scholarly journals Rivaroxaban Limits Complement Activation Compared to Warfarin in Antiphospholipid Syndrome Patients with Venous Thromboembolism

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2328-2328 ◽  
Author(s):  
Deepa Jayakody Arachchillage ◽  
Ian J Mackie ◽  
Maria Efthymiou ◽  
Andrew Chitolie ◽  
Beverley J Hunt ◽  
...  
Keyword(s):  
Venous Thromboembolism ◽  
Antiphospholipid Syndrome ◽  
Complement Activation ◽  
Research Funding ◽  
Alternative Pathway ◽  
Factor Xa ◽  
Therapeutic Modality ◽  
Classical Pathway ◽  
Activation Markers ◽  
Complement Proteins

Abstract Background Complement activation may play a role in the pathogenesis of thrombosis and other pathological processes in the antiphospholipid syndrome (APS). Since coagulation proteases, such as factor Xa, can cleave complement proteins, we investigated complement activation in thrombotic APS patients receiving rivaroxaban, a direct factor Xa inhibitor. Aims To assess markers of complement activation (C3a, C5a, terminal complement complex (SC5b-9) and Bb fragment) in patients with thrombotic APS treated with rivaroxaban or warfarin in a prospective randomised controlled trial. Methods 116 APS patients with previous venous thromboembolism, including 22 with systemic lupus erythematosus (SLE), on long-term warfarin (target INR 2.5) were studied. 59 patients remained on warfarin and 57 (11 with SLE in each group) switched to rivaroxaban (20mg daily). EDTA samples were collected at baseline (all patients on warfarin) and on day 42 (2-4 hours after the last dose of rivaroxaban in patients on rivaroxaban). 5/116 patients were excluded (samples from four patients were haemolysed and one patient withdrew from the trial after randomisation), leaving 111 (55 rivaroxaban and 56 warfarin) patients for analysis at both baseline and day 42. Samples were also collected from 55 normal controls (NC). C3a, C5a SC5b-9 and Bb fragment were assessed using ELISA assay kits (QUIDEL Corp). Results Median (95% CI) C3a, C5a, SC5b-9 and Bb fragment were 48.9 (30.1-100.2) ng/mL, 6.8 (2.2-11.8 ng/mL, 113.9 (50.5-170) ng/mL and 1.1 (0.64-1.86) µg/mL in NC, respectively. APS patients had significantly higher complement activation markers compared to NC at both time points irrespective of the anticoagulant (p<0.0001 for C3a, C5a, SC5b-9 and Bb). There were no differences in the markers between the two patient groups at baseline, or in patients remaining on warfarin at day 42 [median (95% CI) for C3a, C5a, SC5b-9 and Bb fragment levels in patient on warfarin on day 0 vs day 42 were: C3a (ng/mL) 77.2 (33.4-180.1) vs 73.6 (34.7-156), C5a (ng/mL) 10.8 (3.2-19.4) vs 10.3 (3.7-19.8), SC5b-9 (ng/mL) 203.5 (70.5-440.3) vs 214.4 (78.3-470.4) and Bb fragment (µg/mL) 1.3 (0.6-2.8) vs 1.4 (0.7-2.4)]. In 55 patients randomised to rivaroxaban, C3a, C5a and SC5b-9 decreased significantly compared with baseline values on warfarin [day 0 versus day 42: C3a (ng/mL): 82.8 (34.6-146.6) vs 64.0 (29.2-125.1), (p=0.004); C5a (ng/mL):12.0 (4.1-17.9) vs 9.0 (2.4-14.8), p=0.01; SC5b-9 (ng/mL): 201.0 (65.6-350.2) vs 171.5 (55.6-245.5), (p=0.001)]. However, Bb fragment levels were unchanged. Conclusions Complement activation occurs in APS despite anticoagulation with warfarin. Rivaroxaban decreased complement activation compared to warfarin, although levels of the markers did not normalise in the majority of patients. This action of rivaroxaban appears to occur via the classical pathway, since Bb fragment (a marker of alternative pathway activation) was unchanged. The observations in rivaroxaban-treated patients may reflect inhibition of factor Xa cleavage of complement proteins, or inhibition of its pro-inflammatory effects (and consequent complement activation). These data suggest that rivaroxaban may have an additional therapeutic modality in thrombotic APS patients by limiting complement activation. Disclosures Mackie: Volution Immuno Pharmaceuticals (Uk) Ltd: Research Funding. Cohen:Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Honoraria diverted to local charity, Research Funding, Speakers Bureau.

