Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro.

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

Proinflammatory Polyphosphate Increases in Plasma of Obese Children with Insulin Resistance and Adults with Severe Type 2 Diabetes.

Obesity increases the risk of insulin resistance and type 2 diabetes through increased inflammation at cellular and tissue levels. Therefore, study of the molecular elements involved in obesity-related inflammation may contribute to preventing and controlling it. Inorganic polyphosphate is a natural phosphate polymer that has recently been attracting more attention for its role in inflammation and hemostasis processes. Polyphosphates are one of the main constituents of human platelets, which are secreted after platelet activation. Among other roles, they interact with multiple proteins of the coagulation cascade, trigger bradykinin release, and inhibit the complement system. Despite its importance, determinations of polyphosphate levels in blood plasma had been elusive until recently, when we developed a method to detect these levels precisely. Here, we perform cross sectional studies to evaluate plasma polyphosphate in: 25 children, most of them with obesity and overweight, and 20 adults, half of them with severe type 2 diabetes. Our results show that polyphosphate increases, in a significant manner, in children with insulin resistance and in type 2 diabetes patients. As we demonstrated before that polyphosphate decreases in healthy overweight individuals, these results suggest that this polymer could be an inflammation biomarker in the metabolic disease onset before diabetes.

Phosphate Restriction Promotes Longevity via Activation of Autophagy and the Multivesicular Body Pathway.

Nutrient limitation results in an activation of autophagy in organisms ranging from yeast, nematodes and flies to mammals. Several evolutionary conserved nutrient-sensing kinases are critical for efficient adaptation of yeast cells to glucose, nitrogen or phosphate depletion, subsequent cell-cycle exit and the regulation of autophagy. Here, we demonstrate that phosphate restriction results in a prominent extension of yeast lifespan that requires the coordinated activity of autophagy and the multivesicular body pathway, enabling efficient turnover of cytoplasmic and plasma membrane cargo. While the multivesicular body pathway was essential during the early days of aging, autophagy contributed to long-term survival at later days. The cyclin-dependent kinase Pho85 was critical for phosphate restriction-induced autophagy and full lifespan extension. In contrast, when cell-cycle exit was triggered by exhaustion of glucose instead of phosphate, Pho85 and its cyclin, Pho80, functioned as negative regulators of autophagy and lifespan. The storage of phosphate in form of polyphosphate was completely dispensable to in sustaining viability under phosphate restriction. Collectively, our results identify the multifunctional, nutrient-sensing kinase Pho85 as critical modulator of longevity that differentially coordinates the autophagic response to distinct kinds of starvation.

Targeting Protein Kinase C in Glioblastoma Treatment.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor and is associated with a poor prognosis. Despite the use of combined treatment approaches, recurrence is almost inevitable and survival longer than 14 or 15 months after diagnosis is low. It is therefore necessary to identify new therapeutic targets to fight GBM progression and recurrence. Some publications have pointed out the role of glioma stem cells (GSCs) as the origin of GBM. These cells, with characteristics of neural stem cells (NSC) present in physiological neurogenic niches, have been proposed as being responsible for the high resistance of GBM to current treatments such as temozolomide (TMZ). The protein Kinase C (PKC) family members play an essential role in transducing signals related with cell cycle entrance, differentiation and apoptosis in NSC and participate in distinct signaling cascades that determine NSC and GSC dynamics. Thus, PKC could be a suitable druggable target to treat recurrent GBM. Clinical trials have tested the efficacy of PKCß inhibitors, and preclinical studies have focused on other PKC isozymes. Here, we discuss the idea that other PKC isozymes may also be involved in GBM progression and that the development of a new generation of effective drugs should consider the balance between the activation of different PKC subtypes.

Effects of classical PKC activation on hippocampal neurogenesis and cognitive performance: mechanism of action.

