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Journal articles on the topic 'C5-convertase'

Author: Grafiati

Published: 2 July 2021

Last updated: 11 February 2022

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1

Kinoshita, T., A. W. Dodds, S. K. A. Law, and K. Inoue. "The low C5 convertase activity of the C4A6 allotype of human complement component C4." Biochemical Journal 261, no. 3 (August 1, 1989): 743–48. http://dx.doi.org/10.1042/bj2610743.

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We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

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2

Roumenina, Lubka T. "Terminal complement without C5 convertase?" Blood 137, no. 4 (January 28, 2021): 431–32. http://dx.doi.org/10.1182/blood.2020010133.

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3

Krisinger, Michael J., Verena Goebeler, Zhen Lu, Scott C. Meixner, Timothy Myles, Edward L. G. Pryzdial, and Edward M. Conway. "Thrombin generates previously unidentified C5 products that support the terminal complement activation pathway." Blood 120, no. 8 (August 23, 2012): 1717–25. http://dx.doi.org/10.1182/blood-2012-02-412080.

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Abstract The coagulation and complement pathways simultaneously promote homeostasis in response to injury but cause tissue damage when unregulated. Mechanisms by which they cooperate are poorly understood. To delineate their interactions, we studied the effects of thrombin and C5 convertase on C5 in purified and plasma-based systems, measuring release of the anaphylatoxin C5a, and generation of C5b, the initial component of the lytic membrane attack complex. Thrombin cleaved C5 poorly at R751, yielding minimal C5a and C5b. However, thrombin efficiently cleaved C5 at a newly identified, highly conserved R947 site, generating previously undescribed intermediates C5T and C5bT. Tissue factor-induced clotting of plasma led to proteolysis of C5 at a thrombin-sensitive site corresponding to R947 and not R751. Combined treatment of C5 with thrombin and C5 convertase yielded C5a and C5bT, the latter forming a C5bT-9 membrane attack complex with significantly more lytic activity than with C5b-9. Our findings provide a new paradigm for complement activation, in which thrombin and C5 convertase are invariant partners, enhancing the terminal pathway via the generation of newly uncovered C5 intermediates. Delineating the molecular links between coagulation and complement will provide new therapeutic targets for diseases associated with excess fibrin deposition and complement activation.

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4

Leung, Lawrence L., and John Morser. "Plasmin as a complement C5 convertase." EBioMedicine 5 (March 2016): 20–21. http://dx.doi.org/10.1016/j.ebiom.2016.03.015.

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5

Pangburn, M. K., and N. Rawal. "Structure-function studies of the C5 convertase." Biochemical Society Transactions 30, no. 5 (October 1, 2002): A98. http://dx.doi.org/10.1042/bst030a098c.

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6

Pangburn, M. K., and N. Rawal. "Structure and function of complement C5 convertase enzymes." Biochemical Society Transactions 30, no. 6 (November 1, 2002): 1006–10. http://dx.doi.org/10.1042/bst0301006.

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The multisubunit enzymes of the complement system that cleave C5 have many unusual properties, the most striking of which is that they acquire their specificity for C5 following cleavage of another substrate C3. C5 convertases are assemblies of two proteins C4b and C2a (classical or lectin pathways) or C3b and Bb (alternative pathway) and additional C3b molecules. The catalytic complexes (C4b, C2a or C3b, Bb) are intrinsically unstable (t1,2 = 1–3 min) and the enzymes are controlled by numerous regulatory proteins that accelerate this natural decay rate. Immediately after assembly, the bi-molecular enzymes preferentially cleave the protein C3 and exhibit poor activity toward C5 (a Km of approx. 25 μM and a C5 cleavage rate of 0.3-1 C5/min at Vmax). Efficient C3 activation results in the covalent attachment of C3b to the cell surface and to the enzyme itself, resulting in formation of C3b-C3b and C4b-C3b complexes. Our studies have shown that deposition of C3b alters the specificity of the enzymes of both pathways by changing the Km for C5 more than 1000-fold from far above the physiological C5 concentration to far below it. Thus, after processing sufficient C3 at the surface of a microorganism, the enzymes switch to processing C5, which initiates the formation of the cytolytic membrane attack complex of complement.

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7

Takata, Y., T. Kinoshita, H. Kozono, J. Takeda, E. Tanaka, K. Hong, and K. Inoue. "Covalent association of C3b with C4b within C5 convertase of the classical complement pathway." Journal of Experimental Medicine 165, no. 6 (June 1, 1987): 1494–507. http://dx.doi.org/10.1084/jem.165.6.1494.

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The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

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8

Rawal, Nenoo, and Michael K. Pangburn. "C5 Convertase of the Alternative Pathway of Complement." Journal of Biological Chemistry 273, no. 27 (July 3, 1998): 16828–35. http://dx.doi.org/10.1074/jbc.273.27.16828.

