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Identification and Characterization of PF4/Heparin Reactive Antibodies in Heparin-Induce Thrombocytopenia Patients and COVID-19 Patients.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Identification and Characterization of PF4/Heparin Reactive Antibodies in Heparin-Induce Thrombocytopenia Patients and COVID-19 Patients.
Author:	Zhu, Wen.
Published:	Ann Arbor : ProQuest Dissertations & Theses, 2021
Description:	143 p.
Notes:	Source: Dissertations Abstracts International, Volume: 83-04, Section: B.
Notes:	Advisor: Wang, Demin.
Contained By:	Dissertations Abstracts International83-04B.
Subject:	Biology.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28644785
ISBN:	9798460479269

Identification and Characterization of PF4/Heparin Reactive Antibodies in Heparin-Induce Thrombocytopenia Patients and COVID-19 Patients.
Zhu, Wen.

Identification and Characterization of PF4/Heparin Reactive Antibodies in Heparin-Induce Thrombocytopenia Patients and COVID-19 Patients. - Ann Arbor : ProQuest Dissertations & Theses, 2021 - 143 p.

Source: Dissertations Abstracts International, Volume: 83-04, Section: B.

Thesis (Ph.D.)--The Medical College of Wisconsin, 2021.

This item must not be sold to any third party vendors.

Heparin-induced thrombocytopenia (HIT) is a common adverse drug reaction characterized by a platelet count reduction after heparin administration, sometimes accompanied by thrombosis with devastating consequences. HIT is characterized by antibodies specific for conformational epitopes induced in the platelet chemokine-platelet factor 4 (PF4) when it binds to heparin (“HIT antibodies”). Upon injection, heparin binds PF4 to produce circulating PF4/Heparin (PF4/H) complexes which, in some individuals, activate PF4/H recognizing B cells, leading to the production of antibodies that react with PF4/H to produce immune complexes. The Fc domain of antibodies binds to the FcγRIIa receptor on platelets, resulting in FcγRIIa receptor clustering and consequent platelet activation and aggregation. Clinical and laboratory studies indicate there are at least two types of PF4/H reactive antibodies - one of which activates platelets (pathogenic) and a second binds to PF4/H complexes but does not activate platelets (non-pathogenic). However, the molecular basis for this difference in behavior is unknown. This differential antibody function sometimes leads to overdiagnosis and over-treatment. To address this question, we cloned PF4/H reactive antibodies from HIT patients and examined the structural properties of the two types of antibodies. We cloned pathogenic antibodies from HIT patients and found unique characters of the PF4/H specific antibodies, including exclusive Kappa chain usage, biased V(D)J segment usage and combination, long and more charged HCDR3. Most importantly, we identified two pathogenic motifs: RX1-2R/KX1-2R/H or YYYYY in a long HCDR3 (≥20 residues). F(ab’)2 of the pathogenic antibodies and repertoire sequencing were used to confirm the functional relevance of these two motifs and were shown to have the treatment and diagnostic potentials. Nine clones that acquired the RKH or Y5 motif from HIT patient repertoire were positive in PF4/H ELISA, and two can activate platelets in PEA assay. Identification of pathogenic motifs should advance the understanding of HIT pathogenesis and suggest novel approaches to HIT diagnosis and treatment.HIT displays some atypical immune response features. It seems that the immune response acquires both T-cell dependent (TD) and T-cell independent (TI) features. (A previous human study analyzing T cell CDR3 region from HIT patients together with some mouse study