Hippocampal neurogenesis has widely been linked to memory and learning performance. New neurons generated from neural stem cells (NSC) within the dentate gyrus of the hippocampus (DG) integrate in hippocampal circuitry participating in memory tasks. Several neurological and neuropsychiatric disorders show cognitive impairment together with a reduction in DG neurogenesis. Growth factors secreted within the DG promote neurogenesis. Protein kinases of the protein kinase C (PKC) family facilitate the release of several of these growth factors, highlighting the role of PKC isozymes as key target molecules for the development of drugs that induce hippocampal neurogenesis. PKC activating diterpenes have been shown to facilitate NSC proliferation in neurogenic niches when injected intracerebroventricularly. We show in here that long-term administration of diterpene ER272 promotes neurogenesis in the subventricular zone and in the DG of mice, affecting neuroblasts differentiation and neuronal maturation. A concomitant improvement in learning and spatial memory tasks performance can be observed. Insights into the mechanism of action reveal that this compound facilitates classical PKCα activation and promotes transforming growth factor alpha (TGFα) and, to a lesser extent, neuregulin release. Our results highlight the role of this molecule in the development of pharmacological drugs to treat neurological and neuropsychiatric disorders associated with memory loss and a deficient neurogenesis.

A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries.

Neural stem cells are activated within neurogenic niches in response to brain injuries. This results in the production of neuroblasts, which unsuccessfully attempt to migrate toward the damaged tissue. Injuries constitute a gliogenic/non-neurogenic niche generated by the presence of anti-neurogenic signals, which impair neuronal differentiation and migration. Kinases of the protein kinase C (PKC) family mediate the release of growth factors that participate in different steps of the neurogenic process, particularly, novel PKC isozymes facilitate the release of the neurogenic growth factor neuregulin. We have demonstrated herein that a plant derived diterpene, (EOF2; CAS number 2230806-06-9), with the capacity to activate PKC facilitates the release of neuregulin 1, and promotes neuroblasts differentiation and survival in cultures of subventricular zone (SVZ) isolated cells in a novel PKC dependent manner. Local infusion of this compound in mechanical cortical injuries induces neuroblast enrichment within the perilesional area, and noninvasive intranasal administration of EOF2 promotes migration of neuroblasts from the SVZ towards the injury, allowing their survival and differentiation into mature neurons, being some of them cholinergic and GABAergic. Our results elucidate the mechanism of EOF2 promoting neurogenesis in injuries and highlight the role of novel PKC isozymes as targets in brain injury regeneration.

Screening a Protein Array with Synthetic Biotinylated Inorganic Polyphosphate To Define the Human PolyP-ome.

Phenotypes are established by tight regulation on protein functions. This regulation can be mediated allosterically, through protein binding, and covalently, through post-translational modification (PTM). The integration of an ever-increasing number of PTMs into regulatory networks enables and defines the proteome complexity. Protein PTMs can occur enzymatically and nonenzymatically. Polyphosphorylation, which is a recently discovered PTM that belongs to the latter category, is the covalent attachment of the linear ortho-phosphate polymer called inorganic polyphosphate (polyP) to lysine residues. PolyP, which is ubiquitously present in nature, is also known to allosterically control protein function. To date, lack of reagents has prevented the systematic analysis of proteins covalently and/or allosterically associated with polyP. Here, we report on the chemical synthesis of biotin-modified monodisperse short-chain polyP (bio-polyP8-bio) and its subsequent use to screen a human proteome array to identify proteins that associate with polyP, thereby starting to define the human polyP-ome.

Biomarkers of the prothrombotic state in abdominal obesity / Biomarcadores de estado protrombótico en la obesidad abdominal

