CLEC-2/PDPN interaction promoted megakaryocyte expansion. (A) Blood platelet numbers in CLEC-2 WT and cKO mice. CLEC-2 WT, n = 19; CLEC-2 cKO, n = 22. (B) CLEC-2 expression pattern during megakaryocyte differentiation. BM- or fetal liver-derived megakaryocytes were differentiated with TPO in vitro. CLEC-2 expression pattern was validated by ICC. (C) Quantification of BM megakaryocytes in CLEC-2 WT and cKO mice by FACS. The graph on the left indicates the total numbers of CD41⁺ megakaryocytes. The graphs in the middle and on the right show the numbers of megakaryocyte subsets, CD41⁺CD42b⁻ (immature megakaryocytes) and CD41⁺CD42b⁺ (mature megakaryocytes), respectively. CLEC-2 WT, n = 8; CLEC-2 cKO, n = 11. (D-E) Quantification of megakaryocyte expansion by the MegaCult-C colony-formation assay. (D) Autonomous expansion in CLEC-2 WT and cKO megakaryocytes, n = 3 per group. (E) Stimulated expansion with recombinant PDPN (PDPN-rFC2, 20 μg/mL), n = 4 per group. The error bars reflect SEMs. *P < .05. **P < .01. N.S., a nonsignificant difference.