suggests T cell play a role in the pathogenies of the disease. Besides that, IgG1 is the dominant isotype in most HIT patients, further illustrating T cell's importance. And our cloning data showed that although limited, somatic hypermutation (SHM) did play a role in the development of these antibodies. However, the onset and decline of HIT antibodies in patients are rapid. HIT patients don’t seem to have immunological memory. PF4/H complexes have repetitive structures, which is a hallmark for TI-2 antigens. This also indicates that the immune response is T-cell independent. Our lab had a publication a few years ago demonstrating that marginal zone (MZ) B cells play a critical role in the pathogenesis of this disease using a mouse model system. However, MZ B cells in humans are still not fully identified and characterized. Thus, the origin of the B cells and the humoral immune responses in HIT patients are largely unknown. We attempt to understand the origin of the B cells that make the PA and NA antibodies and the nature of the immune response in HIT by analyzing such antibodies' SHM and biological property. We found that PF4/H-binding PA and NA IgGs are largely polyreactive antibodies and contain lower levels of mutations compared to IgG+ memory B cells. Germline conversion of all the PA clones suggested that SHM played a limited role in the development of PA clones. B1 and MZ B cells are innate B cells leading producers of polyreactive natural antibodies. They can respond to toll-like receptor signaling, quickly differentiate into antibody-secreting cells, and undergo IgG class switch extrafollicularly. Polyreactivity identified in the PF4/H-binding PA, and NA IgGs supports the possibility that human B cells producing PF4/H-binding antibodies are innate B cells akin to MZ B cells shown to be a source of PF4/H antibodies in mice. A mutation rate lower than that of IgG+ memory cells in the PF4/H-binding IgGs is also consistent with an extrafollicular (EF) response typical of innate B cells. These observations would help to improve our understanding of the immunological responses and B cell origin in HIT patients.Severe COVID-19 is associated with thrombotic complications and a more robust anti-SARS-CoV2 IgG response, and we investigated the link between the two phenotypes. We found that hospitalized patients with COVID-19 had increased IgG antibody levels targeting a complex consisting of PF4 and heparin (PF4/H), the hallmark of HIT. In COVID-19 patient plasma, we also observed a significant elevation of platelet-activating antibodies that behave like those developed in HIT patients. Strikingly, IgG levels reactive to PF4/H and SARS-CoV-2 spike protein receptor-binding domain (RBD) were significantly correlated, and a large proportion of RBD-binding B cells were cross-reactive to PF4/H. Four out of 42 antibodies cloned from RBD-binding IgG1+ B cells in COVID-19 patients could activate platelets. Of those, three possessed an RKH or Y5 motif in the heavy-chain complementarity determining region (HCDR3) that we recently showed to be prominent among platelet-activating IgG1 antibodies cloned from HIT patients. RKH and Y5 motifs are prevalent in RBD-specific antibodies cloned by others, and three out of six such antibodies we tested were able to activate platelets. IgG1+ and IgG3+ B cells that possess an RKH or Y5 motif were clonally expanded, and their prevalence significantly increased in COVID-19 patients compared to healthy controls. Our study demonstrated that SARS-CoV-2 infection elicits prothrombotic RBD-specific antibodies. We propose an antibody-mediated B-cell-immune thrombosis axis that contributes to thrombotic complications in COVID-19.