Introduction: Central obesity is specifically associated with cardiovascular disease. Nevertheless, the molecular events that promote these conditions remain incompletely defined and risk stratifying patients for cardiovascular disease continues a challenge. Objective: The aim of this study was to assess some cost-efficient haemostatic markers, and its association with central obesity and traditional cardiovascular risk factors, in a cohort of middle aged subjects, without clinical cardiovascular disease, as basis for an improved prevention and intervention. Methods: We studied 307 men, aged 45±7 years, which underwent medical history, physical examination, anthropometric measurements, plasmatic biochemical parameters, plasma concentrations of fibrinogen, prothrombin activity, activated partial thromboplastin time, platelet count and mean platelet volume. Results: Prothrombin activity values were significantly higher in patients with central obesity (103 ± 16 % vs 111 ± 17 %, p<0.001). Across tertiles of fibrinogen (low and high), there was an increase in cholesterol, adjusted for age and body mass index (4.9±0.9 mmol/L vs 5.4±1.1 mmol/L, p< 0.01). High tertile of prothrombin activity showed higher levels of cholesterol (4.8±1.0 mmol/L vs 5.4±0.9 mmol/L , p< 0.05), triglycerides (1.07±0.6 mmol/L vs 1.32±0.9 mmol/L, p< 0.05), and waist circumference (92.8±8.3 cm vs 96.5±8.8 cm, p= ns) . Mean values of cholesterol were higher in low-activated partial thromboplastin time tertile (5.3±0.9 mmol/L vs 4.9±1.1 mmol/L, p<0.01). Participants in the high-mean platelet volume tertile showed higher levels of glycemia (5.7±0.6 mmol/L vs 5.99±0.7 mmol/L, p<0.05). Significant positive correlations were observed between fibrinogen and cholesterol (r=0.198, p<0.001) and triglycerides (r=0.116, p<0.05). Prothrombin activity was positively correlated with waist circumference (r=0.156, p<0.05), glucose (r=0.227, p<0.001), cholesterol (r=0.270, p=0.001), triglycerides (r=0.187, p=0.001) and mean platelet volume (r=0.130, p=0.05). Activated partial thromboplastin time was inversely related cholesterol (r=-0.172, p<0.01) concentrations. Mean platelet volume rose with increasing glucose concentrations (r=0.170, p<0.01). Conclusions: Haemostatic markers studied have shown association with abdominal adiposity and established cardiovascular risk factors. These markers are widely available, relatively inexpensive, and might allow risk stratifying patients for cardiovascular disease and the identification of hypercoagulable state in patients who might deserve preventive measures and are potential tools for assessing the impact of these measures (AU)

Introducción: La obesidad central está especialmente asociada con la enfermedad cardiovascular. No obstante, los mecanismos moleculares que la promueven no están completamente definidos y la estratificación del riesgo de los pacientes para la enfermedad cardiovascular sigue siendo un reto. Objetivo: Evaluar la asociación de la adiposidad central con marcadores hemostáticos coste-eficientes y con los factores de riesgo cardiovascular tradicionales en una cohorte de varones de mediana edad sin enfermedad cardiovascular clínica con objeto de mejorar la prevención y la intervención. Métodos: Estudiamos 307 varones de 45±7 años, a los que se realizó historia clínica, examen físico, mediciones antropométricas y determinaciones plasmáticas bioquímicas y de fibrinógeno, actividad protrombina, tiempo de tromboplastina parcial activada, recuento de plaquetas y volumen plaquetario medio. Resultados: Los valores de actividad protrombina fueron significativamente más elevados en pacientes con obesidad central (103 ± 16 % vs 111 ± 17 %, p<0.001). Los sujetos con cifras de fibrinógeno en el tertil alto presentaban un aumento en las concentraciones plasmáticas de colesterol (4.9±0.9 mmol/L vs 5.4±1.1 mmol/L, p< 0.01), ajustado por edad e índice de masa corporal. El tertil alto de actividad protrombina mostró concentraciones más elevadas de colesterol plasmático (4.8±1.0 mmol/L vs 5.4±0.9 mmol/L, p< 0.05), triglicéridos (1.07±0.6 mmol/L vs 1.32±0.9 mmol/L, p< 0.05), and medida de la circunferencia de la cintura (92.8±8.3 cm vs 96.5±8.8 cm, p= ns) . Las concentraciones medias de colesterol fueron más altas en el tertil bajo de tiempo de tromboplastina parcial activado (5.3±0.9 mmol/L vs 4.9±1.1 mmol/L, p<0.01). Los individuos en el tertil alto de volumen plaquetario medio presentaron glucemia más elevada (5.7±0.6 mmol/L vs 5.99±0.7 mmol/L, p<0.05). Se observaron correlaciones significativas positivas entre fibrinógeno y colesterol (r=0.198, p<0.001) and triglicéridos (r=0.116, p<0.05). La actividad protrombina estuvo positivamente correlacionada con la medida de la circunferencia de la cintura (r=0.156, p<0.05), glucemia (r=0.227, p<0.001), colesterol (r=0.270, p=0.001), triglicéridos (r=0.187, p=0.001) y volumen plaquetario medio (r=0.130, p=0.05). El tiempo de tromboplastina parcial activado estuvo inversamente relacionado con las concentraciones de colesterol (r=-0.172, p<0.01). El volumen plaquetario medio se correlacionó con las concentraciones de glucosa (r=0.170, p<0.01). Conclusiones: Los marcadores hemostáticos estudiados se asocian con la adiposidad abdominal y con los factores de riesgo cardiovascular establecidos. Estos marcadores están disponibles en clínica, son asequibles y podrían ser de ayuda en la estratificación del riesgo cardiovascular de los pacientes y en la identificación del estado hipercoagulable en personas que podrían beneficiarse de medidas preventivas. Además de ser herramientas potenciales para evaluar el impacto de dichas medidas (AU)

