25 results found
Taylor KA, Mahaut-Smith MP, 2021, Ion channels and ion homeostasis in the platelet and megakaryocyte, PLATELETS, ISSN: 0953-7104
Huang Y, Gu B, Salles II, et al., 2021, Fibrinogen-mimicking, multi-arm nanovesicles for human thrombus-specific delivery of tissue plasminogen activator and targeted thrombolytic therapy, Science Advances, Vol: 7, ISSN: 2375-2548
Clinical use of tissue plasminogen activator (tPA) in thrombolytic therapy is limited by its short circulation time and hemorrhagic side effects. Inspired by fibrinogen binding to activated platelets, we report a fibrinogen-mimicking, multi-arm nanovesicle for thrombus-specific tPA delivery and targeted thrombolysis. This novel system is based on the lipid nanovesicle coated with polyethylene glycol (PEG) terminally conjugated with a cyclic RGD (cRGD) peptide. Our experiments with human blood demonstrated its highly selective binding to activated platelets and efficient tPA release at a thrombus site under both static and physiological flow conditions. Its clot dissolution time in a microfluidic system was comparable to that of free tPA. Furthermore, we report a purpose-built computational model capable of simulating targeted thrombolysis of the tPA-loaded nanovesicle and with potential in predicting the dynamics of thrombolysis in physiologically realistic scenarios. This combined experimental and computational work presents a promising platform for development of thrombolytic nanomedicines.
Taylor KA, 2020, EDI(torial): equality, diversity, and inclusion and platelets-2021, PLATELETS, Vol: 32, Pages: 3-4, ISSN: 0953-7104
Khawaja AA, Taylor KA, Lovell AO, et al., 2020, HIV antivirals affect endothelial activation and endothelial-platelet crosstalk., Circulation Research, Vol: 127, Pages: 1365-1380, ISSN: 0009-7330
Rationale: People living with human immunodeficiency virus (PLHIV) on effective antiretroviral therapy are at increased risk of cardiovascular complications, possibly due to off-target drug effects. Some studies have associated antiretroviral therapy with increased risk of myocardial infarction and endothelial dysfunction, but a link between endothelial function and antiretrovirals has not been established. Objective: To determine the effects of antiretrovirals in common clinical use upon in vitro endothelial function in order to better understand cardiovascular risk in PLHIV. Methods and Results: Human umbilical cord vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were pre-treated with the antiretrovirals abacavir sulphate (ABC), tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF). Expression of adhesion molecules, ectonucleotidases (CD39 and CD73), tissue factor (TF), endothelial-derived microparticle (EMP) numbers and phenotype, and platelet activation were evaluated by flow cytometry. TF and ectonucleotidase activities were measured using colourimetric plate-based assays. ABC-treated endothelial cells had higher levels of ICAM-1 and TF expression following TNF-α stimulation. In contrast, TDF and TAF treatment gave rise to greater populations of CD39+CD73+ cells. These cell surface differences were also observed within EMP repertoires. ABC-treated cells and EMP had greater TF activity, whilst TDF- and TAF-treated cells and EMP displayed higher ectonucleotidase activity. Finally, EMP isolated from ABC-treated cells enhanced collagen-evoked platelet integrin activation and α-granule release. Conclusions: We report differential effects of antiretrovirals used in the treatment of HIV upon endothelial function. ABC treatment led to an inflammatory, pro-thrombotic endothelial phenotype that promoted platelet activation. In contrast, TDF and TAF conferred potentially cardioprotective properties associated with
Khawaja AA, Maughan RT, Paschalaki KE, et al., 2020, Modelling endothelial function in vitro and via blood sampling to assess cardiovascular risk in people living with HIV, Publisher: JOHN WILEY & SONS LTD, Pages: 105-105
Taylor KA, Machlus KR, 2020, Blood and Bone: The quarantine chronicles, RESEARCH AND PRACTICE IN THROMBOSIS AND HAEMOSTASIS, Vol: 4, Pages: 727-730
Boustani K, Taylor KA, 2020, Navigating LGBTQ+ discrimination in academia: where do we go from here?, The Biochemist, Vol: 42, Pages: 16-20, ISSN: 0954-982X
<jats:p>Lesbian, gay, bisexual, trans and sexually/gender diverse (LGBTQ+) individuals have long been underrepresented in science, technology, engineering and mathematics (STEM) and these environments have often been portrayed as spaces in which personal identity does not matter. However, for LGBTQ+ individuals, this means suppressing their gender identity and expression and remaining closeted at work, creating an uncomfortable work environment, and this can affect their performance and mental health. Multiple reports have been published within the last decade investigating the experiences of LGBTQ+ people in science. These reports all highlight a common observation that, at some point in their time within science, the majority of individuals have experienced discrimination due to their sexual orientation or gender identity. Here, in our opinion piece, we discuss our experiences of being LGBTQ+ in bioscience, the various types of discrimination that LGBTQ+ scientists may face in academia and some of the existing initiatives and campaigns in place to combat this.</jats:p>
