Platelet functions in lymphatic filariasis patients.

Filariasis is a chronic disease where parasitic worms survive in human hosts even for decades and lead to complications like lymphedema and elephantiasis. Despite the persistent existence of filarial parasites in human hosts, fatal and thrombotic complications are not known, unlike other parasitic diseases like malaria. This suggests that filarial parasites might be affecting the host's platelet functions. This study was conducted to examine platelet functions in confirmed filariasis patients and healthy controls. Results showed that filariasis patients had larger platelets, inhibited aggregation, and slower speed of aggregation, compared to controls. However, in vivo markers of platelet activation and degranulation (beta thromboglobulin and soluble P-selectin) were not affected. Observations suggested that there is increased platelet turnover, cellular apoptosis and inhibited platelet functions in filariasis patients compared to controls. Platelet function inhibition was not associated with the duration of disease, lymphedema-affected organs, or gender of patients. This study confirms that filarial parasites modulate platelet functions in human hosts.

Fatty acid oxidation fuels agonist-induced platelet activation and thrombus formation: Targeting ß-oxidation of fatty acids as an effective anti-platelet strategy.

Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of ß-oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin-stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P-selectin externalization and integrin αIIb ß3 activation. Both etomoxir and trimetazidine impeded agonist-induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose- and incubation time- dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen-epinephrine-induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre-administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride-induced mesenteric thrombosis. In conclusion, ß-oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy-intensive agonist-induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

Non-canonical non-genomic morphogen signaling in anucleate platelets: a critical determinant of prothrombotic function in circulation.

Circulating platelets derived from bone marrow megakaryocytes play a central role in thrombosis and hemostasis. Despite being anucleate, platelets express several proteins known to have nuclear niche. These include transcription factors and steroid receptors whose non-genomic functions are being elucidated in platelets. Quite remarkably, components of some of the best-studied morphogen pathways, namely Notch, Sonic Hedgehog (Shh), and Wnt have also been described in recent years in platelets, which regulate platelet function in the context of thrombosis as well as influence their survival. Shh and Notch pathways in stimulated platelets establish feed-forward loops of autocrine/juxtacrine/paracrine non-canonical signaling that helps perpetuate thrombosis. On the other hand, non-canonical Wnt signaling is part of a negative feedback loop for restricting platelet activation and possibly limiting thrombus growth. The present review will provide an overview of these signaling pathways in general. We will then briefly discuss the non-genomic roles of transcription factors and steroid receptors in platelet activation. This will be followed by an elaborate description of morphogen signaling in platelets with a focus on their bearing on platelet activation leading to hemostasis and thrombosis as well as their potential for therapeutic targeting in thrombotic disorders.

Nattokinase Encapsulated Nanomedicine for Targeted Thrombolysis: Development, Improved in Vivo Thrombolytic Effects, and Ultrasound/Photoacoustic Imaging.

Nattokinase (NK), a potent thrombolytic enzyme that dissolves blood clots, is highly used in the treatment of cardiovascular disorders. However, its effective delivery remains demanding because of stability and bioavailability problems owing to its high molecular weight and proteineous nature. In this research, we have developed novel NK-loaded nontargeted liposomes (NK-LS) and targeted liposomes (RGD-NK-LS and AM-NK-LS) by the reverse phase evaporation method. The physiochemical characterizations (particle size, polydispersity index, zeta potential, and morphology) were performed by a Zetasizer, SEM, TEM, and AFM. The Bradford assay and XPS analysis confirmed the successful surface conjugation of the targeting ligands. Platelet interaction studies by CLSM, photon imager optima, and flow cytometry showed significantly higher (P < 0.05) platelet binding affinity of targeted liposomes. In vitro evaluations were performed using human blood and a fibrinolysis study by CLSM imaging demonstrating the potent antithrombotic efficacy of AM-NK-LS. Furthermore, bleeding and clotting time studies revealed that the targeted liposomes were free from any bleeding complications. Moreover, the in vivo FeCl3 model on Sprague-Dawley (SD) rats using a Doppler flow meter and ultrasound/photoacoustic imaging indicated the increased % thrombolysis and potent affinity of targeted liposomes toward the thrombus site. Additionally, in vitro hemocompatibility and histopathology studies demonstrated the safety and biocompatibility of the nanoformulations.

