title: The integrin PSI domain has an endogenous thiol isomerase function and is a novel target for antiplatelet therapy.
abstract: Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.
authors.forename: Guangheng
authors.initials: G
authors.lastname: Zhu
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China.|Institute of Sun Yat-sen University in Shenzhen, Shenzhen, Guangdong, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.
authors.forename: Emily C
authors.initials: EC
authors.lastname: Reddy
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.
authors.forename: Naadiya
authors.initials: N
authors.lastname: Carrim
authors.affiliation: Department of Physiology, and.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Xiaohong Ruby
authors.initials: XR
authors.lastname: Xu
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Miao
authors.initials: M
authors.lastname: Xu
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.
authors.forename: Yan
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.
authors.forename: Min
authors.initials: M
authors.lastname: Rui
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Tania N
authors.initials: TN
authors.lastname: Petruzziello-Pellegrini
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Department of Physiology, and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Lavalle
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
authors.forename: Tyler W
authors.initials: TW
authors.lastname: Stratton
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.
authors.forename: Xi
authors.initials: X
authors.lastname: Lei
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.
authors.forename: Reheman
authors.identifier: 0000-0002-1973-9695
authors.initials: R
authors.lastname: Adili
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.
authors.forename: Pingguo
authors.initials: P
authors.lastname: Chen
authors.affiliation: Coulter Department of Biomedical Engineering and Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhu
authors.affiliation: Department of Internal Medicine, University of Manitoba, Winnipeg, MB, Canada.
authors.forename: John A
authors.initials: JA
authors.lastname: Wilkins
authors.affiliation: Howard Hughes Medical Institute, David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA; and.
authors.forename: Richard O
authors.initials: RO
authors.lastname: Hynes
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.|Department of Medicine, University of Toronto, Toronto, ON, Canada.
authors.forename: John
authors.initials: J
authors.lastname: Freedman
authors.affiliation: Toronto Platelet Immunobiology Group and Department of Laboratory Medicine, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, University of Toronto, Toronto, ON, Canada.|Canadian Blood Services, Toronto, ON, Canada.|Department of Physiology, and.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.|Department of Medicine, University of Toronto, Toronto, ON, Canada.
authors.forename: Heyu
authors.initials: H
authors.lastname: Ni
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D020134:Catalytic Domain; D015815:Cell Adhesion Molecules; D006801:Humans; D039641:Integrin beta Chains / Q000276:immunology*; D051379:Mice; D009419:Nerve Tissue Proteins; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D019704:Protein Disulfide-Isomerases / Q000276:immunology*; D011994:Recombinant Proteins; D039961:Semaphorins; D013927:Thrombosis / Q000517:prevention & control
chemical_list: D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D039641:Integrin beta Chains; D009419:Nerve Tissue Proteins; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011994:Recombinant Proteins; D039961:Semaphorins; C091529:plexin; D019704:Protein Disulfide-Isomerases