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9

Jongerius, Ilse, Jörg Köhl, Manoj K. Pandey, Maartje Ruyken, Kok P. M. van Kessel, Jos A. G. van Strijp, and Suzan H. M. Rooijakkers. "Staphylococcal complement evasion by various convertase-blocking molecules." Journal of Experimental Medicine 204, no. 10 (September 24, 2007): 2461–71. http://dx.doi.org/10.1084/jem.20070818.

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To combat the human immune response, bacteria should be able to divert the effectiveness of the complement system. We identify four potent complement inhibitors in Staphylococcus aureus that are part of a new immune evasion cluster. Two are homologues of the C3 convertase modulator staphylococcal complement inhibitor (SCIN) and function in a similar way as SCIN. Extracellular fibrinogen-binding protein (Efb) and its homologue extracellular complement-binding protein (Ecb) are identified as potent complement evasion molecules, and their inhibitory mechanism was pinpointed to blocking C3b-containing convertases: the alternative pathway C3 convertase C3bBb and the C5 convertases C4b2aC3b and C3b2Bb. The potency of Efb and Ecb to block C5 convertase activity was demonstrated by their ability to block C5a generation and C5a-mediated neutrophil activation in vitro. Further, Ecb blocks C5a-dependent neutrophil recruitment into the peritoneal cavity in a mouse model of immune complex peritonitis. The strong antiinflammatory properties of these novel S. aureus–derived convertase inhibitors make these compounds interesting drug candidates for complement-mediated diseases.

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10

Fu, Qinglan, D. Channe Gowda, and Peter McPhie. "Methionine modification impairs the C5-cleavage function of cobra venom factor-dependent C3/C5 convertase." IUBMB Life 45, no. 1 (June 1998): 133–44. http://dx.doi.org/10.1080/15216549800202502.

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11

Rawal, Nenoo, and Michael K. Pangburn. "Functional Role of the Noncatalytic Subunit of Complement C5 Convertase." Journal of Immunology 164, no. 3 (February 1, 2000): 1379–85. http://dx.doi.org/10.4049/jimmunol.164.3.1379.

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12

Gorham, Ronald D., Seline A. Zwarthoff, Xiaoguang Xue, Maartje Ruyken, Mihály Józsi, Piet Gros, and Suzan H. M. Rooijakkers. "Functional characterization of alternative pathway C3 and C5 convertase inhibitors." Molecular Immunology 89 (September 2017): 141. http://dx.doi.org/10.1016/j.molimm.2017.06.084.

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13

Ruggenenti, Piero, Erica Daina, Alessia Gennarini, Camillo Carrara, Sara Gamba, Marina Noris, Nadia Rubis, et al. "C5 Convertase Blockade in Membranoproliferative Glomerulonephritis: A Single-Arm Clinical Trial." American Journal of Kidney Diseases 74, no. 2 (August 2019): 224–38. http://dx.doi.org/10.1053/j.ajkd.2018.12.046.

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14

Weiler, John M. "Regulation of C5 convertase activity by properdin, factors B and H." Immunologic Research 8, no. 4 (December 1989): 305–15. http://dx.doi.org/10.1007/bf02935515.

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15

Hong, K., T. Kinoshita, J. Takeda, H. Kozono, P. Pramoonjago, Y. U. Kim, and K. Inoue. "Inhibition of the alternative C3 convertase and classical C5 convertase of complement by group A streptococcal M protein." Infection and Immunity 58, no. 8 (1990): 2535–41. http://dx.doi.org/10.1128/iai.58.8.2535-2541.1990.

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16

Peake, P. W., B. A. Pussell, P. Martyn, V. Timmermans, and J. A. Charlesworth. "The inhibitory effect of rosmarinic acid on complement involves the C5 convertase." International Journal of Immunopharmacology 13, no. 7 (January 1991): 853–57. http://dx.doi.org/10.1016/0192-0561(91)90036-7.

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17

Rawal, Nenoo, and Michael K. Pangburn. "Formation of High Affinity C5 Convertase of the Classical Pathway of Complement." Journal of Biological Chemistry 278, no. 40 (July 23, 2003): 38476–83. http://dx.doi.org/10.1074/jbc.m307017200.

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18

Michelfelder, Stefan, Friedericke Fischer, Astrid Wäldin, Kim V. Hörle, Martin Pohl, Juliana Parsons, Ralf Reski, et al. "The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential." Journal of the American Society of Nephrology 29, no. 4 (January 15, 2018): 1141–53. http://dx.doi.org/10.1681/asn.2017070738.

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The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH−/− mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.

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19

Corvillo, F., M. Bravo García-Morato, P. Nozal, S. Garrido, A. Tortajada, S. Rodríguez de Córdoba, and M. López-Trascasa. "Serum properdin consumption as a biomarker of C5 convertase dysregulation in C3 glomerulopathy." Clinical & Experimental Immunology 184, no. 1 (January 22, 2016): 118–25. http://dx.doi.org/10.1111/cei.12754.

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20

Ebanks, R. "Mouse complement component C4 is devoid of classical pathway C5 convertase subunit activity." Molecular Immunology 33, no. 3 (February 1996): 297–309. http://dx.doi.org/10.1016/0161-5890(95)00135-2.

