OC 34.2 - PAR4 Antibody RC3 Inhibits Platelet Mitochondrial Depolarisation and Procoagulant Platelet Formation in Stable Coronary Artery Disease (CAD) Patients

Monday, June 24, 2024

09:45 – 10:00 ICT

Room: Ballroom B4

Co-Author(s)

HL

Helena Liang

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

Imants Rubenis

Cardiology Advanced Trainee
Concord Repatriation General Hospital
Concord, New South Wales, Australia
SG

Sonali Gnanenthiran

Cardiologist
Concord Repatriation General Hospital
Concord, New South Wales, Australia
Pia Larsson

Senior Research Scientist
Monash University
Melbourne, Victoria, Australia
LK

Leonard Kritharides

Cardiologist
Concord Repatriation General Hospital
Concord, New South Wales, Australia
JH

Justin Hamilton

Senior Researcher
Monash University
Melbourne, Victoria, Australia

Presenting Author(s)

Vivien Chen

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

Background: Human platelets are activated by thrombin via protease-activated receptors, PAR1 and PAR4. PAR4 activation drives the platelet procoagulant response that is pathologically increased in CAD1. Selective inhibition of PAR4 preferentially impairs procoagulant platelet function, leading to marked reductions in thrombin generation and fibrin formation during thrombus formation. However, a common SNP in PAR4 (encoding Thr120 in place of Ala120) renders platelets hyper-reactive and resistant to current PAR4 antagonist (BMS-986120). Humanised anti-PAR4 antibody, RC3, inhibits cleavage at the protease site and may function independent of PAR4 SNP.

Aims: To investigate the effect RC3 on platelet mitochondrial potential (ΔΨm) and procoagulant platelet formation in human subjects with stable CAD by genotype, using ex-vivo assays.

Methods: Blood from angiogram-proven, Gensini-scored, PAR4-genotyped, stable CAD patients was pre-incubated with RC3 or isotype control prior to agonist stimulation and compared with BMS-986120. Platelet ΔΨm was measured by TMRE flow cytometry, procoagulant platelets measured by flow cytometry and confocal microscopy, and fibrin formation and thrombus size assessed by ex-vivo micro-channel imaging.

Results: Platelets from CAD patients (n=52, Table 1) pre-incubated with RC3 had a significant protection from platelet mitochondrial potential (TMRE) loss compared with isotype control or BMS-986120 in response to thrombin or thrombin+CRP stimulation. There was a corresponding reduction in platelet membrane ballooning (Figure 2A), procoagulant platelet response (Figure 2B), ex-vivo thrombus and fibrin formation regardless of PAR4 SNP genotype, antiplatelet therapy or extent of CAD with RC3. RC3 had no effect on platelet ΔΨm, procoagulant platelets or platelet activation in response to CRP, ADP and PAR1 agonist confirming specificity.

Conclusion(s): PAR4 inhibition by humanised monoclonal RC3 selectively reduces thrombin-induced pathological platelet mitochondrial depolarisation and procoagulant platelet formation in stable CAD patients already on antiplatelet therapy, without impacting haemostatic platelet activation, regardless of PAR4 SNP genotype. RC3 may provide a valuable novel antithrombotic to add to CAD secondary prevention therapy.