Platelets are essential players in thrombosis and haemostasis, as they ‘survey’ the
integrity of the vascular system. After vascular injury, platelets are recruited to
the exposed sub-endothelial extracellular matrix that triggers platelet activation
to seal wound sites by the formation of a haemostatic plug, thereby preventing excessive
bleeding. However, if thrombus formation is uncontrolled under pathological conditions,
it may lead to irreversible vessel occlusion and to life-threatening acute ischaemic
disease states, such as myocardial infarction or stroke.[1]
Platelet activation and subsequent thrombus growth involve receptor–ligand interactions,
signaling molecules and cytoskeletal proteins which regulate the cellular mechanotransduction.
In vitro flow chamber systems combined with whole blood from genetically modified
mice or incubated with inhibitors have significantly contributed to a better understanding
of the function of platelet proteins in the thrombotic process under shear conditions.
Most researchers have analysed their generated real-time movies or off-line images
semi-automatically or manually for surface coverage by platelet thrombi (83%), thrombus
volume (78%) and the number of adhered platelets (56%).[2] However, while this method including data evaluation has been used primarily to
characterize the role of platelet proteins in thrombus formation, the mechanobiology
of platelets in this complex environment had been addressed and understood insufficiently.
In this issue, Tunströmer et al introduce and take advantage of a new computational
tool which enables quantitative analysis of platelet intra-thrombus movements.[3] For this purpose, the authors further developed their in-house image processing
script, which has enabled quantification of positional information of individual platelets
within a thrombus so far.[4] They used the acquired positional information of each platelet to track thousands
of platelets in a thrombus at the same time and to measure thrombus contraction.[3]
For this, 5% of platelets were labelled with a platelet-specific antibody and annexin
V was added to whole blood to measure pro-coagulant, phosphatidylserine positive platelets.
The samples were either left untreated or incubated with blocking antibodies against
glycoprotein (GP) IIb/IIIa and GPIbα, or inhibitors blocking P2Y12 or thromboxane
production. Then, blood was perfused in a custom-made flow chamber at 400 s-1 over
a collagen strip. The authors were able to show how the panel of inhibitors affected
thrombus architecture, platelet intra-thrombus movements and thrombus contraction.[3] More specifically, total platelet accumulation was decreased but the ratio of pro-coagulant
platelets to total platelet numbers increased in the presence of inhibitors, particularly
when the receptors GPIIb/IIIa and GPIbα were blocked. Platelet movements were dramatically
shorter for all samples incubated with inhibitors in contrast to control sample. Furthermore,
the authors investigated whether the relative platelet position in a thrombus is a
determinant of platelet movement. Platelet movement trajectories were longer and velocity
was increased on the outer edge of the thrombus compared with platelets in the thrombus
core.
Interestingly, a support vector machine regression approach revealed that initially
platelets contracted to small aggregates, which then conjoined to larger aggregates,
and finally moved in a homogeneous way towards the centre of the thrombus. The contractile
component of platelets strongly contributed to thrombus contraction and was reduced
in the presence of inhibitors. Finally, the authors could show that thrombus contraction
increased during the first 800 seconds of thrombus formation.[3]
Overall, this big set of data supports and conclusively explains the spatial and functional
heterogeneity of a thrombus[5]
[6] with respect to the contractile properties.[3] This advanced analysis tool will be powerful to obtain observer-independent information
of thousands of platelets within a thrombus and to better understand how individual
platelets shape thrombus architecture.
Future studies will show whether this unique analysis tool is also suitable for the
tracking of other platelet sub-populations, for blood with altered platelet count
and size and whether it can be easily used by other researchers. However, knowledge
in programming seems to be required to adapt this method to each user.
