Along the em x /em -axis the movement was symmetrical towards the centre, resulting in a mean net movement close to zero for all conditions throughout the experiments. a platelet staining protocol, designed to enable identification of individual platelets, and image processing, we tracked the movements of a large number of individual platelets during thrombus formation and consolidation. These data were then processed to generate aggregate measures describing the heterogeneous movements of platelets in different areas of the thrombus and at different time points. Applying this model and its potential, to a comparative analysis on a panel of platelet inhibitors, we found that total platelet intra-thrombus movements are only slightly reduced by blocking the interactions between glycoproteins IIb/IIIa and Ib and their ligands or by inhibiting thromboxane synthesis or P2Y12 signalling. In contrast, whereas 30 to 40% of the platelets movements (for the CD42a-labelled CGS 21680 HCl platelets) and 20% (for the pro-coagulant platelets), within a thrombus, are contractile, i.e., towards the centre of GRK1 the thrombus, this contractile component is almost totally abolished in the presence of agents inhibiting these pathways. strong class=”kwd-title” Keywords: flow chambers, thrombosis, platelet aggregation, platelet contraction, fluorescence microscopy Introduction Experimental models of thrombosis give important insights into the events that shape thrombus formation upon vascular injury, and can be used in comparative studies to assess the roles of adhesion molecules 1 or specific platelet receptors, 2 to measure the effects of drugs CGS 21680 HCl 3 4 and to quantify the effects of physical conditions such as shear rate 5 6 on thrombus formation. In the majority of the available thrombosis models, data from images obtained using time-lapse microscopy are converted into quantitative aggregate variables describing how thrombus volume, surface coverage or platelet accumulation (measured as fluorescence intensity) is changed over time. Such an approach carries the implicit assumption that a thrombus can be conceptualized as a homogeneous entity, the composition of which remain largely unchanged during the course of an experiment. However, as new knowledge highlight the heterogenic and dynamic structure of a developing thrombus, 7 8 9 10 the inability of such models to adequately reflect the highly complex processes that shape thrombus formation have become increasingly clear. With the build-up of a platelet plug at a site of vascular injury, platelet recruitment occurs in parallel with thrombus densification due to the generation of platelet contractile forces, causing simultaneous compaction and expansion of the thrombus. The contractile movements of platelets within a thrombus start immediately, independently of fibrin formation, 11 and have important functional implications. 12 The denser platelet plug may also during later CGS 21680 HCl stages affect parameters such as clot elasticity, stiffness 13 and resistance to fibrinolysis. 14 15 Apart from lacking information on platelet contraction, thrombosis models based on measurements of thrombus volume tend to systematically underestimate thrombus build-up of platelets and other blood cells, as the volume expansion resulting from platelet recruitment is partially counteracted by the opposing effects of platelet contraction. Furthermore, it is increasingly evident that not all platelets within a thrombus behave the same; a process of spatial and functional differentiation results in the formation of different intra-thrombus platelet sub-populations with discrete haemostatic activities. 16 Results from studies performed on in vivo mouse models with mechanical vessel ligation or FeCl 3 -induced endothelial injury to provoke thrombus formation have identified spatial clustering of pro-aggregatory and pro-coagulant platelets into functionally distinct thrombus microdomains. 8 Other studies, wherein thrombus formation was studied after laser-induced or micro-puncture injury of the mouse cremaster muscle arterioles, have demonstrated the formation of a stable heterogenic thrombus, characterized by a core of densely packed and highly activated platelets surrounded by a loosely packed shell. 7 Such a thrombus architecture has important implications for the distribution of coagulation factors and secondary platelet mediators such as thromboxane A2 and adenosine diphosphate. 17 These observations strongly suggest that a more complete understanding of thrombosis would require experimental methods capable of mapping the activities and movements of a large number of individual platelets inside the thrombus. We have previously developed a method for counting the individual platelets in a flow chamber model of thrombosis using time-lapse and Z-stack CGS 21680 HCl fluorescence microscopy, 18 enabling quantification and positional information about the platelets within the.