Publications by authors named "Angela Huynh"

9 Publications

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Platelet-activating immune complexes identified in critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia.

J Thromb Haemost 2021 Feb 27. Epub 2021 Feb 27.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, ON, Canada.

Background: Thrombocytopenia and thrombosis are prominent in coronavirus disease 2019 (COVID-19), particularly among critically ill patients; however, the mechanism is unclear. Such critically ill COVID-19 patients may be suspected of heparin-induced thrombocytopenia (HIT), given similar clinical features.

Objectives: We investigated the presence of platelet-activating anti-platelet-factor 4 (PF4)/heparin antibodies in critically ill COVID-19 patients suspected of HIT.

Patients/methods: We tested 10 critically ill COVID-19 patients suspected of HIT for anti-PF4/heparin antibodies and functional platelet activation in the serotonin release assay (SRA). Anti-human CD32 antibody (IV.3) was added to the SRA to confirm FcγRIIA involvement. Additionally, SARS-CoV-2 antibodies were measured using an in-house ELISA. Finally, von Willebrand factor (VWF) antigen and activity were measured along with A Disintegrin And Metalloprotease with ThromboSpondin-13 Domain (ADAMTS13) activity and the presence of anti-ADAMTS13 antibodies.

Results: Heparin-induced thrombocytopenia was excluded in all samples based on anti-PF4/heparin antibody and SRA results. Notably, six COVID-19 patients demonstrated platelet activation by the SRA that was inhibited by FcγRIIA receptor blockade, confirming an immune complex (IC)-mediated reaction. Platelet activation was independent of heparin but inhibited by both therapeutic and high dose heparin. All six samples were positive for antibodies targeting the receptor binding domain (RBD) or the spike protein of the SARS-CoV-2 virus. These samples also featured significantly increased VWF antigen and activity, which was not statistically different from the four COVID-19 samples without platelet activation. ADAMTS13 activity was not severely reduced, and ADAMTS13 inhibitors were not present, thus ruling out a primary thrombotic microangiopathy.

Conclusions: Our study identifies platelet-activating ICs as a novel mechanism that contributes to critically ill COVID-19.
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http://dx.doi.org/10.1111/jth.15283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014456PMC
February 2021

The role of fluid-phase immune complexes in the pathogenesis of heparin-induced thrombocytopenia.

Thromb Res 2020 10 6;194:135-141. Epub 2020 Jun 6.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada; McMaster Centre for Transfusion Research, Hamilton, Ontario, Canada. Electronic address:

Immune complexes assemble on the platelet surface and cause Fc-mediated platelet activation in heparin-induced thrombocytopenia (HIT); however, it is not known if fluid-phase immune complexes contribute to HIT. The objective of this study was to understand the role of fluid-phase immune complexes in platelet activation and HIT. Binding of wild-type and 15 platelet factor 4 (PF4) mutants to platelets was measured using flow cytometry. Platelet activation was measured using the PF4-dependent C-serotonin release assay (PF4-SRA) with KKO and a HIT-patient plasma in the presence of wild-type or PF4 mutants. To activate platelets, we found that a minimal level of wild-type PF4 is required to bind the platelet surface in the presence of KKO (2.67 relative MFI) or HIT-patient plasma (1.71 relative MFI). Only a subset of PF4 mutants was able to support platelet activation, despite having lower surface binding than the minimum binding required of wild-type PF4 (9 mutants with KKO and 2 mutants with HIT-patient plasma). Using individual PF4 mutants, we identified that HIT immune complexes can be formed in fluid-phase and induce platelet activation. Further studies are required to investigate the role of fluid-phase HIT immune complexes in the development of thrombocytopenia and thrombosis associated with clinical HIT.
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http://dx.doi.org/10.1016/j.thromres.2020.06.012DOI Listing
October 2020

Platelet Factor 4 Interactions with Short Heparin Oligomers: Implications for Folding and Assembly.

Biophys J 2020 10 21;119(7):1371-1379. Epub 2020 Apr 21.

