Autocrine regulation of megakaryocyte development

Autocrine components, together with environmental factors, seem to play an important role in the regulation of platelet production. We recently demonstrated that ADP, released by megakaryocytes, regulates proplatelet formation by interacting with P2Y13. The specific P2Y13 inhibitor MRS 2211 inhibited proplatelet formation in a concentration-dependent manner. Megakaryocytes from a patient with severe congenital P2Y12 deficiency showed normal proplatelet formation, which was inhibited by apyrase, cangrelor or MRS 2211 by 50-60%. The platelet count of patients with congenital delta-storage pool deficiency, who lack secretable adenosine diphosphate, was significantly lower than that of patients with other platelet function disorders, confirming the important role of secretable adenosine diphosphate in platelet formation. Additionally, we showed that human megakaryocytes constitutively release Transforming Growth Factor β1 and express its receptors. Importantly, Transforming Growth Factor β1 downstream signaling, through SMAD2/3 phosphorylation, was shown to be active in megakaryocytes extending proplatelets. Furthermore, inactivation of Transforming Growth Factor β1 signaling, by the receptor inhibitors SB431542 and Stemolecule ALK5 inhibitor, determined a significant decrease in proplatelet formation.

Figure 1: Analysis of P2Y13 protein expression in human megakaryocytes in culture and in human platelets. CD61+ human megakaryocytes or washed human platelts were lysed and subjected to western blot analysis. A) P2Y13 receptor expression in human megakaryocytes at different days of culture. B) Expression of CD61 and P2Y13 in human megakaryocytes at day 13 of culture and washed human platelets. The membrane were reprobed with anti–b-actin to ensure equal loading (representative of 3 different experiments). 

Figure 1: Analysis of P2Y13 protein expression in human megakaryocytes in culture and in human platelets. CD61+ human megakaryocytes or washed human platelts were lysed and subjected to western blot analysis. A) P2Y13 receptor expression in human megakaryocytes at different days of culture. B) Expression of CD61 and P2Y13 in human megakaryocytes at day 13 of culture and washed human platelets. The membrane were reprobed with anti–b-actin to ensure equal loading (representative of 3 different experiments). 

The importance of calcium in Megakaryocyte Development

We recently demonstrated, for the first time, that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. We showed that calcium mobilization from intracellular stores exerts two effects: 1) activates signaling cascades that trigger megakaryocyte adhesion on extracellular matrix components and proplatelet formation, 2) promotes extracellular calcium entry which is primarily involved in the regulation of contractile force responsible for megakaryocyte motility. In summary, we report the first evidence that SOCE activators and Ca2+ mobilization are involved in the regulation of human Mk functions. These data, along with the most recent clinical discoveries on calreticulin mutations in myeloproliferative neoplasms, open new perspectives in the study of the signals that in vivo concur in promoting platelet production.

Figure 2: Di Buduo et al., Heamatologica 2014

Figure 2: Di Buduo et al., Heamatologica 2014