Abstract Body: Introduction: Elevated levels of lipoprotein(a) (Lp(a)) are a risk factor for atherothrombotic events, however the direct contribution of Lp(a) to arterial thrombosis remains unclear. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety linked to apolipoprotein(a) (apo(a)). Lp(a) contains proinflammatory oxidized phospholipids (OxPLs) on apo(a) and on the lipoprotein component of Lp(a), and has potential proinflammatory and prothrombotic properties which are not well understood. NETosis is a form of programmed cell death in which neutrophil extracellular traps (NETs), consisting of exteriorized chromatin, are released in response to inflammation, thereby contributing to immunothrombosis. We hypothesized that Lp(a) can stimulate NETosis; we assessed this by measuring the effect of purified Lp(a) on NETosis using an in vitro assay.

Methods: Neutrophils were obtained by differentiation of the HL-60 cells (human promyelocytic leukemia cell line). Neutrophils were treated with phorbol 12-myristate 13-acetate (PMA), a NETosis trigger, in the absence or presence of 250 nmol/L purified Lp(a). NETosis was measured at time points up to 8 hours using a DNA-binding fluorescent dye to measure the extent of chromatin extrusion.

Results: A dose-dependent increase in fluorescence was observed, with the greatest NETosis observed between 15.6 nM and 125 nM PMA treatment. We observed that Lp(a) treatment alone did not stimulate NETosis. However, in the differentiated HL-60 cell line, increased NETosis was observed in response to PMA stimulation in the presence of Lp(a), indicating that Lp(a) can potentiate NETosis. Compared to PMA treatment alone, NETosis was initiated earlier and occurs at a higher rate in the presence of Lp(a).

Conclusion: We demonstrate, for the first time, that Lp(a) can potentiate NETosis. We predict that this effect may contribute to the prothrombotic effects of Lp(a) in the vasculature through stimulation of coagulation as well as inhibition of fibrinolysis. Future studies will also measure the response of PMA-treated neutrophils to purified apo(a) recombinant apo(a) species either containing or lacking proinflammatory OxPLs. Our findings improve our understanding of the pathogenic mechanisms of Lp(a) and contribute a new perspective to the field through exploration of the possible link between proinflammatory OxPLs on Lp(a) and its potential prothrombotic properties.