Xenotropic and polytropic retrovirus receptor 1 regulates procoagulant platelet polyphosphate.

Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis.

NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis.

OBJECTIVE: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1-/-/NOX2-/-/NOX4-/-), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP-a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride-driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. CONCLUSIONS: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

NADPH oxidase 1 is a novel pharmacological target for the development of an antiplatelet drug without bleeding side effects.

Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets, which are circulating cells involved in both hemostasis and thrombosis. Here, the use of Nox1-/- and Nox1+/+ mice as experimental models of human responses demonstrated a critical role of NOX1 in collagen-dependent platelet activation and pathological arterial thrombosis, as tested in vivo by carotid occlusion assays. In contrast, NOX1 does not affect platelet responses to thrombin and normal hemostasis, as assayed in tail bleeding experiments. Therefore, as NOX1 inhibitors are likely to have antiplatelet effects without associated bleeding risks, the NOX1-selective inhibitor 2-acetylphenothiazine (2APT) and a series of its derivatives generated to increase inhibitory potency and drug bioavailability were tested. Among the 2APT derivatives, 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (2APT-D6) was selected for its high potency. Both 2APT and 2APT-D6 inhibited collagen-dependent platelet aggregation, adhesion, thrombus formation, superoxide anion generation, and surface activation marker expression, while responses to thrombin or adhesion to fibrinogen were not affected. In vivo administration of 2APT or 2APT-D6 led to the inhibition of mouse platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion, while tail hemostasis was unaffected. Differently to in vitro experiments, 2APT-D6 and 2APT displayed similar potency in vivo. In summary, NOX1 inhibition with 2APT or its derivative 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis without deleterious effects on hemostasis. These compounds should, therefore, be considered for the development of novel antiplatelet drugs to fight cardiovascular diseases in humans.

A novel combinatorial technique for simultaneous quantification of oxygen radicals and aggregation reveals unexpected redox patterns in the activation of platelets by different physiopathological stimuli.

The regulation of platelets by oxidants is critical for vascular health and may explain thrombotic complications in diseases such as diabetes and dementia, but remains poorly understood. Here, we describe a novel technique combining electron paramagnetic resonance spectroscopy and turbidimetry, which has been utilized to monitor simultaneously platelet activation and oxygen radical generation. This technique has been used to investigate the redox-dependence of human and mouse platelets. Using selective peptide inhibitors of NADPH oxidases (NOXs) on human platelets and genetically modified mouse platelets (NOX1-/- or NOX2-/-), we discovered that: 1) intracellular but not extracellular superoxide anion generated by NOX is critical for platelet activation by collagen; 2) superoxide dismutation to hydrogen peroxide is required for thrombin-dependent activation; 3) NOX1 is the main source of oxygen radicals in response to collagen, while NOX2 is critical for activation by thrombin; 4) two platelet modulators, namely oxidized low density lipoproteins (oxLDL) and amyloid peptide ß (Aß), require activation of both NOX1 and NOX2 to pre-activate platelets. This study provides new insights into the redox dependence of platelet activation. It suggests the possibility of selectively inhibiting platelet agonists by targeting either NOX1 (for collagen) or NOX2 (for thrombin). Selective inhibition of either NOX1 or NOX2 impairs the potentiatory effect of tested platelet modulators (oxLDL and Aß), but does not completely abolish platelet hemostatic function. This information offers new opportunities for the development of disease-specific antiplatelet drugs with limited bleeding side effects by selectively targeting one NOX isoenzyme.

A novel flow cytometry assay using dihydroethidium as redox-sensitive probe reveals NADPH oxidase-dependent generation of superoxide anion in human platelets exposed to amyloid peptide ß.

