Primary platelet adhesion receptors

Thrombotic diseases such as heart attack and stroke remain a major health concern in the Western world despite existing anti-thrombotic drugs. Current studies are revealing structure-function relationships of primary platelet adhesion receptors mediating adhesion, activation and aggregation, and the molecular mechanisms underlying platelet thrombus formation. Platelet adhesion is relevant not only to thrombotic disease, but there is increasing evidence of a specific role for platelets in vascular processes such as inflammation and atherogenesis. This review focuses on recent advances in understanding the molecular basis for platelet thrombus formation, in particular the receptors, glycoprotein (GP)Ib-IX-V and GPVI, that initiate platelet adhesion and activation at high shear stress.     

Determination of ATP impurity in adenine dinucleotides

Adenine dinucleotides (ApnA) are extracellular signal molecules that are released from blood platelets, following stress, into the vascular system. The most abundant and best-characterized ApnA (Ap4A) interacts with a unique receptor on bovine aortic endothelial cells (BAEC) where it induces nitric oxide. Ap4A also interacts with P2 purinoceptors on BAEC to modulate Ca2+ mobilization and prostacyclin release; this behavior can be equally well explained by Ap4A being either a partial agonist to these receptors, or an antagonist in the presence of ATP contamination. To discern between these two possibilities, we have investigated the presence of such contaminants in ApnA preparations. The studies herein indicate that ApnAs (n = 3-6) contain ATP impurities; thus, when characterizing the ApnA interaction with ATP-binding sites, investigators must assure that the response elicited is not partly due to an ATP impurity. We here provide a means for detecting and estimating ATP impurities within Ap4A preparations while also eliminating them; the level of this contamination is estimated to be as low as 0.2%. We applied our method to distinguish the true effect of Ap4A at P2 purinoceptors; our findings are consistent with Ap4A acting as a partial agonist to these receptors. We also applied our method to characterizing the ApnA interaction with luciferase, and found that decontaminated ApnA (n = 4-6) are weak substrates for luciferase.     

Peptide mapping by capillary electrophoresis with Pluronic F127

Separation of peptides and proteins by capillary zone electrophoresis suffers from the interaction of these solutes with the capillary wall which results in the formation of broad peaks and low resolution. To minimize the protein/peptide-capillary wall interaction we tried to use Pluronic F127, a triblock copolymer of the general formula (polyethylene oxide)(x)(polypropylene oxide)(y)(polyethylene oxide)(z) when x=106, y=70 and z=106 which can be considered a surfactant capable of self-association both into isotropic and anisotropic gels. The analytes studied were enzymatic digests (obtained by trypsin or pepsin treatment) of insoluble matrix proteins from avian eggshell. The best separations were obtained by a system exploiting 10% Pluronic F127 in 20 mmol/l phosphate buffer, pH 2.5. Electrophoretic peptide profiles obtained were very complex owing to the complicated nature of the samples (the exact composition of the proteinous insoluble part of the eggshell is still unknown). The separation in phosphate buffer only offered complex maps of incompletely resolved peaks. The use of Pluronic F127 distinctly improved the separation with a considerably better resolution regarding both the number of peaks obtained and the quality of the separation.     

Synthesis and biological evaluation of thienopyrrolizines, a new family of CDK/GSK-3 inhibitors

Fifteen new thieno[2,3-b ]- and thieno[3,4-b]pyrrolizines were synthesized and tested against two protein kinases, CDK1/cyclin B and GSK-3. Among these compounds, 3-(3-hydroxy-4-methoxyphenyl)-8H-thieno[2,3-b]pyrrolizin-8-one 4g was identified as a moderate inhibitor of these kinases. Its molecular modeling study brought to the fore the pivotal role of the 2-methoxyphenol grouping and the interest in replacing it by bioisosteric moieties in future pharmacomodulations.     

Practical aspects of the 2D 15N-[1h]-NOE experiment

The heteronuclear ¹⁵N-NOE experiment was extensively tested with respect to statistical and systematic experimental error. The dependence of signal intensity in the NOE experiment and in the reference experiment on the saturation and relaxation time was experimentally investigated. The statistics of the experimental values were accessed by numerous repetitions of identical set-ups. As a model system a protein of typical size for NMR studies was chosen, i.e., a 120 amino acid residues containing fragment of the F-actin binding gelation factor (ABP-120). The fragment exhibits fast dynamics that are accessible with the ¹⁵N-NOE experiment with various amplitudes. The results of this study show that commonly used parameters are only adequate for accurate measurement of motions with moderate amplitude. Highly flexible parts require longer delay times and thus more experimental time than commonly used. On the other hand, a qualitative or semi-quantitative assessment of a protein's mobility on fast times scales can be obtained from rapidly recorded experiments with unusual short delay times. The findings of this study are of equal importance for highly accurate measurement of the ¹⁵N-NOE as well as for quick identification of mobile or even unstructured residues/parts of a protein.     

