Phd in ANGIO-CARDIO-THORACIC PATHOPHYSIOLOGY AND IMAGING

Thesis title: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) and platelet activation: implications for patients at risk of cardiovascular disease

Background High levels of proprotein convertase subtilisin/kexin 9(PCSK9) is predictive of cardiovascular events (CVEs) in several pathological setting including atrial fibrillation (AF). Moreover, in the association between hypercholesterolemia (HC) and thrombotic risk platelet hyper-reactivity plays an important role. PCSK9 inhibitors (PCSK9i) lower low-density lipoprotein (LDL)-cholesterol, slow atherosclerosis preventing cardiovascular events. While it is known that circulating PCSK9 enhances platelet activation (PA) and that PCSK9i reduce it, the underlying mechanism is not still clarified. In this regard, the hypothesis of this study was that PCSK9 may directly induce platelet activation (PA) and the aim was to evaluate the mechanism involved and the role of PCSK9i. Methods The study was been divided into two parts. In the first part (Study 1) was measured platelet aggregation, recruitment, Thromboxane (Tx)B2 formation and soluble P-selectin (sP-selectin) levels as markers of PA and soluble Nox2-derived peptide (sNox2-dp), hydrogen peroxide (H2O2), isoprostanes and oxidized-LDL (oxLDL) to analyze oxidative stress in 88 patients with FA having PCSK9 values <(n=44) or >(n=44) 1.2ng/ml, balanced for age, sex and cardiovascular risk factors. Furthermore, an in vitro experiment investigated if normal (n=5) platelets incubated with PCSK9 (1.0-2.0 ng/ml) alone or with LDL (50 µg/ml) displayed changes of PA, oxidative stress and downstream signaling. Furthermore, in a multicenter before-after study (Study 2) on 80 heterozygous familial hypercholesterolemia (HeFH) patients on treatment with the maximum tolerated statin dose ± ezetimibe, PA, sNox2-dp and oxidized-LDL (ox-LDL) were measured before and after 6 months of PCSK9i treatment. In vitro study also was performed to investigate the effects of plasma from HeFH patients before and after PCK9i on PA in washed platelets (wPLTs) from healthy subjects (HS). Results (Study 1) PA and oxidative stress markers were significantly higher in patients with PCSK9 levels >1.2ng/ml compared to those with values <1.2ng/ml (p<0.001). Levels of PCSK9 significantly correlated with markers of PA and oxidative stress. Platelets incubation with PCSK9 increased PA, oxidative stress and p38, p47 and PLA2 phosphorylation. These changes were amplified by adding LDL and blunted by CD36 or Nox2 inhibitors. Co-immunoprecipitation analysis revealed an immune complex of PCSK9 with CD36. (Study 2) Compared to baseline, PCSK9i reduced the serum levels of LDL-c, ox-LDL, Thromboxane (Tx) B2, sNox2-dp and PCSK9 (p<0.001). The decrease of TxB2 correlates with that of ox-LDL while ox-LDL reduction correlated with PCSK9 and sNOX2-dp delta. In vitro study demonstrated that wPLTs resuspended in plasma from HeFH after PCSK9i treatment was induced lower PA and sNOX2-dp release than those obtained using plasma before PCSK9i treatment. This reduction was vanished by adding ox-LDL. Indeed, ox-LDL-induced PA was blunted by CD36, LOX1 and Nox2 inhibition. Conclusions We provide the first evidence that PCSK9, at the concentration found in the circulation of AF patients, directly interacts with platelets via CD36 receptor and activating Nox2: this effect is amplified in presence of LDL. Finally, PCSK9i treatment reduces PA modulating Nox2 activity and in turn ox-LDL formation in HeFH patients. All these results suggest that, besides markedly lowering LDL-c levels, PCSK9i treatment also reduces platelet reactivity. These effects on platelets could play a role in the reduction of CV event incidence in patients treated with PCSK9 inhibitors.