PB0740 - Selective inhibition of peptidylarginine deiminase 4 does not disrupt human platelet activation or aggregation ex vivo.

Background: Platelets and neutrophil extracellular traps (NETs), with peptidylarginine deiminase 4 (PAD4) facilitating NET formation, mutually activate each other in thromboinflammation. Expression of the padi4 gene in human platelets led us to investigate a potential role for PAD4 in platelet function. Our previous work revealed no discernible impact on platelet activation in mice lacking PAD4 in platelets. Whether these findings could be translated to human platelets remained unexplored.

Aims: To study protein expression and function of PAD4 in human platelet activation and aggregation, exploring the impact of PAD4 inhibition on these vital processes.

Methods: Washed human platelets were stained for PAD4 and other platelet components (i.e. α-tubulin, CD63) to establish PAD4 protein expression and localization. Functional consequences of selective PAD4 inhibition were evaluated by treating platelet rich plasma (PRP) from healthy donors with GSK484, followed by ex vivo platelet functional testing. Light transmission aggregometry was performed in unstimulated conditions or in the presence of ADP + U46619 (a thromboxane A2 mimetic). Additionally, platelet activation was quantitatively assessed by flow cytometry, using activation markers P-selectin, CD63, and PAC-1, with either ADP + U46619 or TRAP-6 stimulation.

Results: Immunostaining revealed a punctuate pattern of PAD4 expression in human platelets. Absence of proximity of staining with CD63 suggests that PAD4 is not stored in dense granules. Further colocalization studies are ongoing to pinpoint PAD4 expression in platelets. Ex vivo functional testing showed no significant differences in activation markers or aggregation percentages between control platelets and those treated with GSK484, including in unstimulated conditions.

Conclusion(s): These data suggest that GSK484-mediated PAD4 inhibition does not significantly impact ex vivo platelet activation or aggregation. While the broader impact of citrullination by PAD4 on other platelet functions remains unknown, these findings highlight PAD4 as a potential target for mitigating excessive NET formation in various disease contexts without compromising platelet function in hemostasis.