Forced dissociation of selectin-ligand bonds is crucial to such biological processes as leukocyte recruitment, thrombosis formation, and tumor metastasis. Although the bond rupture has been well known at high loading rate r(f) (>or=10(2) pN/s), defined as the product of spring constant k and retract velocity v, how the low r(f) (<10(2) pN/s) or the low k regulates the bond dissociation remains unclear. Here an optical trap assay was used to quantify the bond rupture at r(f)

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Two-dimensional (2D) kinetics of receptorligand interactions governs cell adhesion in many biological processes. While the dissociation kinetics of receptorligand bond is extensively investigated, the association kinetics has much less been quantified. Recently receptorligand interactions between two surfaces were investigated using a thermal fluctuation assay upon biomembrane force probe technique (Chen et al. in Biophys J 94:694-701, 2008). The regulating factors on association kinetics, however, are not well characterized. Here we developed an alternative thermal fluctuation assay using optical trap technique, which enables to visualize consecutive bindingunbinding transition and to quantify the impact of microbead diffusion on receptor-ligand binding. Three selectin constructs (sLs, sPs, and PLE) and their ligand P-selectin glycoprotein ligand 1 were used to conduct the measurements. It was indicated that bond formation was reduced by enhancing the diffusivity of selectin-coupled carrier, suggesting that carrier diffusion is crucial to determine receptor-ligand binding. It was also found that 2D forward rate predicted upon first-order kinetics was in the order of sPs [ sLs [ PLE and bond formation was history-dependent. These results further the understandings in regulating association kinetics of surface-bound receptor-ligand interactions.