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21

Heinen, Stefan, Andrea Hartmann, Nadine Lauer, Ulrike Wiehl, Hans-Martin Dahse, Sylvia Schirmer, Katharina Gropp, et al. "Factor H–related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation." Blood 114, no. 12 (September 17, 2009): 2439–47. http://dx.doi.org/10.1182/blood-2009-02-205641.

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Abstract Homozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.

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22

Borkowska, Sylwia, Malwina Suszynska, Katarzyna Mierzejewska, Marta Budkowska, Daria Salata, Barbara Dolegowska, Janina Ratajczak, Magdalena Kucia, and Mariusz Z. Ratajczak. "Novel Evidence That Crosstalk Between Three Evolutionarily Ancient Proteolytic Enzyme Cascades (coagulation, fibrinolysis, and complement) Plays An Important Role In Mobilization Of Hematopoietic Stem/Progenitor Cells (HSPCs)." Blood 122, no. 21 (November 15, 2013): 903. http://dx.doi.org/10.1182/blood.v122.21.903.903.

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Abstract Background Hematopoietic stem/progenitor cells (HSPCs) residing in BM are released from their niches and circulate under steady-state conditions at detectable levels in the peripheral blood (PB), and their number increases in response to i) systemic or local inflammation, ii) strenuous exercise, iii) stress, iv) tissue/organ injury, and v) pharmacological agents. All these processes involve activation of the complement cascade (ComC), and while mice deficient in the complement protein C3, which is an upstream component of the ComC, are easy mobilizers (Blood 2004; 103:2071), mice deficient in a downstream component of ComC, complement protein 5 (C5), are very poor mobilizers (Leukemia 2009; 23:2052). To explain these observations, it has been suggested that during the mobilization process the C5a cleavage fragment stimulates release of proteolytic enzymes from BM-residing myeloid cells, which attenuates SDF-1–CXCR4 and VLA4–VCAM-1 retention signals in BM niches. In addition, C5a generated in BM sinusoids is a potent chemoattractant for granulocytes and monocytes, which, as the first cells egressing from the BM, play an important role in permeabilization of the BM–PB barrier and thus facilitate the subsequent egress of HSPCs. It is also known that activation of ComC is based on stepwise activation of the cascade of proteolytic pro-enzymes, and thus the lack of upstream C3 should theoretically affect generation of C5 convertase, which is a proteolytic enzyme activating a downstream component of ComC (C5). However, surprisingly, C3–/– mice are easy mobilizers (Blood 2004; 103:2071). Hypothesis To explain how C5 can be activated during the mobilization process even when C3 is missing, we hypothesized that other proteases that are products of the activated coagulation cascade (CoaC) and fibrynolytic cascade (FibC) compensate for the lack of proteolytic activity of ComC-derived C5 convertase. Materials and Methods In our experiments 2-month-old C3-deficient mice (C3–/–) and normal wild type (WT) littermates were mobilized for 6 days by G-CSF in the presence or absence of selected CoaC and FibC inhibitors such as refludan (a direct inhibitor of thrombin) and tranexamic acid (an inhibitor of plasminogen activation). Following mobilization, we measured in PB i) the total number of white blood cells (WBC), ii) the number of circulating clonogenic CFU-GM, and iii) the number of Sca-1+c-kit+lineage– (SKL) cells. In parallel, we measured the activation of C5 by measuring the level of C5a and evaluated activation of CoaC by measuring prothrombin (PT) and activated partial thromboplastin times (APTT) as well as thrombin/antithrombin (TAT) and plasmin/antiplasmin (PAP) complexes. Results We observed that G-CSF-induced mobilization of HSPCs was significantly reduced in easy-mobilizing C3–/– mice if the mice were treated during mobilization with refludan (a CoaC inhibitor) or tranexamic acid (an FibC inhibitor). This reduction correlated with significant inhibition of C5 activation/cleavage. More importantly, we also noticed that inhibitors of CoaC and FibC had a negative effect on mobilization of HSPCs in normal WT animals. The activation of ComC, CoaC, and FibC in mice mobilized with G-CSF was confirmed by an increase in C5a level (Figure 1A) and by measuring PT and APTT time (Figure 1B) as well as TAT and PAP complexes (Figure 1C). Conclusions The data presented in this work demonstrate, for the first time, the existence of crosstalk between all three evolutionarily ancient proteolytic enzyme cascades, ComC, CoaC, and FibC, in the mobilization process of HSPCs. These results also confirm that C5, which plays an important role in egress of HSPCs from the BM, can be activated/cleaved during mobilization not only by ComC-generated C5 convertase but in addition by proteolytic enzymes of CoaC and FibC. Our observations of crosstalk between ComC, CoaC, and FibC may lead to the development of more efficient mobilization strategies in poor mobilizers. Furthermore, since it is known that all these cascades are activated in all the situations in which HSPCs are mobilized from BM into PB (e.g., infections, tissue/organ damage, or strenuous exercise) and show a circadian rhythm of activation due to a drop in blood pH during deep sleep at night, they are involved both in stress-induced as well as in circadian changes in HSPC trafficking in PB. Disclosures: No relevant conflicts of interest to declare.

