Haemostasis in progressive renal failure

This thesis describes a series of clinical and haematological studies of patients with chronic renal failure. My aim was to investigate the role of haemostatic abnormality in two aspects of renal disease: (1) The bleeding tendency of uraemia. (2) The progression of renal failure. (1) In the first part of the thesis I show that anaemia appears to be the most important determinant of uraemic bleeding. The development of a prolonged bleeding time in the early stages of renal failure was documented, and experiments were performed to identify platelet functional abnormalities which might develop in parallel, and hence explain the haemorrhagic defect. The only significant relationship which emerged was a negative correlation between the haematocrit and the bleeding time. This was further investigated by studying platelet function during correction of anaemia with recombinant human erythropoietin. Bleeding time was shortened following erythropoietin, by a degree which correlated with the increase in the haematocrit, but not with any changes in platelet reactivity. A study of platelet aggregation in whole blood showed an aggregation-enhancing effect of uraemic red cells which was not evident with normal erythrocytes. (2) In the second part of the thesis, increased blood viscosity and intravascular haemostatic activation were demonstrated in both diabetic and non-diabetic patients with progressive renal disease, abnormalities which might promote non-immune glomerular injury and hence the progression of renal failure. It was not clear, however, whether these changes represented cause or effect of disease, since in multiple regression analysis, proteinuria emerged as the major independent determinant of progression. A clinical study of antiplatelet therapy in progressive renal failure showed a slowing of progression in half the patients treated, and, in diabetic nephropathy, a significant reduction in proteinuria. These results suggest a partial role for haemostasis in the progression of renal injury

Potential agonists and inhibitors of protein kinase C

Daphnane and tigliane diterpenoid hydrocarbon nuclei were isolated from the latex of Euphorbia poissonii.Pax.Euphorbiaceae using the techniques of liquid-liquid partition, column chromatography, centrifugal liquid chromatography (CLC) and preparative partition and adsorption thin layer chromatography. Six naturally occurring diterpene esters were isolated from the latex and a further eight were semi-synthesised by selective esterification of the C20 primary alcohol of these resiniferonol and 12-deoxyphorbol nuclei. These compounds were characterised by their 1H-NMR, mass spectral, UV and IR properties. Computer-assisted molecular modelling of these compounds in conjunction with standard phorbol ester probes enabled the assessment of their minimum free energy values; resiniferonol derivatives were found to have much lower minimum free energy levels than corresponding 12-deoxyphorbol derivatives. The molecular coordinates of the potent irritant, resiniferatoxin, were also assigned. An in vivo mouse ear erythema assay was used to evaluate the pro-inflammatory response of these compounds and the ability of some resiniferonol derivatives to inhibit erythema induced by phorbol ester and neurogenic (capsaicin) irritants. The pro-inflammatory response induced by resiniferatoxin was found to be of mixed aetiology, comprising of neurogenic and phorbol ester components. The ability of the semi-synthetic compounds to induce epidermal hyperplasia of mouse skin was assessed as a potential correlation for tumour promotion activity. To further understand the biochemical mechanisms of action of these synthetic compounds, their ability to activate or inhibit a mixed isozymic pool of the phorbol ester receptor protein, protein kinase C, isolated from rat brain was studied. The ability of 12-deoxyphorbol-13-O-phenylacetate-20-O-acetate (DOPPA) to selectively activate isozymic forms of protein kinase C isolated from human platelets was also assessed

Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C

Highlights •A range of thiol isomerase enzymes were expressed by HMEC-1 endothelial cells. •Inhibition of thiol isomerases reduced plasminogen activation on HMEC-1 cells. •Exogenous bovine protein disulphide isomerase increased plasminogen activation in HMEC-1 cells. •Inhibition of thiol isomerases also reduced fibrin clot lysis, but not via direct effects on tPA. •HMEC-1 mediated protein C activation was unaffected by thiol isomerase inhibition. Abstract Introduction Cell surface thiol isomerase enzymes, principally protein disulphide isomerase (PDI), have emerged as important regulators of platelet function and tissue factor activation via their action on allosteric disulphide bonds. Allosteric disulphides are present in other haemostasis-related proteins, and we have therefore investigated whether thiol isomerase inhibition has any influence on two endothelial activities relevant to haemostatic regulation, namely activation of protein C and activation of plasminogen, with subsequent fibrinolysis. Materials and Methods The study was performed using the human microvascular endothelial cell line HMEC-1. Thiol isomerase gene expression was measured by RT-PCR and activation of protein C and plasminogen by cell-based assays using chromogenic substrates S2366 and S2251, respectively. Cell mediated fibrinolysis was measured by monitoring absorbance at 405 nm following fibrin clot formation on the surface of HMEC-1 monolayers. Results and Conclusions A variety of thiol isomerase enzymes, including PDI, were expressed by HMEC-1 cells and thiol reductase activity detectable on the cell surface was inhibited by both RL90 anti-PDI antibody and by the PDI inhibitor quercetin-3-rutinoside (rutin). In cell-based assays, activation of plasminogen, but not of protein C, was inhibited by RL90 antibody and, to a lesser extent, by rutin. Fibrin clot lysis occurring on a HMEC-1 monolayer was also significantly slowed by RL90 antibody and by rutin, but RL90-mediated inhibition was abolished in the presence of exogenous tissue plasminogen activator (tPA). We conclude that thiol isomerases, including PDI, are involved in fibrinolytic regulation at the endothelial surface, although not via a direct action on tPA. These findings broaden understanding of haemostatic regulation by PDI, and may aid in development of novel anti-thrombotic therapeutic strategies targeted via the fibrinolysis system

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 μM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) (100 μM) or inhibiting PDI with bacitracin (5 mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 μM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 μM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain

The development of a comprehensive cycle plan for Stellenbosch

The town of Stellenbosch has the potential to grow cycling into a major mode. To date the town has made significant advances in the provision for non-motorised transport (NMT) enabling the creation of a partial network of cycleable town routes. It is also a growing international centre for recreational and competitive cycling. The town is facing challenges in transport terms as a result of decades of myopic private car centred provision as the only transport policy based response. Given the town?s popularity with an expanding university, a growing commercial centre, the wine farming capital and as a tourism node, car based trips into and around the town have grown dramatically. Cycling development is being seen as one major component in a much more sustainable and appropriate range of solutions to transport and urban planning challenges for the town and its catchment. The Cycle Plan details the full programme of measures to advance cycling to become a main mode for local travel. Short term interventions recommend a focus on low income access to bicycles, the development of safe cycling routes and the promotion of school/university and large employer based programmes of travel demand management. Short term network improvements for safe cycling focus on the extension of shared use pathways, the reduction in town centre traffic speeds and the gradual introduction of cycleways in the roadway The action plans recommend a joint public and private approach to funding, recognizing the fiscal constraints and demands on transport budgets. However, the plan should be treated as a first phase input to Stellenbosch area IPTN and Stellenbosch wishes to source PTIS funding in due course to progress with its bold and sustainable plans.Paper presented at the 34th Annual Southern African Transport Conference 6-9 July 2015 "Working Together to Deliver - Sakha Sonke", CSIR International Convention Centre, Pretoria, South Africa.The Minister of Transport, South AfricaTransportation Research Board of the US

Investigating the Diabetic Brain: The Effects of Pioglitazone and Insulin on the Cellular Processes and Pathology of Alzheimer's Disease

Alzheimer’s disease (AD) is the sixth leading cause of death in the US. Some researchers refer to AD as “Type III Diabetes” because of reported glucose metabolism dysfunction. Preclinical studies suggest increasing insulin decreases AD pathology, although the mechanism remains unclear. To sensitize insulin signaling, this study activated Peroxisome Proliferator-Activated Receptor Gamma using intranasal co-administration of pioglitazone (PGZ) and insulin. This method targeted the site of action to reduce peripheral effects and to maximize impact in transgenic mice expressing AD pathology. Data from GC-MS fluxomics analysis suggested that PGZ+Insulin increased glucose metabolism in the brain. Immunohistochemistry with relevant antibodies was used to identify AD pathological markers in the subiculum, indicating that PGZ+Insulin decreased pathology compared to Insulin and Saline. This suggests that increasing glucose uptake in the brain alleviated AD pathology, further clarifying the role of insulin signaling in AD pathology.Gemston

Mechanism of action of novel NO-releasing furoxan derivatives of aspirin in human platelets

