Work performed:
Towards the objective of the action, so-called microPost Array Detectors (mPADs) were fabricated, functionalized for platelet spreading, and imaged by fluorescence microscopy. I programmed a software for the analysis of mPAD images and benchmarked the performance. The benchmarking was used to identify and optimize critical acquisition parameters.
Comparative measurements of contractile forces of single spread platelets on different adhesion proteins were performed. We correlated these with platelet cytoskeletal morphology, as well as with platelet aggregation by Light Transmission Aggregometry (LTA), to assess the effect of modulated myosin activity. We measured Glycoprotein (GP) Ib Levels and traction forces of platelets from healthy donors, and conducted correlative traction force measurements, GPIb receptor counts, morphometric analysis of spread platelets, and LTA with platelets from two patients who were on anti-platelet therapy after ischaemic stroke.
Results:
The usage of a harder elastomer yielded mPADs with higher structural fidelity and a higher modulus. We measured post deflections in three different ways and identified settings for image acquisition and analysis that maximized force resolution or throughput. Force resolution was dependent a.o. on image contrast and was found to vary between samples. A protocol for the stamping of mPAD tops with adhesion proteins and the passivation of remaining surfaces was developed. Platelets spread over 5-20 functionalized mPAD tops and applied 10-50 nN forces. We thus successfully established routine traction force measurements of single platelets on mPADs.
This new technology in our lab revealed that platelets pulled less on vWF than on fibrinogen. The magnitude of pulling forces depended on the number of adhesion receptors on the platelet surface that were available for binding. Lower traction forces were reflected by a lesser actin fibre alignment in the cytoskeleton. The myosin IIa specific inhibitor Blebbistatin dose-dependently reduced actin fibre alignment in spread platelets, whereas it reduced platelet aggregation by only 40% at most. Two stroke patients were recruited and their platelets investigated by several different assays. These case studies yielded preliminary clinical data on the usage of the mPAD technology to assess platelet function in patients receiving anti-platelet therapy .
Exploitation:
No relevant IP arose from ThromboForce. A commercial exploitation of the results is not envisioned. Since the results were merely on the basic science level, they have no effect on public policymaking.
Dissemination:
Intermediate results from ThromboForce have been presented as posters at three international conferences. Manuscripts are in preparation for their publication in high-impact journals. A data management plan was developed that delineates the open access of data accompanying these future publications. The project has also been highlighted in the context of the ‘RCSI Primary Science for Teachers Initiative (PSTI)’ to primary school teachers.