Biomarkers of the prothrombotic state in abdominal obesity.

INTRODUCTION: Central obesity is specifically associated with cardiovascular disease. Nevertheless, the molecular events that promote these conditions remain incompletely defined and risk stratifying patients for cardiovascular disease continues a challenge. OBJECTIVE: The aim of this study was to assess some cost-efficient haemostatic markers, and its association with central obesity and traditional cardiovascular risk factors, in a cohort of middle aged subjects, without clinical cardiovascular disease, as basis for an improved prevention and intervention. METHODS: We studied 307 men, aged 45±7 years, which underwent medical history, physical examination, anthropometric measurements, plasmatic biochemical parameters, plasma concentrations of fibrinogen, prothrombin activity, activated partial thromboplastin time, platelet count and mean platelet volume. RESULTS: Prothrombin activity values were significantly higher in patients with central obesity (103 ± 16 % vs 111 ± 17 %, p<0.001). Across tertiles of fibrinogen (low and high), there was an increase in cholesterol, adjusted for age and body mass index (4.9±0.9 mmol/L vs 5.4±1.1 mmol/L, p< 0.01). High tertile of prothrombin activity showed higher levels of cholesterol (4.8±1.0 mmol/L vs 5.4±0.9 mmol/L , p< 0.05), triglycerides (1.07±0.6 mmol/L vs 1.32±0.9 mmol/L, p< 0.05), and waist circumference (92.8±8.3 cm vs 96.5±8.8 cm, p= ns) . Mean values of cholesterol were higher in low-activated partial thromboplastin time tertile (5.3±0.9 mmol/L vs 4.9±1.1 mmol/L, p<0.01). Participants in the high-mean platelet volume tertile showed higher levels of glycemia (5.7±0.6 mmol/L vs 5.99±0.7 mmol/L, p<0.05). Significant positive correlations were observed between fibrinogen and cholesterol (r=0.198, p<0.001) and triglycerides (r=0.116, p<0.05). Prothrombin activity was positively correlated with waist circumference (r=0.156, p<0.05), glucose (r=0.227, p<0.001), cholesterol (r=0.270, p=0.001), triglycerides (r=0.187, p=0.001) and mean platelet volume (r=0.130, p=0.05). Activated partial thromboplastin time was inversely related cholesterol (r=-0.172, p<0.01) concentrations. Mean platelet volume rose with increasing glucose concentrations (r=0.170, p<0.01). CONCLUSIONS: Haemostatic markers studied have shown association with abdominal adiposity and established cardiovascular risk factors. These markers are widely available, relatively inexpensive, and might allow risk stratifying patients for cardiovascular disease and the identification of hypercoagulable state in patients who might deserve preventive measures and are potential tools for assessing the impact of these measures.

