DSpace Angular :: Browsing by Author "Mezzano, Diego"

Browsing by Author "Mezzano, Diego"

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Clot lysis time in platelet-rich plasma: Method assessment, comparison with assays in platelet-free and platelet-poor plasmas, and response to tranexamic acid
(TAYLOR & FRANCIS INC, 2012) Panes, Olga; Padilla, Oslando; Matus, Valeria; Saez, Claudia G.; Berkovits, Alejandro; Pereira, Jaime; Mezzano, Diego
Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is likely explained by the display of platelet pro-fibrinolytic effects. Focused research is needed to disclose the mechanisms for the relationship between CLT and plasma cholesterol and its potential pathophysiologic and clinical relevance.
Diagnosis of mild platelet function disorders. Reliability and usefulness of light transmission platelet aggregation and serotonin secretion assays
(2009) Quiroga Gutiérrez, Sara Teresita; Goycolea, Manuela; Matus Ritter, Valeria; Zúñiga Contreras, Pamela; Martínez, Carlos; Garrido, Marcelo; Aranda Lauriani, Eduardo Javier; Leighton Puga, Federico; Panes Becerra, Olga Teresa; Pereira Garcés, Jaime Ignacio; Mezzano, Diego
Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey
(2014) Gresele, P.; Harrison, P.; Bur, L.; Falcinelli, E.; Gachet, C.; Hayward, C. P.; Kenny, D.; Mezzano, Diego; Mumford, A. D.; Nugent, D.; Nurden, A. T.; Orsini, S.; Cattaneo, M.
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
(2022) Mezzano, Diego; Harrison, Paul; Frelinger, Andrew L., III; Mumford, Andrew D.; Noris, Patrizia; Lordkipanidze, Marie; Gresele, Paolo
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 delta-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 delta-granule release, and platelet P-selectin expression by flow cytometry and released proteins by ELISA for alpha-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.
Fundamentals for a Systematic Approach to Mild and Moderate Inherited Bleeding Disorders: An EHA Consensus Report
(2019) Rodeghiero, Francesco; Pabinger, Ingrid; Ragni, Margaret; Abdul-Kadir, Rezan; Berntorp, Erik; Blanchette, Victor; Bodo, Imre; Casini, Alessandro; Gresele, Paolo; Lassila, Riitta; Leebeek, Frank; Lillicrap, David; Mezzano, Diego; Noris, Patrizia; Srivastava, Alok; Tosetto, Alberto; Windyga, Jerzy; Zieger, Barbara; Makris, Mike; Key, Nigel
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.
Genotype-phenotype relationship for six common polymorphisms in genes affecting platelet function from 286 healthy subjects and 160 patients with mucocutaneous bleeding of unknown cause
(2009) Martínez, Constantino; Antón, Ana Isabel; Corral, Javier; Quiroga Gutiérrez, Sara Teresita; Panes Becerra, Olga Teresa; Lozano, María Luisa; González Conejero, Rocío; Teruel, Raúl; Navarro Núñez, Leyre; Pereira Garcés, Jaime Ignacio; Mezzano, Diego; Vicente, Vicente; Rivera, José
Health impact of Mediterranean diets in food at work
(CAMBRIDGE UNIV PRESS, 2009) Leighton, Federico; Polic, Gianna; Strobel, Pablo; Perez, Druso; Martinez, Carlos; Vasquez, Luis; Castillo, Oscar; Villarroel, Luis; Echeverria, Guadalupe; Urquiaga, Ines; Mezzano, Diego; Rozowski, Jaime
Objective: To evaluate the feasibility of diet mediterranisation, in a food-at-work context, and its consequence on metabolic syndrome in a mid-age unselected healthy male Population group.
High prevalence of bleeders of unknown cause among patients with inherited mucocutaneous bleeding. A prospective study of 280 patients and 299 controls
(FERRATA STORTI FOUNDATION, 2007) Quiroga, Teresa; Goycoolea, Manuela; Panes, Olga; Aranda, Eduardo; Martinez, Carlos; Belmont, Sabine; Munoz, Blanca; Zuniga, Pamela; Pereira, Jaime; Mezzano, Diego
Background and Objectives
Human platelets synthesize and express functional tissue factor
(AMER SOC HEMATOLOGY, 2007) Panes, Olga; Matus, Valeria; Saez, Claudia G.; Quiroga, Teresa; Pereira, Jaime; Mezzano, Diego
The source and significance of bloodborne tissue factor (TF) are controversial. TF mRNA, protein, and TF-dependent procoagulant activity (PCA) have been detected in human platelets, but direct evidence of TF synthesis is missing. Nonstimulated monocyte-free platelets from most patients expressed TF mRNA, which was enhanced or induced in all of them after platelet activation. Immunoprecipitation assays revealed TF protein (mainly of a molecular weight [Mr] of approximately 47 kDa, with other bands of approximately 35 and approximately 60 kDa) in nonstimulated platelet membranes, which also increased after activation. This enhancement was concomitant with TF translocation to the plasma membrane, as demonstrated by immunofluorescence-confocal microscopy and biotinylation of membrane proteins. Platelet PCA, assessed by factor Xa (FXa) generation, was induced after activation and was inhibited by 48% and 76% with anti-TF and anti-FVIIa, respectively, but not by intrinsic pathway inhibitors. Platelets incorporated [35S]-methionine into TF proteins with Mr of approximately 47 kDa, approximately 35 kDa, and approximately 60 kDa, more intensely after activation. Puromycin but not actinomycin D or DRB (5,6-dichloro-1 -beta-D-ribofuranosylbenzimidazole)inhibited TIF neosynthesis. Thus, human platelets not only assemble the clotting reactions on their membrane, but also supply their own TIF for thrombin generation in a timely and spatially circumscribed process. These observations simplify, unify, and provide a more coherent formulation of the current cellbased model of hemostasis.
