Native SDF-1 enhances MKs in the vascular niche and increases circulating platelets. (A) MKP, MK, and platelet kinetics 24 hours after 1.7 mg (5 μmol) intraperitoneal Diprotin A (red) or vehicle control (gray). Stabilization of SDF-1 with Diprotin A acutely increases circulating platelets with no change in MK or MKP number in the marrow. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments expressed as percent of vehicle control. Mean absolute numbers for vehicle controls: 11 593 MKP/femur, 49 856 MK/femur, 436 × 10³ platelets/μL. (B) MFI/A of surface CXCR4 on primary MKs by imaging flow cytometry of flushed marrow samples prepared 1 hour after treatment with Diprotin A (red) or vehicle control (gray). (C) Quantification of Gp1Bβ+ MKs physically associated with MECA32+ vessels by IHC 1 hour and 24 hours following treatment with Diprotin A (red) or vehicle (gray). Diprotin A-mediated stabilization of SDF-1 acutely increases MK association with the vasculature. (D) Representative images of femoral marrow IHC for Gp1Bβ (MKs, red) and MECA32 (vascular endothelium, green) 24 hours after administration of vehicle (left panel) or Diprotin A (right panel). White arrows indicate examples of MKs physically associated with MECA32+ vessels. Images were processed as described in the “Immunohistochemistry” section. Error bars represent standard error of the mean of ≥3 independent experiments (n = 7-8 total mice per group). Statistical analyses comparing Diprotin A to vehicle controls were performed using a 2-tailed Student’s t test. Bar represents 100 μm (D). *P < .02; **P < .003.