Antiphospholipid Syndrome (APS) Patient Plasmas Consistently Activate Complement: A Novel Approach to Detecting a Pathogenic Mechanism for APS

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 719-719
Author(s):  
Jacob H. Rand ◽  
Xiao-Xuan Wu ◽  
Lucia R Wolgast ◽  
Victor Lei ◽  
Edward M. Conway
Keyword(s):  
Antiphospholipid Syndrome ◽  
Complement Activation ◽  
Conflicts Of Interest ◽  
Alternative Pathway ◽  
Disease Process ◽  
Pathogenic Mechanism ◽  
Phospholipid Vesicles ◽  
Lectin Pathway ◽  
Activation Markers ◽  
Novel Approach

Abstract INTRODUCTION: The antiphospholipid syndrome (APS) has remained an enigmatic condition for over 3 decades. Pathogenic mechanism(s) have not been established, and the disorder has therefore continued to be defined by non-mechanistic assays such as the lupus anticoagulant and antiphospholipid immunoassays. Although evidence from animal models has indicated a role for complement activation in the disease process, it is has been remarkable that activation markers are not consistently detected in human APS plasmas. We hypothesized that this might be due to rapid clearance of activation products and that we might be better able to detect a role for complement in APS through a 2 stage system - i.e. by first allowing APS immune complexes to form on phospholipid vesicles and then measuring whether those vesicles might activate complement in normal serum. METHODS : Complement activation markers - specifically C5a and the C5b-9 membrane attack complex - were measured in plasmas of 5 groups of plasmas (n= 10 for each group): normal healthy controls, APS with clinical histories for thrombosis (8 with venous thromboembolism (VTE), 1 venous stroke, and 1 arterial stroke), cancer patients without thrombosis, systemic lupus erythematosus (SLE) without APS, and VTE without APS. These were compared to measurements of complement activation induced by phospholipid vesicles that were pre-incubated with the same plasmas. Additional experiments were then done to: 1) characterize whether the effects could be reproduced with purified APS vs control IgGs and β2-glycoprotein I (β2GPI), 2) define the complement activation pathways involved with factor B and C2 deficient sera and with serum in the presence of EGTA and MgCl2, and 3) determine whether the activation could be inhibited by the monoclonal anti-C5 antibody, eculizimab. RESULTS: When the plasmas themselves were assayed, we found no significant differences in the plasmas levels of C5a or C5b-9 between APS and controls or among the 5 groups (Fig 1 A, B). In contrast, preincubation of phospholipid vesicles with plasmas resulted in significant elevations of both C5a (Fig 1C) and C5b-9 (Fig 1D) in APS compared to controls. Also, C5a levels in cancer, SLE and VTE were significantly lower than in APS, but above the controls. C5b-9 levels were elevated in APS and the other disease groups compared to controls, with the APS levels that were significantly higher than only the cancer group. The results were confirmed using purified IgGs and β2GPI in place of the whole plasmas. In addition, we found that APS-treated vesicles triggered complement activation via both the classical/lectin pathway and the alternative pathway. Eculizimab completely abrogated the complement activation. CONCLUSIONS: To our knowledge, this is the first demonstration that APS patient plasmas clearly and consistently activate complement. This was achieved through the novel approach of first incubating the plasmas with phospholipid vesicles which serve as platforms for the assembly of APS plasma-derived protein complexes that, in turn, activate complement. In addition, these findings may provide the basis for a mechanistic biomarker for disease activity in APS, by detecting complement activation at the site of injury. * P<0.05 vs Controls. # P<0.05 vs APS Disclosures No relevant conflicts of interest to declare.

