Impaired proplatelet formation of DKO MKs in vitro and in vivo. (A) PPF of BM MKs after lineage depletion and culturing in rHirudin- and TPO-conditioned medium. Proplatelet-forming MKs were counted after enrichment of MKs using a body surface area density gradient. Average of 5 analyzed visual fields per MK culture is shown. Values are mean ± SD (n = 3). Unpaired, 2-tailed Student t test. *P < .05. (B) Intravital 2-photon microscopy of BM MKs in the skull. Blood vessels were visualized with body surface area-FITC and CD105 Alexa F488. MKs were stained with an anti-GPIX antibody derivative conjugated to Alexa F546. Insets and arrows point to proplatelet shafts reaching into sinusoidal vessels. Images are representative of at least n = 5. Scale bars, 50 µm; insets, 20 µm. BM MKs were centrifuged onto glass slides. (C) Proplatelets were visualized using an α-tubulin antibody and phalloidin and analyzed by confocal microscopy (40× objective, Leica TCS SP8) using a 40× objective. Scale bars, 20 µm (upper panels) and 50 µm (lower panels). (D) Quantification of platelets tips per MK and platelet tip size of in vitro–matured WT and DKO MKs. Values are mean ± SD (n = 3). Unpaired, 2-tailed Student t test. ***P < .001.