Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia

Dai, Jing; Madeeva, Daria; Hayes, Vincent; Ahn, Hyun Sook; Tutwiler, Valerie; Arepally, Gowthami M.; Cines, Douglas B.; Poncz, Mortimer; Rauova, Lubica

doi:10.1182/blood-2018-02-830737

Cited by 10 publications

(9 citation statements)

References 46 publications

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“…Third, fibrin accumulation is inhibited by an a-mouse FcRn mAb after vascular injury in a murine model of HIT in which TF is generated in monocytes. 9,41,45 Together, these studies reveal that induction of TF activity requires not only FcgRIIa, as we have previously shown, 42,45 but also FcRn, as we describe here.…”

Section: Discussionsupporting

confidence: 88%

“…Human a-FcRn antibody (SYNT001), mouse a-human PF4/heparin mAb (KKO) or mouse control a-human PF4 monomer mAb (RTO), 40 and mouse a-human FcgRIIa MoAb (clone IV.3) and the corresponding control IgGs (ie, human IgG4 as the negative control for SYNT001 and mouse IgG [TRA] for KKO and for IV.3) were conjugated with Alexa 488, Alexa 568, and Alexa 647 fluorescent dyes, respectively, using antibodylabeling kits (Thermo Fisher, Invitrogen). 41 Cells exposed to HIT ICs or controls were incubated with Alexa 488/SYNT001, Alexa 568/KKO, and Alexa 647/IV.3 mAbs for 1 hour at room temperature. Alexa 488-conjugated human IgG4 and Alexa 568-or Alexa 647-conjugated isotype-matched mouse-irrelevant IgG2b served as the negative controls.…”

Section: Membrane Colocalizationmentioning

confidence: 99%

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FcRn augments induction of tissue factor activity by IgG-containing immune complexes

Cines

Зайцев²,

Rauova

et al. 2020

Blood

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Thromboembolism complicates disorders caused by immunoglobulin G (IgG)–containing immune complexes (ICs), but the underlying mechanisms are incompletely understood. Prior evidence indicates that induction of tissue factor (TF) on monocytes, a pivotal step in the initiation, localization, and propagation of coagulation by ICs, is mediated through Fcγ receptor IIa (FcγRIIa); however, the involvement of other receptors has not been investigated in detail. The neonatal Fc receptor (FcRn) that mediates IgG and albumin recycling also participates in cellular responses to IgG-containing ICs. Here we asked whether FcRn is also involved in the induction of TF-dependent factor Xa (FXa) activity by IgG-containing ICs by THP-1 monocytic cells and human monocytes. Induction of FXa activity by ICs containing IgG antibodies to platelet factor 4 (PF4) involved in heparin-induced thrombocytopenia (HIT), β-2-glycoprotein-1 implicated in antiphospholipid syndrome, or red blood cells coated with anti-(α)-Rh(D) antibodies that mediate hemolysis in vivo was inhibited by a humanized monoclonal antibody (mAb) that blocks IgG binding to human FcRn. IgG-containing ICs that bind to FcγR and FcRn induced FXa activity, whereas IgG-containing ICs with an Fc engineered to be unable to engage FcRn did not. Infusion of an α-FcRn mAb prevented fibrin deposition after microvascular injury in a murine model of HIT in which human FcγRIIa was expressed as a transgene. These data implicate FcRn in TF-dependent FXa activity induced by soluble and cell-associated IgG-containing ICs. Antibodies to FcRn, now in clinical trials in warm autoimmune hemolytic anemia to lower IgG antibodies and IgG containing ICs may also reduce the risk of venous thromboembolism.

