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Platelet tractions through the Glycoprotein (GP) GPIb receptor as a potential marker for platelet reactivity

Periodic Reporting for period 1 - ThromboForce (Platelet tractions through the Glycoprotein (GP) GPIb receptor as a potential marker for platelet reactivity)

Reporting period: 2017-06-15 to 2019-06-14

Problem addressed:
Blood platelets bind to injured sites of blood vessels and aggregate there to stop bleeding. Platelet adhesion and aggregation is an active biomechanical process controlled by platelet biochemistry and signalling. When this process goes out of control, it can result in thrombosis, heart attack, or stroke. Clinical platelet function tests however do not directly address platelet biomechanics. There is thus a need for new assays that can measure how firmly platelets bind to specific components of the vascular bed, and a need to better understand what platelet signalling pathways are modulated by mechanotransduction through the respective adhesion receptors. The initial anchoring of a platelet is mediated by the binding of glycoprotein (GP) Ib receptors on its surface to the protein von Willebrand factor (vWF) that gets immobilized at injured vessel walls. Based on the mechanical force applied through this linkage, the platelet decides whether to bind firmly and aggregate or whether to let go. Recent research has revealed GPIb’s mechanotransduction properties, yet the consequences for platelet biomechanics are not well understood.

Societal importance:
Platelet-mediated thrombosis is a leading cause of mortality and disability in the European Union. Platelet function tests so far have not been able to guide the treatment of patients to prevent cardiovascular events. The current ‘one dose fits all’ strategy is opposed by a substantial variability in the patients’ responses to anti-platelet medication. New insights into fundamental platelet biology and novel assays to measure platelet function are needed to develop better diagnostics for personalizing treatment.

The aim of ThromboForce was to directly measure the mechanical forces that single platelets apply when they bind to vWF. The main objectives were to
i) Establish and optimize a platform for measuring contractile forces of single adhering platelets with high throughput.
ii) Test whether GPIb expression levels affect platelet adhesion forces.
iii) Validate the platform using clinical samples from patients with established cardiovascular disease on antiplatelet therapy.

The action successfully established traction force measurements of single platelets using a newly developed experimental platform and custom software. Platelet traction forces systematically depended on adhesion protein identity, adhesion receptor numbers, and myosin activity. Correlations between traction forces and the more readily available cytoskeletal morphology of spread platelets were established. Preliminary clinical data were obtained from a small number of stroke patients. The results provide new insights into platelet mechanobiology. The established technology helped to establish new collaborations within the platelet scientific community.
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.

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 .

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.

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.
Innovations in the mPAD fabrication process achieved higher feature fidelity and a by 40% higher local density of posts beneath platelets compared to previous studies. The novel analysis enabled the usage of all slices from z-stacks which yielded a superior measurement precision compared to the common ‘top-bottom’ method. Our systematic investigation for the first time highlighted that the force resolution can change from image to image, and our software provides this important information along with statistics on single cell morphology and force generation.

This is to our knowledge the first report that directly compared platelet traction forces on different adhesion proteins. The general finding of higher platelet traction forces on aggregation-related proteins and lower tractions on vascular bed-related proteins implies that the chemistry of the niche dictates the development of platelet contractility. This concept exceeds previous findings that showed an influence of external factors such as substrate stiffness (stiffness sensing) or platelet agonists. Our results are an important step towards a more comprehensive understanding of platelet mechanobiology.

ThromboForce did not develop socio-economic impact; the technology developed is still very much dependent on operation by a specialist and not easily adapted for routine diagnostics. Since the clinical application of the new technique remained preliminary, it is not possible to claim wider societal implications of our findings.