Synthetic flavonoids as novel modulators of platelet function and thrombosis

Cardiovascular diseases represent a major cause of mortality and morbidity in the world and thrombotic conditions such as heart attacks and strokes are caused by unwarranted activation of platelets and subsequent formation of blood clots (thrombi) within the blood vessels during pathological circumstances. Therefore, platelets act as a primary therapeutic target to treat and prevent thrombotic conditions. Current treatments are limited due to intolerance and they are associated with severe side effects such as bleeding complications. Hence, the development of novel therapeutic strategies for thrombotic diseases is an urgent priority. Flavonoids are naturally occurring plant-derived molecules that exert numerous beneficial effects in humans through modulating the functions of distinct cell types. However, naturally occurring flavonoids suffer from several issues such as poor solubility, lipophilicity, and bioavailability, which hinder their efficacy and potency. Despite this, flavonoids act as versatile templates for the design and synthesis of novel molecules for various therapeutic targets. Indeed, several synthetic flavonoids have recently been developed to improve their stability, bioavailability and efficacy including for the modulation of platelet function. Here, we provide insight into the actions of certain natural flavonoids along with the advantages of synthetic flavonoids in the modulation of platelet function, haemostasis and thrombosis

Repurposing cancer drugs, batimastat and marimastat, to inhibit the activity of a group I metalloprotease from the venom of the Western Diamondback rattlesnake, Crotalus atrox

Snakebite envenomation causes over 140,000 deaths every year predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with an incredibly complex pathophysiology due to the vast number of unique toxins/proteins found in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a group I metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity was completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 M, while it is partially potentiated by calcium chloride. Molecular docking studies demonstrate that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites

Evidence for concerted ring opening and C-Br bond breaking in UV-excited bromocyclopropane

Photodissociation of gaseous bromocyclopropane via its A-band continuum has been studied at excitation wavelengths ranging from 230 nm to 267 nm. Velocity-map images of ground-state bromine atoms (Br), spin-orbit excited bromine atoms (Br*) and C3H5 hydrocarbon radicals reveal the kinetic energies of these various photofragments. Both Br and Br* atoms are predominantly generated via repulsive excited electronic states in a prompt photodissociation process in which the hydrocarbon co-fragment is a cyclopropyl radical. However, the images obtained at the mass of the hydrocarbon radical fragment identify a channel with total kinetic energy greater than that deduced from the Br and Br* images, and with a kinetic energy distribution that exceeds the energetic limit for Br + cyclopropyl radical products. The velocity-map images of these C3H5 fragments have lower angular anisotropies than measured for Br and Br*, indicating molecular restructuring during dissociation. The high kinetic energy C3H5 signals are assigned to allyl radicals generated by a minor photochemical pathway which involves concerted C-Br bond dissociation and cyclopropyl ring-opening following single UV-photon absorption. Slow photofragments also contribute to the velocity map images obtained at the C3H5 radical mass, but corresponding slow Br atoms are not observed. These features in the images are attributed to C3H5+ from the photodissociation of the C3H5Br+ molecular cation following two-photon ionization of the parent compound. This assignment is confirmed by 118-nm vacuum ultraviolet ionization studies that prepare the molecular cation in its ground electronic state prior to UV photodissociation

The endogenous antimicrobial cathelicidin LL37 induces platelet activation and augments thrombus formation

Platelet-associated complications including thrombosis, thrombocytopenia and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis, sepsis and inflammatory bowel disease. Despite the reported evidence on numerous mechanisms/molecules that may contribute to the dysfunction of platelets, the primary mechanisms that underpin platelet-associated complications during inflammatory diseases are not fully established. Here, we report the discovery of formyl peptide receptor 2, FPR2/ALX in platelets, and its primary role in the development of platelet-associated complications via ligation with its ligand, LL37. LL37 acts as a powerful endogenous antimicrobial peptide but it also regulates innate immune responses. We demonstrate the impact of LL37 in the modulation of platelet reactivity, haemostasis, and thrombosis. LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail bleeding time in mice. By utilising a pharmacological inhibitor and Fpr2/3 (an orthologue of human FPR2/ALX)-deficient mice, the functional dependence of LL37 on FPR2/ALX was determined. Since the level of LL37 is increased in numerous inflammatory diseases, these results point towards a critical role for LL37 and FPR2/ALX in the development of platelet-related complications in such diseases. Hence, a better understanding of the clinical relevance of LL37 and FPR2/ALX in diverse pathophysiological settings will pave the way for the development of improved therapeutic strategies for a range of thromboinflammatory diseases

