OC 64.4 - Modeling gray platelet syndrome: longitudinal mouse studies and in vitro human iPSC-derived megakaryocytes to investigate clinical and cellular features

Tuesday, July 12, 2022

3:30 PM – 3:45 PM

Location: ExCel Center, ICC Capital Suite Room 14-16

Presentation Format: In-Person

Co-Author(s)

JC

Janine H. Collins

Clinical Research Training Fellow
University of Cambridge
Cambridge, England, United Kingdom
LM

Louisa Mayer

PhD student
University of Cambridge
Cambridge, England, United Kingdom
LK

Laxmikanth Kollipara

res associate
Leibniz-Institut f»r Analytische Wissenschaften _ ISAS _ e.V.
Dortmund, Nordrhein-Westfalen, Germany
AS

Albert Sickmann

President
ISAS
Dortmund, Nordrhein-Westfalen, Germany
HZ

Houjiang Zhou

Chief Scientific Officer
Zhejiang Hisun Pharmaceutical
Taizhou Zhejiang, Jiangsu, China (People's Republic)
GH

Gary Hoffman

Clinical Professor
the university of western australia
Perth, Western Australia, Australia
WE

Wendy Erber

Pro Vice-Chancellor & Executive Dean
the university of western australia
Perth, Western Australia, Australia
Cedric Ghevaert, MD PhD

Professor of Transfusion Medicine
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Jeffrey Cheah Biomedical Centre
Cambridge, England, United Kingdom
WO

Willem H. Ouwehand

Group Coordinator and Principal Investigator
University of Cambridge
Cambridge, England, United Kingdom

Presenting Author(s)

Jose A. Guerrero, PhD

University of Cambridge
Cambridge, England, United Kingdom

Background: Our understanding of the gray platelet syndrome (GPS) has recently expanded with the presence of dysfunctional neutrophils, a pro-inflammatory plasma proteome, autoantibodies and autoimmune disorders. However, the onset and evolution of classical phenotypes such as splenomegaly, emperipolesis and bone marrow fibrosis (BMF), as well as the role of Nbeal2 are still lacking.

Aims: Our aim was to investigate the phenotypes mentioned above.

Methods: Mice aged 3-22 months and genetically-engineered cells (K562, CHRF, iPSC-derived megakaryocytes) using mass-spectrometry (MS) and electron microscopy (EM). For comparison, control and GPS human/murine platelets were used.

Results: Across all ages, spleen size and bone marrow emperipolesis were increased in Nbeal2-/- mice. Mean spleen length was 2.9±0.9cm in Nbeal2-/- mice compared to 1.8±0.4cm in controls (p-value=1.2x10-6). The percentage of megakaryocytes containing neutrophils were 68.2±14.9 and 1.3±1.3 (p-value=4.8x10-13) with 2.24±0.85 and 0.77±0.73 neutrophils/megakaryocyte (p-value=2.237x10-5) in Nbeal2-/- and control mice, respectively. Pathological BMF (≥grade 2) was observed in 40% of Nbeal2-/- mice older than 10 months. In vitro, absence of Nbeal2 was confirmed in all GPS cell models. The number of granules and vacuoles in GPS platelets was statistically different to that of control platelets. However, this level of significance was not reached with the GPS cell models (Fig.1A). A proportion of α-granule proteins identified by MS were lower in GPS platelets and in the in vitro-generated Nbeal2-/- cells when compared to their corresponding controls, e.g. PSEL and PF4, with only a fraction of these proteins reaching statistical significance, e.g. PDGF, THBS1, TIMP1 (Fig.1B&C).

Conclusion(s): While splenomegaly and emperipolesis develop in Nbeal2-/- mice at the earliest age studied, pathological BMF occurs from 10 months. The absence of Nbeal2 from GPS cell models has a smaller effect on the loss of α-granules and their content when compared to platelets suggesting that α-granule loss in GPS might predominantly occur after proplatelet formation or platelet release.