Minimal Role for the Alternative Pathway in Complement Activation By HIT Immune Complexes

Abstract Background: Recent studies show that ultra-large immune complexes consisting of IgG and platelet factor 4 and heparin (P+H) potently activate complement and facilitate complement dependent activation of cellular FcgRIIA (PMID 34189574). In whole blood assays using KKO, a monoclonal anti-PF4/heparin antibody, or antibodies from patients with heparin induced thrombocytopenia (HIT), we showed that classical pathway (CP) inhibition reduced immune complex-mediated complement activation (C3c and soluble C5b-9 generation), cell surface deposition of immune complexes and cellular activation. Aims: As previous studies suggest that the alternative pathway (AP) provides significant amplification (>80%) of the CP pathway, (PMID: 15544620) we compared the effects of AP, CP, and CP/AP inhibitors by KKO and HIT immune complexes in whole blood. Methods: Inhibitors of the CP (BBK32, a borrelia protein inhibitor to C1r), AP (anti-factor B antibody (α-fB), or Factor D (fD inhibitor or fD-INH) Alexion Pharmaceuticals, Boston, MA) or combined AP/CP (C1-esterase inhibitor, C1-INH, Berinert, CSL Behring; or soluble complement receptor 1, sCR1, Alexion) were tested in hemolytic assays of CP or AP to confirm pathway specificity. To examine effects of CP or AP inhibition on complement activation by immune complexes consisting of KKO or HIT IgG, whole blood was pre-incubated with CP, AP or CP/AP inhibitors prior to addition of P+H ± KKO/HIT IgG or isotype controls. WB was incubated for 45 minutes at 37ºC followed by addition of 10mM EDTA to quench further complement activation. Complement activation products (C3c and sC5b-9) and neutrophil degranulation (MMP9) markers were measured using commercial immunoassays. Effects of complement inhibitors on cellular deposition of immune complexes was examined by flow, using previously described methods (PMID 34189574) using fluorescently labeled anti-C3c antibody (Quidel, San Diego, CA) and anti-mouse or human IgG (Biolegend, San Diego, CA). Results: Consistent with prior publications (PMID: 26808924), BBK32 showed marked reduction CP, but not AP-dependent hemolytic assays. The converse was true of AP inhibitors: α-fB and fD-INH prevented AP-dependent, but not CP-dependent hemolysis (data not shown). C1-INH and sCR1 showed activity in both CP- and AP-dependent assays. The CP or CP/AP inhibitors showed potent inhibition of C3c and sC5b-9 generation by KKO and HIT immune complexes, while AP inhibitors had no effect (Figure A for KKO C3c generation; and Table 1 for KKO/HIT C3c generation; sC5b-9 data not shown). For a given CP or CP/AP inhibitor, the concentrations leading to 50% inhibition (IC 50) were generally comparable for KKO and HIT immune complexes for C3c (Figure A and Table 1) and sC5b-9 generation (data not shown), with potency as follows: C1-INH>>BBK32>sCR1 (Table 1). On the other hand, the AP inhibitors, α-fB and fD-INH, showed no inhibitory activity in C3c (Figure A and Table 1)/sC5b-9 (data not shown) generation by KKO or HIT ULICs. As our recent studies indicate that complement activation is critical to cell surface deposition of immune complexes and cellular activation via FcgRIIA, we examined effects of complement inhibitors on IC deposition on B-cells and MMP9 release from neutrophils. CP or CP/AP inhibitors, but not AP inhibitors, reduced cell surface binding of immune complexes (Figure B) as well as MMP9 release (Figure C and Table 1). Conclusion: Together, these studies demonstrate that the AP has a minimal role in supporting complement activation by KKO/HIT ULICs. Future studies should examine CP inhibition as a therapeutic strategy for modulating the cellular activating effects of HIT antibodies. To what extent these findings apply to other immune complexes and/or CP activators requires further study. Funding Agency: NIH HL151730; α-fB antibody, fD inhibitor and sCR1 was provided by Alexion Pharmaceuticals, Boston, MA. BBK32 was provided by Dr. Brandon Garcia, East Carolina University, Greenville, NC. Figure 1 Figure 1. Disclosures Cines: Dova: Consultancy; Rigel: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board. OffLabel Disclosure: C1-esterase inhibitor off label for HIT

