Serotonin secretion by blood platelets: accuracy of high-performance liquid chromatography-electrochemical technique compared with the isotopic test and use in a clinical laboratory.

Background: Mild secretion defects are the most frequent and challenging blood platelet disorders to diagnose. Most δ-granule secretion tests lack validation, are not quantitative, or have unreliable response to weak platelet agonists. Objectives: To compare platelet serotonin secretion by HPLC-electrochemical detection technique (HPLC-ECD) with the reference isotopic test (3H-5-HT), evaluating its performance in clinical laboratories. Methods: The assay validation followed STARD-2015 recommendations. HPLC-ECD measured the nonsecreted serotonin remaining in platelet pellets after aggregation, comparing it with the reference 3H-5-HT assay. We studied subjects with inherited and aspirin-induced blood platelet disorders and assessed the HPLC-ECD operation for routine clinical diagnosis. Results: Calibration curves were linear (R2 = 0.997), with SD for residuals of 3.91% and analytical sensitivity of 5ng/mL. Intra- and interassay imprecision bias ranged between -8.5% and 2.1% and -9% and 3.1%, respectively. Serotonin recovery and stability were >95%, and the variability range of measurements was -5.5% to 4.6%. Statistical differences detected between tests were biologically irrelevant, with bias of 1.48% (SD, 8.43) and CI agreement of -18% to 15%. Both assays distinctly detected platelet secretion induced by 10 µM epinephrine and 4 µmM adenosine diphosphate. However, HPLC-ECD is quantitative and more sensitive to low serotonin content in blood platelets. Reference cutoffs for each agonist were determined in 87 subjects. Initially, the HPLC-ECD requires relatively expensive equipment and trained operators but has remarkably cheap running costs and a turn-around time of 24-36 hours. We have used this diagnostic tool routinely for >8 years. Conclusion: HPLC-ECD assay for platelet serotonin secretion is highly accurate, has advantages over the reference 3H-5-HT test, and is suitable as a clinical laboratory technique.

Expert opinion on the use of platelet secretion assay for the diagnosis of inherited platelet function disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

Assessment of platelet secretion is crucial for diagnosing suspected inherited platelet function disorders (IPFD). A previous survey of the SSC on Platelet Physiology of the ISTH and a comprehensive review highlighted that most of the platelet secretion assays (PSAs) lack standardization and validation. The aim of this study was to provide expert consensus guidance on the use of PSAs for IPFD diagnosis. We surveyed 26 experts from 10 different countries using the RAND/UCLA methodology, to attain a consensus on sensitivity, specificity, feasibility, time to readout, and cost of most PSAs. Answers were then graded in three categories: appropriate, uncertain, and inappropriate. Equivocal or misinterpretable statements required a second and third round survey involving 14 of the original 26 experts. We report here the consolidated results of the entire procedure. There was uniform agreement on several general statements, including that PSAs should be performed in hemostasis laboratories as first line diagnostic tests even in patients with normal platelet aggregation, and should include a δ-granule secretion marker. Among the specific assays examined, lumiaggregometry, other luciferin/luciferase-based assays, high-performance liquid chromatography methods, radiolabeled-serotonin based assays, and whole-mount transmission electron microscopy were rated as appropriate for the measurement of δ-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for α-granule release. For most of the other PSAs, the expert opinions were widely dispersed. Lack of expert consensus on many PSAs clearly indicates an unmet need for rigorous standardization, multicenter comparison of results, and validation of PSAs for clinical laboratory practice.

Platelet activation by charged ligands and nanoparticles: platelet glycoprotein receptors as pattern recognition receptors.

Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.

The collagen receptor glycoprotein VI promotes platelet-mediated aggregation of ß-amyloid.

