Vascular elevation of SDF-1 by IV administration acutely promotes the association of MKs with vasculature and thrombopoiesis. (A) MKP, MK, and platelet kinetics 24 hours after 400 ng IV SDF-1 (blue) or vehicle control (gray). SDF-1-treated mice have an acute increase in 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. Mean absolute numbers for vehicle controls: 16 905 MKP/femur, 45 939 MK/femur, 430 × 103 platelets/μL. (B) MFI of thiozole orange (TO) in platelets identified as CD41+Ter119- by imaging flow cytometry 24 hours after IV SDF-1 or vehicle. Platelets from SDF-1–treated mice have increased TO staining. (C) The proportion of exhausted MKs was identified by imaging flow cytometry and presented as percent of vehicle control for each experiment. Mean proportion of exhausted MKs for vehicle controls: 0.14. (D) Representative images of femoral marrow immunohistochemistry (IHC) for Gp1Bβ (MKs, red) and MECA32 (vascular endothelium, green) 24 hours after IV treatment with vehicle (left panel) or SDF-1 (right panel). White arrows indicate examples of MKs physically associated with MECA32+ vessels. Some MKs express the pan-endothelial antigen recognized by MECA32.57 Images were processed as described in the “Immunohistochemistry” section. (E) Quantification of Gp1Bβ+ MKs physically associated with MECA32+ vessels by manual counting of IHC 1 hour and 24 hours after IV SDF-1 (blue) or vehicle (gray). IV SDF-1 acutely increases MK association with the vasculature. (F) MFI/A of surface CXCR4 on primary MKs by imaging flow cytometry of flushed marrow samples prepared 1 hour and 24 hours after IV SDF-1 (blue) or vehicle (gray). (G) MFI/A of surface CXCR4 on in vitro–derived MKs 1 hour after SDF-1 treatment (blue) by imaging flow cytometry. SDF-1 treatment increases MK surface CXCR4 both in vivo (F) and in vitro (G). Error bars represent standard error of the mean of ≥3 independent experiments (n = 6-18 total mice per group). Statistical analyses comparing SDF-1 to vehicle controls were performed using a 2-tailed Student’s t test. Bar represents 100 μm (D). *P < .04; **P < .005.
