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.

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.

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.

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.

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.

Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

BACKGROUND: The inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine. OBJECTIVES: The mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated. RESULTS: Adenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor. CONCLUSION: Therefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

Is my patient a bleeder? A diagnostic framework for mild bleeding disorders.

Congenital mild bleeding disorders (MBDs) are very prevalent and are the source of frequent diagnostic problems. Most MBDs are categorized as disorders of primary hemostasis (ie, type 1 VWD and platelet function disorders), but mild or moderate deficiencies of clotting factors and some rare hyperfibrinolytic disorders are also included. These patients have abnormal bleeding from the skin and mucous membranes, menorrhagia, and disproportionate hemorrhages after trauma, invasive procedures, and surgery. This review addresses the main problems that physicians and hemostasis laboratories confront with the diagnosis of these patients, including: discerning normal/appropriate from pathological bleeding, the role and yield of screening tests, the lack of distinctive bleeding pattern among the different diseases, the inherent difficulties in the diagnosis of type 1 VWD and the most common platelet functional disorders, improvements in assays to measure platelet aggregation and secretion, and the evidence that most of the patients with MBDs end up without a definite diagnosis after exhaustive and repeated laboratory testing. Much research is needed to determine the pathogenesis of bleeding in MBD patients. Better standardization of current laboratory assays, progress in the knowledge of fibrinolytic mechanisms and their laboratory evaluation, and new understanding of the factors contributing to platelet-vessel wall interaction, along with the corresponding development of laboratory tools, should improve our capacity to diagnose a greater proportion of patients with MBDs.

Nuevos anticoagulantes orales / New oral anticoagulant drugs

Thromboembolic disease (TED) is the leading cause of morbidity and mortality worldwide. The hallmark of oral long-term anticoagulant therapy has been the use of vitamin K antagonists, whose anticoagulant effect is exerted inhibiting vitamin K epoxide reductase. Warfarin and acenocoumarol are the most commonly used. In the last five years several new drugs for long term anticoagulation have been developed, which can inhibit single clotting factors with the purpose of improving drug therapeutic range and, ideally, minimizing bleeding risks. This review addresses the state of the art on the clinical use of inhibitors of activated factor X and thrombin.

Platelet activation in chronic cocaine users: effect of short term abstinence.

Cocaine abuse increases the risk of cardiac and cerebrovascular events, such as myocardial infarction and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood although intravascular thrombus formation has been observed. The aim of this study was to investigate the existence of platelet activation and the effect of short-term abstinence in chronic cocaine consumers. We studied 23 cocaine dependent individuals (aged 20-54 years) who met DSM-IV criteria for cocaine dependence and 20 controls. Samples were obtained at baseline, within 72 h of last drug exposure and after 4 weeks of controlled abstinence. Monocyte-platelet aggregates (MPA) were measured by flow cytometry. Plasma levels of soluble CD40L (sCD40L), Neutrophil-Activating Peptide-2 (NAP-2) and regulated on activation normal T cells expressed and secreted (RANTES) were determined by ELISA. Levels of MPA, sCD40L, NAP-2 and RANTES were significantly higher (all p < 0.05) in cocaine addicts compared to controls at baseline. All the parameters returned to values similar to the control group after 4-weeks' abstinence. Levels of sCD40L and RANTES were associated with an index of intensity of drug consumption (p < 0.02). Our results demonstrate that cocaine use induces platelet activation which is a prominent finding after recent consumption. The persistence over time of this condition may contribute not only to acute thrombotic complications but also to the development of early-onset atherosclerotic process observed in cocaine abusers.

Novel loci involved in platelet function and platelet count identified by a genome-wide study performed in children.