Khawaja A, Taylor K, Lovell A, et al., 2020, Differential effects of HIV antiretroviral drugs upon pro-inflammatory and cardioprotective properties of vascular endothelial cells, Meeting of the British-Pharmacological-Society, Publisher: WILEY, Pages: 2578-2579, ISSN: 0007-1188
Khawaja AA, Taylor KA, Lovell AO, et al., 2019, Distinct pro-inflammatory and cardio-protective effects of antiretroviral drugs in vascular endothelial cells, Publisher: WILEY, Pages: 230-230, ISSN: 1464-2662
Lovell AO, Taylor KA, Winston A, et al., 2019, Investigation of the impact of antiretroviral therapy upon platelet activation to determine HIV-associated cardiovascular risk, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3047-3047, ISSN: 0007-1188
Taylor KA, Mahaut-Smith MP, 2019, A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01, Platelets (London), Vol: 30, Pages: 962-966, ISSN: 0953-7104
TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions.We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5–6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl–-permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic “leak” hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.
Khawaja A, Taylor K, Nelson M, et al., 2019, Abacavir sulphate and tenofovir disoproxil fumarate/alafenamide differentially regulate endothelial dysfunction, Publisher: WILEY, Pages: 24-24, ISSN: 1464-2662
Taylor KA, Smyth E, Rauzi F, et al., 2019, Pharmacological impact of antiretroviral therapy on platelet function to investigate HIV-associated cardiovascular risk, British Journal of Pharmacology, Vol: 176, Pages: 879-889, ISSN: 0007-1188
Background and purposeSome clinical studies have reported increased myocardial infarction in people living with HIV taking the antiretroviral abacavir sulphate (ABC). Given that clinical studies contain confounding variables (e.g. HIV status), we investigated the pharmacological impact of antiretrovirals on platelet function in HIV‐negative volunteers in order to identify mechanisms of increased cardiovascular risk.Experimental approachPlatelets were isolated from healthy volunteers and HIV‐negative subjects enrolled on a Phase I clinical trial and platelet function evaluated using aggregometry and flow cytometry. In vivo platelet thromboembolism was monitored in anaesthetised mice.Key resultsHuman platelet aggregation was unaffected by all antiretrovirals tested but ABC treatment led uniquely to increased platelet granule release. ABC also interrupted nitric oxide (NO)‐mediated inhibition of platelet aggregation and increased in vivo aggregation in mice. An alternative antiretroviral, tenofovir, did not affect platelet function. Furthermore, aggregation and activation of platelets isolated from twenty subjects taking clinically‐relevant doses of tenofovir were comparable to baseline samples.Conclusions and implicationsABC can enhance platelet activation, independently of HIV status suggesting a potential pharmacological effect that is absent with tenofovir. Mechanistically, we propose that ABC enhances platelet degranulation and interrupts NO‐mediated platelet inhibition. The interaction of ABC with NO signalling is supported by data demonstrating ABC‐mediated enhancement of aggregation in vivo and in vitro responses that persist in the presence of NO. Although an association between ABC and platelet activation has not been confirmed in patients, these findings provide evidence of a mechanistic link between platelet activation and antiretroviral therapy.
Ahmed N, Lopes Pires M, Taylor K, et al., 2019, Agonist-evoked increases in intra-platelet zinc couple to functional responses, Thrombosis and Haemostasis, Vol: 119, Pages: 128-139, ISSN: 0340-6245
Background Zinc (Zn2+) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn2+ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated.Objectives This article determines whether cytosolic Zn2+ concentrations ([Zn2+]i) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn2+]i changes on activation markers.Methods Changes in [Zn2+]i were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn2+]i were modelled using Zn2+-specific chelators and ionophores. The influence of [Zn2+]i on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting.Results Increases of intra-platelet Fluozin-3 (Fz-3) fluorescence occurred in response to stimulation by cross-linked collagen-related peptide (CRP-XL) or U46619, consistent with a rise of [Zn2+]i. Fluoresence increases were blocked by Zn2+ chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca2+]i signals. Stimulation of platelets with the Zn2+ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn2+]i, resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin αIIbβ3 activation and release of dense, but not α, granules. Furthermore, Zn2+-ionophores induced externalization of phosphatidylserine.Conclusion These data suggest that agonist-evoked fluctuations in intra-platelet Zn2+ couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn2+ regulates signalling processes, including shape change, αIIbβ3 up-regulation and dense granule release, in a redox-sensitive manner.
