Discovering the amyloid nature of clots removed after an ischaemic stroke

Researchers at the University of Liverpool used the Histology facility to produce wax-free slices from formalin-fixed, wax-embedded thrombi (blood clots). These were taken from patients at the Walton Centre who had experienced an ischemic stroke. It was discovered using fluorogenic stain thioflavin T that such thrombi had a high content of amyloid, explaining why they were so resistant to the normal processes of thrombolysis.

Research challenge and facility involvement

Marie O’Brien at the Histology-SRF used a Leica RM2235 microtome to section wax- embedded clots, then free floated 5um sections through a series of xylene and ethanols to remove the wax. Great care was taken to keep all clots intact.

Samples were placed into u-slide 15-well 3D glass-bottomed well plates in an aqueous buffer for staining and imaging. 

Outcome and impact

It was discovered that thrombi were saturated with proteins that had adopted an amyloid form [1, 2], consistent with a posteriori bioinformatic predictions [3-6] based on the proteins they contain. This explains both why they are so resistant to usual removal techniques, and that they probably form via slow accretion of the fibrinaloid microclots that were discovered some time ago [7, 8].

This has profound implications for our understanding of ischemic stroke and its potential prevention.

Quick details

Principal Investigator:	Professor Douglas Kell
Postdoc:	Dr Justine Grixti
Institution/Department:	Department of Biochemistry, Cell and Systems Biology | Institute of Systems, Molecular and Integrative Biology
Shared Research Facility used:	Histology
Instruments:	Leica RM2235
Publication/outputs:	See references 1 and 2 below
Contact for facility access:	histology@liverpool.ac.uk

References

1. Grixti, J. M., Chandran, A., Pretorius, J. H., Walker, M., Sekhar, A., Pretorius, E. and Kell, D. B. (2024) The clots removed from ischaemic stroke patients by mechanical thrombectomy are amyloid in nature. medRxiv, 10.1101/2024.1111.1101.24316555v24316551.
2. Grixti, J. M., Chandran, A., Pretorius, J. H., Walker, M., Sekhar, A., Pretorius, E. and Kell, D. B. (2025) Amyloid presence in acute ischemic stroke thrombi: observational evidence for fibrinolytic resistance. Stroke. 56, e165-e167.
3. Kell, D. B. and Pretorius, E. (2024) Proteomic evidence for amyloidogenic cross-seeding in fibrinaloid microclots. Int J Mol Sci. 25, 10809.
4. Kell, D. B. and Pretorius, E. (2025) The proteome content of blood clots observed under different conditions: successful role in predicting clot amyloid(ogenicity). Molecules. 30, 668.
5. Kell, D. B., Doyle, K. M., Salcedo-Sora, E., Sekhar, A., Walker, M. and Pretorius, E. (2025) AmyloGram reveals amyloidogenic potential in stroke thrombus proteomes. bioRxiv, 2025.2007.2007.663482.
6. Kell, D. B., Doyle, K. M., Salcedo-Sora, E., Sekhar, A., Walker, M. and Pretorius, E. (2025) AmyloGram reveals amyloidogenic potential in stroke thrombus proteomes. Biochem J, in press.
7. Pretorius, E., Mbotwe, S., Bester, J., Robinson, C. J. and Kell, D. B. (2016) Acute induction of anomalous and amyloidogenic blood clotting by molecular amplification of highly substoichiometric levels of bacterial lipopolysaccharide. J R Soc Interface. 123, 20160539.
8. Kell, D. B. and Pretorius, E. (2017) Proteins behaving badly. Substoichiometric molecular control and amplification of the initiation and nature of amyloid fibril formation: lessons from and for blood clotting. Progr Biophys Mol Biol. 123, 16-41.