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.

Heparin promotes platelet responsiveness by potentiating αIIbβ3-mediated outside-in signaling

“…34 Because of its net negative charge, heparin, like other glycosaminoglycans, is able to bind and concentrate growth factors and chemokines on the cell surface. 35 It has also been exploited to bind and purify numerous protein components of platelet ␣-granules, including von Willebrand factor, 36 fibronectin, 37 Pselectin, 38 thrombospondin, 39 vitronectin, 40 and PF4. 41 Interestingly, and relevant to this discussion, heparin has been shown to bind a number of transmembrane glycoproteins, including the Washed platelets were stirred at 1000 rpm at 37°C alone or in the presence of heparin with or without 6.67 g/mL eptifibatide, lysed, and subjected to immunoblot analysis as described in the legend to Figure 1.…”

Binding Properties of CD94 to Sulfated Glycans and α2,3-NeuAc- Containing Glycoproteins and Mutagenesis Analysis

“…GlcN6S in heparin is critical for interactions with P-and L-selectin [37]. P-selectin binds to heparin with a slow off-rate and a K d of 115 nM according to surface plasmon resonance [38], and 1210 to 580 nM according to quartz crystal microbalance [39].…”

Prospects for the therapeutic application of sulfated polysaccharides of brown algae in diseases of the cardiovascular system: review

“…Thus, Bachelet et al (2009) showed that fucoidan prevented P-selectin binding to sLeX. Simonis et al (2007) demonstrated the specificity of the interaction between P-selectin and fucoidan. Rinetic studies have shown that a much slower dissociation of the fucoidan from the receptor than the physiological ligands is key for inhibitory capacity.…”