Khawaja AA, Taylor KA, Nelson M, et al., 2018, Differential Effects of Antiretroviral Drugs Upon Endothelial Activation and Microparticle Repertoire, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Gillespie S, Holloway PM, Becker F, et al., 2018, The isothiocyanate sulforaphane modulates platelet function and protects against cerebral thrombotic dysfunction, British Journal of Pharmacology, Vol: 175, Pages: 3333-3346, ISSN: 1476-5381
Background and purposePlatelet activation provides a critical link between inflammation and thrombosis. Sulforaphane (SFN), a naturally occurring isothiocyanate, has been shown to display both anti‐inflammatory and anti‐thrombotic actions in the systemic microvasculature. As inflammation promotes thrombosis and vice versa, this study investigates whether SFN is able to reduce inflammatory potentiation of thrombotic events, suppress platelet activation and thrombus formation in the cerebral microvasculature.Experimental approachThrombosis was induced in the murine brain using the light/dye‐injury model, in conjunction with lipopolysaccharide (LPS) treatment, with and without SFN treatment. In vitro and in vivo platelet assays (aggregation, flow and other functional tests) were also employed, using both human and murine platelets.Key ResultsSFN was found to reduce LPS mediated enhancement of thrombus formation in the cerebral microcirculation. In tail bleed experiments LPS treatment prolonged bleeding time, and SFN treatment was found to protect against this LPS‐induced derangement of platelet function. SFN inhibited collagen‐mediated platelet aggregation in vitro and in vivo and the associated adhesion and impaired calcium signalling. Furthermore, Glycoprotein VI (GPVI) is involved in the protective effects observed with SFN treatment.Conclusions and ImplicationsData presented here provides evidence for the use of SFN in preventing stroke in selected high‐risk patient cohorts.
Taylor KA, Emerson M, 2018, Refinement of a mouse cardiovascular model: development, application and dissemination, F1000Research, Vol: 7, ISSN: 2046-1402
European and UK legislation requires all animal procedures to be conductedwith consideration to reduction, refinement and replacement. In this review,3Rs developments are discussed in the field of platelet biology andthromboembolism. Platelet research requires the use of animal models, andmice are widely used in the field. When working , conventional lightin vitrotransmission techniques have been scaled down allowing reduction in animalnumbers. , vascular injury models are widely used and work is ongoing toIn vivodevelop approaches that use fewer animals. Thromboembolic mortalityex vivomodels, which inflict considerable pain and suffering, have also been usedwidely. A published and characterised refinement of this mortality model allowsreal-time monitoring of radiolabelled platelets under general anaesthesia andreduces both the severity level and the numbers of mice used in a typicalexperiment. This technique is more sensitive than the mortality approach andhas opened up new avenues of research, which would not have been feasibleby using death as an end-point. To drive uptake of real-time monitoring, a moresimplistic approach has been developed involving micro-sampling and cellcounting. Thromboembolic mortality models should therefore be consideredobsolete due to the emergence of 3Rs models with improved scientificoutcomes and that can be implemented relatively easily.
Taylor KA, Wilson DGS, Harper MT, et al., 2017, Extracellular chloride is required for efficient platelet aggregation., Platelets, Vol: 29, Pages: 79-83, ISSN: 0953-7104
Anion channels perform a diverse range of functions and have been implicated in ATP release, volume regulation, and phosphatidylserine exposure. Platelets have been shown to express several anion channels but their function is incompletely understood. Due to a paucity of specific pharmacological blockers, we investigated the effect of extracellular chloride substitution on platelet activation using aggregometry and flow cytometry. In the absence of extracellular chloride, we observed a modest reduction of the maximum aggregation response to thrombin or collagen-related peptide. However, the rate of aggregation was substantially reduced in a manner that was dependent on the extracellular chloride concentration and aggregation in the absence of chloride was noticeably biphasic, indicative of impaired secondary signaling. This was further investigated by targeting secondary agonists with aspirin and apyrase or by blockade of the ADP receptor P2Y12. Under these conditions, the rates of aggregation were comparable to those recorded in the absence of extracellular chloride. Finally, we assessed platelet granule release by flow cytometry and report a chloride-dependent element of alpha, but not dense, granule secretion. Taken together these data support a role for anion channels in the efficient induction of platelet activation, likely via enhancement of secondary signaling pathways.
