Figure 3.
Effect of the p.Arg359Trp β1-tubulin variant in platelets and megakaryocytes. The functional and structural consequences of p.Arg359Trp β1-tubulin variant were assessed in members of Pedigree F. Immunofluorescence analysis of β1-tubulin was performed in platelets on poly-l-lysine–coated coverslips under (A) resting (platelet-rich plasma) and (B) spreading (washed platelets) conditions from p.Arg359Trp carriers with (II.1 and III.1) or without thrombocytopenia (III.2), a noncarrier (III.3), and an unrelated healthy control. Platelets were labeled with a β1-tubulin antibody (red) and with fluorescein isothiocyanate-phalloidin (green). Scale bars are 5 μm, and the objective lens is 63×. (C) Bar plot showing the percentage of spread platelets relative to total adhered platelets. Data are means ± SD of values obtained from at least 10 different microscopy fields in patient and control samples. (D) Representative Western blot picture of β1-tubulin levels in platelet lysates from p.Arg359Trp carriers with or without thrombocytopenia, a noncarrier, and 2 healthy controls, using β-actin as internal control. (E) Illustrative images of CD34+ peripheral blood cell–derived megakaryocytes in patients from Pedigree F (p.Arg359Trp carriers with or without thrombocytopenia and a noncarrier) and healthy controls. MKs were labeled with β1-tubulin (green) and rhodamine-phalloidin (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images were acquired in a Carl Zeiss Axio Observer A1 fluorescence microscope with a 63× objective lens. Scale bars are 5 μm. (F) Classification of MKs in family members and controls. Polynucleated cells extending protrusions with terminal tips were considered as proplatelet-forming MKs, whereas those displaying a flattened shape with actin organized in focal adhesion points and fibers as spread MKs. Some MKs were small and isolated, whereas MKs that were present in aggregates of 2 or more MKs were defined clusters. At least 100 MKs from 5 different replicates were analyzed; #P < .05 vs TCP carriers. (G) Microtubule length in CD34+ peripheral blood cell–derived MKs from controls and patients with the p.Arg359Trp β1-tubulin variant was assessed using the ImageJ software. Data are mean ± SD of values obtained from at least 6 different microscopy fields. TCP, thrombocytopenic; non-TCP, nonthrombocytopenic. **P < .005.

Effect of the p.Arg359Trp β1-tubulin variant in platelets and megakaryocytes. The functional and structural consequences of p.Arg359Trp β1-tubulin variant were assessed in members of Pedigree F. Immunofluorescence analysis of β1-tubulin was performed in platelets on poly-l-lysine–coated coverslips under (A) resting (platelet-rich plasma) and (B) spreading (washed platelets) conditions from p.Arg359Trp carriers with (II.1 and III.1) or without thrombocytopenia (III.2), a noncarrier (III.3), and an unrelated healthy control. Platelets were labeled with a β1-tubulin antibody (red) and with fluorescein isothiocyanate-phalloidin (green). Scale bars are 5 μm, and the objective lens is 63×. (C) Bar plot showing the percentage of spread platelets relative to total adhered platelets. Data are means ± SD of values obtained from at least 10 different microscopy fields in patient and control samples. (D) Representative Western blot picture of β1-tubulin levels in platelet lysates from p.Arg359Trp carriers with or without thrombocytopenia, a noncarrier, and 2 healthy controls, using β-actin as internal control. (E) Illustrative images of CD34+ peripheral blood cell–derived megakaryocytes in patients from Pedigree F (p.Arg359Trp carriers with or without thrombocytopenia and a noncarrier) and healthy controls. MKs were labeled with β1-tubulin (green) and rhodamine-phalloidin (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images were acquired in a Carl Zeiss Axio Observer A1 fluorescence microscope with a 63× objective lens. Scale bars are 5 μm. (F) Classification of MKs in family members and controls. Polynucleated cells extending protrusions with terminal tips were considered as proplatelet-forming MKs, whereas those displaying a flattened shape with actin organized in focal adhesion points and fibers as spread MKs. Some MKs were small and isolated, whereas MKs that were present in aggregates of 2 or more MKs were defined clusters. At least 100 MKs from 5 different replicates were analyzed; #P < .05 vs TCP carriers. (G) Microtubule length in CD34+ peripheral blood cell–derived MKs from controls and patients with the p.Arg359Trp β1-tubulin variant was assessed using the ImageJ software. Data are mean ± SD of values obtained from at least 6 different microscopy fields. TCP, thrombocytopenic; non-TCP, nonthrombocytopenic. **P < .005.

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