternative (properdin) and lectin pathways that initiate complement activation are activated by specific properties of some surfaces such as bacterial or yeast cell walls. Cross talk between the various systems is apparent with several well-described interactions such as that between C4-binding protein (C4BP) that regulates complement by inhibiting the classical initiation pathway and protein S, the latter being a cofactor for activated protein C, a regulator of coagulation and inflammation. There are in vitro and in vivo data that suggest coagulation cascade enzymes such as thrombin could cleave C5, carrying out the same reactions as the C5 convertase (Huber-Lang et al., 2006). The cleavage products, however, did not directly generate C5a (Krisinger et al., 2012). A new study (Foley et al., 2016) shows in a venous stasis model that C5a correlates strongly with clot weight implying that pathways initiated during clot formation lead to C5 cleavage. In contrast, there is a very weak correlation with thrombin–antithrombin complexes, suggesting that thrombin is not directly cleaving C5. They then investigated the ability of thrombin, factor Xa and plasmin to cleave C5 by measuring formation of C5a by ELISA. Plasmin, the effector enzyme in the fibrinolytic cascade, was the only enzyme that was able to cleave complement C5 at a similar rate as the canonical C5 convertase formed from complement components. The resultant C5b can assemble into the next stage of the terminal pathway, C5b,6, showing that the C5b generated by plasmin cleavage is functional in assembling the membrane attack complex. The hypothesis that plasmin can act as a C5 convertase was tested in an arterial model in which plasminogen was activated by tissue plasminogen activator (tPA). They found that the levels of C5a were increased, consistent with plasmin activating C5. C5 activation with consequent generation of C5a and the membrane attack complex is a feature of many diseases such as arthritis, sepsis and acute lung injury. Defining the extent of plasmin's role in C5 cleavage outside of its role in thrombotic diseases would clarify if this reaction is important in other contexts. The possibility of exploiting this mechanism as a therapeutic target would need to be approached carefully because of the critical role plasmin plays not just in fibrinolysis but also in tissue repair and inflammatory cell migration (Syrovets et al., 2012). The idea that plasmin is a bona fide C5 convertase has several implications for the links between complement and coagulation. Fibrinolysis occurs after coagulation and therefore, the C5 cleavage products will be present during clot resolution as well as clot formation and progression. However, C5a is a major chemoattractant and activator for neutrophils, leading to elaboration of procoagulant neutrophil extracellular traps (NETs) (Yousefi et al., 2009). Leukocytes attracted to the thrombus can contribute to lysis by secretion of plasminogen activators, thus the precise balance between the prothrombotic and fibrinolytic factors may depend on the disease setting. This study focused on the role of plasmin in C5 cleavage in thrombotic scenarios, but complement plays a key role in eliminating infections. In some infectious diseases such as Yersinia, fibrinogen deficiency leads to decreased survival while plasminogen deficiency increases survival (Degen et al., 2007). The survival advantage conferred by plasminogen deficiency is effectively eliminated in fibrinogen/plasminogen double deficient mice. Thus formation of fibrin prevents the spread of the disease and fibrinolysis promotes it. The Yersinia virulence factor, Pla, can activate plasminogen and many other bacteria express plasminogen activators. It will be important to investigate the role of plasmin in activating the terminal pathway of complement under these circumstances. Foley et al. (Foley et al., 2016) only consider the forward reactions, but these cascades are tightly regulated. The rate of plasminogen activation is closely controlled by the availability of tPA as well as by plasminogen activator inhibitor-1 while plasmin activity is regulated by alpha-2 antiplasmin. Interestingly, carboxypeptidase B2 (TAFI), which can be activated from its zymogen by either thrombin bound on thrombomodulin or by plasmin (Morser et al., 2010), both reduces activation of plasminogen as well as inactivating C3a and C5a, thereby functioning as a regulator of both the enzyme cleaving C5 and one of the products, C5a.

Development and characterization of an innovative heparin coating to stabilize and protect liposomes against adverse immune reactions

Liposomes have been recognized as excellent drug delivery systems, but when they come in direct contact with different blood components they may trigger an immediate activation of the innate immune system. The aim of the present study was to produce long-circulating, blood-compatible liposomes by developing a construct of liposomes covered by a novel unique heparin complex (CHC; 70 heparin molecules per complex) to avoid recognition by the innate immune system. Unilamellar, cationic liposomes were produced by hand extrusion through a 100-nm polycarbonate membrane. Coating of liposomes with the macromolecular CHC was accomplished by electrostatic interactions. Dynamic light scattering as well as QCM-D measurements were used to verify the electrostatic deposition of the negatively charged CHC to cationic liposomes. The CHC-coated liposomes did not aggregate when in contact with lepirudin anti-coagulated plasma. Unlike previous attempts to coat liposomes with heparin, this technique produced freely moveable heparin strands sticking out from the liposome surface, which exposed AT binding sites reflecting the anticoagulant potentials of the liposomes. In experiments using lepirudin-anticoagulated plasma, CHC-coated liposomes, in contrast to non-coated control liposomes, did not activate the complement system, as evidenced by low C3a and sC5b-9 generation and reduced leakage from the liposomes. In conclusion, we show that liposomes can be successfully coated with the biopolymer CHC, resulting in biocompatible and stable liposomes that have significant application potential.

Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.

Abstract TP278: Complement Cascade Inhibition Abrogates tPA-mediated Hemorrhage and Brain Edema Following Ischemic Stroke

Introduction: Tissue plasminogen activator (tPA) remains the only approved pharmacologic treatment for patients with ischemic stroke. Although intravenous thrombolysis improves outcome in a subset of patients, tPA also increases symptomatic hemorrhage and worsens brain edema, thus limiting its clinical applicability. Among several putative mechanisms, plasmin-meditated complement cascade activation likely plays a critical role in the deleterious effects of tPA. Hypothesis: tPA administration promotes C3 cleavage and complement activation in ischemic brain, and pharmacologic inhibition of the C3a receptor abrogates tPA-mediated hemorrhage and brain edema following reperfused stroke. Methods: C3a concentrations were assessed using ELISA. Transient focal cerebral ischemia was induced in mice by 60 minutes of middle cerebral artery occlusion (MCAO), with tPA (10mg/kg) or saline injected intravenously at reperfusion. C3a receptor antagonist (1mg/kg) or vehicle was administered 45 minutes prior to MCAO. Infarct volume was determined at 24 hours by TTC staining, neurological function was assessed on a 5-point scale, and brain hemorrhage was quantified using a spectrophotometric assay. Cerebral edema was determined by calculating the difference in volume between ischemic and non-ischemic hemispheres and normalized to infarct volume. Results: The incubation of tPA with native C3 as well as plasma promotes the generation of C3a. Similarly, intravenous tPA administration dramatically increases cerebral C3a levels in the ischemic hemisphere following MCAO. Intravenous tPA significantly reduces infarct volume (p&lt;0.05) and improves neurological function (p&lt;0.05) in our stroke model. However, tPA also dramatically increases cerebral hemorrhage (p&lt;0.05) and relative cerebral edema (p&lt;0.05). Administration of a C3a receptor antagonist abrogates this hemorrhagic conversion (p&lt;0.05) and brain edema (p&lt;0.05). Conclusions: Intravenous tPA administration promotes complement activation through C3 cleavage in ischemic brain. Pharmacologic inhibition of the C3a receptor protects against the deleterious side effects of tPA and represents a promising therapeutic strategy for stroke patients.

C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells.

Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.