Magnetic coagulometry: towards a new nanotechnological tool for ex vivo monitoring coagulation in human whole blood.

Blood clotting disorders consisting of unwanted blood clot formation or excessive bleeding are some of the main causes of death worldwide. However, there are significant limitations in the current methods used to clinically monitor the dynamics of clot formation in human whole blood ex vivo. Here a new magnetic coagulometry platform for testing ex vivo coagulation is described. This platform exploits the sensitivity of the out-of-phase component of alternating current (AC) magnetic susceptibility (χ'') to variations in mobility and agglomeration of magnetic nanoparticles when trapped during blood clot formation. By labelling human whole blood with magnetic nanoparticles, the out-of-phase component of AC magnetic susceptibility shows that the dynamics of blood clot formation correlates with a decrease in the out-of-phase component χ'' over time activation of coagulation. This is caused by a rapid immobilisation of nanoparticles upon blood coagulation and compaction. In contrast, this rapid fall in the out-of-phase component χ'' is significantly slowed down when blood is pre-treated with three different anticoagulant drugs. Remarkably, the system showed sensitivity towards the effect of clinically used direct oral anticoagulation (DOAC) drugs in whole blood coagulation, in contrast to the inability of clinical routine tests prothrombin time (PT) and partial thromboplastin time (PTT) to efficiently monitor this effect. Translation of this nanomagnetic approach into clinic can provide a superior method for monitoring blood coagulation and improve the efficiency of the current diagnostic techniques.

Developing Biomimetic Hydrogels of the Arterial Wall as a Prothrombotic Substrate for In Vitro Human Thrombosis Models.

Current in vitro thrombosis models utilise simplistic 2D surfaces coated with purified components of the subendothelial matrix. The lack of a realistic humanised model has led to greater study of thrombus formation in in vivo tests in animals. Here we aimed to develop 3D hydrogel-based replicas of the medial and adventitial layers of the human artery to produce a surface that can optimally support thrombus formation under physiological flow conditions. These tissue-engineered medial- (TEML) and adventitial-layer (TEAL) hydrogels were developed by culturing human coronary artery smooth muscle cells and human aortic adventitial fibroblasts within collagen hydrogels, both individually and in co-culture. Platelet aggregation upon these hydrogels was studied using a custom-made parallel flow chamber. When cultured in the presence of ascorbic acid, the medial-layer hydrogels were able to produce sufficient neo-collagen to support effective platelet aggregation under arterial flow conditions. Both TEML and TEAL hydrogels possessed measurable tissue factor activity and could trigger coagulation of platelet-poor plasma in a factor VII-dependent manner. Biomimetic hydrogel replicas of the subendothelial layers of the human artery are effective substrates for a humanised in vitro thrombosis model that could reduce animal experimentation by replacing current in vivo models.

Developing human tissue engineered arterial constructs to simulate human in vivo thrombus formation.

Thrombus formation is highly dependent upon the physico-chemical environment in which it is triggered. Our ability to understand how thrombus formation is initiated, regulated, and resolved in the human body is dependent upon our ability to replicate the mechanical and biological properties of the arterial wall. Current in vitro thrombosis models principally use reductionist approaches to model the complex biochemical and cellular milieu present in the arterial wall, and so researcher have favored the use of in vivo models. The field of vascular tissue engineering has developed a range of techniques for culturing artificial human arteries for use as vascular grafts. These techniques therefore provide a basis for developing more sophisticated 3D replicas of the arterial wall that can be used in in vitro thrombosis models. In this review, we consider how tissue engineering approaches can be used to generate 3D models of the arterial wall that improve upon current in vivo and in vitro approaches. We consider the current benefits and limitations of reported 3D tissue engineered models and consider what additional evidence is required to validate them as alternatives to current in vivo models.

Clot-targeted magnetic hyperthermia permeabilizes blood clots to make them more susceptible to thrombolysis.