Cerebral amyloid angiopathy (CAA) and ß-amyloid (Aß) deposition in the brain parenchyma are hallmarks of Alzheimer's disease (AD). We previously reported that platelets contribute to Aß aggregation in cerebral vessels by secreting the factor clusterin upon binding of Aß40 to the fibrinogen receptor integrin αIIbß3 Here, we investigated the contribution of the collagen receptor GPVI (glycoprotein VI) in platelet-induced amyloid aggregation. Using platelets isolated from GPVI-wild type and GPVI-deficient human donors and mice, we found that Aß40 bound to GPVI, which induced the release of ATP and fibrinogen, resulting in platelet aggregation. Binding of Aß40 to integrin αIIbß3, fibrinogen, and GPVI collectively contributed to the formation of amyloid clusters at the platelet surface. Consequently, blockade of αIIbß3 or genetic loss of GPVI reduced amyloid fibril formation in cultured platelets and decreased the adhesion of Aß-activated platelets to injured carotid arteries in mice. Application of losartan to inhibit collagen binding to GPVI resulted in decreased Aß40-stimulated platelet activation, factor secretion, and platelet aggregation. Furthermore, the application of GPVI- or integrin-blocking antibodies reduced the formation of platelet-associated amyloid aggregates. Our findings indicate that Aß40 promotes platelet-mediated amyloid aggregation by binding to both GPVI and integrin αIIbß3 Blocking these pathways may therapeutically reduce amyloid plaque formation in cerebral vessels and the brain parenchyma of patients.

Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions.

The role of glycoprotein VI (GPVI) in platelets was investigated in 3 families bearing an insertion within the GP6 gene that introduces a premature stop codon prior to the transmembrane domain, leading to expression of a truncated protein in the cytoplasm devoid of the transmembrane region. Western blotting and flow cytometry of GP6hom (homozygous) platelets confirmed loss of the full protein. The level of the Fc receptor γ-chain, which associates with GPVI in the membrane, was partially reduced, but expression of other receptors and signaling proteins was not altered. Spreading of platelets on collagen and von Willebrand factor (which supports partial spreading) was abolished in GP6hom platelets, and spreading on uncoated glass was reduced. Anticoagulated whole blood flowed over immobilized collagen or a mixture of von Willebrand factor, laminin, and rhodocytin (noncollagen surface) generated stable platelet aggregates that express phosphatidylserine (PS). Both responses were blocked on the 2 surfaces in GP6hom individuals, but adhesion was not altered. Thrombin generation was partially reduced in GP6hom blood. The frequency of the GP6het (heterozygous) variant in a representative sample of the Chilean population (1212 donors) is 2.9%, indicating that there are ∼4000 GP6hom individuals in Chile. These results demonstrate that GPVI supports aggregation and PS exposure under flow on collagen and noncollagen surfaces, but not adhesion. The retention of adhesion may contribute to the mild bleeding diathesis of GP6hom patients and account for why so few of the estimated 4000 GP6hom individuals in Chile have been identified.

Platelet glycoprotein VI promotes metastasis through interaction with cancer cell-derived galectin-3.

Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis, but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM) signaling and thereby regulates diverse functions, including platelet adhesion, aggregation, and procoagulant activity. GPVI has been proposed as a safe antithrombotic target, because its inhibition is protective in models of arterial thrombosis, with only minor effects on hemostasis. In this study, the genetic deficiency of platelet GPVI in mice decreased experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses supported that mouse, as well as human GPVI, had platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knockout approach, we identified galectin-3 as the major counterreceptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed galectin-3 uses ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab')2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study revealed a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.

Platelet glycoprotein VI genetic quantitative and qualitative defects.

Platelet membrane glycoprotein VI (GPVI) is increasingly recognized as an important receptor for thrombus formation and growth. Numerous arguments have been published indicating that GPVI plays a major role in thrombosis without being essential for physiological hemostasis. In humans, GPVI deficiencies are rarely reported. These are most often deficiencies occurring in the context of autoimmunity and, more rarely, genetic deficits. The purpose of this review is to compile data on the quantitative and qualitative genetic abnormalities of GPVI.