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23

Rawal, Nenoo, Rema Rajagopalan, and Veena P. Salvi. "Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)." Molecular Immunology 46, no. 15 (September 2009): 2902–10. http://dx.doi.org/10.1016/j.molimm.2009.07.006.

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VICTOR, KIMBERLY D., VIRGINIA PASCUAL, ANN E. STITZEL, GEORGE C. TSOKOS, J. DONALD CAPRA, and ROGER E. SPITZER. "Nucleotide Sequence of a Human Autoantibody to the Alternative Pathway C3/C5 Convertase (C3NeF)." Hybridoma 12, no. 3 (June 1993): 231–37. http://dx.doi.org/10.1089/hyb.1993.12.231.

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25

Avirutnan, Panisadee, Anja Fuchs, Richard E. Hauhart, Pawit Somnuke, Soonjeon Youn, Michael S. Diamond, and John P. Atkinson. "Antagonism of the complement component C4 by flavivirus nonstructural protein NS1." Journal of Experimental Medicine 207, no. 4 (March 22, 2010): 793–806. http://dx.doi.org/10.1084/jem.20092545.

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The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.

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Krishnan, Vengadesan, Karthe Ponnuraj, Yuanyuan Xu, Kevin Macon, John E. Volanakis, and Sthanam V. L. Narayana. "The Crystal Structure of Cobra Venom Factor, a Cofactor for C3- and C5-Convertase CVFBb." Structure 17, no. 4 (April 2009): 611–19. http://dx.doi.org/10.1016/j.str.2009.01.015.

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Lee, D., MJ Smith, JB Tracey, K. Johnson, P. Higgins, CG Yeh, BR Smith, and &NA; Rinder. "C3- AND C5- CONVERTASE INHIBITION PREVENTS MONOCYTE AND PMN ACTIVATION DURING IN VITRO CARDIOPULMONARY BYPASS." Anesthesia & Analgesia 86, Supplement (April 1998): 1SCA. http://dx.doi.org/10.1097/00000539-199804001-00001.

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Doorduijn, D. J., R. D. Gorham, L. van Bloois, E. Mastrobattista, and S. H. M. Rooijakkers. "Creating model systems to study molecular mechanisms of C5 convertase-induced Membrane Attack Complex assembly." Molecular Immunology 89 (September 2017): 160. http://dx.doi.org/10.1016/j.molimm.2017.06.124.

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Lee, D., MJ Smith, JB Tracey, K. Johnson, P. Higgins, CG Yeh, BR Smith, and CS Rinder. "C3- AND C5- CONVERTASE INHIBITION PREVENTS MONOCYTE AND PMN ACTIVATION DURING IN VITRO CARDIOPULMONARY BYPASS." Anesthesia & Analgesia 86, no. 4S (April 1998): 1SCA. http://dx.doi.org/10.1213/00000539-199804001-00001.

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Borodovsky, Anna, Kristina Yucius, Andrew Sprague, James Butler, Shannon Fishman, Tuyen Nguyen, Akshay Vaishnaw, et al. "Development Of RNAi Therapeutics Targeting The Complement Pathway." Blood 122, no. 21 (November 15, 2013): 2471. http://dx.doi.org/10.1182/blood.v122.21.2471.2471.

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Abstract The complement system is a pivotal player in multiple hematological conditions. Antibody blockade of the C5 component of complement has been approved as a treatment for both paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic-uremic syndrome (aHUS), validating C5 as an important therapeutic target. Recently, we developed a robust RNAi therapeutics platform for the delivery of siRNAs to the liver using trivalent GalNAc conjugates, enabling silencing of hepatocyte-expressed genes following subcutaneous (SC) injection. The liver is a major source of C5 and other complement pathway components. The GalNAc conjugate technology allows rapid development of siRNAs targeting multiple members of the complement cascade and evaluation of their silencing in pre-clinical models. To examine the utility of the siRNA approach for targeting complement pathway components we designed and synthesized GalNAc conjugated siRNAs targeting rodent, primate and human C5. Potent siRNA duplexes, showing greater than 95% silencing of C5 mRNA were selected using in vitro screening in human cell lines and mouse primary hepatocytes. C5 silencing and serum hemolytic activity inhibition were evaluated in rodents using single and multi-dose SC treatment regimens. A C5-targeting siRNA conjugate demonstrated a single dose ED50 of 0.625 mg/kg in the mouse with greater than 90% silencing of serum C5 achievable at higher doses. Serum C5 silencing was durable, with recovery starting two weeks after a single SC injection We went on to examine the efficacy of C5 silencing in the rat and observed robust lowering of serum C5 with 2.5 and 5 mg/kg multi-dose regimens, resulting in up to ∼90% inhibition of complement classical pathway hemolytic activity. Evaluation of the translation of this approach to higher species is in progress. Since PNH erythrocyte lysis is thought to be mediated by the activation of the alternative pathway of complement we initiated work on the development of siRNA conjugates targeting Factor B, an essential component of the alternative pathway C3 convertase. siRNAs targeting rodent, primate and human Factor B were identified by in vitro screening and demonstrate >90% silencing of Factor B mRNA in human cell lines and primary mouse hepatocytes. Evaluation of Factor B silencing in rodent models is ongoing. siRNA-mediated silencing of liver-derived complement components is a promising novel therapeutic approach for inhibiting the activity of C5 and other complement pathway targets, with the potential to enable subcutaneous treatment for patients with PNH and related disorders. Disclosures: Borodovsky: Alnylam: Employment. Yucius:Alnylam: Employment. Sprague:Alnylam: Employment. Butler:Alnylam: Employment. Fishman:Alnylam: Employment. Nguyen:Alnylam: Employment. Vaishnaw:Alnylam: Employment. Maier:Alnylam: Employment. Kallanthottathil:Alnylam: Employment. Kuchimanchi:Alnylam: Employment. Manoharan:Alnylam: Employment. Meyers:Alnylam: Employment. Fitzgerald:Alnylam: Employment.

