Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin.

P-selectin, found in storage granules of platelets and endothelial cells, can be rapidly expressed upon stimulation. Mice lacking this membrane receptor exhibit a severe impairment of leukocyte rolling. We observed that, in addition to leukocytes, platelets were rolling in mesenteric venules of wild-type mice. To investigate the role of P-selectin in this process, resting or activated platelets from wild-type or P-selectin-deficient mice were fluorescently labeled and transfused into recipients of either genotype. Platelet-endothelial interactions were monitored by intravital microscopy. We observed rolling of either wild-type or P-selectin-deficient resting platelets on wild-type endothelium. Endothelial stimulation with the calcium ionophore A23187 increased the number of platelets rolling 4-fold. Activated P-selectin-deficient platelets behaved similarly, whereas activated wild-type platelets bound to leukocytes and were seen rolling together. Platelets of either genotype, resting or activated, interacted minimally with mutant endothelium even after A23187 treatment. The velocity of platelet rolling was 6- to 9-fold greater than that of leukocytes. Our results demonstrate that (i) platelets roll on endothelium in vivo, (ii) this interaction requires endothelial but not platelet P-selectin, and (iii) platelet rolling appears to be independent of platelet activation, indicating constitutive expression of a P-selectin ligand(s) on platelets. We have therefore observed an interesting parallel between platelets and leukocytes in that both of these blood cell types roll on stimulated vessel wall and that this process is dependent on the expression of endothelial P-selectin.

Development of labelled platelets as radiopharmaceuticals.

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Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Micro-structure differences in kaolinite suspensions

SEM observations of the aqueous suspensions of kaolinite from Birdwood (South Australia) and Georgia (USA) show noticeable differences in number of physical behaviour which has been explained by different microstructure constitution.. Birdwood kaolinite dispersion gels are observed at very low solid loadings in comparison with Georgia KGa-1 kaolinite dispersions which remain fluid at higher solids loading. To explain this behaviour, the specific particle interactions of Birdwood kaolinite, different from interaction in Georgia kaolinite have been proposed. These interactions may be brought about by the presence of nano-bubbles on clay crystal edges and may force clay particles to aggregate by bubble coalescence. This explains the predominance of stair step edge-edge like (EE) contacts in suspension of Birdwood kaolinite. Such EE linked particles build long strings that form a spacious cell structure. Hydrocarbon contamination of colloidal kaolinite particles and low aspect ratio are discussed as possible explanations of this unusual behaviour of Birdwood kaolinite. In Georgia KGa-1 kaolinite dispersions instead of EE contact between platelets displayed in Birdwood kaolinite, most particles have edge to face (EF) contacts building a cardhouse structure. Such an arrangement is much less voluminous in comparison with the Birdwood kaolinite cellular honeycomb structure observed previously in smectite aqueous suspensions. Such structural characteristics of KGa-1 kaolinite particles enable higher solid volume fractions pulps to form before significantly networked gel consistency is attained.

Influence of smectite suspension structure on sheet orientation in dry sediments : XRD and AFM applications

The structure-building phenomena within clay aggregates are governed by forces acting between clay particles. Measurements of such forces are important to understand in order to manipulate the aggregate structure for applications such as dewatering of mineral processing tailings. A parallel particle orientation is required when conducting XRD investigation on the oriented samples and conduct force measurements acting between basal planes of clay mineral platelets using at. force microscopy (AFM). To investigate how smectite clay platelets were oriented on silicon wafer substrate when dried from suspension range of methods like SEM, XRD and AFM were employed. From these investigations, we conclude that high clay concns. and larger particle diams. (up to 5 μm) in suspension result in random orientation of platelets in the substrate. The best possible laminar orientation in the clay dry film, represented in the XRD 0 0 1/0 2 0 intensity ratio of 47 was obtained by drying thin layers from 0.02 wt.% clay suspensions of the natural pH. Conducted AFM investigations show that smectite studied in water based electrolytes show very long-range repulsive forces lower in strength than electrostatic forces from double-layer repulsion. It was suggested that these forces may have structural nature. Smectite surface layers rehydrate in water environment forms surface gel with spongy and cellular texture which cushion approaching AFM probe. This structural effect can be measured in distances larger than 1000 nm from substrate surface and when probe penetrate this gel layer, structural linkages are forming between substrate and clay covered probe. These linkages prevent subsequently smooth detachments of AFM probe on way back when retrieval. This effect of tearing new formed structure apart involves larger adhesion-like forces measured in retrieval. It is also suggested that these effect may be enhanced by the nano-clay particles interaction.