Fundamentals for a Systematic Approach to Mild and Moderate Inherited Bleeding Disorders: An EHA Consensus Report.

Healthy subjects frequently report minor bleedings that are frequently 'background noise' of normality rather than a true disorder. Nevertheless, unexpected or unusual bleeding may be alarming. Thus, the distinction between normal and pathologic bleeding is critical. Understanding the underlying pathologic mechanism in patients with an excessive bleeding is essential for their counseling and treatment. Most of these patients with significant bleeding will result affected by non-severe inherited bleeding disorders (BD), collectively denominated mild or moderate BD for their relatively benign course. Unfortunately, practical recommendations for the management of these disorders are still lacking due to the current state of fragmented knowledge of pathophysiology and lack of a systematic diagnostic approach. To address this gap, an International Working Group (IWG) was established by the European Hematology Association (EHA) to develop consensus-based guidelines on these disorders. The IWG agreed that grouping these disorders by their clinical phenotype under the single category of mild-to-moderate bleeding disorders (MBD) reflects current clinical practice and will facilitate a systematic diagnostic approach. Based on standardized and harmonized definitions a conceptual unified framework is proposed to distinguish normal subjects from affected patients. The IWG proposes a provisional comprehensive patient-centered initial diagnostic approach that will result in classification of MBD into distinct clinical-pathological entities under the overarching principle of clinical utility for the individual patient. While we will present here a general overview of the global management of patients with MBD, this conceptual framework will be adopted and validated in the evidence-based, disease-specific guidelines under development by the IWG.

Immobilized fibrinogen activates human platelets through glycoprotein VI.

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Nuevos anticoagulantes orales: actualización / New oral anticoagulants: a review

Resumen: Los anticoagulantes orales clásicos del tipo cumarinas han estado disponibles para uso clínico por más de medio siglo. Tienen gran eficacia para tratar o prevenir trombosis y tromboembolias, y son drogas cuyo uso ha aumentado con el mejor conocimiento clínico, el aumento de los factores de riesgo y el envejecimiento de la población. Entre sus desventajas se incluyen la alta variabilidad de su efecto en cada sujeto y entre individuos, la influencia del nivel de ingesta de vitamina K, la necesidad de control periódico del nivel de anticoagulación, su interacción con múltiples drogas. Si bien, el rango terapéutico está estandarizado, es estrecho, haciendo que el tiempo en rango terapéutico sea de ≈ 60%. Por estas limitaciones, se han creado nuevos anticoagulantes orales (NACOs), siendo progresivamente aprobados para uso clínico por agencias internacionales. Genéricamente, son de 2 tipos: inhibidores selectivos de trombina (dabigatrán) o de FXa (rivaroxabán, apixabán, edoxabán y betrixabán). Los NACOs se caracterizan por su dosificación una o dos veces al día, rapidez de acción, corta vida media en la circulación, predictibilidad de su efecto, dosis preestablecidas, sin necesidad de control periódico y con escasa o nula interacción con otras drogas. Estas ventajas no se han traducido en la mayoría de los ensayos en un superior efecto antitrombótico o menor riesgo de sangrado, y en su mayoría (salvo dabigatrán) carecen de antídoto específico demostrado.

