Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies

Lee, Grace M.; Joglekar, Manali; Kuchibhatla, Maragatha; Khandelwal, Sanjay; Rui, Qi; Rauova, Lubica; Arepally, Gowthami M.

doi:10.1182/bloodadvances.2017004408

Cited by 9 publications

(6 citation statements)

References 18 publications

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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%

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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Section: Methodsmentioning

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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“…A mouse monoclonal IgG 2b k anti-PF4/heparin antibody (KKO), a monoclonal IgG 2b k isotype control, a mouse monoclonal IgG 3 k anti-PRT/heparin antibody (ADA), 19 and recombinant human PF4 were isolated, as previously described. 20,21 PMA, a monoclonal IgG 3 isotype, and protamine sulfate were purchased from Sigma (St. Louis, MO), unfractionated heparin (UFH) from Fresenius Kabi (Lake Zurich, IL), enoxaparin from Aventis Pharmaceuticals (Paris, France), and a monoclonal antibody to CD32 (IV.3) from STEMCELL Technologies (Vancouver, BC, Canada).…”

Section: Reagentsmentioning

confidence: 99%

Heterogeneity in neutrophil responses to immune complexes

Duarte¹,

Kuchibhatla

Khandelwal³

et al. 2019

Blood Advances

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“…In the pathogenesis of HIT, antibodies that recognize complexes formed by platelet factor 4 (PF4) and polyanions, such as heparin [ 74 ] and anti-protamine (PRT)/heparin, are implicated [ 75 ]. These immunogenic complexes induce a response in which IgG bind to platelet Fcγ RIIa receptors.…”

Section: Introductionmentioning

confidence: 99%

Mechanisms of Immunothrombosis by SARS-CoV-2

et al. 2021

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SARS-CoV-2 contains certain molecules that are related to the presence of immunothrombosis. Here, we review the pathogen and damage-associated molecular patterns. We also study the imbalance of different molecules participating in immunothrombosis, such as tissue factor, factors of the contact system, histones, and the role of cells, such as endothelial cells, platelets, and neutrophil extracellular traps. Regarding the pathogenetic mechanism, we discuss clinical trials, case-control studies, comparative and translational studies, and observational studies of regulatory or inhibitory molecules, more specifically, extracellular DNA and RNA, histones, sensors for RNA and DNA, as well as heparin and heparinoids. Overall, it appears that a network of cells and molecules identified in this axis is simultaneously but differentially affecting patients at different stages of COVID-19, and this is characterized by endothelial damage, microthrombosis, and inflammation.

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Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies

Cited by 9 publications

References 18 publications

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

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

Heterogeneity in neutrophil responses to immune complexes

Mechanisms of Immunothrombosis by SARS-CoV-2

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