Intraplantar injection of glutamate evokes peripheral adenosine release in the rat hind paw: involvement of peripheral ionotropic glutamate receptors and capsaicin-sensitive sensory afferents

Glutamate receptors have been identified on the peripheral terminals of both primary sensory afferents and sympathetic post-ganglionic neurons, and activation of these receptors produces peripheral sensitization and enhances nociception. Adenosine is an endogenous agent that has a regulatory effect on pain. In brain and spinal cord, adenosine release can be promoted by excitatory amino acids. In the present study, we used in vivo microdialysis to determine whether glutamate also can release adenosine in peripheral tissues. Rats were anesthetized with pentobarbital and microdialysis probes were implanted into the subcutaneous tissue of the plantar aspect of the rat hind paw. Subcutaneous injection of glutamate (50 microL, 0.3-100 micromol) evoked a short-lasting adenosine release immediately following drug injection. Co-administration of either the N-methyl-D-aspartate (NMDA) receptor antagonist, dizocipine maleate (MK-801, 1 nmol) or the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline (CNQX, 10 nmol) with glutamate blocked such release, suggesting an involvement of peripheral ionotropic glutamate receptors in this response. Systemic pre-treatment with capsaicin, a neurotoxin selective for unmyelinated sensory afferents, significantly reduced glutamate-evoked peripheral adenosine release, but release was not affected by systemic pre-treatment with 6-hydroxydopamine, a neurotoxin selective for sympathetic nerve efferents. Neither MK-801 nor CNQX blocked 5% formalin-evoked adenosine release, suggesting adenosine release by formalin is not secondary to ionotropic glutamate receptor activation. We conclude that administration of glutamate evokes peripheral adenosine release, and that peripheral ionotropic glutamate receptors on unmyelinated sensory afferents are involved in such release. The released adenosine may provide a negative feedback control on nociception.     

Analysis of binding of mannosides in relation to Langerin (CD207) in Langerhans cells of normal and transformed epithelia

Tandem-repeat C-type lectins (pattern-recognition receptors) with specificity for mannosides are intimately involved in antigen recognition, uptake, routing and presentation in macrophages and dendritic cells. In Langerhans cells, Langerin (CD207), a type-II transmembrane protein with a single C-type carbohydrate recognition domain attached to a heptad repeat in the neck region, which is likely to establish oligomers with an -coiled-coil stalk, has been implicated in endocytosis and the formation of Birbeck granules. The structure of Langerin harbours essential motifs for Ca²⁺-binding and sugar accommodation. Lectin activity has previously been inferred by diminished antibody binding to cells in the presence of the glycan ligand mannan. In view of the complexity of the C-type lectin/lectin-like network, it is unclear what role Langerin plays for Langerhans cells in binding mannosides. In order to reveal in frozen tissue sections to what extent mannose-binding activity co-localizes with Langerin, we have used a synthetic marker, i.e. a neoglycoprotein carrying mannose maxiclusters, as a histochemical ligand, and computer-assisted fluorescence monitoring in a double-labelling procedure. Mannoside-binding capacity was detected in normal epithelial cells. Double labelling ensured the unambiguous assessment of the binding of the neoglycoprotein in Langerhans cells. Light-microscopically, its localization profile resembled the pattern of immunohistochemical detection of Langerin. This result has implications for suggesting rigorous controls in histochemical analysis of this cell type, because binding of kit reagents, i.e. mannose-rich glycoproteins horseradish peroxidase or avidin, to Langerin (or a spatially closely associated lectin) could yield false-positive signals. To show that recognition of carbohydrate ligands in dendritic cells is not restricted to mannose clusters, we have also documented binding of carrier-immobilized histo-blood group A trisaccharide, a ligand of galectin-3, which was not affected by the presence of a blocking antibody to Langerin. Remarkably, access to the carbohydrate recognition domain of Langerin appeared to be impaired in proliferatively active environments (malignancies, hair follicles), indicating presence of an endogenous ligand with high affinity to saturate the C-type lectin under these conditions.