Nina Dempsey-Hibbert

Thieno[2,3-b]pyridine derivatives are potent anti-platelet drugs, inhibiting platelet activation, aggregation and showing synergy with aspirin

Drugs which inhibit platelet function are commonly used to prevent blood clot formation in patients with Acute Coronary Syndromes (ACS) or those at risk of stroke. The thieno[3,2-c]pyridine class of therapeutic agents, of which clopidogrel is the most commonly used, target the P2Y12 receptor, and are often used in combination with acetylsalicylic acid (ASA). Six thieno[2,3-b]pyridine were assessed for inxa0vitro anti-platelet activity; all derivatives showed effects on both platelet activation and aggregation, and showed synergy with ASA. Some compounds demonstrated greater activity when compared to clopidogrel. These compounds, therefore, represent potential novel P2Y12 inhibitors for improved treatment for patients.

Antimicrobial activity of Ti-ZrN/Ag coatings for use in biomaterial applications

Severely broken bones often require external bone fixation pins to provide support but they can become infected. In order to reduce such infections, novel solutions are required. Titanium zirconium nitride (Ti-ZrN) and Ti-ZrN silver (Ti-ZrN/Ag) coatings were deposited onto stainless steel. Surface microtopography demonstrated that on the silver containing surfaces, Sa and Sv values demonstrated similar trends whilst the Ra, average height and RMS value and Sp values increased with increasing silver concentration. On the Ti-ZrN/Ag coatings, surface hydrophobicity followed the same trend as the Sa and Sv values. An increase in dead Staphylococcus aureus and Staphylococcus epidermidis cells was observed on the coatings with a higher silver concentration. Using CTC staining, a significant increase in S. aureus respiration on the silver containing surfaces was observed in comparison to the stainless steel control whilst against S. epidermidis, no significant difference in viable cells was observed across the surfaces. Cytotoxicity testing revealed that the TiZrN coatings, both with and without varying silver concentrations, did not possess a detrimental effect to a human monocyte cell line U937. This work demonstrated that such coatings have the potential to reduce the viability of bacteria that result in pin tract infections.

Targeting Heat Shock Proteins in Colorectal Cancer

Colorectal cancer (CRC) causes over half a million deaths worldwide and has a particularly poor prognosis when diagnosed at an advanced stage. Heat shock proteins (HSP) have been found to be elevated in CRC patients and HSPB1, HSPA1A and HSPC1 has been shown to have some prognostic value. CRC, in common with all cancers, has important associated oncogene and tumor suppressor gene associations and we show how many of these interact directly with one or more of the HSP. We discuss the current chemotherapeutic options available to the clinician when presented with CRC and how these may be improved with a consideration of the role of HSP in the development of the tumor as well as the response to therapy. Direct manipulation of HSP has the potential to decrease the therapeutic dose of anti-tumor drugs and we propose novel strategies that have the potential to be adapted to the clinic.

The effects of blood conditioning films on the antimicrobial and retention properties of zirconium-nitride silver surfaces

External bone fixation devices provide support and rehabilitation for severely damaged/broken bones, however, this invasive procedure is prone to infection. Zirconium nitride/silver (Ti-ZrN/Ag) coatings were characterised for surface topography, chemical composition, physicochemistry and antimicrobial efficacy (against Staphylococcus aureus and Staphylococcus epidermidis), in the presence of a blood conditioning film. The conditioning film altered the width of the microtopography of the surfaces however, the depth of the features remained relatively constant. The conditioning film also altered the coatings from hydrophobic to hydrophilic/partially hydrophilic surfaces. Following the MATH assay, the presence of a conditioning film reduced affinity towards the hydrocarbons for both microorganisms. The addition of a blood conditioning film reduced the antimicrobial efficacy of the Ti-ZrN/Ag coatings but also reduced the number of retained bacteria. This study suggests that the presence of a pre-defined blood conditioning film may result in surfaces with anti-adhesive properties, potentially leading to a reduction in bacterial retention. This, combined with the antimicrobial efficacy of the coatings, could reduce the risk of infection on biomaterial surfaces.

