Investigating Pim kinase inhibition as a novel anti-thrombotic endothelium targeting strategy

Background and Purpose: Atherothrombosis, a key driver of cardiovascular disease, involves complex interactions between endothelial cells and circulating platelets. While current anti-platelet therapies are effective, they offer limited secondary prevention and are associated with significant adverse effects. Pim kinases have emerged as novel regulators of platelet function and thrombosis and are expressed in endothelial cells. Their inhibition may therefore offer additional therapeutic benefit on thrombosis. This study investigates the effects of PIM1 deletion in mice and pan-Pim kinase inhibitors AZD1208, PIM447, and TP3654 on human coronary artery endothelial cell (HCAEC) function and their potential to modulate thrombogenic responses.

Methodology: Constitutive PIM1-/- mice were used to investigate the impact of PIM1 deletion on coagulation parameters and endothelial phenotype and function. Inhibitor studies were performed to investigate the effect of Pim kinase inhibition on coronary arterial endothelial function. HCAECs were treated with AZD1208, PIM447, and TP3654 (0.1–10µM) and endothelial survival, viability, migration, and angiogenesis assessed using LDH release, MTS assay, wound healing, and tube formation assays, respectively. Expression and release of inflammatory and thrombotic markers were quantified via RNA-Seq, RT-qPCR, and a Luminex multiplex assay. Finally, the impact of Pim inhibitor-conditioned media on platelet adhesion and coagulation was evaluated using in vitro thrombosis and clotting assays.

Key Results: PIM1 deletion in mice was associated with a decrease in PIM2 and PIM3 gene expression in endothelial cells, along with decreased endothelial angiogenesis, and a delay in intrinsic clotting time. In human endothelial cell experiments, expression of the three Pim kinase isoforms was upregulated by TNFα and IL-6 treatment, and Pim kinase inhibitor treatment did not impair HCAEC viability, proliferation, or migration, indicating a lack of cytotoxicity at physiologically achievable concentrations. However, angiogenic capacity of HCAECs was significantly reduced. RNA-Seq analysis demonstrated alterations in expression of endothelial mediators of inflammation and thrombosis following 24-hour inhibitor treatment. Additionally, platelet and leukocyte deposition on confluent treated HCAECs was attenuated under flow and releasate from treated HCAECs delayed intrinsic coagulation pathways.

Conclusion and Implications: Pan Pim kinase inhibitors exhibit anti-inflammatory and anti-thrombotic effects in HCAECs without compromising endothelial integrity or function, supporting further investigation into Pim kinase inhibition as a novel strategy for the prevention of atherothrombosis.