Single platelet variability governs population sensitivity and initiates intrinsic heterotypic behaviours

Droplet microfluidics combined with flow cytometry was used 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 behaviours in health and disease.


Introduction 20
Understanding cellular diversity and interactions provides the key to elucidating system behaviour. It becomes 21 meaningful to investigate cellular diversity and identify even potentially rare phenotypes when amplification 22 mechanisms exist in the system and when there is good reason to predict large-scale variety. Classically, cancer 1-23 3 , immunology 4,5 and stem cells 6,7 with associated cell expansion have been the focus of the large majority of 24 single cell studies.

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In this work we turn our attention to platelets, dispersed sentinels which patrol the vasculature to 26 detect breaches and respond in a coordinated manner using rapid and potent paracrine signalling to collectively 27 form a thrombus. Platelets are also inherently variable 8 , originating from the fragmentation of heterotypic 9 28 megakaryocytes resulting in variously small sub-cellular compartments (60% volume CV) 10 with dissimilar 29 contents and biochemistry 11-13 and, without a nucleus, in a state of decay 14,15 before clearance. Therefore, 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  The 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.

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This 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.

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Droplet microfluidics allows the reliable production of monodisperse droplets in the nanolitre to femtolitre 47 range and has emerged as a powerful tool for packaging single cells in high throughput [33][34][35][36][37]

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The experimental concept is illustrated in Figure 1A along with consideration of the Poisson 77 distribution in Figure 1B which informs the choice of droplet volume and/or platelet concentration required for 78 the efficient encapsulation of single platelets. For a platelet concentration of 25 x 10 6 /mL and with further on-79 chip dilution (x5) with agonist and antibody volumes, this indicates that an 8 pL droplet volume (ø25 μm) 80 produces effective single platelet encapsulation: 3.38% of droplets contain a single platelet, 0.08% contain 81 multiples with a single to multiple ratio of 42. The droplet microfluidic circuit used in this study is shown in 82 Figure 1C and was used to generate 25-μm-diameter droplets ( Figure 1D,E) at 10.4 kHz for single platelet 83 packaging (352 Hz). This allows >100,000 platelets to be encapsulated in the 5 minute collection timeframe. To

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Relative risk analysis was used to determine the significance of the ~20-fold differences between the single and collective

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To evaluate single platelet sensitivity differences a dose response experiment involving stimulating 104 droplet-confined single platelets with convulxin (a GPVI receptor agonist) was undertaken and compared with 105 the stimulation of platelet collectives. Using α IIb β 3 activation (inside-out signalling) as the analytical end-point 106 the platelet collectives produced a sigmoidal response curve emerging at 0.1 ng/mL and saturating at 1 ng/mL 107 concentrations. The signal intensity distribution of the collective population indicates normally distributed 108 functional variety. In comparison, a higher activation threshold is evident with singularly stimulated platelets,

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with activation emerging at 1 ng/mL and saturating at 10 ng/mL levels ( Figure 2A). Extending the analysis to a 110 different pathway, the P-selectin exposure end-point for alpha granule secretion, the same increased activation 111 threshold for singularly stimulated platelets was observed ( Figure 2B). Activation and aggregation density plots 112 for platelets stimulated at 3 ng/mL are shown in Figure 2C and shows the hypersensitive behaviour of the 113 collective response, the correlation between the two end-points and the bimodal distribution for singularly 114 stimulated platelets undergoing population-level transition. Importantly, the hypersensitive sub-population was 115 not observed by platelet collective dilution (up to a further 100-fold dilution), demonstrating the merit of the 116 droplet microfluidics approach for single platelet analysis. To measure the significance in the response 117 differences the relative risk was considered ( Figure 2D,E). At low and high agonist concentrations the relative 118 risk score is insignificant at 1.0, and at 3 ng/mL rises to 53 for α IIb β 3 activation and 6 for P-selectin exposure end-119 points, highlighting the significantly (p<5x10 -5 ) distinct hypersensitivity of collectively stimulated platelets.

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The sensitivity gains emerging from collective platelet behaviour were reproducible, with equivalent

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To confirm that the molecular α IIb β 3 activation and P-selectin end-points represent functional 129 behaviour the dose response study was extended to larger droplets (65 pL; ø50 μm) packaging 0-15 platelets.

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Elevated P-selectin signals relative to bulk conditions are also observed at 10 and 100 ng/mL convulxin. This is 136 indicative of autocrine signalling resulting from the accumulation of degranulation products within the droplets.

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This experiment demonstrates the functional consequence of broad-spectrum sensitivity with cooperation and 138 also that minimalistic platelet cooperation models can be used to understand transition states and the linkage 139 between probabilistic molecular events and collective functional outcomes.

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Collective sensitivity gains are attributed to the existence of low abundance hypersensitive platelets 141 which, upon activation, degranulate to activate platelets in the vicinity that were insensitive to the initial 142 stimulus. These modes of paracrine signalling produce a spatiotemporal corralling effect that drives platelet 143 cooperation to deliver the collective response. Nevertheless, sufficient numbers of activated platelets are 144 required to polarise the entire platelet population into an activated response (e.g. Figure 1A;  Figure 4).

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The study was extended to other agonists; the peptide TRAP-14 functional motif was used in place of 157 thrombin to activate the PAR-1 receptor and as before α IIb β 3 activation and P-selectin aggregation end-points 158 were measured. The median activation threshold was again increased for single platelets stimulated in droplets,

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Further investigations involving large-scale antibody panels for highly multiplexed cytometry or more global 219 proteomic 55 and even transcriptomic screens 56,57 following platelet sorting will be needed to determine the 220 composition of the hypersensitive platelet sub-population.