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A.O. Khan et al.
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Figure 6. Induced pluripotent stem cell megacaryocytes and platelets differentially express TTLL and CCP enzymes to regulate polymodifications during megakaryocyte maturation and platelet production. The loss of tubulin tyrosine ligase like (TTL) TTLL10 is possibly linked to a bleeding phenotype in unrelated human patients. (A) RNA was generated from induced pluripotent stem cell megacaryocytes (iPSC-MK) at different stages of termi- nal differentiation. Day 1 (d1) day 5 (d5) day 5 cells treated with heparin to induce proplatelet formation (d5+Hep) were used to determine whether an upregulation of these enzymes is evident on platelet production. (B) Samples were amplified with house- keeping GAPDH primers (C) A number of TTLL family enzymes were observed, including TTLL1, 2, 3, 4, 5, 6, 7, 10, and 13. Of these, a number appeared to be upregulated in mature and pro- platelet forming cells. (D and E) These samples were taken for- ward and expression was quantified over multiple differentiations using the ΔΔCT method TTLL1, 2, 4, and 10 expression was found to be significantly upregulated on treatment with heparin (**P= 0.0081, *P= 0.0105, *P= 0.0260, ***P=0.0004 respectively). (F to H) A similar panel was performed on cytosolic carboxypepti- dase (CCP) enzymes which reverse polymodifications, with expres- sion of CCP1, 3, 4, 5, and 6 was observed. Statistically significant upregulation of CCP4, and CCP6 were observed on proplatelet production (*P=0.0130, ***P=0.0009). (I) In resting (-) and C- reactive protein (CRP) activated (+) platelets from three healthy donors TTLL7 was the only modifying enzyme found to be consis- tently expressed. (J) Three unrelated families were identified with- in the Genotyping and Phenotyping of Platelets Study (GAPP) cohort, two with frameshift truncations and one with a missense substitution (p.Pro15Argfs*38, p.Val249Glyfs*57, and p.Arg340Trp respectively, and as shown on the protein schemat- ic of TTLL10). (K) All three families present with normal platelet counts and function, but demonstrate an elevated mean platelet volume (MPV) and consistent histories of bleeding including cuta- neous bruising and menorrhagia. (L) Patient A:1:1 was recalled and demonstrated abnormally large platelets on spreading on fib- rinogen when imaged by widefield and single molecule localisa- tion microscopy (tubulin staining, 10 μm scale bar, 5 μm in cropped image). (n=3, standard deviation. Two-Way ANOVA with multiple comparisons performed. Complete unedited gels found in the Online Supplementary Figures S9 and S10).
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haematologica | 2022; 107(1)