Abstracts: Vitamin K inhibitors, coumarins, have been used for more than 50 years with no dispute by other drugs. Coumarins have demonstrated great efficacy in the treatment and prophylaxis of thrombotic and thromboembolic disorders, and their use has increased progressively with the advance of clinical knowledge as well as the increase of risk factors and aging of the population. Limitations of coumarins include great variability intra and inter-individuals, the influence of foods rich in vitamin K, the need for periodical assessment of the anticoagulant level and drug interactions. The therapeutic range is standardized using the INR (International Normalized Ratio). However, the therapeutic window is narrow, with frequent periods of either over or under-dosing, with the concomitant increase of bleeding and thrombotic risks, respectively. Long-term accredited anticoagulant clinics and clinical trials report that, at best, only ≈60% of time in treatment the patients are within the therapeutic range. These limitations have created the need for new oral anticoagulants (NOACs), and several of them have been approved for clinical use by international agencies after exhaustive and specific clinical trials. Generically, NACOs are belong in two types: selective inhibitors of thrombin (dabigatran) or FXa (rivaroxaban, apixaban, edoxaban and betrixaban). NOACs are prescribed once or twice daily, the onset of action is very fast, have a low T1/2 in the circulation, their effects are highly predictable, doses are pre-established, do not need laboratory control and have a low rate of interaction with other drugs. Despite these advantages most clinical trials have shown NOACs to be not inferior with respect to coumarin. However, NOACs have no clear advantages over warfarin in antithrombotic effect or bleeding reduction. Furthermore, most of them (except dabigatran) have no specific antidotes yet.

Human platelet interaction with E. coli O111 promotes tissue-factor-dependent procoagulant activity, involving Toll like receptor 4.

Platelets have a major role in clotting activation and contribute to the innate immune response during systemic infections. Human platelets contain tissue factor (TF) and express functional Toll-like receptor 4 (TLR4). However, the role of TLR4 in triggering the procoagulant properties of platelets, upon challenge with bacteria, is yet unknown. Our hypothesis is that E. coli O111-TLR4 interaction activates platelets and elicits their procoagulant activity. We demonstrated that the strain, but not ultrapure LPS, increased surface P-selectin expression, platelet dependent TF procoagulant activity (TF-PCA) and prompted a faster thrombin generation (TG). Blockade of TLR4 resulted in decreased platelet activation, TF-PCA and TG, revealing the participation of this immune receptor on the procoagulant response of platelets. Our results provide a novel mechanism by which individuals with bacterial infections would have an increased incidence of blood clots. Furthermore, the identification of platelet TF and TLR4 as regulators of the effect of E. coli O111 might represent a novel therapeutic target to reduce the devastating consequences of the hemostatic disorder during sepsis.

Platelet tissue factor activity and membrane cholesterol are increased in hypercholesterolemia and normalized by rosuvastatin, but not by atorvastatin.

BACKGROUND AND AIMS: High plasma LDL-cholesterol (LDL-C) and platelet responses have major pathogenic roles in atherothrombosis. Thus, statins and anti-platelet drugs constitute mainstays in cardiovascular prevention/treatment. However, the role of platelet tissue factor-dependent procoagulant activity (TF-PCA) has remained unexplored in hypercholesterolemia. We aimed to study platelet TF-PCA and its relationship with membrane cholesterol in vitro and in 45 hypercholesterolemic patients (HC-patients) (LDL-C >3.37 mmol/L, 130 mg/dL) and 37 control subjects (LDL-C <3.37 mmol/L). The effect of 1-month administration of 80 mg/day atorvastatin (n = 21) and 20 mg/day rosuvastatin (n = 24) was compared. METHODS: Platelet TF-PCA was induced by GPIbα activation with VWF-ristocetin. RESULTS: Cholesterol-enriched platelets in vitro had augmented aggregation/secretion and platelet FXa generation (1.65-fold increase, p = 0.01). HC-patients had 1.5-, 2.3- and 2.5-fold increases in platelet cholesterol, TF protein and activity, respectively; their platelets had neither hyper-aggregation nor endogenous thrombin generation (ETP). Rosuvastatin, but not atorvastatin, normalized platelet cholesterol, TF protein and FXa generation. It also increased slightly the plasma HDL-C levels, which correlated negatively with TF-PCA. CONCLUSIONS: Platelets from HC-patients were not hyper-responsive to low concentrations of classical agonists and had normal PRP-ETP, before and after statin administration. However, washed platelets from HC-patients had increased membrane cholesterol, TF protein and TF-PCA. The platelet TF-dependent PCA was specifically expressed after VWF-induced GPIbα activation. Rosuvastatin, but not atorvastatin treatment, normalized the membrane cholesterol, TF protein and TF-PCA in HC-patients, possibly unveiling a new pleiotropic effect of rosuvastatin. Modulation of platelet TF-PCA may become a novel target to prevent/treat atherothrombosis without increasing bleeding risks.

