Pitpα^−/−/β^−/−MKs have abnormal α-granule biogenesis and defects in α-granule storage. (A) Electron microscope analysis of pitpα^−/−/β^−/− MKs showing α-granules (Ai-iii). WT MKs have electron-dense α-granules at 15 000× (Ai), while pitpα^−/−/β^−/− MKs have enlarged, empty α-granules at 15 000× (Aii) and 40 000× (magnified view; Aiii). Arrows point to α-granules. WT MKs contain electron-dense MVBs (Aiv), while pitpα^−/−/β^−/− MKs have enlarged, less electron-dense MVBs (Av). An example of massively enlarged MVBs (arrows) in pitpα^−/−/β^−/− MKs (Avi). While not representative, MVBs of this size were noted occasionally. These are significantly larger compared with a typical enlarged pitpα^−/−/β^−/− MK MVB (red arrowheads). All scale bars represent 500 nm. (B-C) ELISA quantification for TSP1 (B) and vWF (C) in MK CM. *P < .05, **P < .005, and ***P < .0005 when compared with control group as determined by Student t test. (D) Western blot analysis of pitpα^−/−/β^−/− MKs lysates probing for major α-granule components, TSP-1 and vWF. (E) Levels of TGF-β precursor propeptide and cleaved TGF-β monomers in pitpα^−/−/β^−/− and pitpα/β WT littermate control MKs lysates were examined by western blot. (F) Densitometry quantification of TGF-β expression detected by western blot (n = 3). (G) Semiquantitative reverse-transcription PCR analysis of TGF-β1 and TGF-β2 expression in littermate control and pitpα^−/−/β^−/− MKs.