Thrombotic disease is the biggest cause of death and disability worldwide, thus improved treatment strategies to prevent excessive thrombosis are highly desirable. In order to find safer/more effective anti-thrombotic treatments a detailed understanding of thrombus formation is central. We aimed to develop a microscopy-based method and analysis strategy that provide detailed information about thrombus dynamics and platelet behaviour in a forming thrombus in the living mouse. To this end, donor platelets were dual labelled with a platelet marker and a calcium dye and injected into a recipient mouse which was subjected to laser-induced vessel injury and 4D confocal intravital imaging using a Nikon A1R confocal microscope platform. High temporal resolution z-stacks were acquired to image thrombus dynamics. Data was analysed using customised image processing/analysis scripts written in the programming language Python. Application of a tracking algorithm allowed us to follow the intra-thrombus movement of thousands of individual platelets over time.
Using this live animal microscopy method we describe in detail the dynamics of thrombus formation as well as how individual platelets behave to form a thrombus in vivo. Determining the 3D position and activation state of platelets provides information about the size, stability, density and reorganisation of different parts of the thrombus. Platelet tracking revealed surprisingly dynamic and coordinated platelet intra-thrombus movement patterns. The increased level of detail obtained using this method will likely increase our understanding of how targeting different platelet activation pathways affects thrombus formation, as well as possibly help us identify potential new targets for anti-thrombotic treatments.