Complement activation and white cell sequestration in postischemic skeletal muscle

1990 ◽  
Vol 259 (2) ◽  
pp. H525-H531
Author(s):  
B. B. Rubin ◽  
A. Smith ◽  
S. Liauw ◽  
D. Isenman ◽  
A. D. Romaschin ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Complement Activation ◽  
Alternative Pathway ◽  
Ischemia Reperfusion ◽  
White Blood Cells ◽  
Classical Pathway ◽  
Myeloperoxidase Activity ◽  
Factor B ◽  
Complement Proteins ◽  
Muscle Ischemia

After skeletal muscle ischemia, tissue damage is augmented during reperfusion. White blood cells (WBCs) and complement proteins may participate in the reperfusion injury. The purpose of this study was to define the kinetics of classical and alternative pathway complement activation and WBC sequestration by postischemic skeletal muscle during the first 48 h of reperfusion in vivo. The isolated canine gracilis muscle model was used. Systemic levels of the complement proteins factor B (alternative pathway) and C4 (classical pathway) were quantitated by hemolytic assay. WBC sequestration was measured by gracilis arterial-venous WBC differences and tissue myeloperoxidase activity. Reperfusion was associated with an 18% decrease in systemic factor B levels but no consistent change in systemic C4 levels. WBCs were sequestered during the first 4 h of reperfusion, and tissue myeloperoxidase activity was elevated 97-fold after 48 h of reperfusion. These results suggest that skeletal muscle ischemia-reperfusion stimulates 1) activation of the alternative but not the classical complement pathway and 2) an immediate and prolonged sequestration of WBCs.

scholarly journals Placental Complement Activation in Fetal and Neonatal Alloimmune Thrombocytopenia: An Observational Study

2021 ◽  
Vol 22 (13) ◽  
pp. 6763
Author(s):  
Thijs W. de Vos ◽  
Dian Winkelhorst ◽  
Hans J. Baelde ◽  
Kyra L. Dijkstra ◽  
Rianne D. M. van Bergen ◽  
...  
Keyword(s):  
Complement Activation ◽  
Histopathological Examination ◽  
Hla Class I ◽  
Unknown Etiology ◽  
Low Grade ◽  
Classical Pathway ◽  
Newly Diagnosed ◽  
Activation Markers ◽  
Amsterdam Criteria ◽  
Alloimmune Thrombocytopenia

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a disease that causes thrombocytopenia and a risk of bleeding in the (unborn) child that result from maternal alloantibodies directed against fetal, paternally inherited, human platelet antigens (HPA). It is hypothesized that these alloantibodies can also bind to the placenta, causing placental damage. This study aims to explore signs of antibody-mediated placental damage in FNAIT. We performed a retrospective study that included pregnant women, their newborns, and placentas. It comprised 23 FNAIT cases, of which nine were newly diagnosed (14 samples) and 14 were antenatally treated with intravenous immune globulins (IVIg) (21 samples), and 20 controls, of which 10 had anti-HLA-class I antibodies. Clinical information was collected from medical records. Placental samples were stained for complement activation markers (C1q, C4d, SC5b-9, and mannose-binding lectin) using immunohistochemistry. Histopathology was examined according to the Amsterdam criteria. A higher degree of C4d deposition was present in the newly diagnosed FNAIT cases (10/14 samples), as compared to the IVIg-treated FNAIT cases (2/21 samples, p = 0.002) and anti-HLA-negative controls (3/20 samples, p = 0.006). A histopathological examination showed delayed maturation in four (44%) placentas in the newly diagnosed FNAIT cases, five (36%) in the IVIg-treated FNAIT cases, and one in the controls (NS). C4d deposition at the syncytiotrophoblast was present in combination with low-grade villitis of unknown etiology in three newly diagnosed FNAIT cases that were born SGA. We conclude that a higher degree of classical pathway-induced complement activation is present in placentas from pregnancies with untreated FNAIT. This may affect placental function and fetal growth.

scholarly journals Infectious diseases associated with complement deficiencies.