The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

Kaolinite platelet orientation for XRD and AFM applications

The structure-building phenomena within clay aggregates are governed by forces acting between clay particles. The nature of such forces is important to understand in order to manipulate the aggregate structure for applications such as settling and dewatering. A parallel particle orientation is required when conducting force measurements acting between the basal planes of clay mineral platelets using atomic force microscopy (AFM). In order to prepare a film of clay particles with the optimal orientation for conducting AFM measurements, the influences of particle concentration in suspension, suspension pH and particle size on the clay platelet orientation were investigated using scanning electron microscopy (SEM) and X-ray diffraction (XRD) methods. From these investigations, we conclude that high clay (dry mass) concentrations and larger particle diameters (up to 5 µm) in suspension result in random orientation of platelets on the substrate. The best possible laminar orientation in the clay dried film as represented in the XRD by the 001/020 intensity ratio of more than 150 and by SE micrograph assessments, was obtained by drying thin layers from 0.2 wt% of -5 µm clay suspensions at pH 10.5. These dried films are stable and suitable for close-approach AFM studies in solution.

Mechanism of interaction of hydrocalumites (Ca/Al-LDH) with methyl orange and acidic scarlet GR

The development of new materials for water purification is of universal importance. Among these types of materials are layered double hydroxides (LDHs). Non-ionic materials pose a significant problem as pollutants. The interaction of methyl orange (MO) and acidic scarlet GR (GR) adsorption on hydrocalumite (Ca/Al-LDH-Cl) were studied by X-ray diffraction (XRD), infrared spectroscopy (MIR), scanning electron microscope (SEM) and near-infrared spectroscopy (NIR). The XRD results revealed that the basal spacing of Ca/Al-LDH-MO was expanded to 2.45 nm, and the MO molecules were intercalated with a inter-penetrating bilayer model in the gallery of LDH, with 49o tilting angle. Yet Ca/Al-LDH-GR was kept the same d-value as Ca/Al-LDH-Cl. The NIR spectrum for Ca/Al-LDH-MO showed a prominent band around 5994 cm-1, assigned to the combination result of the N-H stretching vibrations, which was considered as a mark to assess MO- ion intercalation into Ca/Al-LDH-Cl interlayers. From SEM images, the particle morphology of Ca/Al-LDH-MO mainly changed to irregular platelets, with a “honey-comb” like structure. Yet the Ca/Al-LDH-GR maintained regular hexagons platelets, which was similar to that of Ca/Al-LDH-Cl. All results indicated that MO- ion was intercalated into Ca/Al-LDH-Cl interlayers, and acidic scarlet GR was only adsorped upon Ca/Al-LDH-Cl surfaces.

Nanostructures for sensors, electronics, energy and environment

Nanoscale science is growing evermore important on a global scale and is widely seen as playing an integral part in the growth of future world economies. The daunting energy crisis we are facing could be solved not only by new and improved ways of getting energy directly from the sun, but also by saving power thanks to advancements in electronics and sensors. New, cheap dye-sensitized and polymer solar cells hold the promise of environmentally friendly and simple production methods, along with mechanical flexibility and low weight, matching the conditions for a widespread deployment of this technology. Cheap sensors based on nanomaterials can make a fundamental contribution to the reduction of greenhouse gas emissions, allowing the creation of large sensor networks to monitor countries and cities, improving our quality of life. Nanowires and nano-platelets of metal oxides are at the forefront of the research to improve sensitivity and reduce the power consumption in gas sensors. Nanoelectronics is the next step in the electronic roadmap, with many devices currently in production already containing components smaller than 100 nm. Molecules and conducting polymers are at the forefront of this research with the goal of reducing component size through the use of cheap and environmentally friendly production methods. This, and the coming steps that will eventually bring the individual circuit element close to the ultimate limit of the atomic level, are expected to deliver better devices with reduced power consumption.