PB0363 - Phosphoinositide dynamics in megakaryocyte/platelet granule integrity and platelet functions

Sunday, July 10, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Sonia Severin, PhD

researcher
Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France

Co-Author(s)

MC

Manuella Caux

PhD student
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
RM

Rana Mansour

PhD student
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
JX

Jean-Marie Xuereb

Engineer
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
GC

Gaetan Chicanne

Engineer
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
JV

Julien Viaud

researcher
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
AV

Alicia Vauclard

Assistant engineer
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
FB

Frederic Boal

researcher
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
BP

Bernard Payrastre

professor
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France
HT

Helene Tronchere

researcher
Inserm UMR 1297, Institute of metabolic and cardiovascular diseases
Toulouse cedex 04, Midi-Pyrenees, France

Background: Secretory granules are key elements for platelet functions. Granule biogenesis and integrity are regulated by fine-tuned mechanisms that need to be further characterized. This impedes our understanding of the etiology of hemorrhagic syndromes associated to quantitative or qualitative abnormalities in platelet granules.

Aims: Here, we investigated the role of the phosphoinositide 5-kinase PIKfyve and its lipid products, phosphatidylinositol 5 monophosphate (PtdIns5P) and phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), in granule homeostasis in megakaryocytes and platelets.

Methods: For that, we invalidate PIKfyve by using a specific inhibitor STA 5326 or by silencing using shRNA in the megakaryocytic MEG-01 cell line, which on many points resembles primary MKs and has been validated to study MK granule biogenesis, in imMKCLs and primary MKs and in freshly isolated human platelets.

Results: We show that PIKfyve expression and its lipid product levels increased with megakaryocytic maturation. In megakaryocytes, PtdIns5P and PtdIns(3,5)P2 were found in alpha and dense granule membranes with higher levels in dense granules. Pharmacological inhibition or knock-down of PIKfyve in megakaryocytes decreased PtdIns5P and PtdIns(3,5)P2 synthesis and induced a vacuolar phenotype with a loss of alpha and dense granule identity. Consistently, permeant PtdIns5P and PtdIns(3,5)P2 were able to restore alpha and dense granule integrity following PIKfyve pharmacological inhibition. In platelets, we show that PIKfyve inhibition specifically altered the integrity of dense granules culminating in defects in their secretion, platelet aggregation and thrombus formation.

Conclusion(s): Our data demonstrate that PIKfyve and its products PtdIns(3,5)P2 and PtdIns5P control the maintenance of granule integrity both in megakaryocytes and platelets.