1991 ◽  
Vol 4 (3) ◽  
pp. 359-395 ◽  
Author(s):  
J E Figueroa ◽  
P Densen
Keyword(s):  
Infectious Diseases ◽  
Host Defense ◽  
Complement Activation ◽  
Immune Modulation ◽  
Alternative Pathway ◽  
Membrane Attack Complex ◽  
Host Cells ◽  
Microbial Pathogens ◽  
Classical Pathway ◽  
Cellular Effects

The complement system consists of both plasma and membrane proteins. The former influence the inflammatory response, immune modulation, and host defense. The latter are complement receptors, which mediate the cellular effects of complement activation, and regulatory proteins, which protect host cells from complement-mediated injury. Complement activation occurs via either the classical or the alternative pathway, which converge at the level of C3 and share a sequence of terminal components. Four aspects of the complement cascade are critical to its function and regulation: (i) activation of the classical pathway, (ii) activation of the alternative pathway, (iii) C3 convertase formation and C3 deposition, and (iv) membrane attack complex assembly and insertion. In general, mechanisms evolved by pathogenic microbes to resist the effects of complement are targeted to these four steps. Because individual complement proteins subserve unique functional activities and are activated in a sequential manner, complement deficiency states are associated with predictable defects in complement-dependent functions. These deficiency states can be grouped by which of the above four mechanisms they disrupt. They are distinguished by unique epidemiologic, clinical, and microbiologic features and are most prevalent in patients with certain rheumatologic and infectious diseases. Ethnic background and the incidence of infection are important cofactors determining this prevalence. Although complement undoubtedly plays a role in host defense against many microbial pathogens, it appears most important in protection against encapsulated bacteria, especially Neisseria meningitidis but also Streptococcus pneumoniae, Haemophilus influenzae, and, to a lesser extent, Neisseria gonorrhoeae. The availability of effective polysaccharide vaccines and antibiotics provides an immunologic and chemotherapeutic rationale for preventing and treating infection in patients with these deficiencies.

scholarly journals Time-Dependent Activation of Complement system during Storage of Platelet Product

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4287-4287
Author(s):  
Jian Chen ◽  
Shangbin Yang ◽  
Spero R Cataland ◽  
Haifeng M Wu
Keyword(s):  
Complement System ◽  
Complement Activation ◽  
Research Funding ◽  
Storage Time ◽  
Adverse Reactions ◽  
Alternative Pathway ◽  
Pathway Activation ◽  
The Complement System ◽  
Transfusion Reactions