BACKGROUND: Thrombolysis is a frontline treatment for stroke, which involves the application of tissue plasminogen activator (tPA) to trigger endogenous clot-degradation pathways. However, it is only effective within 4.5 h of symptom onset because of clot contraction preventing tPA permeation into the clot. Magnetic hyperthermia (MH) mediated by tumor-targeted magnetic nanoparticles is used to treat cancer by using local heat generation to trigger apoptosis of cancer cells. OBJECTIVES: To develop clot-targeting magnetic nanoparticles to deliver MH to the surface of human blood clots, and to assess whether this can improve the efficacy of thrombolysis of contracted blood clots. METHODS: Clot-targeting magnetic nanoparticles were developed by functionalizing iron oxide nanoparticles with an antibody recognizing activated integrin αIIbß3 (PAC-1). The magnetic properties of the PAC-1-tagged magnetic nanoparticles were characterized and optimized to deliver clot-targeted MH. RESULTS: Clot-targeted MH increases the efficacy of tPA-mediated thrombolysis in contracted human blood clots, leading to a reduction in clot weight. MH increases the permeability of the clots to tPA, facilitating their breakdown. Scanning electron microscopy reveals that this effect is elicited through enhanced fibrin breakdown and triggering the disruption of red blood cells on the surface of the clot. Importantly, endothelial cells viability in a three-dimensional blood vessel model is unaffected by exposure to MH. CONCLUSIONS: This study demonstrates that clot-targeted MH can enhance the thrombolysis of contracted human blood clots and can be safely applied to enhance the timeframe in which thrombolysis is effective.

Calcium sequestration by human platelet acidic organelles is regulated by the actin cytoskeleton and autocrine 5-hydroxytryptamine.

Human platelets regulate agonist-evoked Ca2+ signalling through Ca2+ release from and sequestration into acidic organelles. Previous studies have pharmacologically characterised the presence of a Ca2+-H+ exchanger in these organelles. This exchanger appears to regulate a secondary plateau phase in agonist-evoked cytosolic Ca2+ signals in fura-2-loaded human platelets. Here we demonstrate that cytochalasin D treatment removes the secondary plateau in ADP-evoked Ca2+ signals elicited in the absence of external Ca2+. This effect was reversed by pretreatment with nigericin, a K+/H+ exchanger that short-circuits the Ca2+-H+ exchanger. Using Fluo-5N- or Lysosensor Green-loaded cells, cytochalasin D was found to enhance Ca2+ sequestration into acidic organelles by preventing their alkalinisation. Additional experiments demonstrated that ADP-evoked alkalinisation of acidic organelles and subsequent slowing of acidic organellar Ca2+ sequestration was mediated by autocrine 5-HT signalling. Enhancing this 5-HT signalling using fluoxetine overcame the inhibitory effect of cytochalasin D on ADP-evoked Ca2+ signals, indicating that cytochalasin D interferes with 5-HT autocrine signalling. The ability of Cytochalasin D to interfere with autocrine 5-HT signalling was downstream of the 5-HT2A receptor as secretion of [3H]-5-HT from ADP-stimulated human platelets was not reduced. These data provide the first evidence that the pH gradient across acidic organelles is dynamically regulated upon human platelet activation, and that this can play a significant role in controlling human platelet function by modulating Ca2+-H+ exchange and so [Ca2+]i.

The Combination of Tissue-Engineered Blood Vessel Constructs and Parallel Flow Chamber Provides a Potential Alternative to In Vivo Drug Testing Models.

Cardiovascular disease is a major cause of death globally. This has led to significant efforts to develop new anti-thrombotic therapies or re-purpose existing drugs to treat cardiovascular diseases. Due to difficulties of obtaining healthy human blood vessel tissues to recreate in vivo conditions, pre-clinical testing of these drugs currently requires significant use of animal experimentation, however, the successful translation of drugs from animal tests to use in humans is poor. Developing humanised drug test models that better replicate the human vasculature will help to develop anti-thrombotic therapies more rapidly. Tissue-engineered human blood vessel (TEBV) models were fabricated with biomimetic matrix and cellular components. The pro- and anti-aggregatory properties of both intact and FeCl3-injured TEBVs were assessed under physiological flow conditions using a modified parallel-plate flow chamber. These were perfused with fluorescently labelled human platelets and endothelial progenitor cells (EPCs), and their responses were monitored in real-time using fluorescent imaging. An endothelium-free TEBV exhibited the capacity to trigger platelet activation and aggregation in a shear stress-dependent manner, similar to the responses observed in vivo. Ketamine is commonly used as an anaesthetic in current in vivo models, but this drug significantly inhibited platelet aggregation on the injured TEBV. Atorvastatin was also shown to enhance EPC attachment on the injured TEBV. The TEBV, when perfused with human blood or blood components under physiological conditions, provides a powerful alternative to current in vivo drug testing models to assess their effects on thrombus formation and EPC recruitment.

