Abstract
Imaging methods based on time-lapse microscopy are important tools for studying the
dynamic events that shape thrombus formation upon vascular injury. However, there
is a lack of methods to translate the vast amount of visual data generated in such
experiments into quantitative variables describing platelet movements that can be
subjected to systematic analysis. In this study, we developed experimental and computational
protocols allowing for a detailed mathematical analysis of platelet movements within
a developing thrombus. We used a flow chamber-based model of thrombosis wherein a
collagen strip was used to initiate platelet adhesion and activation. Combining the
use of 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 platelets) and 20% (for the pro-coagulant platelets),
within a thrombus, are contractile, i.e., towards the centre of the thrombus, this
contractile component is almost totally abolished in the presence of agents inhibiting
these pathways.
Keywords
flow chambers - thrombosis - platelet aggregation - platelet contraction - fluorescence
microscopy