Antimicrobial synergy of cationic grafted poly(para-phenylene ethynylene) and poly(para-phenylene vinylene) compounds with UV or metal ions against Enterococcus faecium

The rise in multidrug resistant bacteria is an area of growing concern and it is essential to identify new biocidal agents. Cationic grafted compounds were investigated for their antimicrobial properties using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests. Synergy testing was carried out using the compounds in the presence of ultraviolet (UV). Fractional inhibitory concentration (FIC) and fractional bactericidal concentration (FBC) tests were carried out using the cationic molecules in conjunction with metal ion solutions of gold, silver, palladium, platinum, rhodium, titanium, tin, vanadium and molybdenum. Individually, the cationic compounds containing quaternary amines, polyphenylene vinylene (PPV) with long polyacrylate grafts (PPV-g-PMETAC (HMw)), polyphenylene ethylene (PPE) with long polyacrylate grafts (PPE-g-PMETAC (HMw)), polyphenylene vinylene (PPV) with short polyacrylate grafts (PPV-g-PMETAC (LMw)) and polyphenylene ethylene (PPE) with short polyacrylate grafts (PPE-g-PMETAC (LMw)) were effective against Enterococcus faecium. The most successful compound under UV was PPV-g-PMETAC (HMw). Following the FICs, palladium and rhodium ion solutions caused a synergistic reaction with all four tested compounds. The presence of conjugated bonds in the cationic molecules increased its antimicrobial activity. These results suggest that the chemical backbone of the compounds, alongside the chain lengths and chain attachment affect the antimicrobial efficacy of a compound. These factors should be taken into consideration when formulating new biocidal combinations.

Primary Colorectal Cells Culture as a Translational Research Model

Preclinical studies are an essential stage for any pharmacological agent hoping to make its way into clinical trials. An ideal preclinical model that can accurately predict clinical response does not exist and the best that the scientific community have at the moment is to select the most relevant study model pertaining to the disease of interest from those available, which includes: cell lines, animal models, and even in-silico methodology. Currently, there is a huge gap between preclinical and clinical trial results, indicating that there is much room for improvement in developing a better model to bridge the translational gap.

161 Btk inhibitors: friends or foes?

Introduction Bruton’s tyrosine kinase (BTK) plays a crucial role in the development and maturation of B-cells. A common side effect of Ibrutinib, a BTK inhibitor approved for the treatment of chronic lymphocytic leukaemia (CLL), is active bleeding in the absence of vascular injury. The mechanisms by which ibrutinib alters haemostasis however are currently unclear. The aim of this study was to investigate the effects of ibrutinib on platelet and endothelial cell function in vitro, to determine the mechanisms that underpin ibrutinib-induced bleeding. Methods Platelet rich plasma (PRP) collected from healthy volunteers was treated with increasing concentrations of ibrutinib for 15u2009min at 37°C, prior to stimulation with collagen (2 µg/ml) or ADP (10 µM). Platelet function and activation were measured by light transmission aggregometry (LTA) and flow cytometry respectively (CD62P+, PAC1+) and platelet morphology analysed using scanning electron microscopy (SEM). Platelet signalling pathways were analysed by Western blotting and the generation of endothelial microvesicles (EMVs) from HUVECs enumerated by flow cytometry, following 24u2009hours treatment with ibrutinib (increasing concentrations from 0.1 µM to 10 µM). Results Ibrutinib significantly reduced collagen-mediated platelet aggregation and activation in a dose-dependent manner (p<0.05). SEM analysis also demonstrated that collagen-mediated shape change and filopodia formation was defective following ibrutinib treatment. Consistent with these findings, signalling downstream of the collagen GPVI receptor was perturbed, with a marked reduction in PLCγ2 phosphorylation. Ibrutinib only exerted mild inhibition of ADP-induced platelet aggregation (p<0.05), which was accompanied by reduced PLA2 activation and inhibition of VASP dephosphorylation. Additionally, our results demonstrated that at low concentrations, ibrutinib reduced the generation of pro-thrombotic EMVs, an effect that is reversed at the highest concentrations (p<0.05). Conclusion Ibrutinib reduces collagen and ADP-mediated platelet aggregation, and activation by reducing phosphorylation of PLCγ2 and PLA2 and inhibiting VASP dephosphorylation. In addition, ibrutinib also appears to alter endothelial cell function by reducing EMV release. Understanding the mechanisms by which ibrutinib alters haemostasis may lead to the identification of novel antithrombotic targets.

187 Novel thienopyridines are potent anti-platelet drugs, inhibiting platelet activation, aggregation and showing synergy with aspirin

