CRLF3 deficiency causes sustained and isolated reduction in platelet count. (A) Platelet counts of male (n = 5-23) and female (n = 5-14) young (12-20 weeks), middle aged (21-40 weeks), and old (>48 weeks) control (WT; blue) and Crlf3^−/− (red) mice. (B) Expression of Crlf3 relative to Gapdh messenger RNA (mRNA) determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) of control (WT; blue) and Crlf3^−/− (red) isolated from in vitro cultured MKs (n = 2). (C) Western blot of platelet lysates against CRLF3 (green) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; red; n = 2). (D) Platelet counts pre- (left; n = 15 WT and 14 Crlf3^−/−) and 16 weeks post–BM transplantation (BMT; right) of control (WT; circles) and Crlf3^−/− (squares) recipient mice that received either control (WT; blue) or Crlf3^−/− (red) donor cells (n = 8 WT → WT; n = 7 all other groups). (E) Chimerism was estimated by expression of Crlf3 relative to Gapdh mRNA isolated from in vitro cultured MKs by qRT-PCR (n = 8 WT → WT; 7 all other groups). (F) Quantification of MKs in hematoxylin and eosin–stained sections of control (WT; blue) and Crlf3^−/− (red) tibias (n = 6). (G) TPO concentration determined by enzyme-linked immunosorbent assay in control (WT; blue) and Crlf3^−/− (red) plasma (n = 5 WT and 6 Crlf3^−/−). (H) Percentage of CD41⁺ cells from control (WT; blue) and Crlf3^−/− (red) in vitro MK cultures (n = 3). (I) Polyploidy of in vitro cultured control (WT; blue) and Crlf3^−/− (red) MKs analyzed by flow cytometry (n = 5). (J) Mature in vitro cultured MKs were purified by bovine serum albumin gradient, seeded onto fibrinogen-coated coverslips and incubated at 37°C for 5 hours to induce proplatelet formation. Fixed samples were stained with CD41 (green) and DAPI (blue) and imaged by fluorescence microscopy. Images are representative of Crlf3^−/− and control (WT) proplatelet-forming MKs. Scale bars are 50 μm. Proplatelet morphology of control (WT; blue) and Crlf3^−/− (red) MKs was assessed by blindly quantifying the number of protrusions per proplatelet-forming MK and number of branches per protrusion (n = 29 WT and 31 Crlf3^−/−). (K) In vitro cultured MKs were seeded onto fibrinogen-coated coverslips and incubated at 37°C for 3 or 5 hours to induce proplatelet formation. After confocal microscopy, percentage of proplatelet forming MKs was determined for control (WT; blue) and Crlf3^−/− (red; n = 3). At least 460 MKs were counted in each condition. (L) Control (WT; blue) and Crlf3^−/− (red) animals were injected with phosphate-buffered saline (PBS; circles) or anti-CD42b (0.6 μg/g body weight; squares) and platelet counts determined by automated hemocytometer 0, 24, 48, 72, and 96 hours postinjection (n = 4 Crlf3^−/− plus CD42b antibody [Ab]; n = 3 all other groups). (M) Control (WT; blue) and Crlf3^−/− (red) mice were injected with 1 mg of NHS-biotin, and percentage of CD41⁺/Ter119⁻/streptavidin⁺ platelets was determined by flow cytometry at 24, 48, 72, 96, and 168 hours postinjection. Percentage of streptavidin⁺ platelets at 24 hours represents 100% biotin-bound platelets (n = 5). Data represent means ± standard deviations. Unpaired 2-tailed Student t test (F-H,J) with correction for multiple comparisons using the Holm-Sidak method (A), 1-way analysis of variance (ANOVA) (D,E), or 2-way ANOVA (I,K-M) with correction for multiple comparisons using the Holm-Sidak method. *P < .05, **P < .01, ***P < .005. HPF, high-powered field; ns, not significant.