OC 09.2 - An Extensional Strain-Sensing Mechanosome Drives Rapid Adhesion-Independent Platelet Activation at Supraphysiological Hemodynamic Gradients

Saturday, July 9, 2022

1:15 PM – 1:30 PM

Location: ExCel Center, ICC Capital Suite Room 1

Presentation Format: In-Person

Presenting Author(s)

Nurul Aisha Zainal Abidin

PhD Student
Monash University
Melbourne, Victoria, Australia

Co-Author(s)

EP

Eric Poon

Research Fellow
St Vincent’s Hospital, University of Melbourne
Fitzroy, Victoria, Australia
CS

Crispin Szydzik

Research Fellow
Australian Centre for Blood Diseases, Monash University
Melbourne, Victoria, Australia
MT

Mariia Timofeeva

PhD Student
Dept of Mechanical Engineering, University of Melbourne
Melbourne, Victoria, Australia
FA

Farzan Akbaridoust

Research Fellow
Dept of Mechanical Engineering, University of Melbourne
Melbourne, Victoria, Australia
RB

Rose J. Brazilek

Clinician
Australian Centre for Blood Diseases, Monash University
Melbourne, Victoria, Australia
FT

Francisco J. Tovar Lopez

Senior Lecturer
School of Engineering, RMIT University
Melbourne, Victoria, Australia
XM

Xiao Ma

PhD Student
Monash Institute of Pharmaceutical Sciences, Monash University
Parkville, Victoria, Australia
CL

Chitrarth Lav

Research Fellow
Dept of Mechanical Engineering, University of Melbourne
Melbourne, Victoria, Australia
IM

Ivan Marusic

Professor
Dept of Mechanical Engineering, University of Melbourne
Melbourne, Victoria, Australia
PT

Philip E. Thompson

Professor
Monash Institute of Pharmaceutical Sciences, Monash University
Parkville, Victoria, Australia
AM

Arnan Mitchell

Position
School of Engineering, RMIT University
Melbourne, Victoria, Australia
AO

Andrew Ooi

Group Leader
Dept of Mechanical Engineering, University of Melbourne
Melbourne, Victoria, Australia
JH

Justin R. Hamilton

Group Leader
Australian Centre for Blood Diseases, Monash University
Melbourne, Victoria, Australia
WN

Warwick S. Nesbitt

Group Leader
Australian Centre for Blood Diseases, Monash University
Melbourne, Victoria, Australia

Background: Severe stenosis or mechanical circulatory support devices impose supraphysiological strain on blood, leading to platelet thromboembolism. Current anti-platelet therapies have limited efficacy, highlighting the need for more effective targeted treatments. Localised shear stress is known to drive thrombus formation mediated by adhesion receptors GPIb/V/IX and integrin αIIbβ3, but the exact mechanics of how platelets respond to haemodynamic gradients in free-flow, independent of adhesion receptors is unknown.

Aims: We investigate the impact of supraphysiological hemodynamics on platelet activation and function and the signalling pathways involved.

Methods: We utilised axisymmetric step and hyperbolic microfluidic geometries that expose platelets to flow accelerations and defined extensional strain, respectively. We assessed real-time calcium signalling by perfusing CAL520-loaded platelets reconstituted +/- red blood cells through these platforms. Confocal imaging at defined regions of interest were normalised against +ve and -ve controls to obtain [Ca2+]c in nM. Platelet aggregation was measured by perfusing DiOC6-labelled whole blood through stepped microchannels selectively coated with VWF, with aggregate dynamics over 180s quantified. Using a pharmacologic approach, we investigated the mechanotransduction mechanism underpinning platelet activation.

Results: Acute supraphysiological shear gradients trigger transient platelet [Ca2+]c flux that is attuned to extensional strain (ε) at flow acceleration, directly affecting immediate downstream platelet aggregation. Pharmacological screening indicates the [Ca2+]c flux occurs independently of adhesion receptor engagement and canonical pathways but is mediated by Piezo1 mechano-sensing coupled to P2X1-dependent signal amplification. We demonstrate a critical role for type II PI3KC2α in coupling the mechanosome to applied ε.

Conclusion(s): ε at flow acceleration plays a critical role in triggering adhesion-independent platelet activation. We describe a novel ‘ε sensing mechanosome’ driven by mechanical activation of Piezo1 coupled to subsequent [Ca2+]c influx through P2X1, which primes platelets for immediate downstream aggregation. The class II PI3KC2α isoform plays a critical role in coupling the mechanosome to externally applied ε.