Reactive oxygen species (ROS) generation is critical in the regulation of platelets, which has important implications in the modulation of hemostasis and thrombosis. Nonetheless, despite several assays have been described and successfully utilized in the past, the analysis of ROS generation in human platelets remains challenging. Here we show that dihydroethidium (DHE) allows the characterization of redox responses upon platelet activation by physiological and pathological stimuli. In particular, the flow cytometry assay that we describe here allowed us to confirm that thrombin, collagen-related peptide (CRP) and arachidonic acid but not adenosine diphosphate (ADP) stimulate superoxide anion formation in a concentration-dependent manner. 0.1unit/ml thrombin, 3 µg/ml CRP and 30 µM arachidonic acid are commonly used to stimulate platelets in vitro and here were shown to stimulate a significant increase in superoxide anion formation. The ROS scavenger N-acetylcysteine (NAC) abolished superoxide anion generation in response to all tested stimuli, but the pan-NADPH oxidase (NOX) inhibitor VAS2870 only inhibited superoxide anion formation in response to thrombin and CRP. The involvement of NOXs in thrombin and CRP-dependent responses was confirmed by the inhibition of platelet aggregation induced by these stimuli by VAS2870, while platelet aggregation in response to arachidonic acid was insensitive to this inhibitor. In addition, the pathological platelet stimulus amyloid ß (Aß) 1-42 peptide induced superoxide anion formation in a concentration-dependent manner. Aß peptide stimulated superoxide anion formation in a NOX-dependent manner, as proved by the use of VAS2870. Aß 1-42 peptide displayed only moderate activity as an aggregation stimulus, but was able to significantly potentiate platelet aggregation in response to submaximal agonists concentrations, such as 0.03 unit/ml thrombin and 10 µM arachidonic acid. The inhibition of NOXs by 10 µM VAS2870 abolished Aß-dependent potentiation of platelet aggregation in response to 10 µM arachidonic acid, suggesting that the pro-thrombotic activity of Aß peptides depends on NOX activity. Similar experiments could not be performed with thrombin or collagen, as NOXs are required for the signaling induced by these stimuli. These findings shed some new light on the pro-thrombotic activity of Aß peptides. In summary, here we describe a novel and reliable assay for the detection of superoxide anion in human platelets. This is particularly important for the investigation of the pathophysiological role of redox stress in platelets, a field of research of increasing importance, but hindered by the absence of a reliable and easily accessible ROS detection methodology applicable to platelets.

The Role of NADPH Oxidases and Oxidative Stress in Neurodegenerative Disorders.

For a number of years, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) was synonymous with NOX2/gp91phox and was considered to be a peculiarity of professional phagocytic cells. Over the last decade, several more homologs have been identified and based on current research, the NOX family consists of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 enzymes. NOXs are electron transporting membrane proteins that are responsible for reactive oxygen species (ROS) generation-primarily superoxide anion (O2●-), although hydrogen peroxide (H2O2) can also be generated. Elevated ROS leads to oxidative stress (OS), which has been associated with a myriad of inflammatory and degenerative pathologies. Interestingly, OS is also the commonality in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). NOX enzymes are expressed in neurons, glial cells and cerebrovascular endothelial cells. NOX-mediated OS is identified as one of the main causes of cerebrovascular damage in neurodegenerative diseases. In this review, we will discuss recent developments in our understanding of the mechanisms linking NOX activity, OS and neurodegenerative diseases, with particular focus on the neurovascular component of these conditions. We conclude highlighting current challenges and future opportunities to combat age-related neurodegenerative disorders by targeting NOXs.

Extracellular Fibrinogen-binding Protein (Efb) from Staphylococcus aureus Inhibits the Formation of Platelet-Leukocyte Complexes.

Extracellular fibrinogen-binding protein (Efb) from Staphylococcus aureus inhibits platelet activation, although its mechanism of action has not been established. In this study, we discovered that the N-terminal region of Efb (Efb-N) promotes platelet binding of fibrinogen and that Efb-N binding to platelets proceeds via two independent mechanisms: fibrinogen-mediated and fibrinogen-independent. By proteomic analysis of Efb-interacting proteins within platelets and confirmation by pulldown assays followed by immunoblotting, we identified P-selectin and multimerin-1 as novel Efb interaction partners. The interaction of both P-selectin and multimerin-1 with Efb is independent of fibrinogen. We focused on Efb interaction with P-selectin. Excess of P-selectin extracellular domain significantly impaired Efb binding by activated platelets, suggesting that P-selectin is the main receptor for Efb on the surface of activated platelets. Efb-N interaction with P-selectin inhibited P-selectin binding to its physiological ligand, P-selectin glycoprotein ligand-1 (PSGL-1), both in cell lysates and in cell-free assays. Because of the importance of P-selectin-PSGL-1 binding in the interaction between platelets and leukocytes, we tested human whole blood and found that Efb abolishes the formation of platelet-monocyte and platelet-granulocyte complexes. In summary, we present evidence that in addition to its documented antithrombotic activity, Efb can play an immunoregulatory role via inhibition of P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes.