Background Management of Acute Coronary Syndromes (ACS) often involves the use of platelet inhibitors. The most commonly used drug, clopidogrel, belongs to a class of thienopyridine molecules which targets the P2Y12 receptor on platelets and it is commonly used in combination with the COX-1 inhibitor acetylsalicylic acid (ASA). However, the effect of these treatments is variable amongst patients, highlighting a need for a refinement of this class of P2Y12 inhibitor. The aim of this study was to assess the efficacy of six novel thienopyridine derivatives synthesised by our group by examining their potential as in-vitro inhibitors of platelet function. Methods Healthy human platelets were isolated and incubated with novel thienopyridine compounds (DJ0081, DJ0199, DJ0021, DJ0206, DJ0171, DJ0097) (10 µM, 30u2009min) prior to stimulation with ADP (10µM) and analysis of alpha granule secretion (CD62P expression), GPIIbIIIa activation (PAC1 expression) and platelet leukocyte aggregate (PLA) formation using flow cytometry. Furthermore, light transmission aggregometry (LTA) was used to assess ADP-stimulated aggregation after these treatments. Synergy with ASA (30 µM) was also analysed by LTA following incubation with ASA and thienopyridine. All results were compared to ADP-stimulated samples and samples treated with clopidogrel (10 µM, 30u2009min) prior to ADP stimulation. Results All six novel compounds demonstrated a significant reduction in ADP-mediated platelet aggregation (p<0.001), CD62P expression (p<0.001), PAC1 expression (p<0.01) and PLA formation (p<0.05). These compounds were also shown to enhance the inhibitory effects of ASA. DJ0171 and DJ0199 were particularly potent, displaying greater inhibitory effect than clopidogrel. Conclusion The study demonstrates the potential for new thienopyridine compounds as modulators of platelet function and points to the possibility of future use in patients at risk of platelet hyperactivity and thrombosis.

189 Altered platelet function and thrombosis in fetal growth restriction

Background Fetal growth restriction (FGR) affects 3%–8% of pregnancies and is associated with increased risk of stillbirth and perinatal morbidity, in addition to increased risk of cardiovascular disease later in life. In severe cases of FGR, vascular resistance in the placenta is increased and blood flow reduced, compromising oxygen and nutrient exchange between mother and fetus. Hypoxia and endothelial dysfunction, have both been demonstrated in FGR, and both have the ability to alter thrombosis and haemostasis. The aim of this study was to determine whether there is increased platelet activation and thrombosis in FGR which may contribute to its pathogenesis. Methods Dual ex-vivo perfusions were performed on placentas from healthy pregnancies (n=10) and those affected by FGR (n=11). Following perfusion (~5u2009hour), placental cross-sections were processed and stained with H and E and Martius Scarlet Blue for blind-randomised histological analysis or immunofluorescence to analyse levels of fibrin and tissue factor. Fetal blood samples were collected from the umbilical vein immediately following delivery (n=10u2009healthy; n=8u2009FGR) and platelet function analysed using the PFA200 Results Placental sections from pregnancies affected by FGR exhibited significantly more thrombi (p<0.01) within the fetal vasculature than healthy placental sections. Thrombi could be observed in both large conduit and small resistance vessels, with many small vessels completely occluded. In FGR, total fibrin deposition in placental tissue was significantly increased (p<0.001), as was fibrin expression specifically located within fetal vessels (p<0.05). In contrast, tissue factor levels remained unaltered. Blood samples from fetal growth restricted babies demonstrated increased closure time in platelet function assays (122±12.6u2009s) compared to healthy controls (90.10±5.46u2009s), and platelet counts were significantly reduced (p<0.05). The red blood cell count, white blood cell count and mean platelet volume were not significantly different. Conclusions These data indicate that in FGR there is increased platelet activation and thrombosis in fetoplacental vessels, which may contribute to increased vascular resistance through the occlusion of small resistance arteries. Anti-thrombotic therapies, which cross the placenta may therefore be beneficial in FGR to prevent placental thrombosis and improve pregnancy outcome.

Surface modification of platelet concentrate bags to reduce biofilm formation and transfusion sepsis

Bacterial contamination of blood products poses a major risk in transfusion medicine, including transfusions involving platelet products. Although testing systems are in place for routine screening of platelet units, the formation of bacterial biofilms in such units may decrease the likelihood that bacteria will be detected. This work determined the surface properties of p-PVC platelet concentrate bags and investigated how these characteristics influenced biofilm formation. Serratia marcescens and Staphylococcus epidermidis, two species commonly implicated in platelet contamination, were used to study biofilm growth. The platelet concentrate bags were physically flattened to determine if reducing the surface roughness altered biofilm formation. The results demonstrated that the flattening process of the platelet bags affected the chemistry of the surface and reduced the surface hydrophobicity. Flattening of the surfaces resulted in a reduction in biofilm formation for both species after 5 days, with S. marcescens demonstrating a greater reduction. However, there was no significant difference between the smooth and flat surfaces following 7 days incubation for S. marcescens and no significant differences between any of the surfaces following 7 days incubation for S. epidermidis. The results suggest that flattening the p-PVC surfaces may limit potential biofilm formation for the current duration of platelet storage time of 5 days. It is hoped that this work will enhance the understanding of how surface properties influence the development of microbial biofilms in platelet concentrate bags in order to devise a solution to discourage biofilm formation.