Background: Proprotein convertase subtilisin/kexin 9 (PCSK9), mainly secreted by the liver and released into the blood, elevates plasma low-density lipoprotein (LDL) cholesterol by degrading LDL receptor. Pleiotropic effects of PCSK9 beyond lipid-metabolism have been shown. However, the direct effects of PCSK9 on platelet activation and thrombosis, as well as the underlying mechanisms, still remain unclear. Methods: We detected the direct effects of PCSK9 on agonists-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, α granule release, spreading, and clot retraction. These studies were complemented by in vivo analysis of FeCl3-injured mouse mesenteric arteriole thrombosis. We also investigated the underlying mechanisms. Using myocardial infarct (MI) model, we explored the effects of PCSK9 on microvascular obstruction and infarct expansion post-MI. Results: PCSK9 directly enhances agonists-induced platelet aggregation, dense granule ATP release, integrin αIIbβ3 activation, P-selection release from α granules, spreading, and clot retraction. In line, PCSK9 enhances in vivo thrombosis in a FeCl3-injured mesenteric arteriole thrombosis mouse model, while PCSK9 inhibitor evolocumab ameliorates its enhancing effects. Mechanism studies revealed that PCSK9 binds to platelet CD36 and thus activates Src kinase and mitogen-activated protein kinase (MAPK)- extracellular signal-regulated kinase 5 and c-Jun N-terminal kinase, increases the generation of reactive oxygen species, as well as activates the p38MAPK/cytosolic phospholipase A2/cyclooxygenase-1/thromboxane A 2 signaling pathways downstream of CD36 to enhance platelet activation. Using CD36 knockout mice, we showed the enhancing effects of PCSK9 on platelet activation are CD36 dependent. Consistently and importantly, aspirin abolishes the enhancing effects of PCSK9 on platelet activation and in vivo thrombosis. Finally, we showed that PCSK9 activating platelet CD36 aggravates microvascular obstruction and promotes MI expansion post-MI. Conclusions: PCSK9 in plasma directly enhances platelet activation and in vivo thrombosis, as well as MI expansion post-MI, by binding to platelet CD36 and thus activating the downstream signaling pathways. PCSK9 inhibitors or aspirin abolish the enhancing effects of PCSK9, supporting the use of aspirin in patients with high plasma PCSK9 levels in addition to PCSK9 inhibitors to prevent thrombotic complications.

Rationale: Targeting inflammation has been shown to provide clinical benefit in the field of cardiovascular diseases. Although manipulating regulatory T-cell function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. IL (interleukin)-35, an immunosuppressive cytokine mainly produced by regulatory T cells, is a novel member of the IL-12 family and is composed of an EBI3 (Epstein-Barr virus–induced gene 3) subunit and a p35 subunit. However, the role of IL-35 in infarct healing remains elusive. Objective: This study aimed to determine whether IL-35 signaling is involved in healing and cardiac remodeling after myocardial infarction (MI) and, if so, to elucidate the underlying molecular mechanisms. Methods and Results: IL-35 subunits (EBI3 and p35), which are mainly expressed in regulatory T cells, were upregulated in mice after MI. After IL-35 inhibition, mice showed impaired infarct healing and aggravated cardiac remodeling, as demonstrated by a significant increase in mortality because of cardiac rupture, decreased wall thickness, and worse cardiac function compared with wild-type MI mice. IL-35 inhibition also led to decreased expression of α-SMA (α-smooth muscle actin) and collagen I/III in the hearts of mice after MI. Pharmacological inhibition of IL-35 suppressed the accumulation of Ly6C low and major histocompatibility complex II low /C-C motif chemokine receptor type 2 − (MHC II low CCR2 − ) macrophages in infarcted hearts. IL-35 activated transcription of CX3CR1 (C-X3-C motif chemokine receptor 1) and TGF (transforming growth factor) β1 in macrophages by inducing GP130 signaling, via IL12Rβ2 and phosphorylation of STAT1 (signal transducer and activator of transcription family) and STAT4 and subsequently promoted Ly6C low macrophage survival and extracellular matrix deposition. Moreover, compared with control MI mice, IL-35–treated MI mice showed increased expression of α-SMA and collagen within scars, correlating with decreased left ventricular rupture rates. Conclusions: IL-35 reduces cardiac rupture, improves wound healing, and attenuates cardiac remodeling after MI by promoting reparative CX3CR1 + Ly6C low macrophage survival.