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Nolasco, Jennifer, Leticia Nolasco, Qi Da, Sonya Cirlos, Zaverio Ruggeri, Joel Moake, and Miguel Cruz. "Complement Component C3 Binds to the A3 Domain of von Willebrand Factor." TH Open 02, no. 03 (July 2018): e338-e345. http://dx.doi.org/10.1055/s-0038-1672189.

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Abstractvon Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (∼280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.

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Rawal, Nenoo, and Michael K. Pangburn. "Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase." Molecular Immunology 44, no. 1-3 (January 2007): 231–32. http://dx.doi.org/10.1016/j.molimm.2006.07.198.

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Rawal, Nenoo, and Michael K. Pangburn. "Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase." Molecular Immunology 44, no. 6 (February 2007): 1105–14. http://dx.doi.org/10.1016/j.molimm.2006.07.282.

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34

Gadeberg, Trine A. F., Dennis Vestergaard Pedersen, and Gregers Rom Andersen. "Structural studies of convertases in the complement system. The properdin-stabilised proconvertase, and the C5 convertase." Molecular Immunology 89 (September 2017): 198. http://dx.doi.org/10.1016/j.molimm.2017.06.208.

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35

Taylor, Ronald P., Margaret A. Lindorfer, Andrew W. Pawluczkowycz, and Charles J. Parker. "A Novel Approach to Treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH): Both Hemolysis and C3 Deposition Are Blocked by a Monoclonal Antibody (mAb) Specific for the Alternative Pathway of Complement (APC) C3/C5 Convertase." Blood 114, no. 22 (November 20, 2009): 157. http://dx.doi.org/10.1182/blood.v114.22.157.157.

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Abstract Abstract 157 In PNH, red blood cells (RBCs) lack key complement control proteins, CD55 and CD59 and are therefore sensitive to complement activation and intravascular hemolysis. These regulatory proteins function at two different steps in the complement cascade; CD55 (decay accelerating factor, DAF) controls the formation and stability of the APC C3 and C5 convertases, while CD59 (membrane inhibitor of reactive lysis, MIRL) blocks formation of the cytolytic membrane attack complex (MAC). The intravascular hemolysis of PNH can be inhibited in vivo by eculizumab, a humanized mAb that binds complement C5, thereby preventing formation of the MAC. However, PNH patients treated with eculizumab continue to manifest evidence of ongoing hemolysis as they remain anemic with an elevated reticulocyte count and low serum haptoglobin concentration, and approximately 50% of eculizumab-treated patients require transfusion. This observation is consistent with the hypothesis that, in patients treated with eculizumab, PNH RBCs undergo extravascular hemolysis as a consequence of C3 opsonization because eculizumab does not compensate for deficiency of DAF. Recent studies (Risitano et al., Blood, 2009) support this hypothesis as patients undergoing treatment with eculizumab were found to have a positive Coombs test for C3 but not IgG, and flow cytometry demonstrated C3 activation and degradation products bound to the PNH RBCs. This process appeared clinically relevant as transfusion requirement correlated with the percentage of C3 opsonized PNH RBCs. These observations suggest that blocking the APC C3/C5 convertase would be a better way to treat the hemolysis of PNH because this approach has the advantage of blocking both extravascular hemolysis by inhibiting C3 opsonization and preventing intravascular hemolysis by inhibiting MAC generation. We have developed a mAb 3E7 and its deimmunized chimeric humanized derivative H17 that specifically block the APC C3/C5 convertase by binding to a neoepitope expressed when complement C3 is activated. In vitro, 3E7/H17 prevents APC-mediated lysis of rabbit RBCs in human serum and blocks deposition of human C3 activation fragments on APC activator substrates such as zymosan (Mol Immunol, 2006; J Immunol, 2007). We now report that mAb H17/3E7 blocks lysis in acidified normal human serum (aNHS) (a process mediated by the APC) of RBCs from patients with PNH (n=5). Representative results for patients 1 and 2 are as follows: 60% and 40% of RBCs were lysed after a one hour incubation at 37°C; lysis was reduced to 10% and 6%, respectively, at 80 ug/ml of mAb H17, and to 1% lysis (both patients) at 170 ug/ml of mAb H17. We also showed that mAb H17/3E7 blocks deposition of C3 activation fragments on PNH RBCs. After lysis in aNHS, blood samples from PNH patients were probed with Al488 mAb 1H8, specific for C3b/iC3b/C3dg. Flow cytometry experiments revealed C3 fragment deposition on lysed cells corresponding to 30,500 molecules of equivalent soluble fluorochrome (MESF) compared to a background signal of 225 MESF on unlysed RBCs in the same sample. Addition of mAb H17 blocked C3 fragment deposition not only on the unlysed cells but also on the small number of recovered ghosts . Importantly, mAb H17/3E7 inhibits the APC specifically. PNH RBCs, opsonized with IgM in serum from a patient with chronic cold agglutinin disease, were lysed in NHS by the classical complement pathway, and this lysis was not inhibited by mAbH17/3E7. Together, these experiments demonstrate that both hemolysis and C3 opsonization of PNH RBCs can be inhibited by a novel mAb that specifically blocks the APC C3/C5 convertase while leaving intact the classical pathway of complement. These findings suggest an approach to therapy of PNH in which both intravascular and extravascular hemolysis can be inhibited while preserving the important immune functions of the classical pathway of complement. Disclosures: No relevant conflicts of interest to declare.

