OC 09.1 - The rich inner life of a thrombus – “next generation” analysis of platelet activity during thrombus formation in vivo

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.

Aims: To develop experimental and analytical protocols that provide an automatized, quantitative analysis of the movements and activation states of large numbers of individual platelets during thrombus formation in vivo.

Methods: Isolated platelets labelled with a platelet marker and a calcium indicator were injected into vehicle- or inhibitor-treated recipient mice used for intravital microscopy. Laser injuries with precisely defined sizes and positions were generated in mesenteric veins and 3D time-lapse data was acquired using a NikonA1R confocal platform. Image data was processed using a neural network trained for platelet segmentation. Pattern recognition algorithms based on gaussian kernel density functions and support vector regression were applied to identify spatiotemporal clusters of platelet activities during thrombus development.

Results: The application of our model enabled accurate spatial and temporal mapping of platelet recruitment and shedding, platelet packing density, platelet-fibrin interactions and platelet intracellular calcium mobilization during thrombus formation. Further, 3D tracking of millions of observations of individual platelets combined with pattern recognition algorithms enabled a systematic and unbiased analysis of how coordinated platelet movements are generated by the propagation of multiple stimulatory waves of platelet signalling within a thrombus. Our results identify discrete clusters of non-canonical platelet activities which shape thrombus architecture.

Conclusion(s): Large-scale tracking of individual platelets during thrombus formation in vivo provides an unprecedented level of detail about the complex processes shaping thrombus formation. The analytical tools developed will help to refine our conceptual understanding of the hemostatic response to injury.