Amyloid ß-peptide-dependent activation of human platelets: essential role for Ca2+ and ADP in aggregation and thrombus formation.

Alzheimer's disease is associated with the accumulation of Aß (amyloid ß)-peptides in the brain. Besides their cytotoxic effect on neurons, Aß-peptides are thought to be responsible for the atherothrombotic complications associated with Alzheimer's disease, which are collectively known as cerebrovascular disease. In the present study, we investigated the effect of Aß-peptides on human platelet signal transduction and function. We discovered that the 25-35 domain of Aß-peptides induce an increase in platelet intracellular Ca2+ that stimulates α-granule and dense granule secretion and leads to the release of the secondary agonist ADP. Released ADP acts in an autocrine manner as a stimulant for critical signalling pathways leading to the activation of platelets. This includes the activation of the protein kinases Syk, protein kinase C, Akt and mitogen-activated protein kinases. Ca2+-dependent release of ADP is also the main component of the activation of the small GTPase Rap1b and the fibrinogen receptor integrin αIIbß3, which leads to increased platelet aggregation and increased thrombus formation in human whole blood. Our discoveries complement existing understanding of cerebrovascular dementia and suggest that Aß-peptides can induce vascular complications of Alzheimer's disease by stimulating platelets in an intracellular Ca2+-dependent manner. Despite a marginal ADP-independent component suggested by low levels of signalling activity in the presence of apyrase or P2Y receptor inhibitors, Ca2+-dependent release of ADP by Aß-peptides clearly plays a critical role in platelet activation. Targeting ADP signalling may therefore represent an important strategy to manage the cerebrovascular component of Alzheimer's disease.

Alternative Methods for the Detection of Superoxide Anion Generation in Platelets.

Reactive oxygen species (ROS) are highly unstable oxygen-containing molecules. Their chemical instability makes them extremely reactive and gives them the ability to react with important biological molecules such as proteins, nucleic acids, and lipids. Superoxide anions are important ROS generated by the reduction of molecular oxygen reduction (i.e., acquisition of one electron). Despite their initial implication exclusively in aging, degenerative, and pathogenic processes, their participation in important physiological responses has recently become apparent. In the vascular system, superoxide anions have been shown to modulate the differentiation and function of vascular smooth muscle cells, the proliferation and migration of vascular endothelial cells in angiogenesis, the immune response, and the activation of platelets in hemostasis. The role of superoxide anions is particularly important in the dysregulation of platelets and the cardiovascular complications associated with a plethora of conditions, including cancer, infection, inflammation, diabetes, and obesity. It has, therefore, become extremely relevant in cardiovascular research to be able to effectively measure the generation of superoxide anions by human platelets, understand the redox-dependent mechanisms regulating the balance between hemostasis and thrombosis and, eventually, identify novel pharmacological tools for the modulation of platelet responses leading to thrombosis and cardiovascular complications. This study presents three experimental protocols successfully adopted for the detection of superoxide anions in platelets and the study of the redox-dependent mechanisms regulating hemostasis and thrombosis: 1) dihydroethidium (DHE)-based superoxide anion detection by flow cytometry; 2) DHE-based superoxide anion visualization and analysis by single platelet imaging; and 3) spin probe-based quantification of superoxide anion output in platelets by electron paramagnetic resonance (EPR).

Antiplatelet drugs do not protect from platelet-leukocyte aggregation in coronary artery disease.

BACKGROUND: Despite advances in cardiovascular medicine, coronary artery disease (CAD) remains a leading cause of mortality. Among the pathophysiological features of this condition, platelet-leukocyte aggregates (PLAs) require further attention, either as diagnostic/prognostic disease markers or as potential interventional targets. OBJECTIVES: In this study, we characterized PLAs in patients with CAD. Primarily, we investigated the association of PLA levels with CAD diagnosis. In addition, the basal levels of platelet activation and degranulation were assessed in patients with CAD and controls, and their correlation with PLA levels was analyzed. Finally, the effect of antiplatelet treatments on circulating PLA numbers, basal platelet activation, and degranulation was studied in patients with CAD. METHODS: Participants were recruited at the Department of Cardiology of the University Heart and Vascular Centre Hamburg Eppendorf. Among patients admitted with severe chest pain, the diagnosis of CAD was made angiographically, and patients without CAD were used as controls. PLAs, platelet activation, and platelet degranulation were assessed by flow cytometry. RESULTS: Circulating PLAs and basal platelet degranulation levels were significantly higher in patients with CAD than in controls. Surprisingly, there was no significant correlation between PLA levels and platelet degranulation (or any other measured parameter). In addition, patients with CAD on antiplatelet therapy did not display lower PLA or platelet degranulation levels compared with those in controls. CONCLUSION: Overall, these data suggest a mechanism of PLA formation that is independent of platelet activation or degranulation and highlights the inefficiency of current antiplatelet treatments for the prevention of basal platelet degranulation and PLA formation.

