OC 55.4 - Identification of the role of ERAD proteins in platelet function through multispecies modeling of a thrombasthenia

Tuesday, June 25, 2024

10:15 – 10:30 ICT

Room: 110 A-C

Presenting Author(s)

Anna R. Dahlgren, PhD

Postdoctoral Research Fellow
University of Michigan
Ann Arbor, Michigan, United States

Co-Author(s)

FC

Francesca Careddu

PhD candidate
University of Pavia
Pavia, Lombardia, Italy
Reheman Adili

Director of Intravital Microscopy Imaging Core
Bloodworks Northwest
Seattle, Washington, United States
EK

Erin Kropp

Hematology/Oncology Fellow
University of Michigan
Ann Arbor, Michigan, United States
QL

Qing Li

Associate Professor of Internal Medicine and Cell & Developmental Biology
University of Michigan
Ann Arbor, Michigan, United States
MH

Michael Holinstat

Associate Professor
University of Michigan
Ann Arbor, Michigan, United States
IB

Ida Biunno

Researcher
University of Pavia
Pavia, Lombardia, Italy
Alessandra Balduini, MD

MD, PhD
University of Pavia, Pavia, Italy
Pavía, Lombardia, Italy
FT

Fern Tablin

Professor Emeritus
University of California Davis
Davis, California, United States
CF

Carrie Finno

Professor of Population Health & Reproduction
University of California Davis
Davis, California, United States
Jordan Shavit, MD, PhD

Professor
University of Michigan
Ann Arbor, Michigan, United States

Background: Endoplasmic reticulum associated degradation (ERAD) is an essential cellular process that clears misfolded proteins. SEL1L and SYVN1 form a protein complex that is the most conserved branch of ERAD. Atypical Equine Thrombasthenia (AET) is a recessive heritable platelet disorder known to affect platelet signaling.

Aims: To determine if the ERAD pathway plays a role in platelet function.

Methods: A whole-genome association study was conducted on five AET-affected and eleven control horses. RT-PCR, flow cytometry, qPCR, and immunofluorescence were used to look at expression and localization of associated genes. Collagen spreading and template bleeding time assays interrogated functional effects of a variant of interest. Immunoblotting of human platelets and megakaryocytes identified protein isoforms. An Mx1-cre;SEL1L mouse model and collagen flow assay, and a CRISPR/Cas9-mediated SEL1L knockout zebrafish line and laser-mediated thrombocyte attachment assay identified how the lack of SEL1L impacts platelet/thrombocyte function. SYVN1 was knocked down in zebrafish embryos using CRISPR/Cas9 and thrombocyte function evaluated.

Results: The association study, RT-PCR, and flow cytometry identified a missense variant in horse SEL1L (p.Ile604Val). SEL1L protein expression was significantly decreased in homozygous mutant horse platelets despite no difference in transcript expression. SEL1L did not localize to the α-granule, but did move to the platelet surface after thrombin activation. Although the template bleeding time was normal, homozygous horse platelets showed defective spreading on collagen. Two SEL1L protein isoforms (p100 and p38) were present during human megakaryopoiesis, but only p100 was delivered to platelets. Murine SEL1L depletion (Mx1-Cre+;SEL1Lfl/fl) led to fewer platelets bound to collagen compared to control siblings. Similarly, fewer thrombocytes from SEL1L-/- and SYVN1 knockdown zebrafish attached to the site of endothelial injury.

Conclusion(s): We show a conserved role for ERAD in platelet function, particularly the ability of platelets/thrombocytes to bind to injured endothelium. Further investigation into this understudied pathway may shed light on human platelet disorders.