Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.

Platelet-collagen interactions play a fundamental role in the process of arterial thrombosis. The major platelet collagen receptor is the glycoprotein VI (GPVI). Here, we determined the effects of a soluble dimeric form of GPVI on platelet adhesion in vitro and in vivo. We fused the extracellular domain of GPVI with the human immunoglobulin Fc domain. The soluble dimeric form of GPVI (GPVI-Fc) specifically bound to immobilized collagen. Binding of GPVI-Fc to collagen was inhibited competitively by soluble GPVI-Fc, but not control Fc lacking the external GPVI domain. GPVI-Fc inhibited the adhesion of CHO cells that stably express human GPVI and of platelets on collagen and attenuated thrombus formation under shear conditions in vitro. To test the effects of GPVI-Fc in vivo, arterial thrombosis was induced in the mouse carotid artery, and platelet-vessel wall interactions were visualized by intravital fluorescence microscopy. Infusion of GPVI-Fc but not of control Fc virtually abolished stable arrest and aggregation of platelets following vascular injury. Importantly, GPVI-Fc but not control Fc, was detected at areas of vascular injury. These findings further substantiate the critical role of the collagen receptor GPVI in the initiation of thrombus formation at sites of vascular injury and identify soluble GPVI as a promising antithrombotic strategy.

Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, were mobile, exhibiting a diffusion constant of 0.6 micro m(2) s(-1). The functionality of receptors on bare glass and on cellulose cushions was compared by measuring adhesion strength to giant vesicles. The vesicles contained lipid-coupled cyclic hexapeptides that are specifically recognized by integrin alpha(IIb)beta(3). To mimic the steric repulsion forces of the cell glycocalix, lipids with polyethylene glycol headgroups were incorporated into the vesicles. The free adhesion energy per unit area deltag(ad) was determined by micro-interferometric analysis of the vesicle's contour near the membrane surface in terms of the equilibrium of the elastic forces. By accounting for the reduction of the adhesion strength by the repellers and from measuring the density of receptors one could estimate the specific receptor ligand binding energy. We estimate the receptor-ligand binding energy to be 10 k(B)T under bioanalogue conditions.

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of epsilon-aminohexanoic acid (epsilonAhx) or of epsilonAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of K(D) = 0.158 microM from ELISA and K(D) = 1.1 microM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido -propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.

Cisplatin-based chemotherapy is the standard therapy used to treat non-small-cell lung cancer. However, its efficacy is largely limited due to the development of drug resistance. The exact mechanism in which cancer cells develop resistance to the drug is not yet fully understood. The purpose of the present study is to test the role of volume-sensitive Cl(-) channels in cisplatin resistance in human lung adenocarcinoma cells (A549 cells) using patch-clamp recording, cell volume measurement and apoptosis assay. The results showed that cisplatin treatment induced an apoptotic volume decrease (AVD) and activated a Cl(-) current that showed properties similar to the volume-sensitive outward rectifying (VSOR) Cl(-) current in wild-type A549 cells. Both the AVD process and VSOR Cl(-) current were blocked by the chloride channel blocker 4,4'-diisothiocyanostilbene-2,2' disulfonic acid. However, the A549/CDDP cells, a model of acquired cisplatin resistance cells, on the other hand, had almost no AVD process and VSOR Cl(-) current when treated with cisplatin. Treatment of A549/CDDP cells with trichostatin A (TSA), a drug that inhibits histone deacetylases, partially restored the VSOR Cl(-) current and increased cisplatin-induced cell apoptosis rate. These results suggest that impaired activity of VSOR Cl(-) channels contributes to the cisplatin resistance in A549/CDDP cells.

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Limited evidence supports the conclusion that CSIs are superior to ABPs for pain relief in the short term; however, this result was reversed in the intermediate and long term. ABPs seemed to be more effective at restoring function in the intermediate term. Because of the small sample size and the limited number of high-quality RCTs, more high-quality RCTs with large sample sizes are required to validate this result.

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