Development of new peptidomimetic NADPH oxidase inhibitors with antithrombotic properties.

The ability of synthetic peptides inhibitors of NOX1 to effectively block the production of ROS by the enzyme was studied with different methodologies. Specifically, taking advantage of our understanding of the active epitope of the regulatory NOX1 subunit NOXA1 as a potent inhibitor of NOX1-derived O2•- formation, a panel of peptidomimetic derivatives of this peptide were designed and synthesized with the aim of improving their activity and increasing their stability in plasma. The results highlighted that improved efficacy and potency was found for both the peptide-peptoid hybrid GS2, whereas stapled peptide AC5 and its precursor showed higher stability despite lower biological potency. This study showed that minimal structural modifications of NOXA1 peptides are required to improve both their potency and stability to finally achieve best candidates as new potential anti-thrombotic drugs.

Human platelets release amyloid peptides ß1-40 and ß1-42 in response to haemostatic, immune, and hypoxic stimuli.

Background: Platelets contain high levels of amyloid ß (Aß) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer's Disease (AD). Objectives: This study aimed to determine whether human platelets release pathogenic Aß peptides Aß1-42 and Aß1-40 and to characterise the mechanisms regulating this phenomenon. Methods and Results: Enzyme-linked immunosorbent assays (ELISAs) revealed that the haemostatic stimulus thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aß1-42 and Aß1-40. Notably, LPS preferentially induced the release of Aß1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective ß secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aß1-40 or Aß1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aß peptides with platelet alpha granules. Conclusions: Taken together, our data suggest that human platelets release pathogenic Aß peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterise this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aß peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aß1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of AD patients.

The amyloid peptide ß disrupts intercellular junctions and increases endothelial permeability in a NADPH oxidase 1-dependent manner.

Alzheimer's disease is the most common form of dementia and is associated with the accumulation of amyloid peptide ß in the brain parenchyma. Vascular damage and microvascular thrombosis contribute to the neuronal degeneration and the loss of brain function typical of this disease. In this study, we utilised a murine model of Alzheimer's disease to evaluate the neurovascular effects of this disease. Upon detection of an increase in the phosphorylation of the endothelial surface receptor VE-cadherin, we focused our attention on endothelial cells and utilised two types of human endothelial cells cultured in vitro: 1) human umbilical vein endothelial cells (HUVECs) and 2) human brain microvascular endothelial cells (hBMECs). Using an electrical current impedance system (ECIS) and FITC-albumin permeability assays, we discovered that the treatment of human endothelial cells with amyloid peptide ß causes a loss in their barrier function, which is oxidative stress-dependent and similarly to our observation in mouse brain associates with VE-cadherin phosphorylation. The activation of the superoxide anion-generating enzyme NADPH oxidase 1 is responsible for the oxidative stress that leads to the disruption of barrier function in human endothelial cells in vitro. In summary, we have identified a novel molecular mechanism explaining how the accumulation of amyloid peptide ß in the brain parenchyma may induce the loss of neurovascular barrier function, which has been observed in patients. Neurovascular leakiness plays an important role in brain inflammation and neuronal degeneration driving the progression of the Alzheimer's disease. Therefore, this study provides a novel and promising target for the development of a pharmacological treatment to protect neurovascular function and reduce the progression of the neurodegeneration in Alzheimer's patients.

A peptide from the staphylococcal protein Efb binds P-selectin and inhibits the interaction of platelets with leukocytes.