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Sandoval, Ana, Rong Ai, John M. Ostresh, and Ronald T. Ogata. "Distal Recognition Site for Classical Pathway Convertase Located in the C345C/Netrin Module of Complement Component C5." Journal of Immunology 165, no. 2 (July 15, 2000): 1066–73. http://dx.doi.org/10.4049/jimmunol.165.2.1066.

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37

Kozono, H., T. Kinoshita, Y. U. Kim, Y. Takata-Kozono, S. Tsunasawa, F. Sakiyama, J. Takeda, K. Hong, and K. Inoue. "Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b." Journal of Biological Chemistry 265, no. 24 (August 1990): 14444–49. http://dx.doi.org/10.1016/s0021-9258(18)77322-5.

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38

Inagi, Reiko, Toshio Miyata, Kenji Maeda, Satoshi Sugiyama, Akio Miyama, and Izumi Nakashima. "FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a." Immunology Letters 27, no. 1 (January 1991): 49–52. http://dx.doi.org/10.1016/0165-2478(91)90243-4.

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39

Shibazaki, Masahiko, Yoshihiro Kawabata, Takashi Yokochi, Akira Nishida, Haruhiko Takada, and Yasuo Endo. "Complement-Dependent Accumulation and Degradation of Platelets in the Lung and Liver Induced by Injection of Lipopolysaccharides." Infection and Immunity 67, no. 10 (October 1, 1999): 5186–91. http://dx.doi.org/10.1128/iai.67.10.5186-5191.1999.

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ABSTRACT We found unique behaviors among platelets within a few minutes of the intravenous injection of lipopolysaccharide (LPS) into mice. Platelets accumulated primarily in the liver at lower doses of LPS, but at higher doses they accumulated largely in the lungs. When the platelets accumulated in these organs were degraded, there was a rapid anaphylactoid shock. The platelet response depended on the strain of mouse and on the source of LPS. Of various LPSs tested, the LPS from the smooth type of Klebsiella O3 (KO3-S LPS) was the most potent at inducing the platelet response and shock. K-76 monocarboxylic acid, an inhibitor of complement C5, effectively prevented the KO3-S LPS-induced degradation (but not accumulation) of platelets and the ensuing rapid shock in BALB/c mice. Moreover, in DBA/2 mice (which are deficient in complement C5), platelets accumulated in the lungs and liver in response toKO3-S LPS but soon returned to the circulation without degradation, and there was no rapid shock. The LPS from the rough type of KO3 induced an accumulation of platelets in the liver and lungs but not a degradation of platelets. On the basis of these results and those reported by other investigators, we propose that in the platelet response to LPS, the lectin pathway to form C3 convertase from C4 and C2 is involved in the rapid accumulation of platelets in the liver and lungs and that the pathway from C5 to C9 is involved in the destruction of platelets and the consequent anaphylactoid shock.

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40

Yu, Jia, Xuan Yuan, Hang Chen, Shruti Chaturvedi, Evan M. Braunstein, and Robert A. Brodsky. "Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition." Blood 136, no. 18 (October 29, 2020): 2080–89. http://dx.doi.org/10.1182/blood.2020008248.

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

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41

Lindorfer, Margaret A., Andrew W. Pawluczkowycz, Elizabeth M. Peek, Kimberly Hickman, Ronald P. Taylor, and Charles J. Parker. "A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement." Blood 115, no. 11 (March 18, 2010): 2283–91. http://dx.doi.org/10.1182/blood-2009-09-244285.

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Abstract The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway–mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.