Human platelets express functional ectonucleotidases that restrict platelet activation signaling.

Platelets play central role in thrombosis and haemostasis. Platelets store adenine nucleotides in their dense granules, which are released upon agonist-stimulation. Level of these nucleotides in extracellular fluid is regulated by activities of ectonucleotidases such as ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) expressed on platelet surface. Here we demonstrate that, expression of surface-bound ectonucleotidases rose significantly in platelets, concomitant with upregulation of their enzymatic activities, when cells were stimulated with thrombin. Interestingly, inhibition of CD73 in thrombin-treated platelets led to enhanced tyrosine phosphorylation of proteins and rise in intracellular free calcium, [Ca2+]i, thus signifying the inhibitory role of the ectonucleotidase on agonist-mediated platelet signaling.

Correction to: Plasma Fibrinogen Is a Natural Deterrent to Amyloid ß-Induced Platelet Activation and Neuronal Toxicity.

Following publication of the original article [1], the author reported an error in Figure 1. The correct version of Figure 1 is as follows.

Platelet HIF-2α promotes thrombogenicity through PAI-1 synthesis and extracellular vesicle release.

Oxygen-compromised environments, such as high altitude, are associated with platelet hyperactivity. Platelets confined within the relatively impervious core of an aggregate/thrombus have restricted access to oxygen, yet they continue to perform energy-intensive procoagulant activities that sustain the thrombus. Studying platelet signaling under hypoxia is, therefore, critical to our understanding of the mechanistic basis of thrombus stability. We report here that hypoxia-inducible factor (HIF)-2α is translated from pre-existing mRNA and stabilized against proteolytic degradation in enucleate platelets exposed to hypoxia. Hypoxic stress, too, stimulates platelets to synthesize plasminogen-activator inhibitor-1 (PAI-1) and shed extracellular vesicles, both of which potentially contribute to the prothrombotic phenotype associated with hypoxia. Stabilization of HIF-α by administering hypoxia-mimetics to mice accelerates thrombus formation in mesenteric arterioles. In agreement, platelets from patients with chronic obstructive pulmonary disease and high altitude residents exhibiting thrombogenic attributes have abundant expression of HIF-2α and PAI- 1. Thus, targeting platelet hypoxia signaling could be an effective anti-thrombotic strategy.

Aerobic glycolysis fuels platelet activation: small-molecule modulators of platelet metabolism as anti-thrombotic agents.

Platelets are critical to arterial thrombosis, which underlies myocardial infarction and stroke. Activated platelets, regardless of the nature of their stimulus, initiate energy-intensive processes that sustain thrombus, while adapting to potential adversities of hypoxia and nutrient deprivation within the densely packed thrombotic milieu. We report here that stimulated platelets switch their energy metabolism to aerobic glycolysis by modulating enzymes at key checkpoints in glucose metabolism. We found that aerobic glycolysis, in turn, accelerates flux through the pentose phosphate pathway and supports platelet activation. Hence, reversing metabolic adaptations of platelets could be an effective alternative to conventional anti-platelet approaches, which are crippled by remarkable redundancy in platelet agonists and ensuing signaling pathways. In support of this hypothesis, small-molecule modulators of pyruvate dehydrogenase, pyruvate kinase M2 and glucose-6-phosphate dehydrogenase, all of which impede aerobic glycolysis and/or the pentose phosphate pathway, restrained the agonist-induced platelet responses ex vivo These drugs, which include the anti-neoplastic candidate, dichloroacetate, and the Food and Drug Administration-approved dehydroepiandrosterone, profoundly impaired thrombosis in mice, thereby exhibiting potential as anti-thrombotic agents.

Regulatory T Cells and Their Association with Serum Markers and Symptoms in Systemic Lupus Erythematosus and Rheumatoid Arthritis.