ISBN: 9798460479269Subjects--Topical Terms:

188667
Biology.
Subjects--Index Terms:

COVID-19

Identification and Characterization of PF4/Heparin Reactive Antibodies in Heparin-Induce Thrombocytopenia Patients and COVID-19 Patients.
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520 $a Heparin-induced thrombocytopenia (HIT) is a common adverse drug reaction characterized by a platelet count reduction after heparin administration, sometimes accompanied by thrombosis with devastating consequences. HIT is characterized by antibodies specific for conformational epitopes induced in the platelet chemokine-platelet factor 4 (PF4) when it binds to heparin (“HIT antibodies”). Upon injection, heparin binds PF4 to produce circulating PF4/Heparin (PF4/H) complexes which, in some individuals, activate PF4/H recognizing B cells, leading to the production of antibodies that react with PF4/H to produce immune complexes. The Fc domain of antibodies binds to the FcγRIIa receptor on platelets, resulting in FcγRIIa receptor clustering and consequent platelet activation and aggregation. Clinical and laboratory studies indicate there are at least two types of PF4/H reactive antibodies - one of which activates platelets (pathogenic) and a second binds to PF4/H complexes but does not activate platelets (non-pathogenic). However, the molecular basis for this difference in behavior is unknown. This differential antibody function sometimes leads to overdiagnosis and over-treatment. To address this question, we cloned PF4/H reactive antibodies from HIT patients and examined the structural properties of the two types of antibodies. We cloned pathogenic antibodies from HIT patients and found unique characters of the PF4/H specific antibodies, including exclusive Kappa chain usage, biased V(D)J segment usage and combination, long and more charged HCDR3. Most importantly, we identified two pathogenic motifs: RX1-2R/KX1-2R/H or YYYYY in a long HCDR3 (≥20 residues). F(ab’)2 of the pathogenic antibodies and repertoire sequencing were used to confirm the functional relevance of these two motifs and were shown to have the treatment and diagnostic potentials. Nine clones that acquired the RKH or Y5 motif from HIT patient repertoire were positive in PF4/H ELISA, and two can activate platelets in PEA assay. Identification of pathogenic motifs should advance the understanding of HIT pathogenesis and suggest novel approaches to HIT diagnosis and treatment.HIT displays some atypical immune response features. It seems that the immune response acquires both T-cell dependent (TD) and T-cell independent (TI) features. (A previous human study analyzing T cell CDR3 region from HIT patients together with some mouse study suggests T cell play a role in the pathogenies of the disease. Besides that, IgG1 is the dominant isotype in most HIT patients, further illustrating T cell's importance. And our cloning data showed that although limited, somatic hypermutation (SHM) did play a role in the development of these antibodies. However, the onset and decline of HIT antibodies in patients are rapid. HIT patients don’t seem to have immunological memory. PF4/H complexes have repetitive structures, which is a hallmark for TI-2 antigens. This also indicates that the immune response is T-cell independent. Our lab had a publication a few years ago demonstrating that marginal zone (MZ) B cells play a critical role in the pathogenesis of this disease using a mouse model system. However, MZ B cells in humans are still not fully identified and characterized. Thus, the origin of the B cells and the humoral immune responses in HIT patients are largely unknown. We attempt to understand the origin of the B cells that make the PA and NA antibodies and the nature of the immune response in HIT by analyzing such antibodies' SHM and biological property. We found that PF4/H-binding PA and NA IgGs are largely polyreactive antibodies and contain lower levels of mutations compared to IgG+ memory B cells. Germline conversion of all the PA clones suggested that SHM played a limited role in the development of PA clones. B1 and MZ B cells are innate B cells leading producers of polyreactive natural antibodies. They can respond to toll-like receptor signaling, quickly differentiate into antibody-secreting cells, and undergo IgG class switch extrafollicularly. Polyreactivity identified in the PF4/H-binding PA, and NA IgGs supports the possibility that human B cells producing PF4/H-binding antibodies are innate B cells akin to MZ B cells shown to be a source of PF4/H antibodies in mice. A mutation rate lower than that of IgG+ memory cells in the PF4/H-binding IgGs is also consistent with an extrafollicular (EF) response typical of innate B cells. These observations would help to improve our understanding of the immunological responses and B cell origin in HIT patients.Severe COVID-19 is associated with thrombotic complications and a more robust anti-SARS-CoV2 IgG response, and we investigated the link between the two phenotypes. We found that hospitalized patients with COVID-19 had increased IgG antibody levels targeting a complex consisting of PF4 and heparin (PF4/H), the hallmark of HIT. In COVID-19 patient plasma, we also observed a significant elevation of platelet-activating antibodies that behave like those developed in HIT patients. Strikingly, IgG levels reactive to PF4/H and SARS-CoV-2 spike protein receptor-binding domain (RBD) were significantly correlated, and a large proportion of RBD-binding B cells were cross-reactive to PF4/H. Four out of 42 antibodies cloned from RBD-binding IgG1+ B cells in COVID-19 patients could activate platelets. Of those, three possessed an RKH or Y5 motif in the heavy-chain complementarity determining region (HCDR3) that we recently showed to be prominent among platelet-activating IgG1 antibodies cloned from HIT patients. RKH and Y5 motifs are prevalent in RBD-specific antibodies cloned by others, and three out of six such antibodies we tested were able to activate platelets. IgG1+ and IgG3+ B cells that possess an RKH or Y5 motif were clonally expanded, and their prevalence significantly increased in COVID-19 patients compared to healthy controls. Our study demonstrated that SARS-CoV-2 infection elicits prothrombotic RBD-specific antibodies. We propose an antibody-mediated B-cell-immune thrombosis axis that contributes to thrombotic complications in COVID-19.
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