BACKGROUND: Genome-wide association studies are currently identifying new loci with potential roles in thrombosis and hemostasis: these loci include novel polymorphisms associated with platelet function traits and count. However, no genome-wide study performed on children has been reported to date, in spite of the potential that these subjects have in genetic studies, when compared to adults, given the minimal degree of confounders, i.e., acquired and environmental factors, such as smoking, physical activity, diet, and drug or hormone intake, which are particularly important in platelet function. DESIGN AND METHODS: To identify new genetic variants involved in platelet reactivity and count, we performed a genome-wide association study on 75 children (8.5±1.8 years) using the Illumina Sentrix Human CNV370-Quad BeadChip containing 320,610 single nucleotide polymorphisms. Functional analyses included assessment of platelet aggregation and granule secretion triggered by different agonists (arachidonic acid, collagen, epinephrine, ADP), as well as platelet count. Associations were selected based on statistical significance and physiological relevance for a subsequent replication study in a similar sample of 286 children. RESULTS: We confirmed previously established associations with plasma levels of factors XII, VII and VIII as well as associations with platelet responses to ADP. Additionally, we identified 82 associations with platelet reactivity and count with a P value less than 10(-5). From the associations selected for further replication, we validated two single nucleotide polymorphisms with mildly increased platelet reactivity (rs4366150 and rs1787566) on the LPAR1 and MYO5B genes, encoding lisophosphatidic acid receptor-1 and myosin VB, respectively; and rs1937970, located on the NRG3 gene coding neuroregulin-3, associated with platelet count. CONCLUSIONS: Our genome-wide association study performed in children, followed by a validation analysis, led us to the identification of new genes potentially relevant in platelet function and biogenesis.

Increased number of circulating endothelial cells and plasma markers of endothelial damage in chronic cocaine users.

BACKGROUND: Cocaine use has been related with the development of accelerated atherosclerosis and with an increased risk of cardiac and cerebrovascular events, such as myocardial infarction, sudden cardiac death, and ischemic stroke. The underlying mechanisms leading to these complications are not fully understood, although thrombus formation and altered vascular function are prominent findings. OBJECTIVES: Our aim was to evaluate markers of endothelial dysfunction in chronic cocaine consumers before and after drug withdrawal. PATIENTS/METHODS: We determined circulating endothelial cells (CECs) and plasma levels of stromal cell-derived factor-1 (SDF-1), monocyte chemotactic protein-1(MCP-1), soluble intracellular adhesion molecule (sICAM), high-sensitivity C reactive protein (hsCRP) and endothelin-1(ET-1), in DSM-IV cocaine addicts at baseline and after one month of cocaine abstinence. RESULTS: Cocaine users showed a strikingly higher numbers of CEC (62.35 ± 18.4 vs 8.25 ± 13.8 CEC/mL) and significantly elevated plasma levels for all the markers evaluated as compared to the control group. After cocaine withdrawal, patients improved SDF-1, ET-1, hsCRP and sICAM levels. However, CEC number and MCP-1 plasma levels remained significantly elevated. All the results were adjusted for blood levels of cholesterol and triglycerides and for smoking habit. CONCLUSIONS: Our results demonstrated that chronic cocaine consumption alters several functions of the endothelium towards a pro-thrombotic condition and that some of those functions remain abnormal even after short-term drug withdrawal. These observations support the notion that endothelial dysfunction may play a key role in the pathogenesis of ischemic vascular disease observed in cocaine abusers.

Daño endotelial y activación del sistema hemostático asociado al uso crónico de cocaína: estudios ex vivo e in vitro / Endothelial damage and activation of the haemostatic system associated to chronic use of cocaine: an ex vivo and in vitro study

Antecedentes: Usuarios crónicos de cocaína tienen riesgo aumentado de presentar infarto de miocardio, angina,muerte súbita y accidentes cerebrovasculares. Aunque la patogenia del daño vascular es mayormente desconocida, se ha encontrado arterioesclerosis prematura y formación de trombos intravasculares. Objetivo: Demostrar evidencia de daño endotelial y activación del sistema hemostático en usuarios crónicos de cocaína. Métodos: Un grupo de 23 pacientes con criterios de dependencia a cocaína DSM-IV; 19 hombres (edad promedio 32 a), con exposición a la droga dentro de 72 h del estudio. Disfunción endotelial se evaluó por enumeración de las células endoteliales circulantes (CEC) y nivel de sICAM . Para activación del sistema hemostático se incluyó: complejos trombina-antitrombina (TAT) y generación de trombina; NAP-2 y RANTES para activación plaquetaria. In vitro, CE en cultivo (HUVEC), se expusieron a plasma de consumidores o controles. Se midió factor von Willebrand (FVW) en el medio y expresión de FvW y factor tisular (FT) sobre las CE. Adhesión plaquetaria estática se evaluó por microscopía. Resultados: En usuarios de cocaína, con respecto a controles, las CEC estaban significativamente elevadas...

