Abstract
Background. During the last two decades, application of fluorescence microscopy to the study of thrombosis in vivo has provided valuable insights into the structural composition of a developing thrombus. However, the level of detail afforded by current experimental and analytical models is insufficient for a detailed characterization of the complex and highly dynamic processes that shape thrombus architecture.
Aim. To (i) develop experimental and analytical protocols to accurately track the activities and activation states of a large number of individual platelets inside a developing thrombus in vivo; and (ii) apply this methodology to provide a quantitative analysis of how different intercellular signalling events contribute to thrombus self-organization by coordinating platelet activities in space and time.
Methods. Platelets isolated from a donor mouse were labelled with a platelet marker and a calcium indicator and then co-injected with specific fluorescent markers and vehicle or different treatments into a recipient mouse used for intravital microscopy. Laser injuries with a precisely defined size and position were generated in mesenteric veins. 3D timelapse data were acquired using a NikonA1R confocal platform. For each treatment group, approximately 15 injuries were imaged and later analysed. Data for around 300 injuries, equating to over four TB of images, were processed and analysed using the MASSIVE M3 high performance computing cluster. Image processing involved training a custom neural network for platelet segmentation and subsequently tracking detected platelets. The activities of individual platelets were quantified using custom measures of movement, activity, and local environment. Aggregate spatiotemporal properties were characterized using kernel density estimation, support vector regression, and topological data analysis.
Results. Platelets display a wide range of activities (e.g. movements, activations states, expression of activation markers, platelet-fibrin interactions) after recruitment to a developing thrombus. By characterizing the aggregate spatiotemporal properties of thrombus formation, we show that these activities are spatiotemporally organized according to a specific and reproducible topology. This organisation results in a highly ordered process of structural differentiation that ensures rapid transition to a stable architecture after an initial phase of rapid thrombus expansion. Inhibition of major components of the intracellular signalling network (e.g. ADP, thromboxane A2, thrombin) resulted in aberrations in the spatiotemporal coordination of platelet behaviour. In this regard, we were able to demonstrate that components of the signalling network have distinct and non-redundant roles in this process.
Conclusion. Our study represents the first systematic attempt to characterize how the coordinated activities of large numbers of individual platelets collectively shape the hemostatic response to injury. This novel analytical framework provides new insights into the regulatory mechanisms that underpin hemostatic function.
Disclosures
Hamilton:CSL: Current Employment.
