Abstract 4373423: Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9)-Mediated Platelet Activation Elicits NETs Formation via Cathepsin-G

Introduction/Background: PCSK9 has been shown to induce neutrophil extracellular trap (NET) formation in experimental models, contributing to thrombotic processes. Heterozygous familial hypercholesterolaemia (HeFH), characterized by elevated LDL-C and increased cardiovascular risk, provides a relevant human model to explore this mechanism. However, the role of PCSK9 in NET formation in humans remains unclear. Research Questions/Hypothesis: This study aimed to determine whether PCSK9 promotes NET formation and thrombotic activation in HeFH patients, and whether PCSK9 inhibitors (PCSK9-I) can reduce these effects. The involvement of cathepsin G and other pathways in NETosis and thrombosis was also investigated. Goals/Aims: The aims were to assess the impact of PCSK9 on NET-associated thrombosis in HeFH. NETosis, platelet activation, and coagulation markers and elucidate the molecular mechanisms by which PCSK9 induces NET formation and thrombus growth. Methods/Approach: 40 HeFH patients on maximal statin ± ezetimibe therapy were studied before (BP) and after (AP) six months of PCSK9-I. 20 healthy subjects (HS) served as controls. Circulating markers of NETosis, platelet and coagulation activation were measured. In vitro, neutrophil-platelet mixtures from healthy donors were incubated with plasma from BP, AP, and HS to assess NET formation, platelet/coagulation activation, and thrombus growth. The effects of recombinant PCSK9 (hrPCSK9) on these processes were also evaluated, with or without inhibitors of PCSK9, CD36, cathepsin G, and PAD4. Results/Data: HeFH patients showed significantly higher NETs, platelet activation, and thrombotic markers than HS, all of which were reduced following PCSK9-I treatment. In vitro, plasma from BP induced greater NETosis, platelet activation, and thrombus formation compared to plasma from AP or HS. Incubation of hrPCSK9 with neutrophil-platelet mixtures enhanced NET formation, cathepsin G release, and thrombus growth. These effects were suppressed by inhibitors of PCSK9, CD36, cathepsin G, and PAD4, supporting a cathepsin G-dependent mechanism of PCSK9-mediated thrombotic process. Conclusions: PCSK9 contributes to NET formation and thrombotic activation in HeFH patients via a mechanism involving cathepsin G. PCSK9-I therapy reduces these effects, revealing a novel antithrombotic benefit beyond lipid lowering. These findings provide new insight into PCSK9’s role in cardiovascular disease and support its inhibition in FH management.