Novel Immunoassay for Complement Activation by PF4/Heparin Complexes

Khandelwal, Sanjay; Johnson, Alexandra M.; Liu, Jian; Keire, David A.; Sommers, Cynthia D.; Ravi, Joann; Lee, Grace M.; Lambris, John D.; Arepally, Gowthami M.

doi:10.1055/s-0038-1660858

Cited by 7 publications

(9 citation statements)

References 10 publications

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“…To measure complement activation and inhibition in vitro, plasma was first incubated with PF4/heparin complexes (25 μg/ml and 0.25 U/mL respectively; formed at a PF4 to heparin molar ratio (PHR) of 6.6). After 1 h of incubation, complement-fixed antigen was captured by KKO, a PF4/heparin specific monoclonal antibody, and complement fragments containing C3 were detected using a biotinylated anti-C3c antibody as previously described 68 .…”

Section: Methodsmentioning

confidence: 99%

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates

et al. 2021

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Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

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Section: Methodsmentioning

confidence: 99%

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates

et al. 2021

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“…The following antibodies were used: anti-C1q (Cell Sciences, Inc., Newburyport, MA), anti-C3c (Quidel, San Diego, CA), anti-MBL (R&D Systems, Minneapolis, MN), and murine IgG1 isotype control (Invitrogen, Carlsbad, CA), fluorescently conjugated anti-human CD19 and conjugated streptavidin (eBioscience, San Diego, CA), and fluorescently conjugated goat anti-human IgM (Jackson Labs, West Grove, PA). Monoclonal antibody KKO (IgG2 b k recognizing PF4/heparin), 28 ADA (IgG3 recognizing PRT sulfate/heparin), 29,30 and 2E4 (monoclonal IgM with polyreactive specificities to single-stranded DNA, b-galactosidase, and other antigens) 31 were developed, purified, and isolated in the laboratory according to published methods. PF4/heparin-specific IgM was isolated using beads coated with PF4 bound to heparin immobilized on diamino-dipropylamine agarose (Thermo Fisher Scientific, Waltham, MA), as previously described.…”

Section: Methodsmentioning

confidence: 99%

“…Antigen-C3c capture ELISA assay and specificity ELISA Antigen-C3c capture enzyme-linked immunosorbent assay (ELISA) was performed as described in supplemental Data, available on the Blood Web site. 30 For studies involving Igs (IgM, IgG, myeloma IgM, monoclonal polyreactive IgM), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), magnesium chloride (MgCl 2 ), or classical/lectin pathway, reagents were added to plasma before incubating with antigen.…”

Section: Immunoglobulin Levelsmentioning

confidence: 99%

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway

Khandelwal

Ravi

Rauova

et al. 2018

Blood

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Abstract The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder “phenotype” (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

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“…Also, C3aR signaling on platelets can further drive thrombotic responses (12). Of note, it has been shown that PF4-heparin complexes, a hallmark of HIT pathology can potently activate C3 in the fluid phase and C3 inhibitors such as Cp40 can abrogate C3 activation triggered by HIT-associated PF4-heparin complexes (13). In line with this, the Cp40-based drug candidate AMY-101 has been shown to effectively decrease both neutrophils and neutrophil extracellular trap (NET) formation, two major drivers of HIT-associated thrombosis, in COVID-19 patients (7,(14)(15)(16).…”

Section: A Proposed Role For Complementmentioning

confidence: 99%