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Background: Polycythaemia vera is a myeloproliferative neoplasm characterised by a high incidence of thrombosis. The contribution of platelets, key players in haemostasis, in this setting is still unclear. So far, the majority of studies have been focussed on specific platelet abnormalities but not on their actual capacity to form thrombi. The aim of this study was to characterise, ex vivo under flow conditions, the capacity of platelets from patients with polycythaemia vera to adhere to collagen and induce thrombus formation., Materials and Methods: Thirty-nine patients and 30 healthy controls were studied. Thrombus formation was induced by perfusing whole blood over a collagen-coated surface, in a parallel-plate flow chamber coupled to a fluorescent microscope. This dynamic system enables platelet adhesion and thrombus formation to be followed in real time and also allows measurements of the extent of the thrombus and platelet surface antigen expression. Laboratory data were analysed in the light of the patients' main haematological parameters and therapies., Results: Platelet adhesion was significantly greater in patients than in control subjects. Patient thrombi were usually larger and more complex than those formed by control platelets. A significant positive correlation was found between platelet adhesion and both the haematocrit and red blood cell count. These parameters remained significantly correlated with platelet adhesion also after multivariable analysis adjusted for gender, age, therapy and JAK2V617F allele burden. Furthermore, subjects with a haematocrit >45% had significantly greater platelet adhesion than subjects with a haematocrit <45%., Discussion: Our data indicate that increased platelet adhesion participates in the thrombotic diathesis of patients with polycythaemia vera, and that the haematocrit level can affect the adhesive and thrombus forming capacities of platelets.

Introduction The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels. Aim To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV. Materials and Methods 86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35 PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4’ at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean ± SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded. Results PLT adhesion was significantly (p < 0.01) greater in ET (44.6 ± 1.6%) and PV patients (49.0 ± 1.9%) compared to controls (37.9 ± 1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n = 23), followed by CalR-positive (n = 16) and triple negative subjects (n = 9), and lowest in the MPL-positive patients (n = 3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p < 0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p < 0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only. Conclusions ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients. Acknowledgement Project funded by “AIRC-IG2013” grant Nr. 14505 from the “Italian Association for Cancer Research” (A.I.R.C.). [ABSTRACT FROM AUTHOR]

Introduction Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are two MPNs characterized by a “clonal” overproduction of one or more blood cell lines, hypercoagulability, and an increased incidence of thrombosis. ROTEM is a point of care global coagulation assay performed in whole blood, able to evaluate platelets and fibrinogen contributions to the clotting process. Until now few studies evaluated the thromboelastometry profile of MPN patients. Aim This study assess the feasibility of using ROTEM to characterize the prothrombotic state of MPN patients and to evaluate whether the thromboelastometry profile varies according to mutational status and/or treatment, and is influenced by hemocromocytometric parameters. Materials and Methods Venous blood samples were collected from 39 ET and 23 PV patients upon informed consent. Analysis was performed using INTEM and EXTEM reagents, to evaluate the intrinsic and extrinsic pathway, respectively. Maximum clot firmness (MCF, [mm]), which reflects the maximum tensile strength of the thrombus, clotting formation time (CFT [sec]), namely the time that clot takes to increase from 2 to 20 mm above baseline, and clotting time (CT [sec]), the time to clot initiation, were recorded. Nineteen healthy subjects acted as a control group. Results ROTEM analysis showed a hypercoagulable profile in MPN patients, who had shorter CFT and higher MCF compared to controls, both with EXTEM and INTEM reagents; no differences were observed in CT parameters. Platelet count was significantly higher in patients compared to controls (p < 0.01). In patients, a strong statistically significant (p < 0.01) correlation was found between platelet count, and MCF [r = 0.650 (ET), r = 0.601 (PV)] or CFT [r = − 0.641 (ET), r = − 0.558 (PV)]. Multivariate analysis, according to blood cell counts, showed that only platelet count was independently associated to ROTEM results. To correct for platelet differences, a ratio between MCF and the respective platelet value (rMCF) was created. Interestingly, rMCF was significantly lower in patients compared to controls (p < 0.01), suggesting a weaker clot formation potential of patients’ samples. Furthermore, rMCF was lower in ET compared to PV (p < 0.05), and in calreticulin-positive subjects (p < 0.05), while was higher in patients under cytoreductive therapy (Hydroxyurea) (p = ns). Conclusions This study confirms, by the ROTEM evaluation, the occurrence of a hypercoagulable state in ET and PV patients. In addition, the ROTEM parameters are significantly influenced by the platelet count. Finally, MCF values corrected for platelet count reveal a lower platelet reactivity in MPN patients, confirming the hypothesis that platelet function is exhausted upon clotting activation. Acknowledgement Project funded by “AIRC-IG2013” grant Nr. 14505 from the “Italian Association for Cancer Research” (A.I.R.C.). [ABSTRACT FROM AUTHOR]