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Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin‐induced thrombocytopenia
Author(s): ,
Angela Huynh
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada
,
Donald M. Arnold
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada. McMaster Centre for Transfusion Research, Hamilton, Canada. Canadian Blood Services, Hamilton, Canada
,
John G. Kelton
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada
,
James W. Smith
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada
,
Peter Horsewood
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada
,
Rumi Clare
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada
,
Alba Guarné
Affiliations:
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada
Ishac Nazy
Affiliations:
Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Canada. McMaster Centre for Transfusion Research, Hamilton, Canada
Correspondence: Ishac Nazy, Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, HSC 3H53, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada|Tel.: +1 905 525 9140 x20242|E‐mail: nazii@mcmaster.ca
ISTH Academy. Nazi I.
Feb 8, 2019; 273373
Ishac Nazi
Ishac  Nazi
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Journal Abstract
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Background
Heparin‐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.
Objectives
To identify PF4 amino acids essential for binding pathogenic HIT antibodies.
Methods
Alanine 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 Conclusions
We 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.
Keyword(s)
CXCL4, heparin, heparin‐induced thrombocytopenia, platelet factor 4, thrombocytopenia, thrombosis
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