Investigating the impact of α-synuclein and PD-L1 deletion on platelet function

Sennett, Christopher

Abstract

Platelet proteomic studies suggest that platelets express over 1,000 proteins, most of which have no known function in platelets. This thesis investigates two such proteins, α-synuclein and Programmed Death Ligand 1 (PD-L1), and their roles in essential platelet functions and associated pathological processes.
Granular secretion is critical for platelet function, contributing not only to haemostasis, but also wound healing and inflammation. The release of granule contents requires the assembly of the Soluble N-ethyl maleimide-sensitive factor (NSF) Attachment Protein Receptor (SNARE) complex. In neurons, α-synuclein is known to regulate granule secretion by facilitating the SNARE complex assembly. Platelet proteomic studies suggest that α-synuclein is highly expressed in platelets, but its function(s) remain unknown. In human platelets, we demonstrate that α-synuclein co-localises with VAMP8, SNAP23, and STX11. Immunoprecipitation shows a significant increase in interaction between α-synuclein and VAMP8/STX11 and STX4 following thrombin stimulation. In α-synuclein knockout mice, we show that α-synuclein facilitates haemostasis, granule secretion, and the immune response to endotoxemia. These mice exhibit a bleeding tendency and prolonged activated partial thromboplastin time (APTT), mirroring in vitro aggregation defects. α-synuclein deletion resulted in poorer outcomes following acute endotoxemia. These findings suggest that α-synuclein regulates platelet secretion by facilitating SNARE complex formation.
In this thesis, we present evidence that PD-L1 is abundantly expressed in platelets. Whilst PD-L1 is recognised as an immunomodulator, recent studies revealed that PD-L1 deletion inhibits platelet function. We generated the first platelet-specific PD-L1 knockout mouse, which exhibited reduced platelet aggregation in vitro. This reduction was accompanied by a significant decrease in platelet activation and secretion. In humans, PD-L1 inhibitors significantly reduced platelet aggregation. Flow cytometric analysis showed that patients with myeloproliferative neoplasms (MPN) have a significantly higher percentage of PD-L1 positive platelets compared to healthy donors. Additionally, incubation of healthy donor platelets with interferon-gamma (IFN-γ) significantly increased PD-L1 expression, a result confirmed by plasma swap experiments where healthy donor platelets upregulated PD-L1 expression after incubation with plasma from MPN patients. These findings suggest that PD-L1 plays a role in platelet activation and can be induced by plasma factors such as IFN-γ.

Citation

Sennett, C. (2025). Investigating the impact of α-synuclein and PD-L1 deletion on platelet function. (Thesis). University of Hull and University of York. https://hull-repository.worktribe.com/output/5182732

• Thesis Type: Thesis
• Deposit Date: May 23, 2025
• Publicly Available Date: Jun 20, 2025
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/5182732
• Additional Information: Centre for Biomedicine; Hull York Medical School; University of Hull and University of York
• Award Date: Apr 2, 2025