PB1289 - Platelet procoagulant and mitochondrial membrane potential phenotyping for characterisation of STIM1 gene variants of uncertain significance in inherited platelet disorders

Tuesday, June 25, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

IT

Ibrahim Tohidi-Esfahani

Consultant haematologist
Haematology Department, Concord Repatriation General Hospital, Sydney, Australia
Pennant Hills, New South Wales, Australia

Co-Author(s)

SW

Shane Whittaker

Senior Research Assistant
ANZAC Research Institute, Concord Repatriation General Hospital, University of Sydney, Sydney, Australia
Concord, New South Wales, Australia
HC

Heather Campbell

Senior Research Assistant
ANZAC Research Institute, Concord Repatriation General Hospital, University of Sydney, Sydney, Australia
Concord, New South Wales, Australia
CL

Christine Shu Mei Lee

Research Fellow
College of Health and Medicine, Australian National University, Canberra, Australia
Canberra, Australian Capital Territory, Australia
HL

Helena Liang

Senior Postdoctoral Research Fellow
ANZAC Research Institute, Concord Repatriation General Hospital, University of Sydney, Sydney, Australia
Concord, New South Wales, Australia
JC

Jennifer Curnow

Haematologist
Westmead Hospital
Sydney, New South Wales, Australia
Marie-Christine Morel-Kopp, PhD

Senior Scientist
Northern Blood Research Centre, Kolling Institute of Medical Research, The University of Sydney, Sydney, Australia
St Leonards, New South Wales, Australia
WS

William Stevenson

Head of Department, Haematologist
Haematology Department, Royal North Shore Hospital, Sydney, Australia
Sydney, New South Wales, Australia
DR

David Rabbolini

Honorary Senior Lecturer
Kolling Institute, Faculty of Medicine and Health, University of Sydney, Sydney, Australia
Sydney, New South Wales, Australia
Vivien Chen

Staff Specialist Haematologist
Concord Repatriation General Hospital
Sydney, New South Wales, Australia

Background: STIM1 regulates store-operated Ca2+ entry, critical for procoagulant platelet formation. The inherited gain-of-function mutation, R304W, causes Stormorken syndrome (SS), with myopathy, macrothrombocytopenia and platelet dysfunction.

Aims: To utilise SS patient intracellular calcium and procoagulant platelet phenotyping to aid characterisation of STIM1 variants of uncertain significance (VUS) within a national referral cohort of inherited platelet disorder (IPD) patients (Sydney Platelet Group, SPG).

Methods: SPG-referred patients with thrombocytopenia and STIM1 VUS on panel-based tier 1/2 IPD gene testing were identified, alongside R304W-confirmed SS patients. Procoagulant platelets were measured by flow cytometry (GSAO-AF647 and P-selectin positive) in citrated whole blood or platelet-rich plasma (PRP), with or without recalcification, agonist stimulation or 100µM STIM1 inhibitor ML9. Cytosolic calcium and mitochondrial membrane potential (MMP) were assessed by confocal microscopy using Fluo-4AM and TMRE in the absence of exogenous calcium.

Results: ML9 reduced healthy donor thrombin +/- cross-linked collagen-related peptide (CRP-XL)-induced procoagulant platelet formation (dual stimulated: vehicle control mean 17.8±3.4%, ML9 3.9±1.3%, n=4, p=0.002), indicating STIM1 is required for procoagulant platelet formation. A high proportion of SS platelets were procoagulant at rest compared to healthy controls (11.6% vs 1.7±0.6%, n=17), with hypersensitive agonist response (Figure 1A). Unlike healthy controls, with absent exogenous calcium, SS platelets maintained procoagulant potential, had increased cytosolic calcium and reduced MMP (Figure 1B-D).

Two patients with macrothrombocytopenia without myopathy, with a STIM1 VUS (patient A: M244K, patient B: T579del), were identified. With exogenous calcium, patient A procoagulant platelet formation matched SS platelets (Figure 1A). Without exogenous calcium, patient A and B had increased response to thrombin and CRP-XL stimulation compared to healthy controls (Figure 1B), and, similar to SS platelets, increased cytosolic calcium and reduced MMP (Figure 1C-D), implicating these STIM1 VUS as gain-of-function variants.

Conclusion(s): The spectrum of STIM1 disorders is widening. Procoagulant platelet phenotyping can give insights into the functional impact of STIM1 VUS.