Introducción: La obesidad central está especialmente asociada con la enfermedad cardiovascular. No obstante, los mecanismos moleculares que la promueven no están completamente definidos y la estratificación del riesgo de los pacientes para la enfermedad cardiovascular sigue siendo un reto. Objetivo: Evaluar la asociación de la adiposidad central con marcadores hemostáticos coste-eficientes y con los factores de riesgo cardiovascular tradicionales en una cohorte de varones de mediana edad sin enfermedad cardiovascular clínica con objeto de mejorar la prevención y la intervención. Métodos: Estudiamos 307 varones de 45±7 años, a los que se realizó historia clínica, examen físico, mediciones antropométricas y determinaciones plasmáticas bioquímicas y de fibrinógeno, actividad protrombina, tiempo de tromboplastina parcial activada, recuento de plaquetas y volumen plaquetario medio. Resultados: Los valores de actividad protrombina fueron significativamente más elevados en pacientes con obesidad central (103 ± 16 % vs 111 ± 17 %, p.

NOA36 protein contains a highly conserved nucleolar localization signal capable of directing functional proteins to the nucleolus, in mammalian cells.

NOA36/ZNF330 is an evolutionarily well-preserved protein present in the nucleolus and mitochondria of mammalian cells. We have previously reported that the pro-apoptotic activity of this protein is mediated by a characteristic cysteine-rich domain. We now demonstrate that the nucleolar localization of NOA36 is due to a highly-conserved nucleolar localization signal (NoLS) present in residues 1-33. This NoLS is a sequence containing three clusters of two or three basic amino acids. We fused the amino terminal of NOA36 to eGFP in order to characterize this putative NoLS. We show that a cluster of three lysine residues at positions 3 to 5 within this sequence is critical for the nucleolar localization. We also demonstrate that the sequence as found in human is capable of directing eGFP to the nucleolus in several mammal, fish and insect cells. Moreover, this NoLS is capable of specifically directing the cytosolic yeast enzyme polyphosphatase to the target of the nucleolus of HeLa cells, wherein its enzymatic activity was detected. This NoLS could therefore serve as a very useful tool as a nucleolar marker and for directing particular proteins to the nucleolus in distant animal species.

Polyphosphate is a novel pro-inflammatory regulator of mast cells and is located in acidocalcisomes.

Polyphosphate (polyP) is a pro-inflammatory agent and a potent modulator of the human blood-clotting system. The presence of polyP of 60 phosphate units was identified in rat basophilic leukemia (RBL-2H3) mast cells using specific enzymatic assays, urea-polyacrylamide gel electrophoresis of cell extracts, and staining of cells with 4,6-diamidino-2-phenylindole (DAPI), and the polyP-binding domain of Escherichia coli exopolyphosphatase. PolyP co-localizes with serotonin- but not with histamine-containing granules. PolyP levels greatly decreased in mast cells stimulated to degranulate by IgE. Mast cell granules were isolated and found to be acidic and decrease their polyP content upon alkalinization. In agreement with these results, when RBL-2H3 mast cells were loaded with the fluorescent calcium indicator fura-2 acetoxymethyl ester to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)), they were shown to possess a significant amount of Ca(2+) stored in an acidic compartment different from lysosomes. PolyP derived from RBL-2H3 mast cells stimulated bradykinin formation, and it was also detected in human basophils. All of these characteristics of mast cell granules, together with their known elemental composition, and high density, are similar to those of acidocalcisomes. The results suggest that mast cells polyP could be an important mediator of their pro-inflammatory and pro-coagulant activities.

Myeloma cells contain high levels of inorganic polyphosphate which is associated with nucleolar transcription.

BACKGROUND: In hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages of blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this study we evaluated the characteristics of intracellular polyphosphate in myeloma cell lines, in primary myeloma cells from patients, and in other human B-cell populations from healthy donors. DESIGN AND METHODS: We have developed a novel flow cytometric method for detecting levels of polyphosphate in cell populations. We also used confocal microscopy and enzymatic analysis to study polyphosphate localization and characteristics. RESULTS: We found that myeloma plasma cells contain higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that high levels of polyphosphate accumulate in the nucleolus of myeloma cells. As the principal function of the nucleolus involves transcription of ribosomal DNA genes, we found changes in the cellular distribution of polyphosphate after the inhibition of nucleolar transcription. In addition, we found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner. CONCLUSIONS: Our results show an unusually high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with nucleolar transcription.