Chemistry Department, University of Massachusetts-Amherst, Amherst, Massachusetts. Electronic address:

Association of platelet factor 4 (PF4) with heparin is a first step in formation of aggregates implicated in the development of heparin-induced thrombocytopenia (HIT), a potentially fatal immune disorder affecting 1-5% of patients receiving heparin. Despite being a critically important element in HIT etiology, relatively little is known about the specific molecular mechanism of PF4-heparin interactions. This work uses native mass spectrometry to investigate PF4 interactions with relatively short heparin chains (up to decasaccharides). The protein is shown to be remarkably unstable at physiological ionic strength in the absence of polyanions; only monomeric species are observed, and the extent of multiple charging of corresponding ions indicates a partial loss of conformational integrity. The tetramer signal remains at or below the detection threshold in the mass spectra until the solution's ionic strength is elevated well above the physiological level, highlighting the destabilizing role played by electrostatic interactions vis-à-vis quaternary structure of this high-pI protein. The tetramer assembly is dramatically facilitated by relatively short polyanions (synthetic heparin-mimetic pentasaccharide), with the majority of the protein molecules existing in the tetrameric state even at physiological ionic strength. Each tetramer accommodates up to six pentasaccharides, with at least three such ligands required to guarantee the higher-order structure integrity. Similar results are obtained for PF4 association with longer and structurally heterogeneous heparin oligomers (decamers). These longer polyanions can also induce PF4 dimer assembly when bound to the protein in relatively low numbers, lending support to a model of PF4/heparin interaction in which the latter wraps around the protein, making contacts with multiple subunits. Taken together, these results provide a more nuanced picture of PF4-glycosaminoglycan interactions leading to complex formation. This work also advocates for a greater utilization of native mass spectrometry in elucidating molecular mechanisms underlying HIT, as well as other physiological processes driven by electrostatic interactions.
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http://dx.doi.org/10.1016/j.bpj.2020.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567982PMC
October 2020

Streptomyces IHF uses multiple interfaces to bind DNA.

Biochim Biophys Acta Gen Subj 2019 11 31;1863(11):129405. Epub 2019 Jul 31.

Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada; Department of Biochemistry, McGill University, Montreal, QC, Canada. Electronic address:

Background: Nucleoid associated proteins (NAPs) are essential for chromosome condensation in bacterial cells. Despite being a diverse group, NAPs share two common traits: they are small, oligomeric proteins and their oligomeric state is critical for DNA condensation. Streptomyces coelicolor IHF (sIHF) is an actinobacterial-specific nucleoid-associated protein that despite its name, shares neither sequence nor structural homology with the well-characterized Escherichia coli IHF. Like E. coli IHF, sIHF is needed for efficient nucleoid condensation, morphological development and antibiotic production in S. coelicolor.

Methods: Using a combination of crystallography, small-angle X-ray scattering, electron microscopy and structure-guided functional assays, we characterized how sIHF binds and remodels DNA.

Results: The structure of sIHF bound to DNA revealed two DNA-binding elements on opposite surfaces of the helix bundle. Using structure-guided functional assays, we identified an additional surface that drives DNA binding in solution. Binding by each element is necessary for both normal development and antibiotic production in vivo, while in vitro, they act collectively to restrain negative supercoils.

Conclusions: The cleft defined by the N-terminal and the helix bundle of sIHF drives DNA binding, but the two additional surfaces identified on the crystal structure are necessary to stabilize binding, remodel DNA and maintain wild-type levels of antibiotic production. We propose a model describing how the multiple DNA-binding elements enable oligomerization-independent nucleoid condensation.

General Significance: This work provides a new dimension to the mechanistic repertoire ascribed to bacterial NAPs and highlights the power of combining structural biology techniques to study sequence unspecific protein-DNA interactions.
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http://dx.doi.org/10.1016/j.bbagen.2019.07.014DOI Listing
November 2019

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia.

Platelets 2019 29;30(8):1017-1021. Epub 2019 Jan 29.