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Mechanism of Complement Activation By PF4/ Heparin Complexes

Blood ◽

10.1182/blood.v128.22.137.137 ◽

2016 ◽

Vol 128 (22) ◽

pp. 137-137

Author(s):

Sanjay Khandelwal ◽

Johnson M Alexandra ◽

C. Garren Hester ◽

Michael M Frank ◽

Gowthami M Arepally

Keyword(s):

B Cells ◽

Complement Activation ◽

Whole Blood ◽

Alternative Pathway ◽

Lectin Pathway ◽

Antigen Binding ◽

C1 Esterase Inhibitor ◽

Heparin Complexes ◽

Esterase Inhibitor

Abstract The mechanisms underlying the PF4/heparin immune response are poorly understood. In recent studies, we showed that PF4/heparin complexes, but not PF4 alone or heparin alone, activate complement (C') in a heparin-dependent manner and lead to selective binding of C'-coated antigen (PF4/heparin complexes) to B cells via CD21. In additional studies, we showed that heparinized patients have circulating B cells with C'-coated PF4/heparin complexes (Khandelwal, Blood 2016). To investigate the mechanism by which PF4/heparin complexes activate complement, we performed studies in whole blood using previously described methods assessing binding of C'-fixed PF4/heparin complexes to B cells in whole blood. We incubated blood with inhibitors or conditions known to selectively block specific complement activation pathways, followed by incubation with PF4 and heparin for 1 hour (hr) at 370 C. Binding of PF4/heparin complexes and C' fragments to B-cells was determined by flow cytometry using the murine monoclonal antibody to PF4/heparin complexes (KKO) and antibodies to specific C' activation products (Khandelwal, Blood 2016). To distinguish activation by classical and lectin pathways from alternative pathway of activation, we incubated blood with C1 esterase inhibitor, a protein that inhibits C' activation by the classical and lectin pathways. As shown in Figure 1, whole blood incubated with PF4/heparin is associated with C' activation as measured by C3c binding to B cells (Figure 1, column 3), but not if blood is incubated with buffer or PF4 alone (Figure 1, columns 1 & 2). With the addition of C1 esterase inhibitor (10 or 20 U/ml) prior to incubation with PF4/heparin complexes, we noted a dose-dependent reduction in C' activation (>85% reduction with 10 and 20 U/ml) suggesting that C' activation by PF4/heparin complexes occurs via the classical or lectin pathways. To further confirm that the alternate pathway is not involved in PF4/heparin mediated C' activation, we used sensitivity of the alternative pathway to Mg2+ using differential chelation with EDTA and EGTA. EDTA, a chelator of both Ca2+ and Mg2+, inhibits complement activation by all three pathways, whereas EGTA, which preferentially sequesters Ca2+ over Mg2+, permits alternative pathway activation. As shown in Figure 2, incubation of PF4/heparin complexes without chelators allowed for maximal antigen binding to B cells (~100%), while incubation with EDTA (Figure 2, column 3) abolished antigen binding, as did EGTA with and without additional Mg2+ supplementation. We next investigated the role of the classical pathway, a pathway triggered by immune complex mediated-binding and activation of C1. By flow cytometry, we were unable to show C1q deposition although we were able to demonstrate C3c/C4c deposition on antigen-coated B cells from healthy donors (data not shown). We also showed that C' activation by PF4/heparin complexes was preserved in human serum low in immunoglobulins (IgG and IgM) as well as plasma from mMT mice lacking circulating immunoglobulins (data not shown). To investigate the role of the lectin pathway, we performed competitive inhibition assays by incubating whole blood with PF4/heparin complexes in the presence of mannan. As shown in Figure 3, we show that mannan (500 mg/mL) inhibited binding of antigen to B cells. In other studies, we show that an anti-MBL antibody partially reduced binding of KKO/C' to B cells. Together, these preliminary studies demonstrate a lack of involvement of the classical and alternative pathways in C' activation and indicate a contribution by the lectin pathway in C' activation by PF4/heparin complexes. Further studies are underway to elucidate the role of the lectin pathway in complement activation by PF4/heparin complexes. Insights from these studies will lead to novel interventions that can block the initial steps of immune activation by heparin. Disclosures Arepally: Biokit: Patents & Royalties.