PURPOSE: Impairment in number and functions of regulatory T cells (Treg) has been found to be associated with many autoimmune diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). This study was conducted to identify and compare Treg by flow cytometry using two different staining approaches. METHODS: Treg were identified by using CD4+CD25+high and CD4+CD25+CD127dim staining approaches in SLE and RA patients and healthy controls. Association of both identified Treg levels with various serum markers and clinical presentation was also examined. RESULTS: Blood CD4+CD25+CD127dim cells levels were 11.4+3.57 %, 9.76+2.37 % and 6.95+1.16 %; while CD4+CD25+high cells were 1.46+1.09 %, 0.95+0.59 % and 1.87+1.14 % in SLE patients, RA patients and healthy controls respectively. Number of CD4+CD25+CD127dim cells was higher than CD4+CD25+high cells in blood samples of all three study groups. Levels of CD4+CD25+CD127dim cells were significantly higher in SLE and RA patients, compared to healthy controls, but this difference was not observed for CD4+CD25+high Treg. CD4+CD25+high levels showed significant correlation with serum C4, IFN-γ and IL-10 levels in healthy subjects and with C4 levels and fever in SLE patients. CD4+CD25+CD127dim levels showed significant association with alopecia and oral ulcers in SLE patients only, but no correlation with measured serum markers. CONCLUSION: Results suggest that both staining approaches detect Treg differently and also that Treg play different role in pathogenesis of SLE and RA.

Modification of histone by glyoxal: recognition of glycated histone containing advanced glycation adducts by serum antibodies of type 1 diabetes patients.

Dicarbonyl compounds react more rapidly, than glucose, with arginine and lysine in proteins to form advanced glycation end products (AGEs) and further produce free radicals which cause DNA damage. AGEs are reliable diagnostic biomarkers for most of the age-related diseases. In the present study histone was modified with glyoxal and it was characterized by various spectral techniques. Binding characteristics of the modified histone towards serum antibodies from type 1 diabetes patients was evaluated by solid phase enzyme immunoassay and the results were compared with normal human subjects. Fluorescence and Fourier transformed infrared analysis of the nuclear protein clearly indicated changes in their respective intensities upon modification with glyoxal. Liquid chromatography together with mass spectrometry showed new peaks and m/z values related to AGE adducts of dihydroimidazolidines/hydroimidazolones. This glyoxal modified protein was recognized by serum antibodies of the diabetes patients while it showed negligible binding with that of normal human subjects. Glyoxal modification of histone causes structural turbulence and formation of advanced glycation adducts in histone. These adducts might be the main antigenic epitope of the modified histone, leading to its recognition by circulating type 1 diabetes antibodies.

Sirtuin Inhibition Induces Apoptosis-like Changes in Platelets and Thrombocytopenia.

Sirtuins are evolutionarily conserved NAD(+)-dependent acetyl-lysine deacetylases that belong to class III type histone deacetylases. In humans, seven sirtuin isoforms (Sirt1 to Sirt7) have been identified. Sirtinol, a cell-permeable lactone ring derived from naphthol, is a dual Sirt1/Sirt2 inhibitor of low potency, whereas EX-527 is a potent and selective Sirt1 inhibitor. Here we demonstrate that Sirt1, Sirt2, and Sirt3 are expressed in enucleate platelets. Both sirtinol and EX-527 induced apoptosis-like changes in platelets, as revealed by enhanced annexin V binding, reactive oxygen species production, and drop in mitochondrial transmembrane potential. These changes were associated with increased phagocytic clearance of the platelets by macrophages. Expression of acetylated p53 and the conformationally active form of Bax were found to be significantly higher in both sirtinol- and EX-527-treated platelets, implicating the p53-Bax axis in apoptosis induced by sirtuin inhibitors. Administration of either sirtinol or EX-527 in mice led to a reduction in both platelet count and the number of reticulated platelets. Our results, for the first time, implicate sirtuins as a central player in the determination of platelet aging. Because sirtuin inhibitors are being evaluated for their antitumor activity, this study refocuses attention on the potential side effect of sirtuin inhibition in delimiting platelet life span and management of thrombosis.

Plasma Fibrinogen Is a Natural Deterrent to Amyloid Beta-Induced Platelet Activation.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, characterized by extensive loss of neurons, and deposition of amyloid beta (Aß) in the form of extracellular plaques. Aß is considered to have critical role in synaptic loss and neuronal death underlying cognitive decline. Platelets contribute to 95% of circulating amyloid-precursor protein that releases Aß into circulation. We have recently demonstrated that, Aß active fragment containing amino acid sequence 25-35 (Aß25-35) is highly thrombogenic in nature, and elicits strong aggregation of washed human platelets in RhoA-dependent manner. In the present study we evaluated the influence of fibrinogen on Aß-induced platelet activation. Intriguingly, Aß failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aß25-35, which could be reversed by increasing doses of Aß. Fibrinogen also attenuated Aß-induced platelet responses like secretion, clot retraction, rise in cytosolic Ca+2 and reactive oxygen species (ROS). Fibrinogen prevented intracellular accumulation of full length amyloid beta peptide (Aß42) in platelets as well as neuronal cells. We conclude that fibrinogen serves as a physiological check against the adverse effects of Aß by preventing its interaction with cells.

