Final Report Summary - FREE-FLOW (Dynamic Microfluidic Deflection Systems for High Throughput Single Platelet Function Testing)
The research has entailed the design and development of a high throughput technology to measure single platelet functional capacity. The technology is required to understand platelet population functional distribution and how this determines homeostasis in health with extrapolation to understand imbalance in disease leading to heart attacks and strokes.
A droplet microfluidic platform was integrated to a multiplexed flow cytometry instrument for high throughput single platelet stimulation and the determination of activation via alphaIIbbeta3 activation (inside out signalling), P-selectin presentation (degranulation) and annexin V exposure (membrane inversion). Critically, droplet encapsulation prevents paracrine cross-talk to definitively ascertain single platelet sensitivities and by comparison with platelet collective experiments understand the global impact.
For 3 different agonists, convulxin, ADP and TRAP14, platelets from a donor have highly variable sensitivities that are not heterogeneous, rather a continuum of sensitivities. This is not due to platelet volume or GP6 receptor count, with efforts on-going to determine if platelet age is a determining factor. Alternatively, the origin, the production of thousands of platelets from a single megakaryocyte, is a highly asymmetric process and make account for the continuous variability.
The consequence of varied sensitivity is that platelet collectives operating together have approx. 10-fold gains in sensitivity than the majority of the platelet population when measured singularly. Thus hypersensitive platelets exist and have been measured, representing hypersensitive sentinels that orchestrate global behaviour. This is at the root of understanding homeostasis and how imbalance can lead to disease resulting in heart attacks and strokes.
Following on from this we went on to address the collagen- and thrombin-activated (COAT) platelet theory, in which functional heterogeneity is thought to be guided extrinsically, as the clot forms in space and time. In droplet microfluidic studies, we determine that intrinsic functional heterogeneity exists to a large degree, and this either augments or replaces the concept of extrinsic heterogeneity.
The basic line of research has involved multiple donors and multiple repeats. Platelet population sensitivities from an individual vary on a day-to-day basis and individuals have differing sensitivities. The results thus promote research extension to (i) determine if platelet population distributions represent a useful prognostic biomarker, (ii) identify the causes of hypersensitivity for the development of novel, possibly personalised anti-platelet therapies. The research has created a paradigm shift in platelet biology, with capacity to monitor predisposition to heart attack and strokes and identify new treatment approaches, to reduce the socio-economic burden.
The funding has allowed the researcher to establish himself as a lecturer in the faculty of medicine at the University of Southampton and by supervision train a platelet biologist on the development and use of microfluidics to extend biological enquiry.
A droplet microfluidic platform was integrated to a multiplexed flow cytometry instrument for high throughput single platelet stimulation and the determination of activation via alphaIIbbeta3 activation (inside out signalling), P-selectin presentation (degranulation) and annexin V exposure (membrane inversion). Critically, droplet encapsulation prevents paracrine cross-talk to definitively ascertain single platelet sensitivities and by comparison with platelet collective experiments understand the global impact.
For 3 different agonists, convulxin, ADP and TRAP14, platelets from a donor have highly variable sensitivities that are not heterogeneous, rather a continuum of sensitivities. This is not due to platelet volume or GP6 receptor count, with efforts on-going to determine if platelet age is a determining factor. Alternatively, the origin, the production of thousands of platelets from a single megakaryocyte, is a highly asymmetric process and make account for the continuous variability.
The consequence of varied sensitivity is that platelet collectives operating together have approx. 10-fold gains in sensitivity than the majority of the platelet population when measured singularly. Thus hypersensitive platelets exist and have been measured, representing hypersensitive sentinels that orchestrate global behaviour. This is at the root of understanding homeostasis and how imbalance can lead to disease resulting in heart attacks and strokes.
Following on from this we went on to address the collagen- and thrombin-activated (COAT) platelet theory, in which functional heterogeneity is thought to be guided extrinsically, as the clot forms in space and time. In droplet microfluidic studies, we determine that intrinsic functional heterogeneity exists to a large degree, and this either augments or replaces the concept of extrinsic heterogeneity.
The basic line of research has involved multiple donors and multiple repeats. Platelet population sensitivities from an individual vary on a day-to-day basis and individuals have differing sensitivities. The results thus promote research extension to (i) determine if platelet population distributions represent a useful prognostic biomarker, (ii) identify the causes of hypersensitivity for the development of novel, possibly personalised anti-platelet therapies. The research has created a paradigm shift in platelet biology, with capacity to monitor predisposition to heart attack and strokes and identify new treatment approaches, to reduce the socio-economic burden.
The funding has allowed the researcher to establish himself as a lecturer in the faculty of medicine at the University of Southampton and by supervision train a platelet biologist on the development and use of microfluidics to extend biological enquiry.