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Complement Regulates the Procoagulant Effects of HIT Immune Complexes

Blood ◽

10.1182/blood-2020-138631 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-12

Author(s):

Sanjay Khandelwal ◽

Lubica Rauova ◽

Ayiesha Barnes ◽

Ann Rux ◽

Serge Yarovoi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Surface ◽

Complement Activation ◽

Immune Complexes ◽

Cell Activation ◽

Alternative Pathway ◽

Classical Pathway ◽

Platelet Factor ◽

Neutrophil Degranulation ◽

Pathway Inhibition

Heparin induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing IgG antibodies bound to multivalent complexes of platelet factor 4 (PF4) and heparin (H). HIT ULICs activate cellular FcγIIA receptors that initiate diverse cellular effector functions including neutrophil degranulation and monocyte expression of tissue factor (TF). Previous studies have shown that HIT ULICs also potently activate complement through the classical pathway (Cines et al., 1980). Whether complement activation contributes to FcγRIIA-dependent prothrombotic pathways has not been addressed in detail. In studies that follow, we describe: 1) robust complement activation by HIT ULICs in plasma and whole blood (WB), 2) cell-surface deposition of complement and IgG triggered by HIT ULICs, 3) complement-dependent neutrophil degranulation and monocyte TF expression, 4) efficacy of proximal, but not terminal, pathway inhibition in regulating monocyte TF expression, and 5) deposition of complement in thrombi formed in "HIT mice" that generate ULICs containing KKO, a HIT-like monoclonal antibody (Arepally et al., 2000). Consistent with prior studies showing involvement of the classical pathway in HIT (Cines et al., 1980), we observed that binding of C1q induced marked enlargement of HIT ULICs in buffer assessed by dynamic light scattering as well as in plasma using confocal microscopy (data not shown). To assess complement activation by HIT ULICs, we incubated WB and plasma with PF4 (25 µg/mL) ± heparin (1 U/mL) in the presence of KKO (or isotype, "ISO"; 50 µg/mL) or HIT IgG (or control IgG, "CON"; 500 µg/mL) and measured C3c with a capture immunoassay as previously described (Khandelwal et al., 2018). KKO (Figure 1A) or HIT ULICs (n=3; HIT1-3, Figure 1B), showed robust generation of C3c in the presence of PF4/heparin, but not antigens alone or with control IgG (ISO/CON). Complement activation by HIT ULICs leads to downstream generation of C5a and formation of sC5b-9 (data not shown). Pre-incubation of plasma or WB with a variety of classical pathway inhibitors, including a C1r inhibitor derived from Borrelia burgdorferi (BBK 32), C1 esterase inhibitor (Berinert, CSL Behring) and anti-C1q antibody (α-C1q Ab; Annexon Biosciences) inhibited C3c generation by KKO ULICs (p <0.001), whereas inhibitors of the alternative pathway (anti-properdin antibody) or C5 inhibitor (α-C5 Ab; Eculizumab, Alexion Pharmaceuticals) did not (data not shown). Incubation of WB with KKO or HIT ULICs, but not ISO or CON IgG, markedly increased deposition of C3 and IgG on neutrophils, monocytes and B cells (data not shown) and lead to cell activation assessed by neutrophil degranulation (MMP9 release) and monocyte TF expression (data not shown). To examine the contribution of complement activation in monocyte TF expression, WB was pre-incubated with α-C1q, α-C5 or IV.3 (a monoclonal antibody to FcγRIIA) or isotype controls prior to addition of HIT ULICs. As shown in Figure 2, the classical pathway inhibitor, α-C1q Ab markedly diminished TF expression (about 70% reduction; p<0.001 vPF4/H/ KKO), as did IV.3 (about 85% reduction; p<0.001 vPF4/H/ KKO) but not α-C5 Ab or ISO antibodies, demonstrating: 1) FcγRIIA independent mechanism of monocyte TF expression and 2) a requirement for proximal rather than terminal complement pathway components in the induction of monocyte TF. We next asked if complement activation facilitates binding of ULICs and promotes subsequent ULIC engagement of FcγRIIA. To examine complement dependent binding of HIT ULICs, we incubated WB with α-C1q Ab prior to addition of KKO ULICs and measured ULIC binding to monocytes and TF expression. As shown in Figure 3, classical pathway inhibition markedly reduced cell-surface IgG (Figure 3A) and monocyte TF expression (Figure 3B). The effects of complement inhibition could not be overcome with increasing amounts of KKO IgG (2-4 fold excess). We observed significant co-localization of complement with KKO ULICs in a cremaster-laser injury model in "HIT mice" and in in situ thrombi formed in uninjured vessels (data not shown). Together, these studies demonstrate an independent role for complement activation in regulating the binding and procoagulant effects of HIT ULICs and identify new non-anticoagulant therapeutic targets that could improve clinical outcomes in this otherwise potentially devastating thrombotic disorder. Disclosures Arepally: Novartis: Consultancy; Alexion: Other; Annexon Biosciences: Consultancy, Other; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biokit: Consultancy, Patents & Royalties; Apotex: Consultancy, Research Funding.

