ENU Mutagenesis Screen Identifies a Novel Regulator of Platelet Production.

The genes that regulate variation in steady-state platelet count are largely unknown. We have attempted to identify novel genes that regulate platelet production by creating heritable models of thrombocytosis in mice by randomly inducing point mutations in the genome with chemical mutagenesis.

Mice with a high platelet count were identified in the third generation of an ENU mutagenesis screen and several different lines of thrombocytotic mice have been established. Subsequent breeding of one line has demonstrated that this mutation, called Plt2, is inherited in an autosomal recessive manner. Automated platelet counts for Plt2 homozygote mice were 1997±642x10⁹/L (mean±2SD; n=51), 140% of those observed for wild-type mice (1487±372; n=56) and Plt2 heterozygote mice (1402±554; n=76). Analysis of Plt2 animals has shown increased numbers of megakaryocyte precursors (as measured by meg-CFC/5x10⁵ cells to maximal cytokine stimulation) in the bone marrow (Plt2 40±20 compared to wild-type (WT) animals 28±8; n=7) and spleen (Plt2 16±6 compared to WT 8±3; n=7, p=0.01). Mature megakaryocytes were increased in frequency in bone marrow sections (Plt2 86.8±28.4 compared to WT 56.8±18.5) and demonstrate a similar maturation profile to control animals when their nuclear ploidy is measured by flow cytometry.

Reciprocal bone marrow transplantation experiments demonstrate that the thrombocytotic phenotype was observed in Plt2 homozygous recipients of either wild type or Plt2 homozygous bone marrow, but not observed in wild type recipients of Plt2 homozygous bone marrow. This data indicates that the Plt2 mutation is acting extrinsically on the haemopoietic system. Despite having an elevated platelet count, Plt2 mutant mice have inappropriately elevated serum thrombopoietin levels (Plt2 5420±2842; n=15) compared to controls (2947 ±2310; n=13; p<0.0001).

Mapping experiments have excluded both thrombopoietin and the thrombopoietin receptor, c-mpl, as the mutated gene. Further experiments will aim to map this mutation, which may be a novel regulator of thrombopoietin.