AIMS: P-selectin is a key surface adhesion molecule for the interaction of platelets with leukocytes. We have shown previously that the N-terminal domain of Staphylococcus aureus extracellular fibrinogen-binding protein (Efb) binds to P-selectin and interferes with platelet-leukocyte aggregate formation. Here, we aimed to identify the minimal Efb motif required for binding platelets and to characterize its ability to interfering with the formation of platelet-leukocyte aggregates. METHODS AND RESULTS: Using a library of synthetic peptides, we mapped the platelet-binding site to a continuous 20 amino acid stretch. The peptide Efb68-87 was able to bind to resting and, to a greater extent, thrombin-stimulated platelets in the absence of fibrinogen. Dot blots, pull-down assays and P-selectin glycoprotein ligand-1 (PSGL-1) competitive binding experiments identified P-selectin as the cellular docking site mediating Efb68-87 platelet binding. Accordingly, Efb68-87 did not bind to other blood cells and captured platelets from human whole blood under low shear stress conditions. Efb68-87 did not affect platelet activation as tested by aggregometry, flow cytometry and immunoblotting, but inhibited the formation of platelet-leukocyte aggregates (PLAs). Efb68-87 also interfered with the platelet-dependent stimulation of neutrophil extracellular traps (NETs) formation in vitro. CONCLUSIONS: We have identified Efb68-87 as a novel selective platelet-binding peptide. Efb68-87 binds directly to P-selectin and inhibits interactions of platelets with leukocytes that lead to PLA and NET formation. As PLAs and NETs play a key role in thromboinflammation, Efb68-87 is an exciting candidate for the development of novel selective inhibitors of the proinflammatory activity of platelets.

Proteomic analysis reveals presence of platelet microparticles in endothelial progenitor cell cultures.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography-tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires "endothelial" characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate.

OBJECTIVE: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis. METHODS AND RESULTS: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin ß(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)ß(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation. CONCLUSIONS: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin ß(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.

Protein Disulphide Isomerase and NADPH Oxidase 1 Cooperate to Control Platelet Function and Are Associated with Cardiometabolic Disease Risk Factors.

BACKGROUND: Protein disulphide isomerase (PDI) and NADPH oxidase 1 (Nox-1) regulate platelet function and reactive oxygen species (ROS) generation, suggesting potentially interdependent roles. Increased platelet reactivity and ROS production have been correlated with cardiometabolic disease risk factors. OBJECTIVES: To establish whether PDI and Nox-1 cooperate to control platelet function. METHODS: Immunofluorescence microscopy was utilised to determine expression and localisation of PDI and Nox-1. Platelet aggregation, fibrinogen binding, P-selectin exposure, spreading and calcium mobilization were measured as markers of platelet function. A cross-sectional population study (n = 136) was conducted to assess the relationship between platelet PDI and Nox-1 levels and cardiometabolic risk factors. RESULTS: PDI and Nox-1 co-localized upon activation induced by the collagen receptor GPVI. Co-inhibition of PDI and Nox-1 led to additive inhibition of GPVI-mediated platelet aggregation, activation and calcium flux. This was confirmed in murine Nox-1-/- platelets treated with PDI inhibitor bepristat, without affecting bleeding. PDI and Nox-1 together contributed to GPVI signalling that involved the phosphorylation of p38 MAPK, p47phox, PKC and Akt. Platelet PDI and Nox-1 levels were upregulated in obesity, with platelet Nox-1 also elevated in hypertensive individuals. CONCLUSIONS: We show that PDI and Nox-1 cooperate to control platelet function and are associated with cardiometabolic risk factors.

Diabetes and Thrombosis: A Central Role for Vascular Oxidative Stress.

Diabetes mellitus is the fifth most common cause of death worldwide. Due to its chronic nature, diabetes is a debilitating disease for the patient and a relevant cost for the national health system. Type 2 diabetes mellitus is the most common form of diabetes mellitus (90% of cases) and is characteristically multifactorial, with both genetic and environmental causes. Diabetes patients display a significant increase in the risk of developing cardiovascular disease compared to the rest of the population. This is associated with increased blood clotting, which results in circulatory complications and vascular damage. Platelets are circulating cells within the vascular system that contribute to hemostasis. Their increased tendency to activate and form thrombi has been observed in diabetes mellitus patients (i.e., platelet hyperactivity). The oxidative damage of platelets and the function of pro-oxidant enzymes such as the NADPH oxidases appear central to diabetes-dependent platelet hyperactivity. In addition to platelet hyperactivity, endothelial cell damage and alterations of the coagulation response also participate in the vascular damage associated with diabetes. Here, we present an updated interpretation of the molecular mechanisms underlying vascular damage in diabetes, including current therapeutic options for its control.