Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/Rf ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments.

Thrombolytic along with anti-platelet activity of crinumin, a protein constituent of Crinum asiaticum.

Several anticoagulants, anti-platelet and thrombolytic medications are used for the treatment of thrombotic disorders. Anti-coagulants and anti-platelet agents prevent the formation of blood clots but do not dissolve existing clots, whereas thrombolytic agents are able to dissolve a clot but emboli can form even after successful treatment. Thus, none of them provide a permanent and complete solution. In this regard a single molecule that could both dissolve the clot and prevent the formation of new clots would be useful in the treatment of thrombotic diseases. Crinumin, a stable and active (in many adverse conditions) serine protease, shows plasmin-like fibrinolytic activity and inhibits platelet aggregation and P-selectin exposure, as established by photography, phase contrast microscopy, whole blood optical Lumi-aggregometry and flow cytometry. Crinumin could be an efficient and inexpensive therapeutic agent for the treatment and prevention of thromboembolic diseases.

Strategies for generating less toxic P-selectin inhibitors: pharmacophore modeling, virtual screening and counter pharmacophore screening to remove toxic hits.

This paper describes the generation of ligand-based as well as structure-based models and virtual screening of less toxic P-selectin receptor inhibitors. Ligand-based model, 3D-pharmacophore was generated using 27 quinoline salicylic acid compounds and is used to retrieve the actives of P-selectin. This model contains three hydrogen bond acceptors (HBA), two ring aromatics (RA) and one hydrophobic feature (HY). To remove the toxic hits from the screened molecules, a counter pharmacophore model was generated using inhibitors of dihydrooratate dehydrogenase (DHOD), an important enzyme involved in nucleic acid synthesis, whose inhibition leads to toxic effects. Structure-based models were generated by docking and scoring of inhibitors against P-selectin receptor, to remove the false positives committed by pharmacophore screening. The combination of these ligand-based and structure-based virtual screening models were used to screen a commercial database containing 538,000 compounds.

Anti-platelet effects of bioactive compounds isolated from the bark of Rhus verniciflua Stokes.

It has previously been shown that EtOAc extracts of Rhus verniciflua Stokes (RVS) inhibit the platelet aggregation response. In this report, bioassay-guided fractionation using ADP-, arachidonic acid-, and collagen-induced human platelet aggregation by a whole blood aggregometer yielded the bioactive compounds isomaltol and pentagalloyl glucose from different highly effective fractions. In addition, column chromatography of fractions from RVS yielded another five compounds: butin, fisetin, sulfuretin, butein and 3,4',7,8-tetrahydroxyflavone. We investigated the effects of bioactive compounds from RVS fractions on several markers of platelet activation using receptor expression on platelet membranes, including glycoprotein IIb/IIIa (CD41), GPIIb/IIIa-like expression (PAC-1) and P-selectin (CD62), and intracelluar calcium mobilization responses by flow cytometry in healthy subjects. Dose-dependent inhibition of platelet aggregation and significantly decreased platelet activation were observed for the isomaltol- and pentagalloyl glucose-treated platelets, respectively. These results show that isomaltol and pentagalloyl glucose from the bark of Rhus verniciflua Stokes have potent anti-platelet activity and emphasize the need to further examine the mechanism of these active compounds for platelet modulation.

Effects of Hypericum perforatum extract in a rat model of ischemia and reperfusion injury.

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants, and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidative stress and exaggerated production of reactive oxygen species play a major role in several aspects ischemia and reperfusion. Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Because polyphenolic compounds have high antioxidant potential, in this study we evaluated the effect of H. perforatum extract on splanchnic artery occlusion (SAO) shock-mediated injury. SAO shock was induced in rats by clamping the superior mesenteric artery and the celiac trunk for 45 min. After 1 h of reperfusion, SAO-shocked rats developed a significant fall in mean arterial blood pressure. Treatment of rats with H. perforatum extract (applied at 25 mg/kg 15 min before reperfusion) significantly reduced a significant fall in mean arterial blood pressure and the migration of polymorphonuclear cells caused by SAO-shock. H. perforatum extract also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase and malondialdehyde caused by SAO shock in the ileum. Immunohistochemical analysis for nitrotyrosine and for poly ADP-ribosylated proteins revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine and poly ADP-ribosylated proteins was markedly reduced in tissue sections obtained from SAO-shocked rats that had received H. perforatum extract. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for intercellular adhesion molecule-1 in the vascular endothelial cells. H. perforatum extract treatment markedly reduced the intensity and degree of P-selectin and intercellular adhesion molecule-1 in tissue section from SAO-shocked rats. H. perforatum extract treatment significantly improved survival. In conclusion, this study demonstrates that H. perforatum extract exerts multiple protective effects in splanchnic artery occlusion-reperfusion shock and suggests that H. perforatum extract may be a candidate for consideration as a therapeutic intervention for ischemia-reperfusion injury.

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin.

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.

Novel mimics of sialyl Lewis X: design, synthesis and biological activity of a series of 2- and 3-malonate substituted galactoconjugates.

A series of potent inhibitors of P-selectin as potential anti-inflammatory agents is reported. These compounds are derivatives of galactocerebrosides bearing a malonate side chain in positions 2 and 3 of the galactose moiety. Based on the binding mode of sialyl Lewis X, the two acidic groups of the malonate are designed to form ionic interactions with two important lysines in the active site of P-selectin, Lys113 and Lys111. On the other hand, the 4- and 6-hydroxy groups on the galactose ring are arranged to chelate the calcium ion in the P-selectin active site. The synthesis and the biological activity of this series of compounds are described. Lead compounds having a greater potency than sialyl Lewis X are identified.