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42

Salerno, C. T., A. P. Dalmasso, D. M. Kulick, M. Guzman, C. G. Yeh, P. J. Higgins, and R. M. Bolman. "A soluble chimeric C3-, and C5-convertase inhibitor can prolong xenograft survival in pig-to-primate cardiac transplantation." Molecular Immunology 35, no. 6-7 (April 1998): 342. http://dx.doi.org/10.1016/s0161-5890(98)90577-6.

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43

Ramos, Theresa N., Meghan M. Darley, Sebastian Weckbach, Philip F. Stahel, Stephen Tomlinson, and Scott R. Barnum. "The C5 Convertase Is Not Required for Activation of the Terminal Complement Pathway in Murine Experimental Cerebral Malaria." Journal of Biological Chemistry 287, no. 29 (June 11, 2012): 24734–38. http://dx.doi.org/10.1074/jbc.c112.378364.

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44

Krishnan, Vengadesan, Yuanyuan Xu, Kevin Macon, John E. Volanakis, and Sthanam V. L. Narayana. "The Crystal Structure of C2a, the Catalytic Fragment of Classical Pathway C3 and C5 Convertase of Human Complement." Journal of Molecular Biology 367, no. 1 (March 2007): 224–33. http://dx.doi.org/10.1016/j.jmb.2006.12.039.

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45

Zarantonello, Alessandra, Henrik Pedersen, Nick S. Laursen, and Gregers R. Andersen. "Nanobodies Provide Insight into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies." Biomolecules 11, no. 2 (February 17, 2021): 298. http://dx.doi.org/10.3390/biom11020298.

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The complement system is part of the innate immune response, where it provides immediate protection from infectious agents and plays a fundamental role in homeostasis. Complement dysregulation occurs in several diseases, where the tightly regulated proteolytic cascade turns offensive. Prominent examples are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and Alzheimer’s disease. Therapeutic intervention targeting complement activation may allow treatment of such debilitating diseases. In this review, we describe a panel of complement targeting nanobodies that allow modulation at different steps of the proteolytic cascade, from the activation of the C1 complex in the classical pathway to formation of the C5 convertase in the terminal pathway. Thorough structural and functional characterization has provided a deep mechanistic understanding of the mode of inhibition for each of the nanobodies. These complement specific nanobodies are novel powerful probes for basic research and offer new opportunities for in vivo complement modulation.

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Tsukasa, Seya, Okada Michiyo, Matsumoto Misako, Hong Kyongsu, Kinoshita Taroh, and John P. Atkinson. "Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP)." Molecular Immunology 28, no. 10 (October 1991): 1137–47. http://dx.doi.org/10.1016/0161-5890(91)90029-j.

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47

Rawal, Nenoo, and Michael K. Pangburn. "C5 convertase of the alternative pathway of complement: Enzymatic properties of the free and surface-bound forms of the enzyme." Molecular Immunology 35, no. 6-7 (April 1998): 392. http://dx.doi.org/10.1016/s0161-5890(98)90779-9.

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48

Tu, Anh-Hue T., Robert L. Fulgham, Mark A. McCrory, David E. Briles, and Alexander J. Szalai. "Pneumococcal Surface Protein A Inhibits Complement Activation by Streptococcus pneumoniae." Infection and Immunity 67, no. 9 (September 1, 1999): 4720–24. http://dx.doi.org/10.1128/iai.67.9.4720-4724.1999.

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ABSTRACT Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the α chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.

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Langemeijer, Saskia, Jun-Ichi Nishimura, Wynne Weston-Davies, Miles A. Nunn, Yuzuru Kanakura, Ian J. Mackie, and Petra Muus. "C5 Polymorphism in a Dutch Patient with Paroxysmal Nocturnal Hemoglobinuria (PNH) and No Asian Ancestry, Resistant to Eculizumab, but in Vitro Sensitive to Coversin." Blood 126, no. 23 (December 3, 2015): 1209. http://dx.doi.org/10.1182/blood.v126.23.1209.1209.

