Germ-line heterozygous mutations in the hematopoietic transcription factor AML1 (RUNX1) have been identified in patients with familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), which is characterized by thrombocytopenia, abnormal platelet function, and propensity to myeloid malignancies. We identified a novel mutation in the AML1 gene in an FPD/AML pedigree characterized by a single nucleotide deletion that generates a frameshift and premature chain termination (Pro218fs-Ter225). Both wild-type and mutant transcripts were expressed in affected individuals by allele-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Thrombopoietin (TPO) binds to the Mpl receptor and is the major regulator of megakaryopoiesis. To explore the mechanisms underlying thrombocytopenia, we studied the TPO/Mpl pathway in this newly identified pedigree. TPO levels were mildly to moderately elevated. On flow cytometry and immunoblotting, Mpl receptor expression was decreased and TPOinduced signaling was impaired. While no mutations were identified in the MPL gene by sequence analysis, low MPL mRNA levels were found, suggesting decreased gene expression. Of particular interest, several AML1-binding motifs are present in the MPL promoter, suggesting MPL is an AML1 target. In conclusion, we identified a C-terminal AML1 mutation that leads to a decrease in Mpl receptor expression, providing a potential explanation for thrombocytopenia in this FPD/AML pedigree. IntroductionFamilial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder characterized by moderate thrombocytopenia, a defect in platelet function, and propensity to develop myeloid malignancy. 1 Germ-line heterozygous mutations in the hematopoietic transcription factor AML1, also known as RUNX1 and CBFA2, have been identified in 12 pedigrees reported so far, including missense, frameshift, and nonsense mutations and a large intragenic deletion. [2][3][4][5][6] AML1 is the DNA-binding subunit of the core binding factor (CBF) transcription complex. Heterodimerization with the non-DNA-binding subunit, CBF␤, increases the affinity of AML1 to DNA and protects it from proteolytic degradation. 7,8 AML1 includes a runt homology domain (RHD), which mediates DNA binding and heterodimerization with CBF␤, and a carboxy (C)-terminal domain responsible for transcriptional activation. 9 The AML1/CBF␤ complex regulates the expression of genes specific to hematopoiesis, including several cytokines and their receptors, such as granulocyte macrophage colony-stimulating factor, interleukin-3, and macrophage colony-stimulating factor receptor. 7 This transcription factor is essential for the establishment of definitive hematopoiesis, as demonstrated in AML1 Ϫ/Ϫ mice, which show a complete absence of fetal liver hematopoiesis and die during embryonic development, precluding analysis of the effects of AML1 in later stages of blood-cell development. 10 The role of this transcription factor in plate...

The frequency of the JAK2V617F mutation in platelets was similar to that reported in granulocytes in the literature, suggesting this mutation does not occur as an isolated event in the megakaryocyte lineage. If confirmed in a larger study, the observed higher frequency of thrombosis in patients younger than 60 might be a useful predictive marker for thrombosis in this subset of patients. Even though this mutation has been predicted to constitutively activate the JAK2 kinase, spontaneous phosphorylation of STAT5 does not seem to be a frequent finding in platelets from ET patients.

Melatonin, an indolamine synthesized in the pineal gland, is known to have antiprostanoid activity. The inhibition of platelet aggregation induced by melatonin has been proposed to take place through the cyclooxygenase pathway. In the present study, we found that melatonin has a marked inhibitory effect on collagen, arachidonic acid (AA), adenosine diphosphate (ADP), epinephrine, and A23187-induced aggregation in platelet-rich plasma. On the other hand, using metrizamide-filtered platelets resuspended in Tyrode's buffer, melatonin fails to suppress AA-induced platelet aggregation and 14C-5-HT release. Under the same conditions, melatonin inhibits collagen-induced platelet activation; however, the addition of threshold doses of AA (0.3 mM) abrogates this effect. These studies suggest that melatonin also inhibits platelet function at a stage preceding the cyclooxygenase-dependent pathway.