High negative charge-to-size ratio in polyphosphates and heparin regulates factor VII-activating protease.

Factor VII-activating protease (FSAP) circulates as an inactive zymogen in the plasma. FSAP also regulates fibrinolysis by activating pro-urokinase or cellular activation via cleavage of platelet-derived growth factor BB (PDGF-BB). As the Marburg I polymorphism of FSAP, with reduced enzymatic activity, is a risk factor for atherosclerosis and liver fibrosis, the regulation of FSAP activity is of major importance. FSAP is activated by an auto-catalytic mechanism, which is amplified by heparin. To further investigate the structural requirements of polyanions for controlling FSAP activity, we performed binding, activation and inhibition studies using heparin and derivatives with altered size and charge, as well as other glycosaminoglycans. Heparin was effective in binding to and activating FSAP in a size- and charge density-dependent manner. Polyphosphate was more potent than heparin with regard to its interactions with FSAP. Heparin was also an effective co-factor for inhibition of FSAP by plasminogen activator inhibitor 1 (PAI-1) and antithrombin, whereas polyphosphate served as co-factor for the inhibition of FSAP by PAI-1 only. For FSAP-mediated inhibition of PDGF-BB-induced vascular smooth muscle cell proliferation, heparin as well as a polyphosphate served as efficient co-factors. Native mast cell-derived heparin exhibited identical properties to those of unfractionated heparin. Despite the strong effects of synthetic polyphosphate, the platelet-derived material was a weak activator of FSAP. Hence, negatively charged polymers with a high charge-to-size ratio are responsible for the activation of FSAP, and also act as co-factors for its inhibition by serine protease inhibitors.

Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2-dependent cyclin E expression.

Hyperhomocysteinemia (HHcy)-abnormally elevated plasma levels of homocysteine (Hcy)-has been associated with the development of neurodegenerative dementia and mild cognitive impairment. This association suggests that HHcy might facilitate memory loss in the elderly. As memory loss can occur through a deteriorated neurogenic capacity, we have studied the effects of Hcy on neural progenitor cells (NPCs) both in vitro and in vivo. We show that Hcy exerts an antiproliferative effect on basic fibroblast growth factor (bFGF) -stimulated NPCs isolated from the postnatal subventricular zone (SVZ), accompanied by inactivation of the extracellular signal-regulated kinase (Erk1/2) and inhibition of Erk1/2-dependent expression of cyclin E. Using a mice model we show that, under normal folate conditions, HHcy exerts an inhibitory effect on adult brain neurogenesis. This inhibition occurs in the caudal areas of the dentate gyrus (DG) of the hippocampus, a neurogenic area mainly involved in learning and memory performance, and in the SVZ, recently implicated in olfactory learning performance. In both areas reduced number of proliferative neuroblasts were found. Since neuroblasts are primarily bFGF-responsive progenitors already committed to a neuronal phenotype, our results strongly suggest that excess Hcy inhibits neurogenesis in the DG and SVZ by inhibiting the bFGF-dependent activation of Erk1/2 in these cells.

Organellar proteomics of human platelet dense granules reveals that 14-3-3zeta is a granule protein related to atherosclerosis.

Dense granules, a type of platelet secretory organelle, are known to accumulate high concentrations of small molecules such as calcium, adenine nucleotides, serotonin, pyrophosphate, and polyphosphate. Protein composition of these granules has been obscure, however. In this paper, we use proteomics techniques to describe, for the first time, the soluble protein composition of platelet dense granules. We have isolated highly enriched human platelet dense granule fractions that have been analyzed using two proteomics methods. Using this approach, we have identified 40 proteins, and most of them, such as actin-associated proteins, glycolytic enzymes, and regulatory proteins, have not previously been related to the organelle. We have focused our efforts on studying 14-3-3zeta, a member of a conserved family of proteins that interact with hundreds of different proteins. We have demonstrated that 14-3-3zeta is localized mostly on dense granules and that it is secreted after platelet activation. As some proteins secreted from activated platelets could promote the development of atherosclerosis and thrombosis, we have studied the expression of 14-3-3zeta in sections of human abdominal aorta of patients with aneurysm, identifying it at the atherosclerotic plaques. Together, our results reveal new details of the composition of the platelet dense granule and suggest an extracellular function for 14-3-3zeta associated with atherosclerosis.

