Anagrelide Modulates Proplatelet Formation Resulting in Decreased Number and Increased Size of Platelets

We retrospectively evaluated 48 essential thrombocythemia (ET) patients who were treated in our institute (male/female, 14/34, median age, 61.5 years). In 14 patients treated with anagrelide (ANA), the degree of platelet count reduction (median, -56.6%) was strongly correlated with increase of mean platelet volume (MPV) (median, +11.7%) (R = 0.777). This correlation was not observed in ET patients treated with hydroxycarbamide alone (R = 0.245). The change in size of platelets strongly suggested that ANA affected the final process of platelet production. Thus, we hypothesized that ANA modifies the process by which platelets are released from proplatelets. To verify the association in an in vitro setting, we compared MEG-01 cells treated with PMA ± ANA. The number of platelet-like particles (PLPs) was decreased (P < 0.05) and the size of PLPs estimated by using flow cytometry was significantly increased when MEG-01 cells were treated with PMA + ANA (P < 0.05 vs PMA alone), recapitulating the clinical findings. The cytoplasmic protrusions extending from MEG-01 cells were shorter and thicker and the number of proplatelets was decreased when MEG-01 cells were treated with PMA + ANA (P < 0.01 vs PMA alone). Western blotting analysis showed that ANA treatment resulted in increased phosphorylation of MLC2 and reduced phosphorylation of focal adhesion kinase (FAK). The morphological change of proplatelets were reversed by blebbistatin, a specific inhibitor of myosin II. These findings indicated that ANA modulates the FAK-RhoA-ROCK-MLC2-myosine IIA pathway and suppresses proplatelet maturation, leading to a decrease in platelet count and increase in MPV.