Circulating Apoptotic Endothelial Cells

model. To compare the translational efficiencies of normal and mutant TPO mRNAs, the DG mutation was introduced into the 58-UTR of TPO cDNA by recombinant polymerase chain reaction (PCR) and subcloned into the pcDNA3 vector, as previously described.2 Mutant and control TPO mRNAs were transcribed in vitro using T7 polymerase and translated in reticulocyte lysate in the presence of 35S-methionine (Fig 1B). Translation of normal TPO mRNA was strongly repressed, as previously described.2 In contrast, the DG-mutant mRNA was translated with high efficiency, producing amounts of TPO protein comparable to an artificial construct with a deletion of all but the last 7 nucleotides of the 58-UTR (DUTR). Consistent with presence of an extended N-terminus resulting from translation initiation at AUG7, the translation product of the DG-mutant migrated higher than the normal TPO protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Previous mutational analysis demonstrated that AUG7 is used very efficiently, whereas AUG5 and AUG6 are weak inhibitors of translation.2 Thus, the DG-mutation improves the efficiency of TPO translation in reticulocyte lysates by eliminating the inhibitory effect normally exerted by uORF7. The DG mutation also improved translation of TPO transcripts originating from promoter 1 (P1) and of a rare P1-variant that lacks exon 2 (not shown). To demonstrate that the addition of 23 amino acids to the TPO signal peptide resulting from the DG frameshift in uORF7 does not interfere with secretion of a biologically active TPO protein, we transfected COS cells with TPO cDNA expression constructs and measured TPO protein concentrations in tissue culture supernatants (Fig 1C). Cells transfected with a construct carrying the DG mutation secreted 7-fold more TPO protein, as determined by enzyme-linked immunosorbent assay (ELISA), than cells transfected with the corresponding normal construct (solid bars). A similar increase was detected using a TPO bioassay (open bars), demonstrating that the extended signal peptide can ensure secretion of biologically active TPO. Our results show that derepression of translation is responsible for overproduction of TPO protein in individuals carrying the DG mutation in this Japanese HT family. A different TPO gene mutation, which also results in derepression of TPO mRNA translation, was previously described as the cause of HT in a Dutch family.3 These data suggest that increased efficiency of TPO mRNA translation might be a common mechanism in the pathogenesis of HT and illustrate the importance of translational repression for normal platelet homeostasis.