Affinity and kinetics of P-selectin binding to heparin

Jian-Guo Wang; Jian-Guo Geng

doi:10.1160/TH03-01-0045

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2003; 90(02): 309-316
DOI: 10.1160/TH03-01-0045

Platelets and Blood Cells

Schattauer GmbH

Affinity and kinetics of P-selectin binding to heparin

Jian-Guo Wang

¹Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

,

Jian-Guo Geng

¹Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

› Institutsangaben

Abstract

Summary

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 (IC₅₀ = 3 µM heparin) and neutralized the binding of P-selectin to A375 cells (IC₅₀ = 4 µM 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 × 10^-3 s^-1 and the calculated on rate (k _on) was 2.75 × 10⁴ 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.

Keywords

P-selectin - heparin - heparan sulfate proteoglycans - affinity - kinetics

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Referenzen

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