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation.

Human platelets use a rise in cytosolic Ca2+ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca2+ signals to trigger each of these independently is unknown. Other cells create local Ca2+ signals to activate Ca2+-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca2+ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca2+ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca2+ to isolate a cytosolic Ca2+ nanodomain created by Ca2+ release from the DTS. In the absence of any detectable rise in global cytosolic Ca2+ concentration, thrombin stimulation triggered Na+/Ca2+ exchanger (NCX)-dependent Ca2+ removal into the extracellular space, as well as Ca2+-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca2+ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca2+ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca2+ nanodomain contained within the MC that can control shape change independently of platelet aggregation.

Controlling human platelet activation with calcium-binding nanoparticles.

Human platelets aggregate at sites of blood vessel damage in response to a rise in their cytosolic calcium concentration. Controlling these cytosolic calcium rises would provide a method to inhibit platelet activation and prevent the unwanted blood clots that causes heart attack and strokes. Previously we have predicted that calcium accumulation within the lumen of an infolded portion of the platelet plasma membrane called the open canalicular system (OCS) is essential for maintaining this cytosolic calcium rise. Due to its nanometer dimensions of the OCS, it has been difficult to measure or interfere with the predicted luminal calcium accumulation. Here we utilise iron oxide magnetic nanoparticles coated with the known calcium chelator, citrate, to create calcium-binding nanoparticles. These were used to assess whether an OCS calcium store plays a role in controlling the dynamics of human platelet activation and aggregation. We demonstrate that citrate-coated nanoparticles are rapidly and selectively uptaken into the OCS of activated human platelets, where they act to buffer the accumulation of calcium there. Treatment with these calcium-binding nanoparticles reduced thrombin-evoked cytosolic calcium rises, and slowed platelet aggregation and clot retraction in human platelets. In contrast, nanoparticles that cannot bind calcium have no effect. This study demonstrates that the OCS acts as a key source of calcium for maintaining cytosolic calcium rises and accelerating platelet aggregation, and that calcium-binding nanoparticles targeted to the OCS could provide an anti-platelet therapy to treat patients at risk of suffering heart attacks or strokes.

Batch anaerobic digestion of deproteinated malt whisky pot ale using different source inocula.

A novel process has been developed for the selective removal of protein from pot ale with recovered protein holding potential as a value-added by-product for the whisky industry. The purpose of this work was to assess the effect of deproteination on pot ale physicochemical characterisation and anaerobic digestion (AD) treatment. Pot ales were taken from five malt whisky distilleries and tested untreated, after centrifugation/filtration and after deproteination at laboratory or pilot scale. At laboratory scale, the deproteination process removed around 20% of total chemical oxygen demand (tCOD) from untreated pot ale and at least 30% dissolved copper from centrifuged pot ale. Biochemical methane potential of untreated, filtered and deproteinated pot ale obtained at pilot scale has been determined using two types of inocula from different source. Average methane yield values of 554±67, 586±24 and 501±23 Nl CH4 kg-1 VS were obtained for untreated, filtered and deproteinated pot ale respectively. A significant difference in methane yield was only observed for untreated pot ale using the two types of inocula. Specifically, when using a non-adapted inoculum untreated pot ale biogas yield was significant lower suggesting inhibition of the AD process. As no significant differences were found for treated pot ale (filtered and deproteinated) with the two inocula it suggests, deproteination may have a positive effect on AD start-up. The results present a clear case for continuation of this work and evaluating the effect on continuous AD.

The role of biomarkers in the diagnosis and risk stratification of acute coronary syndrome.

Coronary artery disease is a growing concern. Although traditional biomarkers, such as troponins and creatine kinase, play a central role in the diagnosis, risk stratification and management of coronary artery disease, they are unable to detect myocardial ischemia in the absence of necrosis. Therefore, early detection of ischemia in patients presenting with acute coronary syndrome still remains a burning question. High-sensitivity troponin is evolving as a reliable biomarker in this regard and has been absorbed into clinical practice. Biomarkers are currently the focus of immense interest as it not only helps with diagnosis and management but also helps to understand the pathophysiology of the disease process. In addition, analysis using a multimarker strategy has also proven to be a very useful tool in risk stratification. This review will focus on the biomarkers and its application in the diagnosis and risk stratification of acute coronary syndrome.

