Iron Deficiency Correction in Myeloproliferative Neoplasms Reduces Thrombosis Risk Via Decreased P-Selectin

Erythrocytosis increases blood viscosity, which is considered a risk factor for thrombosis. The primary treatment goal of erythrocytosis is to keep hematocrit (Hct) in Polycythemia Vera (PV) below 45 %. This is usually accomplished through phlebotomies resulting in inevitable iron deficiency (ID). Major cause of morbidity and mortality in PV and Chuvash erythrocytosis (CE), a congenital disorder of augmented hypoxia-inducible factors (HIFs), is thrombosis; yet, prevalence of thromboses is not increased in other acquired and congenital erythrocytoses. In Framingham study of healthy non-smokers, higher Hct was not associated with an increased risk of strokes (Kannel 1987). Furthermore, phlebotomies and ID in CE and in another congenital disorder of augmented HIFs signaling from gain of function EPAS1 (encodes HIF-2a) mutation, increases rather than decreases thromboses (Gordeuk 2020). Iron is required for enzymatic activity of prolyl hydroxylase enzymes (PHD encoded by EGLN genes); which is the principal negative regulator of HIFs. ID stabilizes HIFs, leading to overexpression of target genes associated with inflammation and thrombosis (Zhang 2015).

Both PV and Essential Thrombocythemia (ET) are associated with increased HIFs activity, resulting in increased tissue factor and other prothrombotic and proinflammatory factors in neutrophils and platelets; especially higher in those with history of thromboses (Gangaraju 2020; Reeves 2022). We hypothesized that ID in PV and ET increases the risk of thrombosis by augmented HIFs. We found that these prothrombotic and proinflammatory genes further increase in ID of PV and ET (Song ASH 2021) However, a plethora of other clinical determinants among individuals increase thromboses. Thus, we analyzed iron deficient PV (n=17), ET (n=3), myelofibrosis (MF) (n=2), and erythrocytosis (n=8) subjects, serving as their own controls, before and after correcting their ID. Although we did not anticipate changes in JAK2V617F allele burden by iron replacement, we measured the allele burden in the clonal neutrophils of 12 patients. Results showed a decrease in 6 patients (50%), an increase in 3 patients (25%), and no change in the remaining 3 patients (25%). All patients have been treated with either hydroxyurea, interferon alpha, or JAK inhibitors to control blood counts before iron replacement. Expression levels of prothrombotic genes (F3, THBD, SELP, SERPINE1, THBS1), anti-thrombotic gene (KLF2) (Song, 2022), inflammatory genes (IL1B, CXCL8, IL15, TNF, IL6), and HIF-targeted genes (EDN1, LDHA, SLC2A1, VEGFA) in neutrophils and platelets of erythrocytosis patients with ID were higher in MPNs with ID. After correction of ID following iron replacement, expression levels of HIF target genes LDHA (34.4% in neutrophils, p= 0.0254) and SLC2A1 (32.5% in neutrophil, p=0.0176; 25.8 % in platelets, p=0.0138) were decreased while EDN1 increased in both neutrophils and platelets. In neutrophils, IL6 (32.5 %, p=0.0226), IL1B (30.1 %, p=0.0271), and IL15 (28%, p=0.049) decreased. IL6 (30.1 %, p=0.0025) and IL15 (22 %, p=0.0282) transcript levels were also decreased but IL1B and CXCL8 increased in platelets after iron replacement. We measured IL6, TNF, IL1B, and IL8 protein levels in plasma and found that only TNF protein was reduced (20 %, p= 0.0466) after iron replacement.

Transcriptional factor KLF2 downregulates thromboses, its deletion in neutrophils causes massive thromboses in mice (Nayak ASH 2018). We showed that KLF2 transcript decreased in PV and ET neutrophils and platelets, is lower in those with history of thromboses (Song 2023), and decreased in iron deficient PV and ET patients. KLF2 transcript increased after iron replacement (61% in neutrophils, p=0.110; 72 % in platelets, p=0.0189). F3 and THBD transcript levels increased in platelets. SELP (encodes p-selectin), one of the prothrombotic genes, decreased in both platelets (42.2 %, p=0.0087) and neutrophils (30.7 %, p=0.0068).

We report that iron replacement therapy in PV and ET decreases inflammatory and SELP gene expression, correlating with reduced HIF transcriptional activity. These suggest that treatment of ID in PV and ET patients may help lower the risk of thrombosis. Anemia of inflammation and cancer induces hepcidin, causing iron sequestration from circulation to the storage ““functional ID” (Ganz, 2011). Whether it also increases HIF activity, and thrombosis risk remains to be determined.