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Section: Discussionsupporting

confidence: 88%

Section: Membrane Colocalizationmentioning

confidence: 99%

FcRn augments induction of tissue factor activity by IgG-containing immune complexes

Cines

Зайцев²,

Rauova

et al. 2020

Blood

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“…Heparin is a widely used anticoagulant and about 3-5% of patients exposed to heparin can develop a severe drug reaction known as heparin-induced thrombocytopenia (HIT) [1,2]. HIT occurs when antibodies against multimolecular complexes of platelet factor 4 (PF4, CXCL4) and heparin activate platelets and monocytes [3][4][5][6][7][8][9]. PF4 is a tetrameric chemokine with four cysteines, which form two disulfide bonds.…”

Section: Introductionmentioning

confidence: 99%

Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin‐induced thrombocytopenia

Huynh

Arnold

Kelton

et al. 2019

Journal of Thrombosis and Haemostasis

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Many patients produce antibodies but few lead to heparin‐induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non‐pathogenic antibodies. Summary BackgroundHeparin‐induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti‐PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. ObjectivesTo identify PF4 amino acids essential for binding pathogenic HIT antibodies. MethodsAlanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet‐activating murine monoclonal anti‐PF4/heparin antibody (KKO) and HIT patient sera (n = 9). Results and ConclusionsWe identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n = 45) and non‐pathogenic antibodies (EIA‐positive but not platelet activating, n = 28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4.

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“…The tendency to a decrease in monocyte levels is especially noteworthy given that neutrophil counts increased in almost all patients, often dramatically. Recently, Dai et al, 20 reporting on a passive immunization murine model of HIT whereby acute thrombocytopenia was triggered by infusing a HIT-like monoclonal antibody (KKO) to human PF4 (hPF4) þ /FcγIIA receptor (FcγIIA)þ mice, found that acute declines in monocyte levels were observed in mice following an intravenous infusion of KKO (the authors did not report on whether changes in neutrophil counts occurred or not). However, as we also involved a tendency to decreasing monocyte counts in patients with non-HIT thrombosis, the significance if any of mildly declining monocyte levels in our patients with HIT is unclear.…”

Section: Discussionmentioning

confidence: 99%

“…18,19 A recent study noted occurrence of transient monocytopaenia in a passive murine immunization HIT model. 20 Other investigators found that HIT antibodies also activate polymorphonuclear leukocytes (neutrophils), leading to leukocyte degranulation. 21 Despite these laboratory studies involving isolated cells and animal models, there is to our knowledge no reported clinical study evaluating changes in leukocyte counts during the clinical course of HIT.…”

Section: Introductionmentioning

confidence: 99%

Neutrophil and Monocyte Counts in Heparin-Induced Thrombocytopenia

Hui

Sheppard

et al. 2019

Thromb Haemost

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Background Heparin-induced thrombocytopenia (HIT) antibodies activate platelets, monocytes and neutrophils. Despite these findings, it is unknown whether white blood cell (WBC) counts, including neutrophils and monocytes, are altered during HIT. Materials and Methods We evaluated changes in total WBC counts (including WBC subsets), in 50 post-cardiac surgery patients with serologically confirmed HIT (30 patients with HIT-associated thrombosis). Daily leukocyte counts were compared with those measured one day prior to HIT onset; WBC increases were classified as mild (20.0–49.9%), moderate (50.0–99.9%) or major (≥ 100% increase). We also compared changes in WBC counts in HIT patients with and without HIT-associated thrombosis, and non-HIT patients with thrombosis. Results Most (34/50 [68.0%]) patients with HIT developed WBC count increases (mild, 35.3%; moderate, 44.1%; major, 20.6%). The peak WBC count occurred on day 4 (median) of HIT, which corresponded to day 10 (median) post-surgery. Absolute neutrophil counts increased in most patients (38/50 [76.0%]); whereas absolute monocyte counts rose in some patients, the overall tendency was for the monocyte count to decrease during HIT. Unexpectedly, we found that the increase in total WBC counts, as well as in neutrophils, was seen mainly in patients who developed HIT-associated or non-HIT-associated thrombosis; in contrast, no difference in monocyte levels was seen in patients with or without thrombosis. Conclusion Leukocytosis and neutrophilia are commonly observed in patients with HIT, particularly in patients with HIT-associated thrombosis, as well as non-HIT patients with thrombosis. Thus, leukocytosis/neutrophilia should not infer automatically a diagnosis of infection or inflammation, when evaluating thrombocytopenia in heparin-exposed patients.

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Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia

Cited by 10 publications

References 46 publications

FcRn augments induction of tissue factor activity by IgG-containing immune complexes

FcRn augments induction of tissue factor activity by IgG-containing immune complexes

Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin‐induced thrombocytopenia

Neutrophil and Monocyte Counts in Heparin-Induced Thrombocytopenia

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