Ruthenium-conjugated chrysin analogues modulate platelet activity, thrombus formation and haemostasis with enhanced efficacy

The constant increase in cardiovascular disease rate coupled with significant drawbacks of existing therapies emphasise the necessity to improve therapeutic strategies. Natural flavonoids exert innumerable pharmacological effects in humans. Here, we demonstrate the effects of chrysin, a natural flavonoid found largely in honey and passionflower on the modulation of platelet function, haemostasis and thrombosis. Chrysin displayed significant inhibitory effects on isolated platelets, however, its activity was substantially reduced under physiological conditions. In order to increase the efficacy of chrysin, a sulfur derivative (thio-chrysin), and ruthenium-complexes (Ru-chrysin and Ru-thio-chrysin) were synthesised and their effects on the modulation of platelet function were evaluated. Indeed, Ru-thio-chrysin displayed a 4-fold greater inhibition of platelet function and thrombus formation in vitro than chrysin under physiologically relevant conditions such as in platelet-rich plasma and whole blood. Notably, Ru-thio-chrysin exhibited similar efficacy to chrysin in the modulation of haemostasis in mice. Increased bioavailability and cell permeability of Ru-thio-chrysin compared to chrysin were found to be the basis for its enhanced activity. Together, these results demonstrate that Ru-thio-coupled natural compounds such as chrysin may serve as promising templates for the development of novel anti-thrombotic agents

Development and characterisation of a novel, megakaryocyte NF-κB reporter cell line for investigating inflammatory responses

Background Due to the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. Objectives The primary objective of this study is to develop and functionally characterise a novel megakaryocyte NF-κB reporter cell line in order to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. Methods A Meg-01-NF-κB-GFP-Luc (Meg-01R) cell line was developed by inserting a reporter NF-κB-GFP-Luc cassette into normal Meg-01 cells to produce luciferase following activation of NF-κB to enable easy detection of pro-inflammatory and reparative signalling. Results and conclusions Meg-01 and Meg-01R cells have comparable characteristics including the expression of both GPIb and integrin β3. Meg-01R cells responded to various inflammatory molecules as measured by NF-κB-dependent bioluminescence. For example, inflammatory molecules such as TNFα and Pam3CSK4 increased NF-κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg-01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg-01R cells were able to respond to LPS (non-ultrapure) although it was not able to react to ultrapure LPS due to the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterisation of a novel megakaryocyte NF-κB reporter cell line (Meg-01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF-κB activity

Optically Transparent Anionic Nanofibrillar Cellulose Is Cytocompatible with Human Adipose Tissue-Derived Stem Cells and Allows Simple Imaging in 3D.

The anti-inflammatory and immunomodulatory properties of human mesenchymal stromal cells (MSCs) are a focus within regenerative medicine. However, 2D cultivation of MSCs for extended periods results in abnormal cell polarity, chromosomal changes, reduction in viability, and altered differentiation potential. As an alternative, various 3D hydrogels have been developed which mimic the endogenous niche of MSCs. Nevertheless, imaging cells embedded within 3D hydrogels often suffers from low signal-to-noise ratios which can be at least partly attributed to the high light absorbance and light scattering of the hydrogels in the visible light spectrum. In this study, human adipose tissue-derived MSCs (ADSCs) are cultivated within an anionic nanofibrillar cellulose (aNFC) hydrogel. It is demonstrated that aNFC forms nanofibres arranged as a porous network with low light absorbance in the visible spectrum. Moreover, it is shown that aNFC is cytocompatible, allowing for MSC proliferation, maintaining cell viability and multilineage differentiation potential. Finally, aNFC is compatible with scanning electron microscopy (SEM) and light microscopy including the application of conventional dyes, fluorescent probes, indirect immunocytochemistry, and calcium imaging. Overall, the results indicate that aNFC represents a promising 3D material for the expansion of MSCs whilst allowing detailed examination of cell morphology and cellular behaviour.Peer Reviewe

Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae

Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity

Modern microwave methods in solid state inorganic materials chemistry: from fundamentals to manufacturing

No abstract available