Background: chronic cocaine users have an increased risk of developing myocardial infarction, angina, suddendeath and stroke. Although the pathogenesis of this effect is not completely known, premature atheromatosis and intravascular thrombosis appear to be involved.Aim: to provide evidence for the presence of endothelial damage and activation of the haemostatic system in chronic cocaine users. Methods: 23 subjects (19males, overall mean age 32) with DSM-IV criteria for cocaine dependency and exposure to the drug within 72 hours were studied. Endothelial dysfunction was determined by circulating endothelial cell counts (CEC) and sICAM levels. Thrombin-antithrombin complexes (TAT) and thrombin generation were used to characterize haemostatic status. In vitro, platelet activation was studied by NAP-2 and RANTES. EC in culture (HUVEC) were exposed to plasma from cocaine users and controls. Von Willebrand factor was measured in the culture media as well as its expression along with that of tissue factor in EC. Platelet adhesion was evaluated by microscopy. Results: Compared to controls, EC were significantly increased in cocaine users...

La atorvastatina no modifica el incremento agudo de los niveles de p-selectina y fibrinógeno inducido con esfuerzo físico máximo / Atovastatin does not modify the acutely increased levels of P-selectin and fibrinogen induced by maximal physical exercise

Introducción: Las estatinas han demostrado disminuir los eventos cardiovasculares en sujetos con y sin enfermedad aterosclerótica establecida. Se ha demostrado, que sus efectos benéficos no sólo dependen de la reducción del colesterol, sino que también podrían ser secundarios a otros efectos de las estatinas, como su efectos de reducción de inflamación y/ o trombogénesis entre otros. Sin embargo, no existen trabajos que demuestren que las estatinas sean capaces de frenarla activación de la cascada de inflamación y/o trombogénesis. Objetivos: Determinar el efecto de la administración oral de atorvastatina por 7 días sobre los niveles plasmáticos de proteína C- reactiva ultrasensible (PCR us), fibrinógeno y P-selectina, pre y post prueba de esfuerzo máximo inmediato y a las 24 horas de su ejecución. Métodos: Ensayo clínico en 50 hombres sanos (18 a 50 años), randomizado atorvastatina 80 mg/día - placebo por 7 días, doble ciego. Muestras tomadas en sangre para PCRus, fibrinógeno y P-selectina, perfil lipídico, creatin kinasa y transaminasas hepáticas, pre y post test de esfuerzo, y a las 24 horas. Los resultados para datos continuos se expresan como medias +/- desviación estándar, test de student para muestras independientes, ANOVA para muestras repetidas. Programa estadístico SPSS 14.0. Resultados: Un grupo de 44 sujetos completaron el estudio: atorvastatina 80 mg (n=24) o placebo (n=20). En el grupo atorvastatina, después de una semana de tratamiento, los niveles de LDLc disminuyeron en 38 por ciento (LDL basal: 97 +/- 27 mg/dL vs LDL post: 62 +/- 31 mg/dL, p < 0.001). Sin embargo, no se observaron cambios en ese mismo período en los niveles de PCRus, fibrinógeno y P-selectina con respecto a placebo. Los niveles de fibrinógeno se elevaron 8 por ciento entre la etapa pre y post ejercicio inmediato (341 +/- 56 mg/dL vs 368 +/- 65 mg/dL, p<0.001), retornando a los niveles basales a las 24 horas; no hubo diferencias entre atorvastatina - placebo...