Retrograde Coronary Chronic Total Occlusion Intervention Using a Novel Reverse Controlled Antegrade and Retrograde Subintimal Tracking.

Chronic total occlusion (CTO) is a frequent finding in patients with coronary artery disease and when clinically indicated, remains one of the most challenging subsets for intervention. Although successful recanalization rates of CTO have remained suboptimal in percutaneous coronary intervention, evolving techniques including retrograde approach, have raised hopes for improving the overall success. Reverse controlled antegrade and retrograde subintimal tracking is the most commonly used out of various retrograde approaches. In this review, the author has thrown light on a step-by-step approach of this technique, its standardization, various modifications along with complications.

Amyloid ß peptide stimulates platelet activation through RhoA-dependent modulation of actomyosin organization.

Platelets contribute to 95% of circulating amyloid precursor protein in the body and have widely been employed as a "peripheral" model of neurons in Alzheimer's disease. We sought to analyze the effects of amyloid ß (Aß) on platelets and to understand the underlying molecular mechanism. The Aß active fragment containing amino acid sequence 25-35 (Aß(25-35); 10-20 µM) was found to induce strong aggregation of human platelets, granule release, and integrin activation, similar to that elicited by physiological agonists. Platelets exposed to Aß(25-35) retracted fibrin clot and displayed augmented adhesion to collagen under arterial shear, reflective of a switch to prothrombotic phenotype. Exposure of platelets to Aß peptide (20 µM) resulted in a 4.2- and 2.3-fold increase in phosphorylation of myosin light chain (MLC) and MLC phosphatase, respectively, which was reversed by Y27632, an inhibitor of Rho-associated coiled-coil protein kinase (ROCK). Aß(25-35)-induced platelet aggregation and clot retraction were also significantly attenuated by Y27632. Consistent with these findings, Aß(25-35) elicited a significant rise in the level of RhoA-GTP in platelets. Platelets pretreated with reverse-sequenced Aß fragment (Aß(35-25)) and untreated resting platelets served as controls. We conclude that Aß induces cellular activation through RhoA-dependent modulation of actomyosin, and hence, RhoA could be a potential therapeutic target in Alzheimer's disease and cerebral amyloid angiopathy.

Regulatory role of proteasome in determination of platelet life span.

Limit of platelet life span (8-10 days) is determined by the activity of a putative "internal clock" composed of Bcl-2 family proteins, whereas the role of other molecular players in this process remains obscure. Here, we sought to establish a central role of proteasome in platelet life span regulation. Administration of mice with inhibitors of proteasome peptidase activity induced significant thrombocytopenia. This was associated with enhanced clearance of biotin-labeled platelets from circulation and reduction in average platelet half-life from 66 to 37 h. Cells pretreated in vitro with proteasome inhibitors exhibited augmented annexin V binding and a drop in mitochondrial transmembrane potential indicative of apoptotic cell death and decreased platelet life span. These cells were preferentially phagocytosed by monocyte-derived macrophages, thus linking proteasome activity with platelet survival. The decisive role of proteasome in this process was underscored from enhanced expression of conformationally active Bax in platelets with attenuated proteasome activity, which was consistent with pro-apoptotic phenotype of these cells. The present study establishes a critical role of proteasome in delimiting platelet life span ostensibly through constitutive elimination of the conformationally active Bax. These findings bear potential implications in clinical settings where proteasome peptidase activities are therapeutically targeted.

Melatonin regulates splenocytes proliferation via IP3-dependent intracellular Ca2+ release in seasonally breeding bird, Perdicula asiatica.

