Affinity and kinetics of P-selectin binding to heparin.

P-selectin (CD62P), expressed on stimulated endothelial cells and activated platelets, reacts with P-selectin glycoprotein ligand-1 (PSGL-1, CD162) for leukocyte rolling. It also binds to heparin and heparan sulfate proteoglycans (HSPGs), which attenuates P-selectin mediated adhesions of leukocytes and cancer cells. Here we report that P-selectin mediated adhesion, but not rolling, of the HSPGs bearing human malignant melanoma A375 cells under shear stress. To understand its underlying molecular mechanism, we measured the biophysical properties of this interaction. Heparin inhibited the adhesion of A375 cells to immobilized P-selectin under flow (IC(50) = 3 microM heparin) and neutralized the binding of P-selectin to A375 cells (IC50 = 4 microM heparin). Using surface plasmon resonance technique, we found that P-selectin bound to heparin with a dissociation constant (K(d)) of 115 +/- 6 nM. The measured off rate (k(off)) was 3.15 +/- 0.34 x 10(-3) s(-1) and the calculated on rate (k(on)) was 2.75 x 10(4) M(-1) s(-1). Taken together, our data suggest that the very slow k(off) and the reduced k(on), but apparently not the K(d), are responsible for adhesion, but not rolling of A375 cells, to P-selectin under flow.