Abstract
Introduction: Platelets are key drivers of thrombus formation following the rupture of atherosclerotic plaques, leading to acute myocardial infarction. In cardiovascular diseases, particularly coronary artery disease (CAD) and peripheral artery disease (PAD), there is a heightened risk of thrombosis. This highlights the need to probe understanding of platelet function in atherosclerotic disease and the mechanisms driving increased platelet activation while on dual antiplatelet therapy (DAPT) in this high-risk group. Therefore, this thesis aimed to develop a comprehensive method to characterise platelet activation by spectral flow cytometry
Methods: We stimulated platelets from whole blood of healthy volunteers using adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP-6), and arachidonic acid (AA) to induce maximal, submaximal and minimal platelet activation responses. These agonists target independent activation pathways, enabling a thorough assessment of platelet activation. We optimised a spectral flow cytometry panel that incorporated key activation markers P-selectin (CD62p), glycoprotein IIb/IIIa (PAC1), CD63 (a marker of dense and lysosomal degranulation), and CD154 (a marker of platelet-leukocyte interactions), alongside platelet-specific identifiers CD42a and CD42b, and red blood cell specific lineage marker CD235a. Additionally, we optimised acquisition of platelets within whole blood by adjusting threshold parameters and implemented regular washing steps. This approach allowed us to evaluate platelet activation, adhesion, and aggregation comprehensively while keeping acquisition between experiments clean and consistent.
Results: Our results demonstrated heterogeneity in platelet responses, with varying levels of marker expression correlating with concentration and agonist type. CD62p and PAC1 exhibited substantial upregulation in response to ADP stimulation. In contrast, TRAP-6 and AA displayed greater variation in marker expression, with CD62p expression more consistently activated than PAC1. Maximal, submaximal and minimal concentrations were identified for all agonists across 3 healthy volunteers. CD154 expression required implementing a sequential staining step for resolution of a positive population. Triggering on a fluorescent threshold alongside implementing washing steps substantially reduced the event rate and carryover observed when acquiring platelets in whole blood.
Concentrations which achieved 90% platelet activation for ADP, TRAP-6 and AA were tested in three patients with myocardial infarction (MI). The results indicated substantial inter-individual variability across activation markers for each agonist. Platelet activation occurred upon stimulation with all agonists despite administration standard-of-care DAPT.
Conclusion: Our optimised protocol aimed to provide a robust method for characterising platelet activation across different pathways to offer insight into thrombotic risk in patients with MI, despite the use of DAPT. These findings contribute to the development of a more standardised approach for characterising increased platelet activation in clinical research. Future work will focus on extending this analysis to a larger cohort of MI patients and others with high thrombotic risk to explore links between platelet activation and these pathologies.