Melatonin plays an important role in the immune regulation of birds. Both endogenous and exogenous melatonin modulates lymphocyte proliferation via its specific membrane receptors, Mel(1a), Mel(1b) and Mel(1c), though the mechanisms behind this process are poorly understood. We investigated the differences in melatonin membrane receptor Mel(1a), Mel(1b) and Mel(1c) expression by western blot and reverse transcription reaction and the in vitro effect of melatonin on the intracellular Ca(2+) concentration ([Ca2+]i) in splenocytes of the Indian Jungle Bush Quail, Perdicula asiatica. We used a non-selective melatonin receptor antagonist for Mel(1a) and Mel(1b), luzindole, and the selective Mel(1b) blocker, 4P-PDOT to check the specific role of melatonin receptor on ([Ca2+]i). The expression of Mel(1a), Mel(1b) and Mel(1c) receptors mRNA and protein was upregulated by melatonin (10(-7) M) with a significant high rise in ([Ca2+]i), which was differentially blocked by supplementation of antagonist, luzindole (10(-7) M) and 4P-PDOT (10(-7) M). Furthermore, we noted in vitro effect of melatonin and 2-aminoethoxydiphenyl borate (2-APB), a cell-permeable antagonist of inositol 1, 4, 5-trisphosphate (IP3) receptor to check the rise in ([Ca2+]i) through the IP3 pathway. Significantly low ([Ca2+]i) was noted in melatonin and 2-APB pretreated splenocytes when compared with splenocytes where 2-APB was absent. Thus, our data suggest that melatonin through its membrane receptor induced the elevation of ([Ca2+]i) via IP(3)-dependent pathway for splenocyte proliferation in P. asiatica.

An Investigation of Size Distribution and Calcium Signaling in Human Platelets.

Background Platelets are thin disc-shaped blood cells that play a major role in hemostasis, maintenance of vascular integrity, and blood coagulation. Large platelets are more reactive and seen in patients with cardiovascular disease. This study aims to analyze the changes in platelet size of ex vivo activated platelets which phenotypically simulates that of a patient at risk of cardiovascular disease and elucidate the calcium signaling pathway responsible for this change. Methodology Platelets were isolated from adult human blood by differential centrifugation. Calcium was mobilized into platelets by treatment with calcium ionophore A23187 in the presence of Ca2+. Platelet size distribution was analyzed using Coulter Counter Multisizer 4. The following signaling parameters were studied: intracellular Ca2+ measurement (using Fura-2/AM by fluorescence spectrophotometry), Ca2+-dependent thiol protease calpain assay (using fluorogenic substrate t-butoxycarbonyl-Leu-metchloromethylcoumarin in fluorescence microplate reader), platelet-derived microparticles (using FACS Calibur flow cytometry), and cytoskeletal protein talin expression (by western immunoblotting). Results When adult platelets were treated with A23187 and Ca2+, two subcellular populations (<2 µm and between 2-4 µm) were noted. The mean size of the second cell population was significantly higher than that of resting platelets (2.94 ± 0.13 µm vs. 2.82 ± 0.15 µm, t = 4.605, p = 0.00). A23187 treatment led to elevated intracellular Ca2+, release of platelet-derived microparticles, increase in calpain activity, and cytoskeletal talin degradation. These events were inhibited by calpeptin (a specific calpain inhibitor). Conclusions Elevated calcium caused talin degradation by calpain activity. Breakdown of this cytoskeletal protein leads to relative swelling of cells reflected by the increase in platelet size.

Nanogold-coated stent facilitated non-invasive photothermal ablation of stent thrombosis and restoration of blood flow.

In-stent restenosis (ISR) and stent thrombosis (ST) are the most serious complications of coronary angioplasty and stenting. Although the evolution of drug-eluting stents (DES) has significantly restricted the incidence of ISR, they are associated with an enhanced risk of ST. In the present study, we explore the photothermal ablation of a thrombus using a nano-enhanced thermogenic stent (NETS) as a modality for revascularization following ST. The photothermal activity of NETS, fabricated by coating bare metal stents with gold nanorods generating a thin plasmonic film of gold, was found to be effective in rarefying clots formed within the stent lumen in various in vitro assays including those under conditions mimicking blood flow. NETS implanted in the rat common carotid artery generated heat following exposure to a NIR-laser that led to effective restoration of blood flow within the occluded vessel in a model of ferric chloride-induced thrombosis. Our results present a proof-of-concept for a novel photothermal ablation approach by employing coated stents in the non-invasive management of ST.