Correction to: Islet-intrinsic effects of CFTR mutation.

Unfortunately, due to a tagging error, Dr Fiona N. Manderson Koivula's name is shown incorrectly as Koivula FN on PubMed. Her name appears correctly in the html and pdf versions of the paper.

A Real-Time Monitoring System to Assess the Platelet Aggregatory Capacity of Components of a Tissue-Engineered Blood Vessel Wall.

Native blood vessels contain both an antiaggregatory intimal layer, which prevents platelet activation in the intact vessel, and a proaggregatory medial layer, which stimulates platelet aggregation upon vascular damage. Yet, current techniques for assessing the functional properties of tissue-engineered blood vessels may not be able to assess the relative effectiveness of both these pro- and antiaggregatory properties of the vessel construct. In this study, we present a novel technique for quantitatively assessing the pro- and antiaggregatory properties of different three-dimensional blood vessel constructs made using a layered fabrication method. This technique utilizes real-time measurements of cytosolic Ca(2+) signaling to assess platelet activation in fluorescently labeled human platelet suspensions using fluorescence spectrofluorimetry, while also permitting examination of thrombus formation upon the surface of the construct using fluorescent imaging of DiOC6-labeled platelets. Experiments using this method demonstrated that type I collagen hydrogels, commonly used as scaffolds for vascular tissue engineering, were unable to support significant platelet activation, while type I and III neo-collagen secreted from human coronary artery smooth muscle cells cultured within these hydrogels as the medial layer were able to support thrombus formation. The incorporation of an intimal layer consisting of human umbilical vein endothelial cells on top of the medial layer inhibited platelet activation and aggregation. These data demonstrate that the methodology presented here is able to quantitatively compare the capacity of different constructs to trigger or prevent platelet activation. As such, this technique may provide a useful tool for standardizing the assessment of the functional properties of tissue-engineered blood vessel constructs developed using different culturing techniques.

TRP-Na(+)/Ca(2+) Exchanger Coupling.

Na(+)/Ca(2+) exchangers (NCXs) have traditionally been viewed principally as a means of Ca(2+) removal from non-excitable cells. However there has recently been increasing interest in the operation of NCXs in reverse mode acting as a means of eliciting Ca(2+) entry into these cells. Reverse mode exchange requires a significant change in the normal resting transmembrane ion gradients and membrane potential, which has been suggested to occur principally via the coupling of NCXs to localised Na(+) entry through non-selective cation channels such as canonical transient receptor potential (TRPC) channels. Here we review evidence for functional or physical coupling of NCXs to non-selective cation channels, and how this affects NCX activity in non-excitable cells. In particular we focus on the potential role of nanojunctions, where the close apposition of plasma and intracellular membranes may help create the conditions needed for the generation of localised rises in Na(+) concentration that would be required to trigger reverse mode exchange.

Islet-intrinsic effects of CFTR mutation.

Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD. Summary • CF is an autosomal recessive disorder caused by mutations in the CFTR gene • The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline • Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell • Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population.

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca²âº signalling in human platelets.

Rises in cytosolic Ca(2+) concentration ([Ca(2+)]cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca(2+)]cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca(2+)]cyt (Ca(2+) buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca(2+) or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca(2+) signalling, we here monitor Ca(2+) flux around the platelet by measuring net Ca(2+) fluxes to or from the extracellular space and the intracellular Ca(2+) stores, which act as the major sources and sinks for Ca(2+) influx into and efflux from the cytosol, as well as monitoring the cytosolic Na(+) concentration ([Na(+)]cyt), which influences platelet Ca(2+) fluxes via Na(+)/Ca(2+) exchange. The intracellular store Ca(2+) concentration ([Ca(2+)]st) was monitored using Fluo-5N, the extracellular Ca(2+) concentration ([Ca(2+)]ext) was monitored using Fluo-4 whilst [Ca(2+)]cyt and [Na(+)]cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca(2+)]cyt in the absence of extracellular Ca(2+). PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca(2+) release and Ca(2+) removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca(2+)]cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na(+)]cyt which would be expected to reduce Ca(2+) removal via the Na(+)/Ca(2+) exchanger (NCX). Thrombin-evoked rises in [Na(+)]cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn(2+) quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca(2+)]cyt following SERCA inhibition and either removal of extracellular Na(+) or inhibition of Na(+)/K(+)-ATPase activity by removal of extracellular K(+) or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca(2+)]cyt by acceleration of SERCA activity, whilst rises in [Ca(2+)]cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na(+)/K(+)-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na(+)]cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca(2+) signalling.

