EWVDV-containing peptides inhibit HSO₃-Le^a binding to human P-selectin.

Competition studies of HSO₃-Le^a-PAA-biotin (0.3 μg/mL) binding to P-selectin by synthetic peptides were performed in an ELISA. (A) HSO₃-Le^a-PAA-biotin binding is inhibited by blocking antibodies AK-4, WASP 12.2, and by rPSGL-Ig, but not by non–blocking antibody AC1.2. (B) Binding of HSO₃-Le^a-PAA-biotin is inhibited by EWVDV-containing peptides, but not by control peptides. IC₅₀ values are dependent on the length of the peptide. Bold letters indicate the amino acids that differ from the original TM11 sequence. (○) EWVDV, IC₅₀ = 158 μM; (▵) EWVKV; (▴) VEWVDV, IC₅₀ = 119 μM; (■) DVEWVDVS, IC₅₀ = 32 μM; (♦) DVEAVDVS; (●) CDVEWVDVSC, IC₅₀ = 7.6 μM. (C) Lineweaver-Burke plot of HSO₃-Le^a-PAA-biotin binding in the presence and absence of 50 μM CDVEWVDVSC. Linear regression analysis reveals that both curves cross the y-axis through the same point (E450 = 1.01 ± 0.01 for control and 1.08 ± 0.03 for inhibitor) indicative of competitive inhibition.