Overexpression of a Zn2+-sensitive soluble exopolyphosphatase from Trypanosoma cruzi depletes polyphosphate and affects osmoregulation.

We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn2+, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5'-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation.

Inorganic polyphosphate and specific induction of apoptosis in human plasma cells.

BACKGROUND AND OBJECTIVES: Inorganic polyphosphate (polyP), a ubiquitous phosphate polymer with ATP-like bonds, has recently been related to a variety of functions including blood coagulation and cell proliferation. We investigated the effects of polyP in the biology of human plasma cells (PC), responsible for the production and maintenance of antibodies in response to antigens. DESIGN AND METHODS: The U266 myeloma cell line was used to study whether polyP affects immunoglobulin (Ig) secretion and survival. Different human cell lines were used to test the specificity of polyP on viability. We analyzed Ig secretion of PC from bone marrow and peripheral blood after polyP addition. A conventional tetanus toxoid booster immunization was used to increase the proportion of PC in order to examine the ex vivo effects of polyP. We also tested the effects of polyP on primary myeloma cells. Ig secretion and apoptosis were determined by ELISA and FACS respectively. RESULTS: Addition of polyP to human PC produced an unexpected inhibition of Ig secretion and stimulation of apoptosis. PolyP generated apoptosis specifically in PC, myeloma (malignant PC) cell lines, primary myeloma cells, and B lymphoid cell lines. Normal B cells, T cells, total blood mononuclear cells, and non-lymphoid cell lines were not affected by polyP. In the U266 myeloma cell line, polyP induced externalization of phosphatidylserine, activation of caspase-3, and arrest of the cell cycle. The protective effects of interleukin-6 did not overcome the polyP-induced apoptosis Interpretation and CONCLUSIONS: Taken together, our results suggest for the first time the relevance of the use of polyP to the humoral immune response and open prospects for polyP as a novel therapy for myeloma.

The acidocalcisome Ca2+-ATPase (TgA1) of Toxoplasma gondii is required for polyphosphate storage, intracellular calcium homeostasis and virulence.

A large proportion of intracellular Ca2+ in Toxoplasma gondii tachyzoites is stored within acidocalcisomes. These organelles are characterized by their acidic nature and high calcium and polyphosphate (polyP) content. The activity of a Ca2+/H+-ATPase named TgA1 may be important for the accumulation of Ca2+ in these organelles. This enzyme belongs to a group of plasma membrane Ca2+-ATPase (PMCA) that lack a calmodulin-binding domain and have vacuolar localization. To investigate the role of this enzyme, we have generated T. gondii mutants deficient in TgA1 through gene disruption. Proliferation of these mutants decreased dramatically because of deficient cell invasion. In addition, these cells had reduced virulence in a mouse model. Biochemical analysis revealed that the cell polyP content was drastically reduced, and the basal calcium levels were increased and unstable. Microneme secretion under the conditions of stimulation by ionophores was altered. Complementation of null mutants with TgA1 restored most functions. In summary, these results establish a link between TgA1, calcium homeostasis, polyP storage and virulence.

Polyphosphate content and fine structure of acidocalcisomes of Plasmodium falciparum.

Although acidocalcisomes have been well characterized morphologically in other apicomplexan parasites, no such characterization has been done in Plasmodium spp. Here, we report that Plasmodium falciparum merozoites possess electron-dense organelles rich in phosphorus and calcium, as detected by X-ray microanalysis of intact cells, which are similar to the acidocalcisomes of other apicomplexans, but of more irregular form. In agreement with these results malaria parasites possess large amounts of short- and long-chain polyphosphate (polyP), which are associated with acidocalcisomes in other organisms. PolyP levels were highest in the trophozoite stage of the parasite. Treatment of isolated trophozoites with chloroquine resulted in a significant hydrolysis of polyP. Taken together, these results provide evidence that acidocalcisomes from Plasmodium falciparum do not differ significantly from acidocalcisomes of other apicomplexan parasites.