Attenuation of Atherosclerosis with PAR4 Deficiency: Differential Platelet Outcomes in apoE-/- vs. Ldlr-/- Mice

Wadding-Lee, C.; Jay, M.; Shearer, S. M.; Benson, T.; Spuzzillo, A.; Howatt, D. A.; Thompson, J.; Daugherty, A.; Mackman, N.; Owens, A. P.

doi:10.1101/2024.08.01.606266

bioRxiv · 10.1101/2024.08.01.606266

Attenuation of Atherosclerosis with PAR4 Deficiency: Differential Platelet Outcomes in apoE-/- vs. Ldlr-/- Mice

Wadding-Lee, C.·Jay, M.·Shearer, S. M.·Benson, T.·Spuzzillo, A.·Howatt, D. A.·Thompson, J.·Daugherty, A.·Mackman, N.·Owens, A. P.

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Abstract

ObjectiveCardiovascular disease (CVD) is a significant burden globally and, despite current therapeutics, remains the leading cause of death. Platelet inhibitors are of interest in CVD treatment to reduce thrombus formation post-plaque rupture as well their contribution to inflammation throughout the progression of atherosclerosis. Protease activated receptor 4 (PAR4) is a receptor highly expressed by platelets, strongly activated by thrombin, and plays a vital role in platelet activation and aggregation. However, the role of PAR4 Approach and ResultsMice on a low-density lipoprotein receptor-deficient (Ldlr-/-) background were bred with Par4 deficient (Par4-/-) mice to create Ldlr-/-/Par4+/+ and Ldlr-/-/Par4-/- cousin lines. Mice were fed high fat (42%) and cholesterol (0.2%) Western diet for 12 weeks for all studies. Bone marrow transplant (BMT) studies were conducted by irradiating Ldlr-/-/Par4+/+ and Ldlr-/- /Par4-/- mice with 550 rads (2x, 4 hours apart) and then repopulated with Par4+/+ or Par4-/- bone marrow. To determine if the effects of thrombin were mediated solely by PAR4, the thrombin inhibitor dabigatran was added to the Western diet. Ldlr-/-/Par4-/- given dabigatran did not further decrease their atherosclerotic burden. Differences between apolipoprotein E deficient (apoE-/-) and Ldlr-/- platelets were assessed for changes in reactivity. We observed higher PAR4 abundance in arteries with atherosclerosis in human and mice versus healthy controls. PAR4 deficiency attenuated atherosclerosis in the aortic sinus and root versus proficient controls. BMT studies demonstrated this effect was due to hematopoietic cells, most likely platelets. PAR4 appeared to be acting independent of PAR1, as there werer no changes with addition of dabigatran to PAR4 deficient mice. apoE-/- platelets are hyperreactive compared to Ldlr-/- platelets. ConclusionsHematopoietic-derived PAR4, most likely platelets, plays a vital role in the development and progression of atherosclerosis. Specific targeting of PAR4 may be a potential therapeutic target for CVD. HighlightsO_LIDeficiency of protease-activated receptor 4 attenuates the development of diet-induced atherosclerosis in a Ldlr-/- mouse model. C_LIO_LIPAR4 deficiency in hematopoietic cells is atheroprotective. C_LIO_LIPAR4 deficiency accounts for the majority of thrombin-induced atherosclerosis in a Ldlr-/- mouse model. C_LIO_LIThe examination of platelet-specific proteins and platelet activation should be carefully considered before using the apoE-/- or Ldlr-/- mouse models of atherosclerosis. C_LI

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Wadding-Lee, C., Jay, M., Shearer, S. M., Benson, T., Spuzzillo, A., Howatt, D. A., Thompson, J., Daugherty, A., Mackman, N., Owens, A. P.. 2024-08-07. Attenuation of Atherosclerosis with PAR4 Deficiency: Differential Platelet Outcomes in apoE-/- vs. Ldlr-/- Mice. https://doi.org/10.1101/2024.08.01.606266

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