1. Incorporation of a nitric oxide (NO)-releasing moiety in aspirin can overcome its gastric side effects. 2. We investigated the NO-release patterns and antiplatelet effects of novel furoxan derivatives of aspirin (B8 and B7) in comparison to existing antiplatelet agents. 3. Cyclooxygenase (COX) activity was investigated in purified enzyme using an electron paramagnetic resonance-based technique. Concentration–response curves for antiplatelet agents±the soluble guanylate cyclase inhibitor, ODQ (50 μM) were generated in platelet-rich plasma (PRP) and washed platelets (WP) activated with collagen using turbidometric aggregometry. NO was detected using an isolated NO electrode. 4. The furoxan derivatives of aspirin (B8, B7) and their NO-free furazan equivalents (B16, B15; all 100 μM) significantly inhibited COX activity (P<0.01; n=6) in vitro and caused aspirin-independent, cGMP-dependent inhibition of collagen-induced platelet aggregation in WP. B8 was more potent than B7 (PRP IC(50)=0.62±0.1 μM for B8; 400±89 μM for B7; P<0.0001. WP IC(50)s=0.6±0.1 and 62±10 μM, respectively). The NO-free furazan counterparts were less potent antiplatelet agents (WP IC(50)s=54±3 μM and 62±10 μM, respectively; P<0.0001, B8 vs B16). Of the hybrids investigated, only B8 retained antiplatelet activity in PRP. 5. NO release from furoxan–aspirin hybrids was undetectable in buffer alone, but was accelerated in the presence of either plasma or plasma components, albumin (4%), glutathione (GSH; 3 μM) and ascorbate (50 μM), the effects of which were additive for B7 but not B8. NO generation from furoxans was greatly enhanced by platelet extract, an effect that could largely be explained by the synergistic effect of intracellular concentrations of GSH (3 mM) and ascorbate (1 mM). 6. We conclude that the decomposition of furoxan–aspirin hybrids to generate biologically active NO is catalysed by endogenous agents which may instil a potential for primarily intracellular delivery of NO. The blunting of the aspirin effects of furoxan hybrids is likely to be due to loss of the acetyl moiety in plasma; the observed antiplatelet effects are thereby primarily mediated via NO release. Compounds of this class might represent a novel means of inhibiting platelet aggregation by a combination of NO generation and COX inhibition

Inhibition of transforming growth factor α (TGF-α)-mediated growth effects in ovarian cancer cell lines by a tyrosine kinase inhibitor ZM 252868

The modulating effects of the epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor ZM 252868 on cell growth and signalling have been evaluated in four ovarian carcinoma cell lines PE01, PE04, SKOV-3 and PE01CDDP. Transforming growth factor α (TGF-α)-stimulated growth was completely inhibited by concentrations ≥ 0.3 μM in the PE01 and PE04 cell lines and by ≥ 0.1 μM in SKOV-3 cells. TGF-α inhibition of PE01CDDP growth was reversed by concentrations ≥ 0.1 μM ZM 252868. TGF-α-stimulated tyrosine phosphorylation of both the EGF receptor and c-erbB2 receptor in all four cell lines. The inhibitor ZM 252868, at concentrations ≥ 0.3 μM, completely inhibited TGF-α-stimulated tyrosine phosphorylation of the EGF receptor and reduced phosphorylation of the c-erbB2 protein. EGF-activated EGF receptor tyrosine kinase activity was completely inhibited by 3 μM ZM 252868 in PE01, SKOV-3 and PE01CDDP cells. These data indicate that the EGF receptor-targeted TK inhibitor ZM 252868 can inhibit growth of ovarian carcinoma cells in vitro consistent with inhibition of tyrosine phosphorylation at the EGF receptor. © 1999 Cancer Research Campaig

Exploring the case for a central support centre for transport planning and analysis in South Africa

Paper presented at the 23rd Annual Southern African Transport Conference 12 - 15 July 2004 "Getting recognition for the importance of transport", CSIR International Convention Centre, Pretoria, South Africa. Improving South Africa’s land transport efficiency is critical to lowering transaction costs, improving accessibility for economic and social development and for the development of sustainable urban environments. Given the enormous planning challenge for authorities involved in planning and managing transport and the support needs from the private consulting sector, R&D establishments and academic institutions, which would seem to warrant a centralised planning support response. Internationally support centres for transport planning are thriving. This paper briefly sets down the need, possible role and functions of a Transport Analysis Support Centre for South Africa to improve the quality of planning and implementation across all sectors.This paper was transferred from the original CD ROM created for this conference. The material on the CD ROM was published using Adobe Acrobat technology. The original CD ROM was produced by Document Transformation Technologies Postal Address: PO Box 560 Irene 0062 South Africa. Tel.: +27 12 667 2074 Fax: +27 12 667 2766 E-mail: [email protected] URL: http://www.doctech.co.z