OC 75.2 - GP1b-FilaminA interaction regulates megakaryocyte localization and budding during platelet biogenesis

Wednesday, July 13, 2022

10:30 AM – 10:45 AM

Location: ExCel Center, ICC Capital Suite Room 10&11

Presentation Format: In-Person

Presenting Author(s)

ME

Marc L. Ellis, MBBS

PhD Candidate
Heart Research Institute, University of Sydney
Sydney, New South Wales, Australia

Co-Author(s)

AT

Antoine Terreaux

PhD student
WEHI
Melbourne, Victoria, Australia
RS

Rhyll Smythe

Research Assistant
Heart Research Institute, The University of Sydney
Sydney, New South Wales, Australia
AE

Anita Eckly

Research Director EFS
UMR_S 1255 EFS INSERM
Strasbourg, Alsace, France
SC

Sue Cranmer

Clinical Trials Administration Manager
Monash University
Melbourne, New South Wales, Australia
JM

Jessica Maclean

Postdoctoral Scientist
Heart Research Institute, The University of Sydney
Sydney, New South Wales, Australia
IA

Imala Alwis

Senior Research Fellow
Heart Research Institute, The University of Sydney
Sydney, New South Wales, Australia
JP

Jose Perdomo

Research Fellow
University of NSW
Kogarah, New South Wales, Australia
FP

Freda Passam

Principal Investigator
Heart Research Institute, University of Sydney
Sydney, New South Wales, Australia
ST

Samir Taoudi

Principal Investigator
WEHI
Melbourne, Victoria, Australia
FL

François Lanza

PhD
Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, F-67065 Strasbourg, France
Strasbourg, Alsace, France
YY

Yuping Yuan

Senior Research Fellow
Heart Research Institute, University of Sydney
Melbourne, Victoria, Australia
JS

Jackson Shaun

Director Cardiovascular Research; Group Leader Thrombosis
Heart Research Institute, University of Sydney
Sydney, New South Wales, Australia

Background: Glycoprotein Ib(alpha) is a critical receptor on platelets and megakaryocytes and is anchored to the membrane skeleton by Filamin A. While GPIb and FlnA have well defined roles in platelet biogenesis, the critical nature of the interaction of these proteins in megakaryocyte biology has not been evaluated in vivo. We sought to determine the influence of disruption of the GPIba/FlnA interaction on in vivo platelet biogenesis, particularly focusing on the recently described phenomenon of megakaryocyte membrane budding.

Aims: Investigate the role of the GPIba/FlnA interaction and functional consequence of disruption of this interaction in a transgenic human GPIba mouse model.

Methods: We generated mice with either a wild-type (WT) or FlnA-binding mutant (FW) human GPIba transgene within a GPIba-null mouse line. Platelet counts, platelet clearance, GPIba and GPIbb expression and proplatelet formation were assessed. We examined ultrastructure of megakaryocytes using BM TEM. Megakaryocyte distribution and budding were assessed using confocal and STED microscopy from bone marrow cryosections.

Results: Mice expressing mutant FW GPIba transgene exhibited a macrothrombocytopenia with platelet counts ranging from 150-200 x 10^6/mL with increased platelet volume. GPIb surface expression was preserved, albeit at lower levels than transgenic controls. Platelet clearance was normal and differentiation of FW megakaryocytes to proplatelets in vitro was preserved. Pulmonary proplatelets and bone marrow megakaryocytes numbers were normal in FW mice. The most striking defect in FW mice was the presence of dysregulated megakaryocyte budding, resulting in enlarged megakaryocyte buds that were ectopically released into the bone marrow interstitium. Expression of the cytoplasmic tail of GPIba in megakaryocytes was sufficient to restore normal megakaryocyte localization and budding and correct the macrothrombocytopenia.

Conclusion(s): These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated megakaryocyte budding. The GPIba/FlnA interaction is dispensable for megakaryocyte budding, however it plays a major role in regulating megakaryocyte localization and budding morphogenesis.