Abstract Platelet transfusion is known for carrying a high incidence of clinically significant transfusion reactions such as febrile nonhemolytic transfusion reaction. The mechanism responsible for these transfusion-associated adverse events, however, is poorly understood. In this study, we hypothesize that prolonged in vitro storage activates the complement system in the platelet product that in turn causes a high frequency of transfusion reactions. Fresh platelet units obtained from three blood donors were stored on a temperature controlled platelet rotator between 22-24 C°. An aliquot of platelet product was obtained using sterile techniques from each unit on day 2 through day 7. The platelet product from each collection was then immediately centrifuged to obtain platelet poor plasma for the study of complement activation levels. For all study samples, C4d levels were assayed to evaluate the activation of the classical pathway, factor Bb levels were measured to determine the status of the complement alternative pathway, C3a levels were used to examine common pathway activation, and C5a and C5b-9 were assayed for determination of the terminal pathway activation of the complement system. The reference range for each complement factor was determined using citrated plasma from 40 healthy donors. As shown in table 1, both C4d and C3a demonstrated time-dependent increases relevant to storage time. On day 7, C4d and C3a levels were five-fold higher than their baseline levels measured on day 2. In contrast, factor Bb levels remained stable and within the normal range throughout the study. Over a storage span of seven days, the terminal complement factors C5a and C5b-9 were also significantly increased, although not as dramatically as C4d and C3a. Figure 1 illustrates a progressive increase of C3 activation in all three study donors over the time of storage (2-7 days). This report, for the first time, provides strong evidence that substantial complement activation occurs in the platelet products under standard storage conditions. A longer storage time of platelet product in vitro is accompanied by a remarkable elevation of complement activation biomarkers. By examining the pattern of complement profiles in the stored platelets, we further demonstrated that the activation of the classic pathway, rather than alternative pathway, appears to be the driving event that leads up to a level of over-reactivity of the complement system. Given the fact that complement hyperactivation is known to disrupt host homeostasis and cause disease, the adverse reactions seen in platelet recipients is likely related to the infusion of C3a and C5a which are known to be potent inflammatory cytokines. The observations from this study therefore provide a new perspective in understanding the pathophysiology responsible for adverse reactions from platelet transfusions. Further studies will be required to fully evaluate the clinical impact of complement activation in transfused platelet products. Figure 1 Figure 1. Disclosures Cataland: Alexion Corporation: Honoraria, Research Funding, Speakers Bureau. Wu:Alexion Corporation: Honoraria, Research Funding, Speakers Bureau.

scholarly journals Multi-organ complement deposition in COVID-19 patients

2021 ◽  
Author(s):  
Paolo Macor ◽  
Paolo Durigutto ◽  
Alessandro Mangogna ◽  
Rossana Bussani ◽  
Stefano D'Errico ◽  
...  
Keyword(s):  
Complement Activation ◽  
Alternative Pathway ◽  
Tissue Level ◽  
Spike Protein ◽  
Classical Pathway ◽  
Limited Information ◽  
Alveolar Cells ◽  
Complement Components ◽  
Alternative Pathways ◽  
Activation Products

Background: Increased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at tissue level. The mechanisms and pathways of local complement activation remain unclear. Methods: We performed immunofluorescence analyses of autopsy specimens of lungs, kidney and liver from nine COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG and spike protein of SARS-CoV-2. Findings: Lung deposits of C1q, C4, C3 and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. Factor B deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on hepatic artery and portal vein of the liver. Interpretation. Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease.

scholarly journals Bacteremia, Not Coagulation Proteases Contribute to In Vivo Complement Activation in Sepsis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 275-275
Author(s):  
Ravi Shankar Keshari ◽  
Robert Silasi-Mansat ◽  
Cristina Lupu ◽  
Fletcher B. Taylor ◽  
Florea Lupu
Keyword(s):  
Complement Activation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Coagulation Cascade ◽  
Activation Markers ◽  
E Coli ◽  
Complement Proteins ◽  
Direct Activation

