The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular...
Investigations into the nature of platelet functional variety and consequences for homeostasis require new methods for resolving single platelet phenotypes. Here we combine droplet microfluidics with flow cytometry for high throughput single platelet function analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains scaled with agonist potency (convulxin > TRAP-14>ADP) and reduced the collagen and thrombin activation threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite future discoveries into the nature of hypersensitive platelets and how community effects produce population level responses in health and disease.
9Droplet microfluidics combined with flow cytometry was used for high throughput single platelet function 10 analysis. A large-scale sensitivity continuum was shown to be a general feature of human platelets from 11 individual donors, with hypersensitive platelets coordinating significant sensitivity gains in bulk platelet 12 populations and shown to direct aggregation in droplet-confined minimal platelet systems. Sensitivity gains 13 scaled with agonist potency (convulxin>TRAP-14>ADP) and reduced the collagen and thrombin activation 14 threshold required for platelet population polarization into pro-aggregatory and pro-coagulant states. The 15 heterotypic platelet response results from an intrinsic behavioural program. The method and findings invite 16 future discoveries into the nature of hypersensitive platelets and how community effects produce population 17 level behaviours in health and disease. 18 19 30 platelet activation represents an ideal system for investigating cellular diversity and consequences for 31 homeostatic system control. Indeed, the nature and functional consequences of platelet diversity has been a 32 matter of enquiry for almost half a century 8,10,11 . More recently, the discovery that dual stimulation with 33 collagen and thrombin 16,17 polarises platelets into distinct pro-coagulant and pro-aggregatory phenotypes 8,18-24 34 has renewed interest on the topic of platelet diversity. In particular, the pro-coagulant platelets have been 35 further characterised 25-30 , revealing diverse functions that either represent multiple pro-coagulant 36 subpopulations or a unified, yet versatile pro-coagulant subpopulation 21 . The bifurcation of the platelet 37 population into the two phenotypes further creates debate regarding intrinsic versus extrinsic functional 38 programming 8 . Allied to this, subjects with reduced GPVI levels showed reduced thrombus formation 31 , 39 implicating platelet heterogeneity with increased activity by platelets with elevated GPVI levels 32 . Overall, a 40 complex picture is emerging, with precision methods required to accurately delineate subpopulations and their 41 functional roles to inform our understanding of platelet interactions governing thrombus formation.
2The paracrine signalling inherent to platelet activation represents a technical challenge for measuring 43 single platelet behaviour without interference by the secretion products of activated platelets in the vicinity.
44This implies the requirement for confinement, discretising the analysis into single platelet measurements. The 45 other requirement is throughput to effectively resolve the functional structure of the platelet population.
46Droplet microfluidics allows the reliable production of monodisperse droplets in the nanolitre to femtolitre 4 aggregation responses to be investigated. Dual fluorescent imaging (P-selectin and CD63) with brightfield overlay of minimal 100 platelet collectives stimulated with 1 ng/mL convulxin (H) and dose response violin plots of minimal platelet collectives 101 comp...
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