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University , Hamilton , Ontario , Canada.

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = -0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.
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http://dx.doi.org/10.1080/09537104.2018.1562169DOI Listing
February 2020

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

J Thromb Haemost 2019 02;17(2):389-399

Department of Medicine, Michael G. DeGroote School of Medicine, McMaster University, Hamilton, Ontario, Canada.

Essentials 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: 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.
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http://dx.doi.org/10.1111/jth.14369DOI Listing
February 2019

Direct binding and internalization of diverse extracellular nucleic acid species through the collagenous domain of class A scavenger receptors.

Immunol Cell Biol 2018 10 26;96(9):922-934. Epub 2018 Apr 26.

Biochemistry and Biomedical Sciences, McMaster Immunology Research Centre, Michael DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada.

Nucleic acids are potential pathogen-associated or danger-associated molecular patterns that modulate immune responses and the development of autoimmune disorders. Class A scavenger receptors (SR-As) are a diverse group of pattern recognition receptors that recognize a variety of polyanionic ligands including nucleic acids. While SR-As are important for the recognition and internalization of extracellular dsRNA, little is known about extracellular DNA, despite its association with chronic infections and autoimmune disorders. In this study, we investigated the specificity of and requirement for SR-As in binding and internalizing different species, sequences and lengths of nucleic acids. We purified recombinant coiled-coil/collagenous and scavenger receptor cysteine-rich (SRCR) domains that have been implicated as potential ligand-binding domains. We detected a direct interaction of RNA and DNA species with the coiled-coil/collagenous domain, but not the SRCR domain. Despite the presence of additional surface receptors that bind nucleic acids, SR-As were found to be sufficient for nucleic acid binding and uptake in A549 human lung epithelial cells. Moreover, these findings suggest that the coiled-coil/collagenous domain of SR-As is sufficient to bind nucleic acids independent of species, sequence or length.
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http://dx.doi.org/10.1111/imcb.12052DOI Listing
October 2018

Development of a high-yield expression and purification system for platelet factor 4.

Platelets 2018 May 27;29(3):249-256. Epub 2017 Nov 27.

a Department of Medicine , Michael G. DeGroote School of Medicine, McMaster University , Hamilton , Ontario , Canada.

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.
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http://dx.doi.org/10.1080/09537104.2017.1378808DOI Listing
May 2018

A naturally occurring transcript variant of MARCO reveals the SRCR domain is critical for function.

Immunol Cell Biol 2016 08 18;94(7):646-55. Epub 2016 Feb 18.

Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

Macrophage receptor with collagenous structure (MARCO) is a class A scavenger receptor (cA-SR) that recognizes and phagocytoses a wide variety of pathogens. Most cA-SRs that contain a C-terminal scavenger receptor cysteine-rich (SRCR) domain use the proximal collagenous domain to bind ligands. In contrast, the role of the SRCR domain of MARCO in phagocytosis, adhesion and pro-inflammatory signaling is less clear. The discovery of a naturally occurring transcript variant lacking the SRCR domain, MARCOII, provided the opportunity to study the role of the SRCR domain of MARCO. We tested whether the SRCR domain is required for ligand binding, promoting downstream signaling and enhancing cellular adhesion. Unlike cells expressing full-length MARCO, ligand binding was abolished in MARCOII-expressing cells. Furthermore, co-expression of MARCO and MARCOII impaired phagocytic function, indicating that MARCOII acts as a dominant-negative variant. Unlike MARCO, expression of MARCOII did not enhance Toll-like receptor 2 (TLR2)-mediated pro-inflammatory signaling in response to bacterial stimulation. MARCO-expressing cells were more adherent and exhibited a dendritic-like phenotype, whereas MARCOII-expressing cells were less adherent and did not exhibit changes in morphology. These data suggest the SRCR domain of MARCO is the key domain in modulating ligand binding, enhancing downstream pro-inflammatory signaling and MARCO-mediated cellular adhesion.
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http://dx.doi.org/10.1038/icb.2016.20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4980223PMC
August 2016