Background: Chronic statin therapy is known to decrease ínflammation and platelet aggregation. However, little data exist regarding acute effect of statins upon these variables. Exercise can be used to induce ínflammation and platelet aggregation. Aim: to determine the acute effect of atorvastatin upon plasma levels of ultra sensitive C reactive protein (US-PCR), fibrinogen and P selectin before, immediately after and 24 hr following a maximal exercise test in healthy subjects. Methods: This was a double blind, randomized prospective study Fifty healthy male subjects (aged 18to 50years) received atorvastatin 80 mg or placebo daily for 7 days. US-PCR, fibrinogen, P-selectin, blood lipids, total creatin-kinase (CK) and transaminases were determined pre and immediately after maximal treadmill exercise. Repeat determinations were performed 24 following the test. Results were analyzed using the SPSS statistical package, and are expressed as mean +/- SD. Student's t and repeated measures ANOVA were used as appropriate. Results: 44 subjects completed the study (atorvastatin =24; placebo= 20). LDL cholesterol decreased from 97 +/- 27 to 62 +/- 31 mg/dl in the atorvastatin group (p<0.001). US-PCR, After 1 week, Fibrinogen and P-selectin were not significantly modified from baseline, and no differences were observed between groups (atorvastatin vs. control). However, fibrinogen increased 8 percent from baseline to immediately post exercise (341 +/- 6 vs. 368 +/- 65mg/dl (95 percent CI. 21/.3 - 33.6). 24hr after exercise, fibrinogen levels returned to baseline. Similar changes were observed for P-selectin (25 +/- 5, 28 +/- 1.7 ng/dl, baseline and post exercise respectively p<0.01), again returning to baseline 24hr after exercise. No significant changes were observed for US-PCR after exercise in neither group. CK increased 43 percent in the atorvastatin group and 12 percent in controls (NS). Conclusion: Atorvastatin...

Circulating platelet-derived microparticles in systemic lupus erythematosus. Association with increased thrombin generation and procoagulant state.

The risk for thrombosis is significantly increased in systemic lupus erythematosus (SLE), affecting both venous and arterial vessels. Activated platelets are known to participate in thrombus formation and growth. A general feature of activated cells is the shedding of microparticles (MP) which support coagulation by exposure of negatively charged phospholipids and possibly tissue factor (TF). In this work we characterized circulating MP in patients with SLE and their relationship with a procoagulant state. Thirty patients with SLE (aged 15-72 years, mean age 38 years) and 20 healthy controls (aged 22-54 years, mean age 34 years) were studied; patients fulfilled 4 revised criteria for SLE. The number and cellular source of circulating MP were determined by flow cytometry using double labeling with specific monoclonal antibodies and annexin V. Thrombin generation was measured as the endogenous thrombin potential (ETP) without the addition of exogenous phospholipids and TF; under these conditions the generation of thrombin depended directly on the number of MP present in plasma. Thrombin anti-thrombin (TAT) and plasmin-antiplasmin (PAP) complexes were measured by ELISA. Compared to the controls, circulating MP were significantly elevated in the patient group (1218 +/- 136 vs 653 +/- 74 x 10(3)/ml plasma, p: 0.0007). In both groups, most of these MP were of platelet origin (927 +/- 131 vs 517 +/- 72 x 10(3)/ml plasma, p:0.009 ). ETP was higher among patients as compared to the controls (804 +/- 64 vs 631 +/- 37 nM thrombin, p: 0.025). Plasma levels ofTAT in patients and controls were 3.4 +/- 0.8 and 1.4 +/- 0.5 microg/L, respectively (p:0.04), and of PAP complexes were 62.5 +/- 14 and 24.05 +/- 2.5 microg/ml, respectively (p: 0.014). The number of platelet-derived MP correlated significantly with thrombin generation (r: 0.42; p: 0.038) and TAT levels (r: 0.40; p: 0.035). We did not find an association of circulating MP with disease activity nor with the presence of antiphospholipid antibodies. The increased number of circulating platelet-derived microparticles and their association with high ETP and activation of the coagulation system suggest that these microparticles play an important role in the pathogenesis of the prothrombotic state in SLE patients.

