Hereditary thrombocythemia (HT) has been described as a rare benign disorder caused by mutations in the thrombopoietin (THPO) or the c-Mpl receptor genes. Here we report two families with HT resulting from a THPO c.13+1 G>C mutation in the splice donor of intron 3. In one family there were coexisting distal limb defects, whereas in the other one member developed early-onset multiple myeloma. These observations, together with previously reported patients, suggest that THPO gain of function may dysregulate the hemangioblast and disturb vasculogenesis and hematopoietic development. Overstimulation of the THPO pathway might therefore predispose to clonal hematopoietic disease and to congenital abnormalities.

Key Points• The c-Mpl activity in downstream signaling and in platelet homeostasis can be functionally separated.• The c-Mpl platelet homeostasis depends on correct processing and surface expression of the receptor, whereas downstream signaling does not.The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis. (Blood. 2015;125(7):1159-1169) IntroductionPlatelet production is stimulated by the interaction of the cytokine thrombopoietin (THPO) with its receptor c-Mpl on megakaryocytes and their progenitors with subsequent activation of several downstream pathways, including the Janus kinase/signal transducer and activator of transcription pathway, the phosphatidylinositol 3-kinase pathway, and the mitogen-activated protein kinase pathway. The homeostasis of platelet numbers in the blood is maintained by a negative feedback loop, which requires the clearance of THPO from the plasma by c-Mpl-carrying megakaryocytes and platelets.1 In addition, disruption of the signaling pathway by inactivating THPO or c-Mpl mutations can cause serious thrombocytopenia.2-4 By contrast, activating THPO or c-Mpl mutations can cause hereditary thrombocythemia (HT). [5][6][7][8][9][10][11][12] Interestingly, decreased expression of c-Mpl can also result in HT by high THPO levels, although the mechanism for this observation remains unknown. [13][14][15] Genotype analyses in HT have previously identified the transmembrane S505N, the extracellular N35K, and the P106L c-Mpl mutations to occur in Japanese, African American and Arabic populations, respectively. 5,9,12,[16][17][18] Furthermore, the c-Mpl S505N mutation in the transmembrane region and the juxtamembrane c-Mpl W515L and W515...

2374 Thrombopoiesis is tightly regulated by the interaction between thrombopoietin (TPO) and its receptor c-Mpl. Receptor binding also leads to the clearance of TPO from the plasma thus establishing a negative feedback loop. However, it is still an open question how the receptor activates its downstream pathway. Alternative models posit that ligand binding either results in receptor dimerization in the plasma membrane or leads to conformational change of preformed receptor dimers. Several mutations in the TPO and the c-Mpl receptor genes have been linked to either hereditary thrombocytopenia or thrombocytosis. We focused on mutations in the extracellular part of the c-Mpl receptor, where ligand binding and receptor dimerization occur. Mutated homozygous c-Mpl R102P and compound heterozygous R102P/F104S receptors cause severe hereditary thrombocytopenia. In contrast, the homozygous c-Mpl P106L mutation was found in patients with hereditary thrombocytosis. We now addressed the question of how the disparate phenotype of mutations in the same domain of the c-Mpl receptor can be explained. We first functionally analyzed and compared normal with mutated R102P, F104S and P106L c-Mpl receptors in transfected HeLa and BA/F3 cells and found that the normal and the F104S c-Mpl receptors are glycosylated normally by the Golgi apparatus and reach the plasma membrane. In contrast, the R102P and P106L mutated receptors are not fully glycosylated, do not reach the plasma membrane and are atypically distributed in the ER. Functional analysis of the TPO/c-Mpl signaling pathway in BA/F3 cells showed decreased phosphorylation of Stat3, Stat5 and Erk1/2 with the R102P and F104S mutants when compared to normal. By contrast, TPO/c-Mpl signaling was up-regulated in cells transfected with the P106L-mutated receptor. Moreover, the P106L mutant, but not the other mutant receptors, enhanced ligand-independent growth of transfected BA/F3 cells. Despite of their opposite function, the TPO plasma levels of patients carrying both, homozygous R102P and P106L mutations were elevated 10 to 20-fold compared to normal and heterozygous individuals. This finding, together with their impaired glycosylation and inability to reach the plasma membrane, suggests that these mutants do not bind and internalize their ligand. TPO binding and degradation thus requires the receptor to be expressed at the plasma membrane, whereas, surprisingly, c-Mpl P106L activated its signaling pathway in a ligand independent fashion. Correct receptor processing and function can thus be separated. This indicates that TPO binding is required for regulation but that the constitutive activation of c-Mpl P106L is a likely direct consequence of premature receptor dimerization in the ER, auto-phosphorylation and subsequent activation of downstream targets. Disclosures: No relevant conflicts of interest to declare.