Analysis of the mechanism of anagrelide-induced thrombocytopenia in humans

Abstract

Anagrelide is a new therapeutic compound recently demonstrated to have a rapid and selective thrombocytopenic effect in humans. The effects of anagrelide were evaluated in plasma clot and liquid suspension cultures of optimally stimulated normal human peripheral blood megakaryocyte progenitors in order to determine the mechanism of its thrombocytopenic activity. In plasma clot cultures, at clinically relevant, therapeutic concentrations (5 to 50 ng/mL), anagrelide exerted no significant inhibitory effect on megakaryocyte colony numbers or colony size. Only at anagrelide concentrations of 10 to 500 times therapeutic doses did anagrelide inhibit megakaryocyte colony development: an anagrelide concentration of 5 micrograms/mL reduced colony numbers by 57% and colony size by 31%. In contrast, lower, therapeutic anagrelide concentrations exerted profound effects in liquid culture on megakaryocyte cytoplasmic maturation, size, and DNA content. When present for the entire 12-day culture duration, anagrelide induced left- shifted megakaryocyte maturation and reduced both megakaryocyte ploidy and megakaryocyte diameter. Anagrelide, at concentrations of 5 to 50 ng/mL, shifted the modal cultured megakaryocyte morphologic stage from III to II, reduced the model ploidy value from 16N to 8N, and decreased the mean megakaryocyte diameter by up to 22%, from 27.6 microns to 21.6 microns. Megakaryocyte diameter was significantly reduced in most instances, even when analyzed as a function of morphologic stage. When anagrelide was added to the cultures after 6- and 9-day delays (during the final 6 and 3 days, respectively, of culture), similar inhibitory effects on megakaryocyte maturation stage and ploidy distribution were observed. However, the magnitude of the left-shift in ploidy appeared to be less as the duration of anagrelide exposure was reduced. Conversely, megakaryocyte diameter was not significantly affected by the shorter 3- and 6-day anagrelide exposures. These data indicate that therapeutic concentrations of anagrelide influence primarily the postmitotic phase of megakaryocyte development, decreasing platelet production by reducing megakaryocyte size and ploidy, as well as by disrupting full megakaryocyte maturation. Inhibition of megakaryocyte diameter appears to require more prolonged anagrelide exposure than inhibition of maturation stage and ploidy. The molecular mechanisms responsible for the inhibitory effects of anagrelide on megakaryocytopoiesis remain to be defined.

Effects of Anagrelide on Megakaryopoiesis and Platelet Production

“…43 EFFECT OF ANA ON MEGAKARYOPOIESIS Experimental studies were in keeping with the finding that in cell cultures ANA results in a left-shifting of megakaryocyte maturation with a decrease in ploidy. 38 These data confirm the histological evaluation of routinely (hematoxylin and eosin) stained BM slides that showed an increased number of immature megakaryocytes 39,44 consistent with a prevalence of a low-ploidy population. 45,46 However, applying flow cytometry and fluorescence-assisted cell sorting (FACs), both a decrease in cell size (modal ploidy) and also in cell mass (number of megakaryocytes) were demonstrated.…”

“…First, ANA exerts no effect on the circulating half-time of platelets, indicating that it does not enhance their premature destruction. [37][38][39] For this reason, research has focused on issues such as megakaryocyte maturation and differentiation as well as apoptosis (programmed cell death). In addition to the effects on megakaryopoiesis, recently a stimulating influence of this agent concerning the complex pathomechanisms responsible for the generation myelofibrosis [40][41][42] has been reported that caused considerable concern among clinicians.…”

Anagrelide as a New Platelet-Lowering Agent in Essential Thrombocythemia: Mechanism of Action, Efficacy, Toxicity, Current Indications

“…The effect of anagrelide on human megakaryocytopoiesis has been shown to be concentration depen dent. 48,49 In four patients with ET and one with chronic granulocytic leukemia (CGL) being treated with standard doses of anagrelide, we did not detect any change in bone marrow cellularity or the number of megakaryocytes per high-power field. 48 A similar observation was reported earlier.…”

Anagrelide for Gastrointestinal Stromal Tumor

“…Anagrelide hydrochloride targets the PDE3 enzyme family and reduces the peripheral blood platelet numbers by inhibiting megakaryopoiesis in the bone marrow (19,20), and is used for the treatment of essential thrombocythemia (21,22). Anagrelide inhibits the cAMP phosphodiesterase activity elevating the cAMP levels in platelets (23,24).…”