PF684 ANAGRELIDE MODULATES PROPLATELET FORMATION RESULTING IN DECREASED NUMBER AND INCREASED SIZE OF PLATELETS
Authors: Miyashita N. , Onozawa M. , Hayasaka K. , Kunishima S. , Teshima T. 
Published: June 2019

Background:

In patients with essential thrombocythemia (ET) who have high risk factors for thrombotic events, cytoreductive therapy with anagrelide (ANA) or hydroxycarbamide (HC) is one of the treatment options. Despite the clinically evident platelet‐lowering effect of ANA, the detailed mechanism of its effect remains to be elucidated. In addition to the previously reported inhibition of megakaryocytic differentiation, we hypothesized that ANA affects the formation of proplatelets, the final process of platelet release.

Aims:

To determine platelet size change and proplatelet formation with ANA treatment, we retrospectively analyzed patients’ data and analyzed data obtained from an experimental model using a human megakaryocytic cell line (MEG‐01 cells), an accepted model of thrombopoiesis.

Methods:

We retrospectively evaluated 48 patients diagnosed with ET in Hokkaido University Hospital (male/female: 14/34, age, 18 to 87 years; median age, 61.5 years; ANA only, 3 patients; ANA after HC due to insufficient decrease in platelets, 11; HC only, 18; observed without ANA or HC, 16). We analyzed changes in platelet counts and mean platelet volume (MPV) after administering each medicine. This study was approved by the Institutional Review Board of Hokkaido University Hospital. Megakaryocytic differentiation of MEG‐01 cells was induced by PMA treatment with or without ANA. Morphological change of proplatelets and size of platelet‐like particles (PLPs) were analyzed.

Results:

The figure shows the relationship between decrease in platelet count and MPV change in each patient at the time of maximum response to ANA or HC. In patients treated with ANA (n = 14), the degree of platelet count reduction (‐83.4% to −27.1%; median, −56.6%) was strongly correlated with increase of MPV (‐6.6% to +28.7%; median, +16.7%) (left panel; R = 0.777, P = 0.001). On the other hand, in patients treated with HC (n = 29), the degree of platelet count reduction (‐78.7% to −20.8%; median, −55.5%) was not correlated with change of MPV (‐10.3% to +12.9%; median, +1.0%) (right panel; R = 0.245, P = 0.21). PMA‐induced megakaryocytic differentiation of MEG‐01 cells promoted production of PLPs. The number of PLPs was decreased in PMA + ANA‐treated MEG‐01 cells (P < 0.01 vs. PMA alone). PLP size measured by flow cytometry was significantly increased in PMA + ANA‐treated MEG‐01 cells (P < 0.05 vs. PMA alone). These results are consistent with the clinical findings. MGG staining of MEG‐01 cells revealed that ANA modified the formation of cytoplasmic protrusions (proplatelets). We compared morphological parameters including the length, width and number of protrusions. The protrusions were shorter and thicker and the number of protrusions was decreased in PMA + ANA‐treated MEG‐01 cells (P < 0.01 vs. PMA alone).

The FAK‐RhoA‐ROCK‐MLC2‐myosin IIA pathway is an important pathway for the formation of proplatelets and production of platelets from megakaryocytes. We evaluated the effect of ANA on phosphorylation of FAK and MLC2. Western blot analysis showed that ANA treatment caused reduced phosphorylation of FAK and increased phosphorylation of MLC2 (PMA vs. PMA+ANA 5 μM, P < 0.01), which presumably resulted in suppression of actin remodeling.

Summary/Conclusion:

ANA reduces platelet counts and increases platelet size in ET patients. Analysis of ANA‐treated MEG‐01 cells suggested that suppression of proplatelet formation through modulation of the FAK‐RhoA‐ROCK‐MLC2‐myosin IIA pathway is a key mechanism. Morphological observation suggested that ANA induces premature release of platelets, resulting in increased platelet size.

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