Abstract

Platelet activation is an important step in arterial thrombosis, the acute complication of atherosclerosis. However, current diagnostic techniques for platelet function have been shown to be inadequate to predict thrombosis. In spite of this, many patients are prescribed aspirin to prevent (further) occurrence of arterial thrombosis, introducing bleeding risk. Platelets have been shown to be heterogeneous in a number of features such as size, volume and density, and this variety may underpin overall system behaviour. Functional heterogeneity has been suggested in several studies but current methods are not suitable to reliably study single platelet function. Such a method should provide a high throughput means to profile large platelet populations for the identification of, potentially rare, hyperactive platelets. In addition, the technique must prevent paracrine signalling (platelet-mediated activation of neighbouring platelets), necessitating the isolation of single platelets. This study adapts a droplet microfluidics approach to investigate single platelet functionality.

Here, single platelet sensitivity is studied by adding the agonist (convulxin, specific ligand of the GPVI receptor for collagen) during encapsulation in droplets. After an incubation period the platelets are retrieved from the droplets into fixative, followed by flow cytometry analysis of markers for activation. The PAC-1 antibody is used to identify the active conformation of the Î ± IIbβ3 receptor, important for aggregation and adhesion, and anti-CD62P (p-selectin) to identify degranulation of the platelets. Platelets are identified with CD42b, which is a platelet specific receptor.

Platelets are individually encapsulated in monodisperse (CV of 1–4%) water-in-oil droplets with a mean volume of 14 pL and a diameter of 30 μm. Droplets are produced with a throughput of 4 kHz, with droplets containing a single platelet produced at a rate of 0.25 kHz (following a Poisson distribution). With this method an intrinsic variation in the platelet response to convulxin is observed, that is unrelated to the size of the platelet.

The research entailed the development of a method capable of measuring the intrinsic variation in platelet function. The method will be used to compare the sensitivity profiles of people with known risk factors for arterial thrombosis with the profiles of people with low risk. This presents the possibility to identify a novel prognostic biomarker. Furthermore, a better understanding of the functional heterogeneity of platelets could be used to identify new targets to aid the rational design of new therapeutics. Ideally, this would target only hyperactive platelets, while preserving normal haemostasis.