ICln, a novel integrin alpha(11b)beta3-associated protein, functionally regulates platelet activation.

A critical role for the conserved -integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin IIb3. To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with IIb3 by surface plasmon resonance. The affinity of this interaction was 82.2 ± 24.4 nM in a cell free assay. ICln co-immunoprecipitates with IIb3 in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 µM to 5 mM), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10–100 µM) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.

Structural basis for the platelet-collagen interaction - The smallest motif within collagen that recognizes and activates platelet glycoprotein VI contains two glycine-proline-hydroxyproline triplets

Collagen-related peptide is a selective agonist for the platelet collagen receptor Glycoprotein VI. The triple helical peptide contains ten GPO triplets/strand (single letter amino acid nomenclature, where O is hydroxyproline) and so over-represents GPO compared with native collagen sequence. To investigate the ability of Glycoprotein VI to recognize GPO triplets in a setting more representative of the collagens, we synthesized a set of triple helical peptides containing fewer GPO triplets, varying their number and spacing within an inert (GPP)(n) backbone. The adhesion of recombinant human Glycoprotein VI ectodomain, like that of human platelets, to these peptides increased with their GPO content, and platelet adhesion was abolished by the specific anti-Glycoprotein VI-blocking antibody, 10B12. Platelet aggregation and protein tyrosine phosphorylation were induced only by cross-linked peptides and only those that contained two or more GPO triplets. Such peptides were less potent than cross-linked collagen-related peptide. Our data suggest that both the sequences GPOGPO and GPO center dot center dot center dot center dot center dot center dot center dot center dot center dot GPO represent functional Glycoprotein VI recognition motifs within collagen. Furthermore, we propose that the (GPO)(4) motif can support simultaneous binding of two glycoprotein VI molecules, in either a parallel or anti-parallel stacking arrangement, which could play an important role in activation of signaling.

Differential roles of integrins alpha(2)beta(1), and alpha(IIb)beta(3) in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin alpha(2)beta(1) and possibly to other receptors. In this study, we have compared the role of integrins alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A(2) (TxA(2)) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and alpha(IIb)beta(3) activation. This adhesion was substantially mediated by alpha(2)beta(1). The alpha(IIb)beta(3) antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that alpha(IIb)beta(3) outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA(2) greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to alpha(2)beta(1).

Platelet plasminogen activator inhibitor 1 in patients with type II diabetes

To compare platelet plasminogen activator inhibitor 1 (PAI-1) concentration in type II diabetic patients and healthy control subjects.

Characterization of a ligand-attenuated binding site on glycoprotein IIb/IIIa

We describe an epitope on the platelet integrin, GPIIb/IIIa, identified by the monoclonal antibody, 4F8, which is attenuated by small-molecule GPIIb/IIIa ligands. 4F8 did not bind to the ligand binding pocket as it did not compete with a radiolabelled antagonist, H-3-SC-52012. This indicates that the 4F8 epitope behaves as a ligand-attenuated binding site (LABS). Ligand-induced attenuation of 4178 was an active process as it was prevented by pretreating platelets with cytochalasin D and reduced by prostaglandin E-1 or inhibition of protein kinase C. Disappearance of the epitope was required for full platelet activation as 4F8 prevented platelet aggregation without inhibiting fibrinogen binding. These results suggest a model where disappearance of the 4F8 epitope is a secondary event required for full

P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

NO-releasing zeolites and their antithrombotic properties

Transition metal-exchanged zeolite-A adsorbs and stores nitric oxide in relatively high capacity (up to 1 mmol of NO/g of zeolite). The stored NO is released on contact with an aqueous environment under biologically relevant conditions of temperature and pH. The release of the NO can be tuned by altering the chemical composition of the zeolite, by controlling the amount of water contacting the zeolite, and by blending the zeolite with different polymers. The high capacity of zeolite for NO makes it extremely attractive for use in biological and medical applications, and our experiments indicate that the NO released from Co-exchanged zeolite-A inhibits platelet aggregation and adhesion of human platelets in vitro.

High-capacity hydrogen and nitric oxide adsorption and storage in a metal-organic framework

Gas adsorption experiments have been carried out on a copper benzene tricarboxylate metal-organic framework material, HKUST-1. Hydrogen adsorption at 1 and 10 bar (both 77 K) gives an adsorption capacity of 11.16 mmol H-2 per g of HKUST-1 (22.7 mg g(-1), 2.27 wt %) at 1 bar and 18 mmol per g (36.28 mg g(-1), 3.6 wt %) at 10 bar. Adsorption of D-2 at 1 bar (77 K) is between 1.09 (at 1 bar) and 1.20(at < 100 mbar) times the H-2 values depending on the pressure, agreeing with the theoretical expectations. Gravimetric adsorption measurements of NO on HKUST-1 at 196 K (1 bar) gives a large adsorption capacity of similar to 9 mmol g(-1), which is significantly greater than any other adsorption capacity reported on a porous solid. At 298 K the adsorption capacity at 1 bar is just over 3 mmol g(-1). Infra red experiments show that the NO binds to the empty copper metal sites in HKUST-1. Chemiluminescence and platelet aggregometry experiments indicate that the amount of NO recovered on exposure of the resulting complex to water is enough to be biologically active, completely inhibiting platelet aggregation in platelet rich plasma.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.