Increased Platelet Thrombus Formation Under Flow Condition In Myeloproliferative Neoplasms (MPN)
Authors: Vignoli Alfonso , van der Meijden Paola , Marchetti Marina , Giaccherini Cinzia , Tessarolo Serena , Swieringa Frauke , Cate Hugo ten , Finazzi Guido , Rambaldi Alessandro , Heemskerk Johan , Falanga Anna 
Published: October 2019

Abstract

Background
Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are MPNs characterized by a high incidence of both arterial and venous thrombosis, and microcirculatory disturbances. Platelet abnormalities have been variably involved in the thrombotic diathesis of these patients, without providing conclusive evidence. Remarkably, no studies have explored so far the MPN platelet thrombus formation capacity underflow conditions.

Aim
In order to evaluate whether and to what extent the MPN platelet membrane abnormalities may influence their interactions with the vessel wall components in a dynamic system, we wanted to characterize the ET and PV platelet adhesion capacity under flow conditions, by using an in vitro system based on a parallel flow chamber connected to the EVOS fluorescence microscope. The effects of the V617F JAK2 mutational status, cytoreductive therapy, and circulating von Willebrand Factor (vWF) on platelet adhesion in this system were also explored.

Methods
Nine ET (3M/6F; age 63 years, range 60-81) and 6 PV (3M/4F; age 74 years, range 58-82) patients were enrolled into the study upon informed consent. Eleven healthy subjects (5M/6F; age 44 years, range 35-55) acted as a control group (CTR). Peripheral venous whole blood was withdrawn in sodium citrate, recalcified in the presence of heparin, and perfused over a collagen-coated surface for 4 min at a shear rate of 1000 s-1. Platelets were then stained with an anti-CD62P (P-selectin)-FITC antibody as a platelet activation index, and annexin V-AlexaFluor647 as a measure of the procoagulant phosphatidylserine (PS) expression. After staining, images of adherent platelets in random fields were taken using phase contrast and fluorescence imaging with the EVOS fluorescence microscope system. Results were the mean±SD of the percentage of area covered by all platelets, or as the % of adherent platelets positive to either P-selectin or annexin V. In parallel, plasma vWF antigen and activity levels were measured by ELISA. Statistical analysis was performed by SPSS software package.

Results
Platelet adhesion was significantly greater (p<0.05) in both ET (49.3±14.5%) and PV patients (55.3±12.7%) compared to controls (31.6±7.3%). Among patients, platelet adhesion was significantly (p<0.05) increased in those positive for the V617F JAK2 mutation compared to the negative ones, with the highest values in the homozygous subjects. Patients on hydroxyurea (HU) therapy (n=7) had significantly lower platelet adhesion (45.2±13.0%) compared to non-HU-treated patients (56.9±10.4%; p<0.05).The % platelet adhesion directly related to either platelet (r=0.623, p=0.001) and leukocyte (r=0.506, p<0.01) counts, but not to plasma vWF levels. Multivariate regression analysis adjusted for age, sex, and HU therapy, confirmed platelet count and V617F JAK2 mutation as significant determinants of platelet adhesion. Although adhesion was increased, the platelet surface expression of P-selectin and PS was reduced in ET (P-sel: 70±11% pos. platelets; PS: 11±6% pos. platelets) and PV (P-sel: 66±13%; PS: 9±5%) patients compared to controls (P-sel: 74±8%; PS: 20±8%).

Conclusions
The data show for the first time, in MPN patients, an increased platelet adhesion capacity to collagen under flow conditions, indicating a greater thrombus formation potential. This phenomenon is likely not related to the expression of membrane P-selectin and phosphatidylserine, but is significantly influenced by the V617F JAK2 mutation burden and is sensitive to the cytoreductive treatment with HU. Prospective studies are worth to establish the role of the EVOS assay in assessing the levels of thrombotic risk in MPN patients.

Disclosures:
No relevant conflicts of interest to declare.