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Targeted Complement Inhibitors Protect against Posttransplant Cardiac Ischemia and Reperfusion Injury and Reveal an Important Role for the Alternative Pathway of Complement Activation

The Journal of Immunology ◽

10.4049/jimmunol.1001504 ◽

2010 ◽

Vol 185 (11) ◽

pp. 7007-7013 ◽

Cited By ~ 29

Author(s):

Carl Atkinson ◽

Songqing He ◽

Keeley Morris ◽

Fei Qiao ◽

Sarah Casey ◽

...

Keyword(s):

Reperfusion Injury ◽

Complement Activation ◽

Alternative Pathway ◽

Cardiac Ischemia ◽

Ischemia And Reperfusion ◽

Ischemia And Reperfusion Injury ◽

Complement Inhibitors ◽

Alternative Pathway Of Complement

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Breakdown of C3 Covalently Bound to F(ab')(2) Immune Complexes after Complement Activation by the Alternative Pathway

Complement ◽

10.1159/000467881 ◽

1986 ◽

Vol 3 (2) ◽

pp. 53-62 ◽

Cited By ~ 8

Author(s):

L.C. Antón ◽

J.M. Alcolea ◽

G. Marqués ◽

P. Sánchez-Corral ◽

F. Vivanco

Keyword(s):

Complement Activation ◽

Immune Complexes ◽

Alternative Pathway ◽

Covalently Bound

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Recruitment of Human C1 Esterase Inhibitor Controls Complement Activation on Blood StagePlasmodium falciparumMerozoites

The Journal of Immunology ◽

10.4049/jimmunol.1700067 ◽

2017 ◽

Vol 198 (12) ◽

pp. 4728-4737 ◽

Cited By ~ 11

Author(s):

Alexander T. Kennedy ◽

Lakshmi C. Wijeyewickrema ◽

Alisee Huglo ◽

Clara Lin ◽

Robert Pike ◽

...

Keyword(s):

Complement Activation ◽

C1 Esterase Inhibitor ◽

Esterase Inhibitor

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Complement-dependent Clearance of Apoptotic Cells by Human Macrophages

Journal of Experimental Medicine ◽

10.1084/jem.188.12.2313 ◽

1998 ◽

Vol 188 (12) ◽

pp. 2313-2320 ◽

Cited By ~ 464

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.