Fibrin and D-dimer bind to monomeric GPVI.

Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, we show that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a KD of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs.

Strain auricular izquierdo y biomarcadores cardíacos como predictores de accidente cerebrovascular en pacientes con fibrilación auricular de reciente comienzo / Left atrial strain and biomarkers for stroke in patients with recent onset atrial fibrillation

Introducción: La miopatía y fibrosis auricular representan el sustrato protrombótico y proarrítmico en pacientes con fibrilación auricular (FA). Estudios recientes muestran relación entre el strain auricular izquierdo (SAI), eventos cardiovasculares y recurrencia en pacientes con FA. La asociación entre SAI y bio-marcadores cardíacos como predictores de accidente cerebrovascular silente (ACVs) en pacientes con FA de reciente comienzo (FArc) no ha sido estudiada. Objetivo: Determinar si la asociación entre SAI y biomarcadores cardíacos contribuye a la predicción de ACV en pacientes con FArc. Métodos: Se realizó un estudio prospectivo que permitió reclutar 57 pacientes con FArc (primer episodio de < de 8 semanas de evolución). Obtenido consentimiento informado (CI) se realizó recolección de datos clínicos y muestras de sangre para determinación de Pro-BNP, Dimero-D y GDF-15. Se realizó resonancia nuclear magnética cerebral (RNMc) y ecocardiograma transtorácico (ETT) durante los primeros 3 días de inclusión y en ritmo sinusal. Para la evaluación de SAI se consideró la curva de deflexión positiva durante la sístole ventricular (SAIs), derivada de speckle tracking, considerando el promedio de 5 ciclos. Se utilizó Mann Whitney U test y Spearman Rho para análisis estadístico. Resultados: La edad promedio fue 70±8,2 años y el 70% fueron hombres. El CHA2DS2-VASc score promedio fue 3,1±1 y el promedio de pro-BNP, Di-mero-D y GDF-15 fue 96,1±12,4 pg/ml, 990±140 ng/ ml y 12 ng/ml respectivamente. 15% de los pacientes (n=9) presentaban ACVs en la RNMc al momento del diagnóstico. Se observó, además, que los pacientes con ACV presentaban un SAIs más bajo que los pacientes sin eventos (5,5±1,1% y 14,6±7,3% respectivamente p=0.04). Adicionalmente, se encontró una correlación significativa entre SAIs y pro-BNP, Dimero-D y GDF-15. Conclusiones: En este trabajo se evidenció que el 15% de los pacientes con FArc presenta ACVs al momento del diagnóstico. El SAIs bajo se correlaciona de forma inversa con los biomarcadores de sobrecarga, trombogénesis, fibrosis auricular y presencia de ACV silente. Estos resultados pueden ser utilizados para una mejor estratificación del riesgo de ACV en pacientes con FA.