Pericellular Ca(2+) recycling potentiates thrombin-evoked Ca(2+) signals in human platelets.

We have previously demonstrated that Na(+)/Ca(2+) exchangers (NCXs) potentiate Ca(2+) signaling evoked by thapsigargin in human platelets, via their ability to modulate the secretion of autocoids from dense granules. This link was confirmed in platelets stimulated with the physiological agonist, thrombin, and experiments were performed to examine how Ca(2+) removal by the NCX modulates platelet dense granule secretion. In cells loaded with the near-membrane indicator FFP-18, thrombin stimulation was observed to elicit an NCX-dependent accumulation of Ca(2+) in a pericellular region around the platelets. To test whether this pericellular Ca(2+) accumulation might be responsible for the influence of NCXs over platelet function, platelets were exposed to fast Ca(2+) chelators or had their glycocalyx removed. Both manipulations of the pericellular Ca(2+) rise reduced thrombin-evoked Ca(2+) signals and dense granule secretion. Blocking Ca(2+)-permeable ion channels had a similar effect, suggesting that Ca(2+) exported into the pericellular region is able to recycle back into the platelet cytosol. Single cell imaging with extracellular Fluo-4 indicated that thrombin-evoked rises in extracellular [Ca(2+)] occurred within the boundary described by the cell surface, suggesting their presence within the open canalicular system (OCS). FFP-18 fluorescence was similarly distributed. These data suggest that upon thrombin stimulation, NCX activity creates a rise in [Ca(2+)] within the pericellular region of the platelet from where it recycles back into the platelet cytosol, acting to both accelerate dense granule secretion and maintain the initial rise in cytosolic [Ca(2+)].

Salicylic acid-releasing polyurethane acrylate polymers as anti-biofilm urological catheter coatings.

Biofilm-associated infections are a major complication of implanted and indwelling medical devices like urological and venous catheters. They commonly persist even in the presence of an oral or intravenous antibiotic regimen, often resulting in chronic illness. We have developed a new approach to inhibiting biofilm growth on synthetic materials through controlled release of salicylic acid from a polymeric coating. Herein we report the synthesis and testing of a ultraviolet-cured polyurethane acrylate polymer composed, in part, of salicyl acrylate, which hydrolyzes upon exposure to aqueous conditions, releasing salicylic acid while leaving the polymer backbone intact. The salicylic acid release rate was tuned by adjusting the polymer composition. Anti-biofilm performance of the coatings was assessed under several biofilm forming conditions using a novel combination of the MBEC Assay™ biofilm multi-peg growth system and bioluminescence monitoring for live cell quantification. Films of the salicylic acid-releasing polymers were found to inhibit biofilm formation, as shown by bioluminescent and GFP reporter strains of Pseudomonas aeruginosa and Escherichia coli. Urinary catheters coated on their inner lumens with the salicylic acid-releasing polymer significantly reduced biofilm formation by E. coli for up to 5 days under conditions that simulated physiological urine flow.

A key role for dense granule secretion in potentiation of the Ca2+ signal arising from store-operated calcium entry in human platelets.

Recent work has demonstrated a role for Na(+)/Ca(2+) exchange in potentiation of the Ca(2+) entry elicited through the human platelet store-operated channel by controlling a Mn(2+)-impermeable Ca(2+) entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na(+)/Ca(2+) exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn(2+)-impermeable Ca(2+) entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca(2+) signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca(2+) signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets.

Two mechanisms for store-operated Ca(2+) entry (SOCE) regulated by two independent Ca(2+) stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP(3) receptor (IP(3)RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca(2+) entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca(2+)-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca(2+) stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca(2+) stores and the DTS, the major releasable Ca(2+) store in platelets. These findings were confirmed by using Sr(2+) as a surrogate for Ca(2+) entry. Colchicine attenuated the coupling between IP(3)RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP(3)RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca(2+). These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca(2+) stores.