Abstract Bacterial sepsis induces strong activation of coagulation, complement and fibrinolytic systems that contribute to disseminated intravascular coagulation, organ damage and death. While the contact of the blood with pathogens or pathogen-associated molecular patterns (PAMPs) can trigger the activation of both systems, a bidirectional complement-coagulation crosstalk is believed to occur. Although the role of complement activation products as positive regulators of coagulation is documented, direct activation of the complement proteins by thrombin or other hemostatic proteases was alluded but not demonstrated in vivo. Here we aimed to: (i) determine if in vivo generation of thrombin and other hemostatic proteases can activate the complement proteins and (ii) discriminate between the direct effect of the pathogen/PAMPs vs. hemostatic proteases on complement activation in a clinically relevant model of sepsis. We have compared the time-course of complement activation markers (C3b, C5a and C5b-9 terminal complex) in plasma of baboons exposed to 1010 cfu/kg (LD100) E. coli vs. intravenous infusion of factor Xa/PC:PS, a potent procoagulant stimulus. In baboons challenged with LD100 E. coli, complement activation markers C3b, C5a and C5b-9 reached maximum levels after 2 hrs (see figure). Complement activation coincided with the peak of bacteremia and LPS, but not with markers of thrombin generation (TAT and fibrinogen consumption; see figure) or fibrinolysis (FDP, D-dimers), which reached peak levels after 6 hours. Differently, infusion of FXa/PC:PS (36.6 pmol/L FXa and 56.3 nmol/L PC/PS per kg body weight) induced a rapid burst of thrombin and almost full consumption of fibrinogen during the first 10 min post-infusion, with no increase of complement activation markers. Based on these data we conclude that in vivo activation of the coagulation cascade does not support complement activation as was postulated by previous in vitro studies. Therefore, we conclude that pathogens and PAMPs are the main activators of the complement during sepsis while direct activation by hemostatic proteases is minor or absent. Figure Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Complement-dependent Clearance of Apoptotic Cells by Human Macrophages

1998 ◽  
Vol 188 (12) ◽  
pp. 2313-2320 ◽  
Author(s):  
Dror Mevorach ◽  
John O. Mascarenhas ◽  
Debra Gershov ◽  
Keith B. Elkon
Keyword(s):  
Cell Surface ◽  
Complement Activation ◽  
Apoptotic Cell ◽  
Alternative Pathway ◽  
Systemic Circulation ◽  
Classical Pathway ◽  
Apoptotic Cells ◽  
Complement Components ◽  
Serum Factors ◽  
Systemic Autoimmunity

Apoptotic cells are rapidly engulfed by phagocytes, but the receptors and ligands responsible for this phenomenon are incompletely characterized. Previously described receptors on blood- derived macrophages have been characterized in the absence of serum and show a relatively low uptake of apoptotic cells. Addition of serum to the phagocytosis assays increased the uptake of apoptotic cells by more than threefold. The serum factors responsible for enhanced uptake were identified as complement components that required activation of both the classical pathway and alternative pathway amplification loop. Exposure of phosphatidylserine on the apoptotic cell surface was partially responsible for complement activation and resulted in coating the apoptotic cell surface with C3bi. In the presence of serum, the macrophage receptors for C3bi, CR3 (CD11b/CD18) and CR4 (CD11c/CD18), were significantly more efficient in the uptake of apoptotic cells compared with previously described receptors implicated in clearance. Complement activation is likely to be required for efficient uptake of apoptotic cells within the systemic circulation, and early component deficiencies could predispose to systemic autoimmunity by enhanced exposure to and/or aberrant deposition of apoptotic cells.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 158-158 ◽  
Author(s):  
Terry Robert Schaid ◽  
Julia Nguyen ◽  
Chunsheng Chen ◽  
Fuad Abdulla ◽  
Trevor Killeen ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Complement Activation ◽  
Research Funding ◽  
Annexin V ◽  
Dorsal Skin ◽  
Cell Disease ◽  
Activation Markers ◽  
Isotype Control ◽  
Skin Fold

Abstract Introduction Innate 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 Results Whole 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. Conclusions These 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. Disclosures Chen: Imara: Research Funding. Belcher:Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:Imara: Research Funding; CSL-Behring: Research Funding.

scholarly journals Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement

2010 ◽  
Vol 84 (18) ◽  
pp. 9128-9139 ◽  
Author(s):  
Elizabeth A. Moulton ◽  
Paula Bertram ◽  
Nanhai Chen ◽  
R. Mark L. Buller ◽  
John P. Atkinson
Keyword(s):  
Immune Response ◽  
Complement Activation ◽  
Catalytic Domain ◽  
High Titer ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Ectromelia Virus ◽  
Virus Inhibitor ◽  
Infected Cells

ABSTRACT Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

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