Polimorfismo del receptor P2Y12 de ADP plaquetario en pacientes con enfermedad coronaria y variabilidad en agregación plaquetaria: resultados preliminares / Platelet P2Y12 ADP receptor polymorphism in patients with coronary artery disease and varying degrees of platelet

Introducción: Uno de los mecanismos que explicaría la variabilidad en la agregación plaquetaria observada en la respuesta inhibitoria a clopidogrel es el polimorfismo del receptor P2Y12 de ADP plaquetario, específicamente, el haplotipo H2 y H1/H2. No se ha descrito la prevalencia del haplotipo H1/H2 del receptor plaquetario en pacientes con enfermedad coronaria. Objetivo: Evaluar la presencia del haplotipo H1/H2 del receptor P2Y12 en un grupo de pacientes con enfermedad coronaria Métodos: Estudio prospectivo en pacientes sometidos en forma electiva a angioplastía coronaria con stent. Todos recibieron aspirina y una dosis de carga de clopidogrel de 600 mg, seguido de dosis de mantención de 75 mg/día. En todos se midió agregación plaquetaria previo a la dosis de carga de clopidogrel (día 0) y luego entre 6º y 8º día det ratamiento. La agregación plaquetaria se expresó de acuerdo al porcentaje de cambio respecto del valor basal. Se utilizó test t student pareado para evaluar el porcentaje de cambio. Se amplificó el segmento de interés del ADN de los pacientes mediante PCR y se determinó la presencia del haplotipo H1/H2 usando enzimas de restricción. Resultados: Se enrolaron 40 pacientes, 34 (85 por ciento) hombres, edad promedio 61 +/-12 años. El promedio de agregación plaquetaria, previo y durante terapia con clopidogrel fue de 64 +/-10 por ciento y 41 +/-14 por ciento, respectivamente (p<0.0001) frente a ADP 8 µM. La respuesta de agregación a clopidogrel presentó una distribución normal según el test de Kolmogorov-Smirnov (p=0.58). Los pacientes se estratificaron de acuerdo al porcentaje de cambio en cuartiles y el cuartil de menor cambio representó una diferencia menor a 10 por ciento. De estos pacientes, 30 por ciento (3 pacientes) tenían el haplotipo H1/H2. En total, se demostró la presencia del haplotipo H1/H2 en 4 (10 por ciento) pacientes...

Background: One factor influencing the variability in the anti aggregation effect of clopidogrel is the polymorphism of the platelet P2Y12 ADP receptor, specifically the H2 and H1/H2 haplotypes. The prevalence of the H1/H2 haplotype has not been described in patients with coronary artery disease. Aim: To evaluate the presence of H1/H2 haplotype of P2Y12 receptor in patients with coronary artery disease. Methods: A prospective study was conducted in patients undergoing elective PTCA with stenting. All received aspirin followed by a loading dose of clopidogrel 600 mg and a maintenance dose of 75 mg daily. Platelet aggregation was measured prior to the loading clopidogrel dose and at 6 and 8 days post treatment. Platelet aggregation was indicated as the percent change over the basal value. The Student’s t test was used to evaluate the response. The DNA segment involved was amplified by PCR and the H1/H2 haplotype was determined using restriction enzymes. Results: Fourty patients (85 percent males) with mean age 61 years (SD 12) were studied. Mean platelet aggregation changed from a basal value of 64+/-10 to a post clopidogrel value of 41 +/-14 percent (p<0.0001) with an ADP level of 8 µM. The platelet aggregation response was normal according to Kolmogorov-Smirnov. The lowest quartile of platelet aggregation showed a <10 percent change. Three patients in this group (30 percent) had the H1/H2 haplotype. The overall incidence of this haplotype was 10 percent (4 patients). Conclusion: Clopidogrel does not induce a significant decreased platelet aggregation in 25 percent of patients subjected to coronary angioplasty. A third of this patients exhibited the H1/H2 haplotype for the P2Y12 receptor. This group of patients might be at increased risk from subsequent cardiovascular events.

