Human CB HSC specifically bind to the cryptic site in N-terminal trOPN via α₉β₁ and α₄β₁ and is found endogenously bound to CB HSC. (A) The peptide, SVVYGLR-FITC, representing the cryptic site in human trOPN, was used to demonstrate specific interactions between N-terminal trOPN and human HSC/HPC (CD34⁺ cells; red line) compared with the scrambled peptide control (black line). Blocking antibodies specific to α₉β₁ (orange line) and α₄ (green line) demonstrated OPN binding via both α₉β₁ and α₄β₁. Data are representative of 6 biologic repeats. (B) Similarly, the peptide, SLAYGLR-FITC, representing the cryptic site in murine trOPN, was demonstrated specific interactions between N-terminal trOPN and murine HSC/HPC (LSK cells; red line) compared with the scrambled peptide control (black line). (C) CD34⁺CD38⁺ and CD34⁺CD38⁻ human CB cell lysates were immunoprecipitated using specific α₄ and α₉β₁ antibodies and bound proteins immunoblotted with anti-OPN antibody (LF-124). An N-terminal cleaved form of OPN (approximately 32 kDa) is bound to both α₄β₁ and α₉β₁ on CB CD34⁺CD38⁺ cells (→, lanes 1 and 4) and CD34⁺CD38⁻ (→, lanes 2 and 5). No staining is seen with CD34⁺CD38⁺ lysates immunoprecipitated using uncoupled beads and immunoblotted with LF-124 (lanes 3 and 6). (D) The GFP⁺ MSCV vector was used to transduce CHO cells to express human α₄. (E) Parental α₉⁺ (black) or GFP⁺ α₄⁺ (green) CHO cells expressed equivalent levels of integrin when labeled with individual antibodies. (F) CHO cells expressing human α₉β₁ (GFP⁻, blue) or human α₄β₁ (GFP⁺, blue) were used in a 50:50 mix to determine the binding of OPN cleaved by MMP-3 (F), MMP-7 (G), or thrombin (H). There was no evidence of MMP-3 or MMP-7 cleaved OPN binding, however, trOPN bound equivalently to both integrins and in a dose-dependent manner (red, 30 μg/mL; green, 7.5 μg/mL; and black, secondary alone).