title: Kinetic analysis of heparin and glucan sulfates binding to P-selectin and its impact on the general understanding of selectin inhibition.
abstract: P-Selectin, expressed on activated endothelial cells and platelets, is a high kinetic adhesion receptor involved in leukocyte rolling of the inflammatory response, or in tumor cell binding in the course of metastasis. Thus, P-selectin inhibition is a promising therapeutic target. The anti-inflammatory and anti-metastatic activities of heparin have partly been related to the inhibition of P-selectin binding. Here we apply a quartz crystal microbalance (QCM) biosensor to determine the kinetic constants of heparin and other sulfated polysaccharides binding to immobilized P-selectin. Binding kinetics of the derivatives were correlated with their inhibitory capacity in a P-selectin cell rolling assay. Three commercial heparins differ in cell rolling inhibition and display slightly different affinities (KD 1.21 x 10(-6) M to 5.86 x 10(-7) M). Inhibitory capacity appears to be mainly driven by a slow off-rate from the receptor (2.27 x 10(-3) s-1 to 1.23 x 10(-3) s-1). To correlate the impact of binding kinetics on inhibitory capacity structurally, we analyzed six semisynthetic glucan sulfates. They display different degrees of sulfation (DS), which has a strong influence on inhibitory activity. Kinetic data illustrate that the inhibitory capacity correlates excellently with the off-rate of these polysaccharides (R = 0.99), while the association (on-rate) affects activity to a lesser extent. In general, the consideration of binding kinetics sheds new light on the mechanism of selectin inhibition. A much slower dissociation of the inhibitors from the receptor than the physiological ligands is key for inhibitory capacity. Structurally, highly charged compounds with a slow off-rate, such as heparin or glucan sulfates, appear as potent candidates for P-selectin inhibition.
authors: Department of Pharmacy, Rheinische Friedrich Wilhelms University Bonn, An der Immenburg 4, 53121 Bonn, Germany.
authors: Dirk
authors: D
authors: Simonis
authors: Juliane
authors: J
authors: Fritzsche
authors: Susanne
authors: S
authors: Alban
authors: Gerd
authors: G
authors: Bendas
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D002448:Cell Adhesion / Q000502:physiology; D002464:Cell Migration Inhibition; D002465:Cell Movement / Q000502:physiology; D005936:Glucans / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019007:P-Selectin / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism*; D011791:Quartz / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020298:U937 Cells
chemical_list: D005936:Glucans; D008024:Ligands; D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011509:Proteoglycans; D011994:Recombinant Proteins; C036090:heparin proteoglycan; D011791:Quartz; C078962:glucan sulfate; D006493:Heparin