Distinctive effects of red wine and diet on haemostatic cardiovascular risk factors

The aim of this study was to compare the effects of Mediterranean-type diet (MD), high-fat diet (HFD), and red wine supplementation on plasma concentration of emergent haemostatic cardiovascular risk factors (HCVRF) and on variables of primary haemostasis (bleeding time, plasma von Willebrand factor and platelet aggregation/secretion). In a controlled prospective intervention study, two groups (21 healthy males each) received either MD or HFD during 90 days. Between days 30-60, both diets were supplemented with 240 ml/day of red wine. After adjusting by baseline values, MD was associated with: lower plasma fibrinogen (p =0.03), factor VIIc (p=0.034) and factor VIIIc (p=0.0057); higher levels of protein S (p=0.013); longer bleeding time (p=0.017); and marginal increases in platelet serotonin aggregation and secretion after stimulation with epinephrine. Red wine supplementation, in both diets, resulted in decreased plasma fibrinogen (p=0.001) and factor VIIc (p=0.05), and in increased t-PA (p=0.01) and PAI-1 (p=0.0003). The effects of wine on antithrombin III (p=0.01) were divergent: there was a decrease in the HFD group but it increased slightly in the MD group. No effects of diet or wine were detected in plasma protein C, C-reactive protein or von Willebrand factor. BT did not change significantly with wine supplementation. Wine intake resulted in a significant increase in ex vivo platelet aggregation and secretion after stimulation with collagen (1 and 2 µg/ml, p ≤ 0.01). MD and moderate consumption of red wine have complementary, mostly beneficial effects on haemostatic CV risk factors. The longer BT in individuals on MD, obtained independently of red wine, denotes less interaction of platelets with the vascular wall, which could be beneficial from the point of view of CV risk.

Platelet aging in vivo is associated with activation of apoptotic pathways: studies in a model of suppressed thrombopoiesis in dogs.

The mechanism(s) involved in the clearance of senescent platelets are largely unknown. We have recently demonstrated that platelet aging in vivo is associated with loss of membrane phospholipid asymmetry, a universal phenomenon in cells undergoing apoptosis. Thus, we postulated that senescent platelets may exhibit programmed cell death changes. which may trigger their removal from circulation. Since platelets contain the apoptosis machinery as well as mitochondria, a key organelle in the regulation of apoptosis, we studied the appearance of apoptotic-like changes during platelet aging in vivo. To investigate this, we assessed changes in mitochondrial membrane potential (deltapsi) in circulating canine platelets during decline in platelet count after suppression of thrombopoiesis by estradiol injection, a validated model to obtain circulating platelets of increasing mean age. Phosphatidylserine (PS) exposure was determined by flow cytometry by binding of FITC-labeled annexin V. Mitochondrial deltapsi was studied with the cationic lipophilic dye DIOC6 (3) and the J-aggregate-forming cation JC-1 and analysis by flow cytometry. The proportion of platelets with exposed PS rose significantly with age, from 2.88% before to 6.7%, 8 days after estradiol injection. By flow cytometry it was demonstrated a significant decreased in DIOC6 (3) fluorescence (median fluorescence intensity 791+/-98 vs 567+/-102 day 0 vs day 8 post injection of estradiol, respectively; n: 11; p <0.01), consistent with mitochondrial deltapsi collapse. JC-1 has the unique property of forming J-aggregates under high mitochondrial deltapsi (red fluorescence, FL2) whereas the monomeric form fluoresces in green (FL1). Aged platelets in vivo, loaded with JC-1, exhibited a significant increase in FL1/FL2 ratio (2.5+/-1.7 vs 4.7+/-1.6, day 0 vs day 8 post injection of estradiol, respectively; n: 13; p <0.05), confirming the mitochondrial deltapsi alteration. The results show that platelet aging in vivo is associated with a decrease in mitochondrial deltapsi and PS exposure. In conclusion, our data provide for the first time, evidence that platelet senescence is associated with changes characteristics of apoptosis, which may promote their removal from circulation.