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.

The effects of anagrelide on human megakaryocytopoiesis

“…This apparent selective activity of anagrelide on human megakaryocytopoiesis has been studied in vitro with various concentrations of anagrelide. Our finding of the inhibition of CFU-M growth at supratherapeutic concentrations of anagrelide (Fig 3) is consistent with earlier observations (Mazur et al, 1992). In the latter study a decrease in colony size was also noted only at high concentrations of the drug.…”

“…Another possibility is that anagrelide might interfere with the intracellular processes that lead to irregular surface events, such as the formation of pseudopods (Leven & Yee, 1987). These observations are similar to those of other investigators who demonstrated that anagrelide induced a shift to the left in the distribution of morphological stages and a decrease in cell size and ploidy of megakaryocytes (Mazur et al, 1992).…”

“…At therapeutic concentrations, anagrelide does not appear to shorten platelet survival or to inhibit the proliferation of CFU-M in vivo. These observations, including the lack of effect of anagrelide on platelet survival in normal subjects (Abe Andes et al, 1984) and the concentrationdependent effect of anagrelide on human megakaryocytopoiesis (Mazur et al, 1992), have been recognized by other investigators.…”

Comparison of the biological activities of anagrelide and its major metabolites in haematopoietic cell cultures

“…Previous findings have suggested that anagrelide acts primarily on the postmitotic phase of MK maturation (Mazur et al, 1992;Solberg et al, 1997). More recently work by another laboratory has shown that in vivo anagrelide also reduces MK cell numbers (Tomer, 2002).…”

“…Initial studies in humans indicated that anagrelide does not sig-nificantly affect platelet-survival time (Abe et al, 1984) nor that it acts by inducing haematopoietic progenitor cell toxicity (Silverstein et al, 1988). Later on, in vitro studies of human megakaryocytopoiesis suggested that, in vivo, the thrombocytopenic activity of this compound results primarily from an inhibitory effect on the postmitotic phase of megakaryocyte (MK) maturation (Mazur et al, 1992). However, notwithstanding its clinical success and these pioneering cellular studies, unravelling the primary mechanism of action by which anagrelide reduces platelet count has remained elusive .…”