Blood Cells Derived Microparticles and Vascular Markers Study in Essential Thrombocythaemia Patients
Authors: Piccin Andrea , Pusceddu Irene , Marcheselli Luigi , Murphy Ciaran , Eakins Elva , Amaddii Giovanni , Frajria Carlo Moesender , Veneri Dino , Perbellini Omar , Pacquola Enrica , Gottardi Michele , Gherlinzoni Filippo , Favaro Flaviano , Faggian Diego , Plebani Mario , Pizzolo Giovanni , Murphy William , Cortelazzo Sergio 
Published: October 2019

Abstract
Abstract 5193
Essenthial thrombocythemia (ET) patients are at risk of developing an arterious or venous thrombotic event. Recent studies have shown that there is no clear association between platelet (PLT) count and thrombotic events, but that white cell count plays a role. Qualitative PLT abnormalities, activation of endothelial cells (EC) and PLT may be involved in these processes. Following activation of blood cells, plasma membrane vescicles are released, these are known as microparticles (MP) (<1. 5Î¼m of size). They may originate from PLT (PMP), EC (EMP) or red cells (RMP). MP may affect endothelial modulators such as vasorelaxant nitric oxide (NO), adrenomedullin (ADM) and endothelin-1 (ET-1), a potent vasocostrictor agent. Previous studies [Trappenburg MC et al 2009] demonstrated that MP are present in elevated numbers in ET. However, the relationship between MP with treatment and MP with endothelial modulators is unknown. We investigated the relationship between MP, NO, ADM and ET-1 in ET patients on treatment with hydroxyurea (HU), anagrelide (AN) and aspirin(ASA) only. 52 patients with ET diagnosis were studied: 18 on HU, 15 on AN, and 19 on ASA. Platelet-poor-plasma aliquotes were stored at −80Â°C and subsequently processed. Samples were analysed for MP numbers (absolute total values) and functional markers (percentage values) by flow cytometry, as previously described [Jy W et al JTH 2004]. PMP was identified using CD62P, CD36, and CD63, EMP using CD105 and RMP using CD235a. Endothelial modulator markers NO, ADM and ET-1 were measured by ELISA. Statistical analysis included Kruskal-Wallis test, Bonferroni test, correlation matrix, principal component (PCA) and partial least square regression analysis (PLSR).
Total-MP were increased in all patients groups compared to controls (p<0. 01). Strong correlations were seen for tot-MP and %MP CD62P (R=0. 89, p<0. 05), tot-MP and %MPCD36 (R=0. 97, p<0. 05), tot-MP CD62P+ and CD36+ (R=0. 85, p<0. 05) and for tot -MP TF+ and %MP CD105+ (R=0. 66, p<0. 05). Bonferroni test, showed higher MP total number in the AN group compared to the HU and the ASA group (p <0. 05). The percentage of CD63 MP was higher than control both in the HU and in the AN group (p <0. 05), while CD36 MP% was higher in the AN group compared to the HU group (p <0. 05). NO and ADM values were higher in the HU group, while ET-1 values were lower in the AN group (p <0. 05). The principle components (PC) responsible for value variations were PC1 [positive loading for WCC, PMP and EMP] reflecteing PLT and EC activation, and PC4 [positive loadings for PLT, PMP; negative loadings for RMP, NO]. Both PC1 and PC4 were statistically significant for treatment.
This study confirms previous findings that MP are present in ET. We also showed that MP are affected by treatment and that MP are mainly PMP and express CD62P and CD36, a marker of immature PLT, which suggests that reticulated PLT partecipate in these processes. We also showed that HU increase NO and ADM levels, while AN reduces ET-1. As NO and ET-1 have antagonistic actions on endothelial cells, we suggest that HU and AN may have overlapping effects on endothelium, although using different biochemichal pathways.

The finding that treatment influences PC1 (PMP and EMP) supports the idea that in ET, treatment impacts directly on MP. The finding that NO and RMP have a negative loading within PC4, suggests that they have an antagonist-action to PMP and EMP in ET.

Disclosures:
Pizzolo: Hoffmann-La Roche: Consultancy, Honoraria.