Introduction: Atrial myopathy and fibrosis constitute a pro-arrhythmic and pro-thromboembolic substrate in patients with atrial fibrillation (AF). Recent studies using left atrial strain (LAS) have shown that LAS contributes to predict AF recurrence in patients with paroxysmal AF. The association between LAS and cardiac biomarkers in predicting silent stroke (SS) in patients with new AF has not been studied. Aim: The association of LAS and cardiac biomarkers contribute to predict SS in patients with new AF. Methods: We have prospectively evaluated 57 consecutive patients with new AF (first episode with less than 8 weeks of evolution). Baseline clinical characteristics and blood samples for determinations of Pro-BNP, D-Dimer and GDF-15 were obtained. Brain magnetic resonance (BMRI) and 2D Echo were performed within 3 days. In sinus rhythm, the positive deflection during ventricular systole of the LAS curve derived from speckle tracking was considered (mean of 5 cycles) (LASS). Mann Whitney U test and Spearman Rho were used for statistical analysis. Results: Mean age was 70±8,2 years, 70% were men. The mean CHA2DS2-VASc score was 3,1±1. Mean pro-BNP, D-Dimer and GDF-15 were 96,1±12,4 pg/ml, 990±140 ng/ml and 12 ng/ml, respectively. Fifteen percent of patients (n=9) had evidence of previous SS in BMRI. Patients with SS had significantly less LASS than patients without events (5,5±1,1% and 14,6±7,3% respectively p=0,04). In addition, a significant correlation between LASs and pro-BNP, D-Dimer and GDF-15 was found. Conclusion: Evidence of SS was found in 15% of patients with new AF. This was associated with LASs impairment, which was inversely correlated with cardiac biomarkers of LV overload, thrombogenesis and LA fibrosis. These findings could be utilized for a better risk stratification of stroke in patients with new AF.

Inhibition of angiogenesis by platelets in systemic sclerosis patients.

INTRODUCTION: Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by microvascular damage, inflammation, and fibrosis. It has become increasingly evident that platelets, beyond regulating hemostasis, are important in inflammation and innate immunity. Platelets may be an important source of proinflammatory and profibrotic cytokines in the vascular microenvironment. In this study, we sought to assess the contribution of platelet-derived factors in patients with SSc to the angiogenesis of human dermal microvascular endothelial cells (DMVECs) in a tubule formation assay and to characterize the secretion of profibrotic and proinflammatory cytokines in these platelets. METHODS: We analyzed platelets obtained from 30 patients with SSc and 12 healthy control subjects. Angiogenesis was evaluated in vitro with a DMVEC tubule formation assay on Matrigel and platelet-derived angiogenic factors such as vascular endothelial growth factor (VEGF), 165b isoform (VEGF165b), and cytokine secretion was evaluated. Platelet serotonin content was also determined. RESULTS: When DMVECs were incubated with SSc platelet releasates, tubule formation was significantly inhibited (p < 0.01, t test), and higher expression of endothelin-1 in these cells was observed compared with control subjects (p < 0.05, Mann-Whitney U test). In SSc platelet releasates, VEGF165b was significantly higher (p < 0.05, t test), and the VEGF165b/VEGF ratio was increased compared with that of control subjects. Higher secretion of transforming growth factor ß (p < 0.01, t test) and CD40L (p < 0.01, t test) was observed compared with control subjects. Also, intraplatelet serotonin levels were lower in platelets obtained from patients with diffuse SSc compared with patients with limited SSc and control subjects (p < 0.05, t test). CONCLUSIONS: Our findings suggest that antiangiogenic factors such as VEGF165b, together with proinflammatory and profibrotic factors secreted by platelets, can contribute to the progression of peripheral microvascular damage, defective vascular repair, and fibrosis in patients with SSc.

Increased RhoA/Rho-Kinase Activity and Markers of Endothelial Dysfunction in Young Adult Subjects with Metabolic Syndrome.