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Titanium Is a Highly Thrombogenic Biomaterial: Possible Implications for Osteogenesis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614630 ◽

1999 ◽

Vol 82 (07) ◽

pp. 58-64 ◽

Cited By ~ 142

Author(s):

Jaan Hong ◽

Joakim Andersson ◽

Kristina Nilsson Ekdahl ◽

Graciela Elgue ◽

Niklas Axén ◽

...

Keyword(s):

Whole Blood ◽

Direct Contact ◽

Titanium Surface ◽

Specific Inhibitor ◽

Intrinsic Pathway ◽

C1 Esterase Inhibitor ◽

Biomaterial Surfaces ◽

Chamber Model ◽

Esterase Inhibitor

SummaryTitanium has superior osteointegrating properties compared to other biomaterials. The mechanism for this is unknown. During the initial phase of bone implantation the biomaterial comes into direct contact with whole blood. In this study we use a newly developed in vitro chamber model to compare different commonly used biomaterials in contact with whole blood. These materials were selected with respect to their different osteointegrating properties in order to correlate these properties with the response to whole blood. In the presence of 3 IU/ml of heparin only titanium induced macroscopic clotting. This was reflected by the generation of thrombin-antithrombin which was much increased in blood in contact with titanium compared with steel and PVC. The coagulation activation caused by titanium was triggered by the intrinsic pathway because the generation of FXIIa-AT/C1 esterase inhibitor paralleled that of thrombin-antithrombin, and both thrombinantithrombin complex and FXIIa-AT/C1 esterase inhibitor generation were abrogated by corn trypsin inhibitor, which is a specific inhibitor of FXIIa. The binding of platelets was increased on the titanium surface compared to the other biomaterial surfaces and the state of platelet activation was much more pronounced as reflected by the levels of β-thromboglobulin and PDGF. This study indicates that titanium is unsuitable as a biomaterial in devices which are in direct contact with blood for a prolonged period. Furthermore, PDGF and other α-granule proteins e.g. TGF-β, are known to be potent promotors of osteogenesis which suggests that the pronounced thrombogenic properties of titanium might contribute to the good osteointegrating properties.

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Complement activation during bypass in acquired C1 esterase inhibitor deficiency

The Annals of Thoracic Surgery ◽

10.1016/0003-4975(91)90921-c ◽

1991 ◽

Vol 52 (3) ◽

pp. 541-543 ◽

Cited By ~ 22

Author(s):

Robert S. Bonser ◽

Jitendra Dave ◽

John Morgan ◽

Cliff Morgan ◽

Edward Davies ◽

...

Keyword(s):

Complement Activation ◽

C1 Esterase Inhibitor ◽

Esterase Inhibitor

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SARS-CoV-2 triggers complement activation through interactions with heparan sulfate

10.1101/2022.01.11.475820 ◽

2022 ◽

Author(s):

Martin W Lo ◽

Alberto A Amarilla ◽

John D Lee ◽

Eduardo A Albornoz ◽

Naphak Modhiran ◽

...

Keyword(s):

Functional Analysis ◽

Cell Surface ◽

Heparan Sulfate ◽

Alternative Pathway ◽

Monocyte Activation ◽

Surface Deposition ◽

Factor B ◽

The Relationship ◽

The Complement System ◽

Free Plasma

The complement system has been heavily implicated in severe COVID-19 with clinical studies revealing widespread gene induction, deposition, and activation. However, the mechanism by which complement is activated in this disease remains incompletely understood. Herein we examined the relationship between SARS-CoV-2 and complement by inoculating the virus in lepirudin-anticoagulated human blood. This caused progressive C5a production after 30 minutes and 24 hours, which was blocked entirely by inhibitors for factor B, C3, C5, and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface deposition of complement and interactions with heparan sulfate. Additional functional analysis revealed that complement-dependent granulocyte and monocyte activation was delayed. Indeed, C5aR1 internalisation and CD11b upregulation on these cells only occurred after 24 hours. Thus, SARS-CoV-2 is a non-canonical complement activator that triggers the alternative pathway through interactions with heparan sulfate.

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