Figure 3. Loss of function at the Tpm4 locus causes macrothrombocytopenia in mice.

(A) Schematic representation of the Plt53 mutation (A to G substitution at nucleotide g.72,147,268) in the first dinucleotide of the donor splice site in exon 7 of Tpm4, which is predicted to result in a protein bearing 8 intron-encoded amino acids followed by premature truncation due to the presence of an in-frame stop codon at nucleotide g.72,147,291. (B) Top panel: Use of an antibody directed against the TPM4 C-terminus (δ/9d, AB5449, Millipore) demonstrates that TPM4.2 is not expressed in Tpm4^Plt53 platelets. Middle panel: Use of an antibody directed against the TPM4 N-terminus (δ/1b) reveals residual expression of a truncated TPM4.2 protein from the Tpm4^Plt53 allele (orange arrow). Bottom panel: β-Actin loading control. Results are representative of 3 independent experiments. (C) The Plt53 mutation causes macrothrombocytopenia. Reduced platelet count and increased platelet size in Tpm4^Plt53/+ and Tpm4^Plt53/Plt53 mice compared with Tpm4^+/+ mice (n = 17). (D) Platelet count and size in Tpm4.2 WT (+/+), heterozygous (+/–), and homozygous knockout (–/–) mice (n = 10–15) on a C57BL/6 background. Measurements were performed using an Advia hematology analyzer. One-way ANOVA, unpaired 2-tailed Student’s t test with Bonferroni correction for multiple comparisons. *P < 0.05, ***P < 0.001.