Platelet Adhesion Under Flow Condition in Patients with Essential Thrombocythemia (ET) and Polycythemia Vera (PV): Analysis According to the Mutational Status
Authors: Vignoli Alfonso , Tessarolo Serena , Marchetti Marina , Gamba Sara , Piras Francesca , Finazzi Guido , van der Meijden Paola , Swieringa Frauke , ten Cate Hugo , Heemskerk Johan , Rambaldi Alessandro , Falanga Anna 
Published: October 2019

Abstract
Background: ET and PV are two myeloproliferative neoplasms characterized by a high incidence of both arterial and venous thrombosis, and microcirculatory disturbances. So far, no studies have been performed to characterize ET and PV patient platelet (PLT) adhesion properties in flow conditions, an index of thrombus formation capacity in vitro (Platelets 2012; 23(3): 229-42).
Aim: In this study, in a cohort of patients with ET or PV, we wanted to evaluate the PLT adhesiveness in vitro by a dynamic method and to estimate whether this property is influenced by any of the three somatic mutations, i.e. JAK2 V617F, Calreticulin (CalR) and MPL, typical of these diseases, of which, the JAK2 V617F mutation has a significant association with thrombosis occurrence, while CalR and MPL mutations have not. Further, the relations of PLT adhesion to main hematological parameters and to cytoreductive hydroxyurea (HU) therapy were also considered.
Patients and Methods: Sixty-nine patients, i.e. 47 ET (20 M/27 F; mean age 60 years, range 33-86) and 22 PV (13 M/9 F; mean age 65 years, range 46-80), and 26 healthy controls (13 M/13 F; 44 years, range 27-61) were included into the study. For the adhesion assay, peripheral venous whole blood was drawn in sodium citrate, recalcified in the presence of heparin, and perfused over a collagen-coated surface for 4 min at a shear rate of 1,000 s-1. PLTs were then stained with an anti-CD62P (P-selectin)-FITC antibody as a PLT activation index, and annexin V-AlexaFluor647 to detect PLT procoagulant phosphatidylserine expression. After staining, images of adherent platelets in random fields were taken using phase contrast and fluorescence imaging with the EVOS® fluorescence microscope (Life Technologies). Results are expressed as the mean±SEM of the percentage of area covered by all PLTs (% coverage), or as the % of adherent PLTs positive for either P-selectin or phosphatidylserine. Main hematological parameters (i.e. PLT count, WBC count, RBC count, Hb, Hct, MCV, MPV), therapeutic regimens, and mutational status (JAK2 V617F, CalR, or MPL) were also recorded. Statistical analysis was performed by SPSS® (IBM) software package.
Results: PLT adhesion was significantly (p<0.01) greater in both ET (44.6±2% coverage) and PV patients (45.6±2.7%) compared to controls (36±2.1%). The analysis of PLT adhesion according to the mutational status shows that, in ET patients, PLT adhesion was greatest in JAK2 V617F mutation carriers (n=22), followed by triple negative (n=7), and CalR-positive (n=15). The only 3 MPL-positive ET patients had the lowest PLT adhesion values. In PV patients, those with >50% JAK2 V617F allele burden carriers (n=8; 52.5±2% coverage) presented a significantly higher PLT adhesion than those with <50% allele burden (n=14; 42.4±3.9, p<0.05). Notably, in both ET and PV patients, while the overall PLT adhesion was significantly increased, the percentage expression of P-selectin by adherent PLTs (~ 75%) was not statistically different from that of control subjects. Differently, the expression of procoagulant phosphatidylserine on adherent PLTs was reduced in both ET (p<0.001) and PV patients (p=n.s.) compared to controls. Among hematological parameters, in ET patients, a significant (p<0.001) correlation was found between PLT adhesion with PLT count, but not with WBC count, RBC count, Hb, Hct, MCV, or MPV. As regard to treatments, a significant reduction in PLT adhesion was associated with cytoreductive treatment with HU (p<0.05) in patients with PV, but not with ET.
Conclusions: ET and PV platelets show a greater thrombus formation capacity in vitro in a dynamic flow condition, as measured by an increase in adhesion to collagen. This capacity is significantly augmented according to the JAK2 V617F mutational allele burden and decreased by HU therapy. A prospective study of the PLT thrombus formation potential in a dynamic model is ongoing to evaluate the predictive value of this parameter on the thrombotic risk of ET and PV patients.
Project funded by "AIRC IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research (A.I.R.C.)".

Disclosures
No relevant conflicts of interest to declare.