Investigating protein arginine methylation in platelets and hair

Marsden, Alistair James

Abstract

Post-translational modification (PTM) of platelet proteins has long been known to have an effect on their ability to aggregate and form a thrombus. Arginine methylation (ArgMe) is catalysed by protein arginine methyltransferases (PRMTs) which transfer methyl groups to arginine residues in proteins. An increase in protein ArgMe is observed in many disease states including cancers, and cardiovascular disease (CVD) where the proteolytic product ADMA is well-known to correlate with an increased risk of CVD. To combat this risk, inhibitors of PRMTs have been developed and are currently in clinical trials as promising anti-cancer therapies, however the off-target effect of these inhibitors on other body systems is unknown. In this thesis, the primary hypothesis is that proteins in platelets and hair are modified by ArgMe, with the secondary hypothesis that the ArgMe of these proteins is important to cellular function. To investigate this, the arginine methylome (ArgMeOme) of platelets and hair was identified using bioinformatics data mining of proteomic datasets. This data was then corroborated by mass spectrometry evidence, identifying many proteins modified by ArgMe in platelets, and a majority protein of interest, keratin-83, in hair. In platelets the identified proteins were highly enriched in gene ontology terms associated with platelet function including aggregation and degranulation. These terms were investigated further by incubating platelets ex vivo with the PRMT inhibitor Furamidine. Platelet aggregation and adhesion were interrogated using light-transmission aggregometry and spreading analysis. This identified a reduction in platelet aggregation but not adhesion, when stimulated with thrombin and collagen agonists. This was further tested using flow cytometry analysis of key platelet receptors involved in aggregation and thrombus formation, identifying a reduction in expression of the membrane associated fibrinogen receptor αIIbβ3. Using the monoclonal antibody PAC-1 against active αIIbβ3, a further reduction in active fibrinogen receptor was identified. Mass spectrometry analysis of platelet membrane components has confirmed bioinformatics results identifying αIIbβ3 as modified by ArgMe. Using donor hair samples, proteins modified by ArgMe were identified by western blot, with a band at the keratin-83 molecular weight correlating with levels of vADMA in the serum measured by ELISA. The identity of this band was confirmed as keratin-83 by mass spectrometry evidence, finding 8 sites of methylation. Overall, this work opens new avenues for research on ArgMein platelets and hair, including the unique opportunity to repurpose ArgMe inhibitors currently in clinical trials as cancer therapies, as novel antiplatelet drugs. Taken alongside the possibility to develop the understanding of keratin-83 ArgMe in hair as a biomarker of CVD, there is the exciting possibility to use the understanding of ArgMe in platelets and hair to improve the longitudinal management of CVD patients and start reducing the global burden of CVD.

Citation

Marsden, A. J. (2023). Investigating protein arginine methylation in platelets and hair. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4386508

• Thesis Type: Thesis
• Deposit Date: Sep 12, 2023
• Publicly Available Date: Sep 14, 2023
• Public URL: https://hull-repository.worktribe.com/output/4386508
• Additional Information: Hull York Medical School; The University of Hull and the University of York
• Award Date: Aug 22, 2023