title: Heat shock protein 70 regulates platelet integrin activation, granule secretion and aggregation.
abstract: Molecular chaperones that support protein quality control, including heat shock protein 70 (Hsp70), participate in diverse aspects of cellular and physiological function. Recent studies have reported roles for specific chaperone activities in blood platelets in maintaining hemostasis; however, the functions of Hsp70 in platelet physiology remain uninvestigated. Here we characterize roles for Hsp70 activity in platelet activation and function. In vitro biochemical, microscopy, flow cytometry, and aggregometry assays of platelet function, as well as ex vivo analyses of platelet aggregate formation in whole blood under shear, were carried out under Hsp70-inhibited conditions. Inhibition of platelet Hsp70 blocked platelet aggregation and granule secretion in response to collagen-related peptide (CRP), which engages the immunoreceptor tyrosine-based activation motif-bearing collagen receptor glycoprotein VI (GPVI)-Fc receptor-γ chain complex. Hsp70 inhibition also reduced platelet integrin-αIIbβ3 activation downstream of GPVI, as Hsp70-inhibited platelets showed reduced PAC-1 and fibrinogen binding. Ex vivo, pharmacological inhibition of Hsp70 in human whole blood prevented the formation of platelet aggregates on collagen under shear. Biochemical studies supported a role for Hsp70 in maintaining the assembly of the linker for activation of T cells signalosome, which couples GPVI-initiated signaling to integrin activation, secretion, and platelet function. Together, our results suggest that Hsp70 regulates platelet activation and function by supporting linker for activation of T cells-associated signaling events downstream of platelet GPVI engagement, suggesting a role for Hsp70 in the intracellular organization of signaling systems that mediate platelet secretion, "inside-out" activation of platelet integrin-αIIbβ3, platelet-platelet aggregation, and, ultimately, hemostatic plug and thrombus formation.
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon;
authors.forename: Rachel A
authors.identifier: http://orcid.org/0000-0003-4188-1935
authors.initials: RA
authors.lastname: Rigg
authors.affiliation: Department of Cell, Developmental & Cancer Biology, School of Medicine, Oregon Health & Science University, Portland, Oregon;
authors.forename: Laura D
authors.initials: LD
authors.lastname: Healy
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon; Department of Medical Physics, Universite des Sciences et Technologies de Lille, Villeneuve d'Ascq, France; and.
authors.forename: Marie S
authors.initials: MS
authors.lastname: Nowak
authors.affiliation: Department of Medical Physics, Universite des Sciences et Technologies de Lille, Villeneuve d'Ascq, France; and.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Mallet
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon; School of Chemical, Biological, and Environmental Engineering, Oregon State University, Corvallis, Oregon.
authors.forename: Marisa L D
authors.initials: ML
authors.lastname: Thierheimer
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon;
authors.forename: Jiaqing
authors.initials: J
authors.lastname: Pang
authors.affiliation: Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, Portland, Oregon; Department of Cell, Developmental & Cancer Biology, School of Medicine, Oregon Health & Science University, Portland, Oregon; Division of Hematology and Medical Oncology, School of Medicine, Oregon Health & Science University, Portland, Oregon;
authors.forename: Owen J T
authors.initials: OJ
authors.lastname: McCarty
authors.affiliation: Knight Cardiovascular Institute, School of Medicine, Oregon Health & Science University, Portland, Oregon; aslanj@ohsu.edu.
authors.forename: Joseph E
authors.identifier: http://orcid.org/0000-0002-8873-0387
authors.initials: JE
authors.lastname: Aslan
mesh_terms: D001792:Blood Platelets / Q000378:metabolism*; D005434:Flow Cytometry; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D015539:Platelet Activation / Q000502:physiology*; D010974:Platelet Aggregation / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D018840:HSP70 Heat-Shock Proteins; D016023:Integrins
keywords: chaperones; hemostasis; integrin; platelets; thrombosis