BACKGROUND: Metabolic syndrome, a chronic condition associated with higher risk of cardiovascular diseases, is increasingly prevalent in young adults. Dyslipidemia, proinflammatory cytokines, endothelial dysfunction signs, and RhoA/Rho-kinase (ROCK) activation are considered risk factors of cardiovascular diseases. The occurrence of these factors in young patients with metabolic syndrome but without type 2 diabetes or hypertension has not been fully studied. The objective of this study was to evaluate young subjects with enlarged waist circumference and dyslipidemia but without type 2 diabetes or hypertension,for markers associated with a higher risk of cardiovascular diseases. METHODS: Thirty-two male patients aged 31 ± 1.3 years diagnosed with metabolic syndrome according to the National Cholesterol Education Program Adult Treatment Panel III guide for enlarged waist circumference, elevated triglycerides, and low HDL levels, but with blood pressure and fasting glucose within normal ranges, were evaluated for RhoA/ROCK activity in leukocytes, serum fatty acid methyl esters profile, proinflammatory cytokines, and oxidative stress markers in addition to thrombin generation and biochemical analysis. Age- and gender-matched healthy subjects were equivalently evaluated. RESULTS: Patients showed higher RhoA/ROCK activity, elevated levels of interleukin-6, soluble CD40L, monocyte chemoattractant protein, and high-sensitivity C-reactive protein (P < 0.001) as well as parameters of endogenous thrombin generation potential (P < 0.05) compared with healthy subjects. Increased thiobarbituric acid reactive substances, advanced oxidation protein product, and insulin levels and low nitric oxide biodisponibility (P < 0.001) were also found in patients as compared with controls. Palmitic acid was one of the saturated fatty acids found to be significantly elevated in patients compared with control subjects (P = 0.0087). CONCLUSIONS: Increased markers of cardiovascular risk are already present in young adults with metabolic syndrome but without type 2 diabetes or hypertension.

Serum Ferritin Is Associated with Metabolic Syndrome and Red Meat Consumption.

BACKGROUND AND AIMS: Hyperferritinemia has been related with a wide spectrum of pathologies, including diabetes, cardiovascular disease, neurodegenerative disorders, and metabolic syndrome. The aim of this study was to investigate the association between hyperferritinemia and iron consumption. METHODS AND RESULTS: Serum ferritin concentration was evaluated in 66 presumed healthy men, along with other clinical and biochemical markers of chronic diseases. A three-day food questionnaire was applied for nutrition information. Hyperferritinemia was a condition found in 13.4% of the volunteers analyzed. Significant correlations were found between serum ferritin concentration and metabolic syndrome parameters (HDL cholesterol, triglycerides, and fasting glucose) as well as an increase of the serum ferritin mean value with the number of risk factors of metabolic syndrome. Also, oxidative stress markers (carbonyl groups, AOPP, and glycated hemoglobin), hepatic damage markers (GGT, SGOT), and parameters related to insulin resistance (HOMA, blood insulin, and blood glucose) correlate significantly with serum ferritin. Volunteers had an excessive iron intake, principally by bread consumption. Analyses of food intake showed that red meat consumption correlates significantly with serum ferritin. CONCLUSION: Red meat consumption, metabolic syndrome, and chronic disease markers are associated with hyperferritinemia in a population of Chilean men.

Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells.

BACKGROUND: An increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and "Metastasis Initiating Cell (MIC)" marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients. METHODS: With informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay. RESULTS: The co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity. CONCLUSIONS: We present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders.

Measurement of platelet granule release to detect inherited platelet secretion disorders (IPSDs) is essential for the evaluation of patients with abnormal bleeding and is necessary to distinguish which granule sub-types are affected and whether there is abnormal granule bio-synthesis or secretion. The radioactive serotonin incorporation and release assay, described before 1970, is still considered the "gold standard" test to assess platelet δ-granule release, although is unsuitable for clinical diagnostic laboratories. Luciferin-based assays, such as lumiaggregometry, are the most widely performed alternatives, although these methods do not distinguish defects in δ-granule biosynthesis from defects in secretion. Platelet α-granule release is commonly evaluated using flow cytometry by measuring surface exposure of P-selectin after platelet activation. However, this assay has poor sensitivity for some α-granule disorders. Only few studies have been published with more recently developed assays and no critical reviews on these methods are available. In this review, we describe the rationale for developing robust and accurate laboratory tests of platelet granule release and describe the characteristics of the currently available tests. We identify an unmet need for further systematic evaluation of new assays and for standardisation of methodologies for clinical diagnostic laboratories.