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Abstract Eculizumab (Soliris®) is a humanized monoclonal antibody that targets complement factor C5and inhibits the production of C5a and formation of the terminal complement membrane attack complex. It is registered for the treatment of PNH and aHUS. Eculizumab results in significant reduction of hemolysis in PNH, improves symptoms and reduces the incidence of PNH related thrombosis . A poor response, defined as sustained high levels of LDH during treatment with eculizumab irrespective of improvement of clinical symptoms, has been reported in a subgroup of PNH patients (Nishimura et al, NEJM 2014;370:632-639). These patients had a genetic variant of C5 which occurs in approximately 3.5% of the Japanese and 1% of the Chinese Han populations and interferes with binding of eculizumab to C5. We describe the first patient with no known Asian ancestry with a poor response to eculizumab, who subsequently had a good in vitro response to protein rEV576 (syntheticOrnithodoros moubata complement C5 binding) or coversin. Coversin is a recombinant small protein derived from a tick salivary molecule which binds to C5 and, through steric hindrance, interferes with the access of C5 convertase to the active site and thus prevents cleavage to C5a and C5b in a similar fashion to eculizumab. Our 30-year old male patient commenced treatment with Eculizumab because of PNH (granulocyte clone size: 90%), severe haemolysis (LDH 3-6x ULN, and peak value of 17xULN), transient renal failure, extreme fatigue and erectile dysfunction. He had no history of thrombosis and no underlying bone marrow disease. During eculizumab treatment (dosed 600 mg iv every 7 days, weeks 1-4 and 900 mg biweekly starting in week 5) he felt better, seemed less fatigued and experienced less erectile dysfunction. However, laboratory examination showed sustained elevated markers of hemolysis. Other causes of hemolysis were excluded. Underdosing of eculizumab was ruled out by demonstrating sustained high LDH levels at different time points in between subsequent eculizumab infusions and by measuring trough levels of eculizumab (>100ug/ml). In vitro terminal complement complex blockage by eculizumab through antibody-coated chicken red blood cell lysis was indicative of ongoing active hemolysis in our patient's serum. The presence of Human Anti-Drug Antibodies was excluded using an illuminescent MSD®assay. Treatment was discontinued when the patient experienced increased hemolysis (LDH 9x ULN) and macroscopic hemoglobinuria one day after receiving a dose of 900 mg eculizumab. As expected, discontinuation did not result in further increase of hemolysis parameters or clinical change. To investigate whether a mutation of complement C5 might explain the eculizumab resistance in our patient, DNA analysis of the coding region of C5 was performed. This showed a single C5 heterozygous missense mutation, c.2653C>A, which predicts p.Arg885Ser. This new mutation was very similar to variants previously found in Japanese patients (c.2654G>A, which predicts p.Arg885His) and an Argentinian patient with Asian ancestry (c.2653C>T, which predicts p.Arg885Cys), indicating the importance of this amino acid in C5 recognition by eculizumab. The same mutation was demonstrated in the DNA of our patient's healthy father. Since coversin binds to an epitope on C5 remote from the eculizumab binding site, we hypothesised that it might block C5 cleavage in our patient. Serum samples from our patient and 6 healthy controls were spiked with ascending doses of either eculizumab or coversin and complement activity was measured using a commercially available CH50 Equivalent ELISA (Quidel Corporation ®). In agreement with our in vivo observation eculizumab was incapable of inhibiting CH50 activity in the patient's serum beyond approximately 75%, even at concentrations of 100ug/ml. In contrast coversin, even in concentrations of 10 ug/ml inhibited complement activity completely, both in serum of our patient and serum of healthy controls. We conclude that coversin may prove a useful alternative to eculizumab for patients with resistance due to C5 polymorphisms. Figure 1. Change in serum complement C5 activity in response to ascending doses of coversin (Cov) and eculizumab (Ecu). R2 = patient sample, NC3 = normal control Figure 1. Change in serum complement C5 activity in response to ascending doses of coversin (Cov) and eculizumab (Ecu). R2 = patient sample, NC3 = normal control Disclosures Nishimura: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Weston-Davies:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Nunn:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mackie:Volution Immuno Pharmaceuticals (Uk) Ltd: Research Funding. Muus:Alexion Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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50

Kim, Y. U., T. Kinoshita, H. Molina, D. Hourcade, T. Seya, L. M. Wagner, and V. M. Holers. "Mouse complement regulatory protein Crry/p65 uses the specific mechanisms of both human decay-accelerating factor and membrane cofactor protein." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 151–59. http://dx.doi.org/10.1084/jem.181.1.151.

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Normal host cells are protected from the destructive action of complement by cell surface complement regulatory proteins. In humans, decay-accelerating factor (DAF) and membrane cofactor protein (MCP) play such a biologic role by inhibiting C3 and C5 convertases. DAF and MCP accomplish this task by specific mechanisms designated decay-accelerating activity and factor I cofactor activity, respectively. In other species, including mice, structural and/or functional homologues of these proteins are not yet well characterized. Previous studies have shown that the mouse protein Crry/p65 has certain characteristics of self-protecting complement regulatory proteins. For example, Crry/p65 is expressed on a wide variety of murine cells, and when expressed on human K562 erythroleukemic cells, it prevents deposition of mouse C3 fragments on the cell surface during activation of either the classical or alternative complement pathway. We have now studied factor I cofactor and decay-accelerating activities of Crry/p65. Recombinant Crry/p65 demonstrates cofactor activity for factor I-mediated cleavage of both mouse C3b and C4b. Surprisingly, Crry/p65 also exhibits decay-accelerating activity for the classical pathway C3 convertase strongly and for the alternative pathway C3 convertase weakly. Therefore, mouse Crry/p65 uses the specific mechanisms of both human MCP and DAF. Although Crry/p65, like MCP and DAF, contains tandem short consensus repeats (SCR) characteristic of C3/C4 binding proteins, Crry/p65 is not considered to be a genetic homologue of either MCP or DAF. Thus, Crry/p65 is an example of evolutionary conservation of two specific activities in a single unique protein in one species that are dispersed to individual proteins in another. We propose that the repeating SCR motif in this family has allowed this unusual process of evolution to occur, perhaps driven by the use of MCP and DAF as receptors by human pathogens such as the measles virus.

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