VEGF-A is required for the activation of HSCs and MPP2 in response to acute thrombocytopenia. (A) Representative graphs of pVEGFR-2 on the surface of HSCs, MPP2, PreMegs, and MPP3/4 2 hours after platelet depletion. (B) Quantitative PCR analysis of Vegf-a expression in ECs (Ctrl., n = 11; Depl., n = 13) and Mks (Ctrl., n = 5; Depl., n = 4) 2 hours after platelet depletion. (C) Concentration of VEGF-A in the BM supernatant 2 hours after platelet depletion (Ctrl., n = 7; Depl., n = 13). (D) Mean fluorescence intensity (MFI) of pVEGFR-2 on Mks (Ctrl., n = 7; Depl., n = 7; minimum 60 Mks per condition) and BM-ECs (Ctrl., n = 6; Depl., n = 8). (E) Concentration of VEGF-A in the BM supernatant of Pf4-cre⁺; iDTR mice 2 hours after platelet depletion (Ctrl., n = 5; Depl., n = 5). (F) Concentration of PDGF-BB in the BM supernatant of mice treated with anti-VEGFR-2 antibody prior to platelet depletion. Samples were collected 2 hours after platelet depletion (Ctrl., n = 6; Depl., n = 5). (G) Concentration of total SCF in the BM of anti-VEGFR-2–treated mice 2 hours after platelet depletion (Ctrl., n = 6; Depl., n = 5). (H) Frequency of m-SCF⁺ Mks after PDGFRb blocking antibody treatment and platelet depletion at 2 hours, as analyzed by fluorescence activated cell sorting (Ctrl., n = 5; Depl., n = 5). (I) Cell cycle analysis of CD41⁻ HSCs after VEGFR-2 signaling blockade in control and thrombocytopenic mice. Anti-VEGFR-2 antibodies were injected 10 minutes prior to thrombocytopenia. Cells were analyzed 12 hours after platelet depletion (Ctrl., n = 6; Depl., n = 6; Ctrl.+blocking, n = 4; Depl.+blocking, n = 9).