Cleavage of osteopontin by thrombin has been reported to enhance cell adhesion. We asked whether thrombin could regulate the ␣ v ␤ 3 -mediated adhesion of platelets and B lymphocytes to this substrate. Although there was no difference in the extent or the avidity of thrombin-and ADP-stimulated platelet adhesion to intact or thrombin-cleaved human osteopontin, both the extent and avidity of phorbol ester-stimulated B cell adhesion to thrombin-cleaved osteopontin was significantly increased. Thus, these data suggest that the ability of ␣ v ␤ 3 to recognize osteopontin can be differentially regulated in a cell-specific manner. To localize the ␣ v ␤ 3 binding site on osteopontin, we measured cell adhesion to the two thrombin cleavage products of osteopontin and to a series of nested RGD-containing osteopontin peptides cross-linked to albumin. Whereas ADP-stimulated platelets adhered to the amino-terminal but not the carboxyl-terminal osteopontin fragment and to the osteopontin peptide RGDSVVYGLR, phorbol ester-stimulated B cells did not adhere to this peptide, although they did so in the presence of 1 mM Mn 2؉ . Thus, our data confirm that thrombin cleavage enhances the accessibility of the binding motif for ␣ v ␤ 3 on osteopontin, but this enhancement is also a function of the activation state of ␣ v ␤ 3 . Moreover, they indicate that the sequence RGDS-VVYGLR contains sufficient information to specify activation-dependent ␣ v ␤ 3 -mediated platelet and lymphocyte adhesion.The initial event in the formation of a hemostatic platelet plug or a platelet thrombus is platelet adhesion to components of the subendothelial matrix of a damaged blood vessel (1). The acidic phosphorylated glycoprotein osteopontin (OPN) 1 is a component of the subendothelial matrix of blood vessels involved by atherosclerosis, in which it surrounds areas of dystrophic calcification. We reported that platelets and B lymphocytes adhere to OPN-coated surfaces and that their adhesion is mediated by the integrin ␣ v ␤ 3 (2). Unlike other cells that adhere to OPN, however, the adhesion of platelets and B cells to OPN requires agonist stimulation (2), indicating that the activation state of ␣ v ␤ 3 on platelets and lymphocytes, like that of the homologous platelet integrin ␣ IIb ␤ 3 , is regulated by cellular agonists.The most potent physiologic platelet agonist is thrombin (3). It is noteworthy that OPN contains a potential thrombin cleavage site six amino acids downstream from an RGDS motif (4) and that thrombin cleavage has been reported to enhance the ability of OPN to support the attachment and spreading of a number of cultured cells in vitro (5). We postulated that not only might thrombin regulate the activation state of platelet ␣ v ␤ 3 , but it might also regulate the ability of OPN to serve as a substrate for platelet adhesion. To address this possibility, we cleaved recombinant human OPN with thrombin and compared the ability of the cleaved protein and the isolated cleavage products to support platelet adhesion. In addition, because agonis...

Anti-bacterial activities were compared for two series of voltage-dependent pore-formers: (i) alamethicin (Alm) and its synthetic analogs (Alm-dUL) where alpha-amino-isobutyric acid residues (Aibs) were replaced by leucines and selected key residues substituted and (ii) homologous voltage sensors of the electric eel sodium channel (repeats S4L45 (III) and S4L45 (IV)). Spiroplasma melliferum, a bacterium related to the mycoplasmas, was used as a target cell. The data show that with respect to growth inhibition, cell deformation and plasma membrane depolarization, the highest efficient peptide remained natural Alm although the minimal inhibitory concentrations of its Leu analogs were within the same range as the parent molecule, except for Alm-dUL P14A. Thus, as for the pore-forming activity observed in artificial membranes and for the toxicity towards mammalian cells, proline-14 proved to be a critical residue for the anti-bacterial activity of alamethicin. Regarding the sodium voltage sensors, their anti-bacterial efficiency was at least 10 times lower although they promoted spiroplasma cell agglutination. The anti-bacterial activities of the peptides were correlated with their pore-forming properties, especially with the apparent and mean number of monomers per conducting aggregate () when both peptide families were considered and, secondly, with mean open times (tau(o)) within each family. This suggests that although they may form 'raft-like' structures, the mechanism underlying anti-bacterial activity of Alm and its active analogs, as well as the S4L45 voltage sensors with the S. melliferum plasma membrane, is predominantly through pore-formation according to the 'barrel-stave' mechanism.

