Activated platelets are molecular drivers of endothelial-to-mesenchymal transition in calcific aortic valve disease

Abstract

Bicuspid aortic valves (BAV) predispose to premature CAVD development. The morphology of BAVs induces hemodynamic disturbances, readily activating circulating platelets. Activated platelets release soluble and vesicular mediators (collectively termed platelet releasate [PR]) from their granules, including TGF-β, a prominent mediator of endothelial-to-mesenchymal transition (EndMT). Nonetheless, the direct contribution of platelets to the onset of CAVD remains elusive. We hypothesize that patients with BAVs versus tricuspid aortic valves (TAV) present with increased platelet activation and that their PR triggers distinct molecular signaling pathways leading to the induction of EndMT in valvular endothelial cells (VECs).

Whole blood from CAVD patients undergoing surgical valve replacement was collected, platelets were isolated by sequential centrifugation and activated with 1U/mL thrombin to generate PR. Platelet proteomics revealed a pre-activated or “spent” platelet phenotype in patients with BAVs, indicated by reduced levels of granule proteins such as platelet factor 4 (PF4) or glycoprotein IIb/IIIa. Simultaneously, Nanoparticle Tracking, Flow Cytometry and immunoassays revealed augmented levels of plasma extracellular vesicles (EVs) and platelet activation markers (PF4, CD62P) in patients with BAVs. Co-culture of VECs with PR or autologous plasma (to investigate the contribution of acute/local platelet activation and constitutive activation by circulating factors, respectively) induced phenotypic and molecular changes indicative of EndMT. Proteomic analysis of activated VECs will delineate conserved and divergent molecular signaling pathways driving EndMT in BAVs and TAVs.

These findings support that platelet activation differentially contributes to CAVD onset and may provide the opportunity to elucidate early diagnostic biomarkers and develop targeted therapies.