DROPLET MICROFLUIDICS FOR DETECTING HYPER-REACTIVE PLATELETS

Abstract

Introduction: Cardiovascular Diseases (CVDs) are the main cause of morbidity and mortality globally. Of these, elevated platelet reactivity is a common cause of heart attacks and strokes. Platelet function assay typically measures platelet bulk response to an agonist, potentially overlooking hyper-reactive platelets, which can drive thrombus formation and present a risk of heart attacks and strokes. To overcome this challenge, we have combined droplet microfluidics with flow cytometry for high-throughput single-platelet function analysis to identify hyper-reactive platelets. Here we evaluate the technology's suitability for characterising models of elevated platelet reactivity and extend this to the measurement of platelets from at-risk patients as a first step towards assessing the candidacy of the technology for predicting thrombosis risk. Objectives: Use the droplet microfluidics and flow cytometry method to identify hyper-reactive platelets, enabling us to understand the role of hyper-reactive platelets in health, in models of disease (priming) and clinical samples. Evaluate the candidacy of the method for predicting thrombosis risk in at-risk clinical cohorts (chest infection and fractured neck of femur). Material and methods: Platelet-Rich Plasma (PRP) was obtained from healthy donors (IRAS: 320501/REC: 22/LO/0801) by centrifugation. Droplets were generated containing PRP, with or without priming with Thrombopoietin (TPO), as well as convulxin concentrations as the agonist. Platelets were released from the droplets and fixed for cytometry analysis using P-selectin and PAC-1 end-points. For the small clinical study (chest infection and fractured neck of femur), ethics approvals were obtained (IRAS: 321105 /REC: 14/SC/0211) and droplets were generated only containing PRP and analyzed as described above. Results and discussion: We detected hyper-reactive platelets by comparing the responses of single platelets confined in droplets (single) with bulk platelet responses (collective) experiments. In bulk experiments, these hyper-reactive platelets cooperate by paracrine signalling to increase system-level sensitivity, highlighting the importance of hyper-reactive platelets. TPO priming increased platelet reactivity to ADP, but not to convulxin, implying the coupling of priming pathways with P2Y12 receptor activation by ADP, but not GPVI receptor activation by convulxin. By test-driving droplet cytometry with at-risk patient cohorts, we found one chest infection patient with elevated platelet reactivity and one fractured neck of the femur patient with increased platelet sensitivity. Bulk cytometry did not detect these characteristics, indicating that paracrine signalling masks hyper-reactive and hyper-sensitive platelets. Conclusion: Droplet cytometry can be used to detect hyper-reactive platelets by excluding paracrine signalling and warrants further investigation to assess the potential for predicting thrombosis risk in the clinic.