Abstract
Introduction
Precision mapping of the functional structure of platelet populations holds great promise for the identification of hyper-reactive subtypes that are likely to be disease drivers, having value in prognostics and as therapeutic targets. However, the ability to measure the intrinsic functional capacity of individual platelets is confounded by potent paracrine cross-talk, resulting in phenotypic remodeling of the entire platelet population, and in doing so obscuring the identity of hyper-reactive platelets.
Methods
To address this we have developed a droplet microfluidics strategy for single platelet confinement to exclude paracrine signaling. Consideration of the Poisson distribution was used for high throughput single platelet encapsulation and the preparation of minimal platelet collectives serving as digital models for understanding the role of hyper-reactive platelets coordinating system-level behavior by paracrine signaling. Platelets are retrieved from the droplets for phenotyping using standard flow cytometry. In addition, we have incorporated a staggered herringbone micromixing element for accurate agonist and antibody dispensing in droplets.
Results
The methodology was used for characterizing sensitivity distributions from healthy blood donors in response to convulxin (agonist of the GPVI receptor, the major platelet receptor for collagen). P-selectin exposure and αIIbβ3 integrin activation were used as analytical end-points to demonstrate the existence of hyper-reactive platelets that direct 20-fold gains in system level sensitivity.
Conclusions
The analytical workflow represents an enabling tool for the accurate classification of platelet subtypes and description of their underlying biology.
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Abbreviations
- PDMS:
-
Poly(dimethylsiloxane)
- PRP:
-
Platelet rich plasma
- HEPES:
-
Sodium 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonate
- BSA:
-
Bovine serum albumin
- CVX:
-
Convulxin
- PBS:
-
Phosphate buffered saline
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Acknowledgments
The authors are grateful to the blood donors, and also to George Devitt and Dianne M. Lopez for access to a fluorescent plate reader. The research was funded by Marie Curie (333721, J.W.), the British Heart Foundation (FS/13/67/30473, M.S.A.J.), the National Institute of Health (R01 HL101972) and the Medical Research Council (MC_PC_15078, M.S.A.J.).
Author Contributions
MS.A.J., S.I.R.L., P.M.H. and J.W. undertook the research. M.S.A.J., S.I.R.L. and J.W. prepared the figures and manuscript. P.M.H., N.A.E. and O.J.T.M. revised and edited the manuscript. All authors approved the final version of the manuscript.
Conflict of Interest
MS.A.J., S.I.R.L., P.M.H., N.A.E., O.J.T.M. and J.W. declare no conflicts of interest.
Research Involving Human and Animal Rights
All human subject research was carried out in accordance with institutional guidelines approved by the University of Southampton Ethics Committee ((ERGO 5538) and South Central – Hampshire B National Research Ethics Service (REC: 14/SC/0211)). No animal studies were carried out by the authors for this article.
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Jongen, M.S.A., Holloway, P.M., Lane, S.I.R. et al. Droplet Microfluidics with Reagent Micromixing for Investigating Intrinsic Platelet Functionality. Cel. Mol. Bioeng. 14, 223–230 (2021). https://doi.org/10.1007/s12195-020-00665-6
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DOI: https://doi.org/10.1007/s12195-020-00665-6