PAR1 Antagonist Specifically Inhibits Thrombin-Induced PAR1 Activation and Thrombo-Inflammation in Philadelphia Negative Myeloproliferative Neoplasms
Authors: Uslu-Bicak Ildeniz , Nalcaci Meliha , Sozer Selcuk 
Published: December 2024

Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPNs) are a group of clonal disorders characterized by high morbidity and mortality, driven by the uncontrolled proliferation of myeloid cell lines from hematopoietic stem cells, and associated with a significant risk of thrombosis.

This study aimed to elucidate the relationship between JAK2V617F and protease-activated receptor 1 (PAR1), investigating the expression and activation levels of PAR1 alongside JAK2V617F across various stages of differentiation in circulating hematopoietic stem/progenitor cell (HSPC) subgroups, including multipotent progenitor cells (MPPs), erythro-myeloid restricted progenitors (EMPs), hematopoietic and endothelial progenitor cells (HSCs/EPCs), and endothelial progenitor cells (EPCs). This interaction may contribute to the hypercoagulable state observed in Ph-MPNs.We also examined the functional effects of PAR1 agonists (thrombin), a PAR1 selective antagonist, and a JAK2 inhibitor (ruxolitinib) on Ph-MPN cells. Mononuclear cells (MNCs) were isolated from the peripheral blood of patients diagnosed with Ph-MPN (n=13), cord blood (CB) samples (n=5). Using cell sorting, specific compartments including CD133+/-CD34-/- and PAR1+/-CD34+/- were isolated from the CD45- and propidium iodine (PI) negative population of MNCs. PAR1 expression and JAK2V617F status in each compartment were analyzed using real-time qRT-PCR and allele-specific nested PCR. PAR1 expression changes were studied post-treatment with thrombin (1U/1hr), PAR1 antagonist (80uM/1hr), and ruxolitinib (300uM/3hrs) using real-time qRT-PCR.

Surface expression analysis of PAR1 and CD34 in Ph-MPN cells revealed that most PAR1+ cells (~95%) also expressed CD34+, with a smaller PAR1+ population (~5%) not expressing CD34 in the CD45- PI-, all of which were JAK2V617F+. Relative PAR1 expression in MNCs from Ph-MPN samples was significantly higher than in CB (p=0.0005). Detailed analysis showed increased PAR1 expression in EMP, HSC/EPC, and EPC subsets of Ph-MPN cells compared to CB, all were JAK2V617F+. MPPs showed no significant change in PAR1 expression relative to CB.

Following thrombin-induced PAR1 activation and the inhibitory effects of PAR1 antagonist on cultured Ph-MPN CD45-CD34+/depleted populations were tested. Thrombin itself cleaves the PAR1 receptor therefore the cell surface expression of PAR1 is diminished after thrombin (1U) treatment. Since the PAR1 antagonist blocks the thrombin binding to the PAR1 receptor, the expression of cell surface PAR1 expression (%) in Ph-MPN CD34+ cells were determined to be significantly reduced after PAR1 antagonist treatment. (*P&lt;0.05, **P&lt;0.01, respectively). The combined effect of PAR1 antagonist and ruxolitinib on MNCs from Ph-MPN (n=11) and healthy volunteers (HV) (n=11) showed that PAR1 antagonist significantly downregulated PAR1 expression in Ph-MPN MNCs compared to HV (p&lt;0.0001). Ruxolitinib alone did not significantly affect PAR1 expression but significantly downregulated it when combined with PAR1 antagonist (p=0.0008). The other PAR-pathway related genes including CCL2, CSF2, GJA1, IL1B, CXCL8, NAB2, TNF, and MMP2 that have roles in thrombo-inflamation revealed significant downregulation (&gt;2-fold) after treatment.

This study highlights the novel finding that PAR1 expression is significantly elevated in primitive hematopoietic subpopulations in Ph-MPNs, suggesting its role in disease progression and the increased risk of thrombosis. This innovative aspect of our research sheds light on a potential new target for therapeutic intervention. The use of PAR1 antagonists, particularly in combination with JAK2 inhibitors like ruxolitinib, has shown promise in reducing PAR1 expression and potentially mitigating thrombotic events in Ph-MPN patients.

–Funding:The Research Fund of Istanbul University (Project No: TDK-2021-38358 and TSA-2017-25370) and TUBITAK (Project No. 112S483)