Distinct Hemostatic Profile of Leukocytes in Essential Thrombocythemia (ET) Carrying the JAK2 V617F Mutation.
Authors: Falanga Anna , Marchetti Marina , Balducci Donatella , Vignoli Alfonso , Russo Laura , Guerini Vittoria , Rambaldi Alessandro , Barbui Tiziano 
Published: April 2020

Abstract
The pathogenesis of the thrombotic diathesis of patients with ET is not completely clarified. Activation of polymorphonuclear leukocyte (PMN) occurs in these patients and is associated with a hypercoagulable state and increased number of circulating platelet/PMN aggregates. Further, increased procoagulant Tissue Factor (TF) expression in ET PMN is also reported. Recently, a gain-of-function JAK2 mutation (V617F) has been described in a high proportion of bcr/abl-negative myeloproliferative disorders. Specifically, in subjects with ET the mutation is present in about 50% of cases and retrospective data suggest an association with a higher rate of complications, including thrombosis. Aim of this study was to evaluate whether ET patients carrying the JAK2 mutation possess PMN with different hemostatic characteristics compared to ET subjects without JAK2 mutation and healthy controls. Twenty ET patients, 10 with and 10 without JAK2 mutation (median age: 50 years, range 32–61; platelet count: median 782 x 109/L, range 474–1,565 x 109/L), not receiving cytoreductive therapy (classified as low risk group), were included in the study; 16 age matched healthy subjects acted as controls. Expression of CD11b, TF and fibrinogen on PMN surface as well as PMN-platelet mixed aggregates (defined as the percentage of CD11b-positive PMN co-expressing a platelet-specific marker, i.e. CD42b or CD62P) were evaluated by whole blood flow-cytometry in both basal condition and after in vitro PMN stimulation by f-MLP. In washed isolated PMN samples the level of TF-mRNA was determined by RT-PCR. In basal conditions, significantly (p&lt;0.01) increased levels of PMN/platelet aggregates (both CD42 and CD62P pos. PMN) compared to controls were found, independently from JAK2 mutation. Differently, PMN from JAK2 mutation carriers expressed significantly higher surface TF (18.2±9 %pos. cells) and fibrinogen (12.3±7 %pos. cells) antigens compared to non-carrier (TF: 11.6±5 %; fibrinogen: 7.2±2 %pos. cells) and control subjects (TF: 11.1±3 %; fibrinogen: 7.1±3 %pos. cells). In vitro stimulation with f-MLP increased the proportion of platelet/PMN aggregates (CD11b/CD42 and CD11b/CD62P) in all ET patients and controls. However, in JAK2 mutation carriers the levels of both CD62P and CD42b positive PMN were significantly greater than those of non-carrier ET patients and controls (p&lt;0.05). Similarly, TF and fibrinogen levels on stimulated PMN surface were more elevated in the JAK2 mutation positive group compared to both the mutation negative and control groups. The analysis of PMN TF-mRNA showed a significantly higher expression in the whole ET patient group compared to controls, independently from the presence of JAK2 mutation. In conclusions, these data indicate that the expression of JAK2 mutation in ET patients may confer to PMN a different hemostatic phenotype in terms of increased interaction with platelets and increased expression of surface TF and fibrinogen, suggesting a new link of this mutation with a prothrombotic status.