Pugh N, Maddox BD, Bihan D, et al., 2017, Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions., Thrombosis and Haemostasis, Vol: 117, Pages: 1588-1600, ISSN: 0340-6245
The platelet receptors glycoprotein (Gp)VI, integrin α2β1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca(2+) ([Ca(2+)]i) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca(2+)]i measurements using confocal imaging. All three collagen receptors coupled to [Ca(2+)]i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca(2+)]i signals leading to real-time increases in integrins α2β1- and αIIbβ3-mediated platelet adhesion. αIIbβ3-dependent platelet aggregation was dependent on P2Y12 signalling. Co-engagement of α2β1 and GpIb/V/IX generated transient [Ca(2+)]i spikes and low amplitude [Ca(2+)]i responses that potentiated GpVI-dependent [Ca(2+)]i signalling. Therefore α2β1, GpIb/V/IX and GpVI synergize to generate [Ca(2+)]i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for αIIbβ3 in stable platelet adhesion and aggregation.
Watson BR, White NA, Taylor KA, et al., 2016, Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner, Metallomics, Vol: 8, Pages: 91-100, ISSN: 1756-5901
Following platelet adhesion and primary activation at sites of vascular injury, secondary platelet activation is induced by soluble platelet agonists, such as ADP, ATP, thrombin and thromboxane. Zinc ions are also released from platelets and damaged cells and have been shown to act as a platelet agonist. However, the mechanism of zinc-induced platelet activation is not well understood. Here we show that exogenous zinc gains access to the platelet cytosol and induces full platelet aggregation that is dependent on platelet protein tyrosine phosphorylation, PKC and integrin αIIbβ3 activity and is mediated by granule release and secondary signalling. ZnSO4 increased the binding affinity of GpVI, but not integrin α2β1. Low concentrations of ZnSO4 potentiated platelet aggregation by collagen-related peptide (CRP-XL), thrombin and adrenaline. Chelation of intracellular zinc reduced platelet aggregation induced by a number of different agonists, inhibited zinc-induced tyrosine phosphorylation and inhibited platelet activation in whole blood under physiologically relevant flow conditions. Our data are consistent with a transmembrane signalling role for zinc in platelet activation during thrombus formation.
Taylor KA, Pugh N, 2016, The contribution of zinc to platelet behaviour during haemostasis and thrombosis, METALLOMICS, Vol: 8, Pages: 144-155, ISSN: 1756-5901
Osman S, Taylor KA, Allcock N, et al., 2016, Detachment of surface membrane invagination systems by cationic amphiphilic drugs, Scientific Reports, Vol: 6, ISSN: 2045-2322
Several cell types develop extensive plasma membrane invaginations to serve a specificphysiological function. For example, the megakaryocyte demarcation membrane system (DMS)provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitateexcitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurementsand electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause completeseparation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulininhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs alsocaused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of humanplatelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinaseinhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cellmembrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surfacedetachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a largeclass of pharmaceutical compounds.
Mahaut-Smith MP, Taylor KA, Evans RJ, 2016, Calcium Signalling through Ligand-Gated Ion Channels such as P2X1 Receptors in the Platelet and other Non-Excitable Cells, CALCIUM ENTRY PATHWAYS IN NON-EXCITABLE CELLS, Editors: Rosado, Publisher: SPRINGER INT PUBLISHING AG, Pages: 305-329, ISBN: 978-3-319-26972-6
Taylor KA, Wright JR, Mahaut-Smith MP, 2015, Regulation of Pannexin-1 channel activity, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 43, Pages: 502-507, ISSN: 0300-5127
Taylor KA, Wright JR, Vial C, et al., 2014, Amplification of human platelet activation by surface pannexin-1 channels, Journal of Thrombosis and Haemostasis, Vol: 12, Pages: 987-998, ISSN: 1538-7836
BackgroundPannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ~1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown.ObjectiveTo determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques.MethodsPanx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca2+ influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL−1 apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels.ResultsPANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca2+ influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors.
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