The emergence of combinatorial and parallel library synthesis as an important strategy for the generation and optimization of pharmaceutical lead structures has resulted in major and exciting new challenges in the field of synthetic methodology. [1] The preparation of libraries with significant structural and functional diversity requires synthetic reactions that are not only highly effective, but also generally applicable. Since solid-phase synthesis is the method of choice for the generation of libraries, the development of methods applicable to immobilized substrates adds yet another dimension to the challenge.Despite the enormous recent progress in solid-phase synthesis, [2] stereoselective reactions have played a relatively minor part. [3,4] A possible explanation is that absolute stereocontrol is not important in library synthesis. Indeed, if the goal is to generate as many compounds as possible and the screening process allows for the evaluation of mixtures and the deconvolution of individual components, then stereochemical control may not be necessary or even desirable. However, if both specificity and affinity for a target are sought, then evaluation of stereoisomerically pure compounds may be essential. For example, cyclic compounds that contain the amino acid sequence Arg-Gly-Asp (RGD) can switch specificities to different targets as a result of subtle structural and stereochemical variation of the peptide backbone. [5,6] With the goal of developing enantioselective methods for solidphase syntheses and highlighting their possible utility for lead discovery and optimization, we describe here the asymmetric catalytic ring opening of polymer-bound meso-epoxides with trimethylsilyl azide (TMSN 3 ), [7] and the transformation of the resulting products into stereochemically diverse templates for the synthesis of compounds with RGD pharmacophores.The reaction of the TentaGel S PHB-immobilized [8] epoxy ester 2 a with an excess of TMSN 3 in the presence of the [(salen)CrN 3 ] complex [9] (R,R)-1 proceeded to completion within 24 h, as monitored by IR spectroscopy (Scheme 1). The Scheme 1. Asymmetric ring opening of polymer-bound meso-epoxides catalyzed by [(salen)CrN 3 ] (1). product was isolated, excess reagents and catalyst were removed by filtration, and the product was desilylated with a dilute methanolic solution of trifluoroacetic acid to afford resin-bound azido alcohol 3 a. Transesterification with MeOH/DMF/Et 3 N (9/1/1) then provided soluble 4 a in greater than 95 % yield with 92 % ee, as established by 1 H NMR spectroscopy and gas chromatography. The scope of this solidphase asymmetric reaction was evaluated on a series of other meso-epoxides with five-membered rings (Scheme 2). In all cases, epoxide ring opening occurred cleanly to provide the corresponding azido alcohol derivatives with good to excellent enantiomeric excesses. O O O N O O O O O O O O O O O O O O O O O O O 91% ee 77% ee 92% ee 94% ee 85% ee 94% ee Scheme 2. Enantioselectivities of the asymmetric ring opening of some polymer-bound...

A 34-mer peptide, encompassing the S4 and S45 segments of domain IV of the electric eel voltage-dependent sodium channel, was synthesized in order to test the potential implication of S45 in the gating or permeation pathway. The secondary structure of peptide S4-S45 assessed by circular dichroism was found mainly helical, both in organic solvents and in lipid vesicles, especially negatively-charged ones. The macroscopic conductance properties of neutral and negatively-charged Montal-Mueller planar lipid bilayers doped with S4-S45 were studied and compared with those of S4. With regard to voltage-dependence, the most efficient system was S4-S45 in neutral bilayers. Voltage thresholds for exponential conductance development were found to correlate with the background or "leak" conductance. Assuming that the latter reflects interfacial peptide concentration, the mean apparent number of monomers per conducting aggregate could be estimated to be 3-5. In single-channel experiments, the most probable events had amplitudes of 8 pS and 5 pS in neutral and negatively-charged bilayers respectively. Ionic selectivity under salt gradients conditions, both at macroscopic and single-channel levels, was in favour of sodium ions (PNa/PK = 3). These properties compare favourably to previous reports dealing with peptide modelling transmembrane segments of voltage-dependent ionic channels. Specifically, when compared to S4 alone, the reduced unit conductance and the increased selectivity for sodium support the implication of the S45 region in the inner lining of the open configuration of sodium channels.