Increased number of circulating endothelial cells and plasma markers of endothelial damage in chronic cocaine users
(PERGAMON-ELSEVIER SCIENCE LTD, 2011) Saez, Claudia G.; Olivares, Paulina; Pallavicini, Julio; Panes, Olga; Moreno, Natalia; Massardo, Teresa; Mezzano, Diego; Pereira, Jaime
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.
Influence of the F12-4 C > T polymorphism on hemostatic tests
(LIPPINCOTT WILLIAMS & WILKINS, 2010) Corral, Javier; Anton, Ana I.; Quiroga, Teresa; Gonzalez Conejero, Rocio; Pereira, Jaime; Roldan, Vanessa; Vicente, Vicente; Mezzano, Diego
The common F12 - 4 C>T polymorphism significantly regulates plasma levels of FXII, the first element of the intrinsic pathway of coagulation. Due to the robust effects that this pathway has on blood coagulation in vitro, the objective of our study was to evaluate the influence of this polymorphism on different hemostatic tests. We studied 46 hemostatic parameters in 566 participants: 280 patients with mucocutaneous bleeding and 286 controls. The F12 - 4T allele, associated with reduced levels of FXII (P<0.001), also significantly delayed the activated partial thromboplastin time (aPTT) expressed as aPTTr (ratio sample plasma/normal pooled plasma). Thus, both patients and controls carrying the T allele had higher aPTTr than C/C homozygous individuals (P<0.001). Interestingly, 92% of healthy controls who had prolonged aPTTr carried the F12 - 4T allele. Moreover, individuals with the F12 - 4T allele also had less thrombin generation (assessed by endogenous thrombin potential, thrombin peak and time to achieve the peak of thrombin) using a test with low tissue factor concentration and explicit contact phase activation. Finally, both patients and controls carrying the F12 - 4T allele also displayed significantly lower FIXc and FVIIc levels than C/C individuals (P<0.01). For all associations except for FVIIc, a gene-dosage effect was observed, and homozygous TT individuals had the farthest values. Our study reveals a significant effect of the F12 - 4 C>T polymorphism on hemostatic tests widely used in routine clinical practice. Blood Coagul Fibrinolysis 21: 632-639 (c) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Is my patient a bleeder? A diagnostic framework for mild bleeding disorders
(AMER SOC HEMATOLOGY, 2012) Quiroga, Teresa; Mezzano, Diego
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.
Laboratory Assessment of Familial, Nonthrombocytopenic Bleeding: A Definitive Not Possible Mucocutaneous Diagnosis Is Often Not Possible
(THIEME MEDICAL PUBL INC, 2008) Pereira, Jaime; Quiroga, Teresa; Mezzano, Diego
Patients with inherited mucocutaneous bleeding (MCB) pose frequent and significant diagnostic challenges. Bleeding symptoms are frequent among the otherwise healthy population, and the clinical distinction between normal subjects and patients with genuine bleeding disorders is complex. Screening or global laboratory assays are nonspecific and have low sensitivity to detect mild bleeding disorders. Moreover, there are inherent difficulties in diagnosing von Willebrand disease and platelet function defects, the best-characterized and most frequent disorders of primary hemostasis. On the other hand, some patients with moderate to severe clotting factor deficiencies and those with increased fibrinolysis usually present with MCB. Finally, in a significant proportion of patients, the definitive diagnosis is not possible even after an extensive laboratory workup. This article reviews the clinical and laboratory approach to the diagnosis of patients presenting with MCB, the limitations of the available methodologies to evaluate the clinical significance of bleeding, and the diagnostic yield of global and specific hemostasis tests used to investigate these patients.
Novel loci involved in platelet function and platelet count identified by a genome-wide study performed in children
(FERRATA STORTI FOUNDATION, 2011) Guerrero, Jose A.; Rivera, Jose; Quiroga, Teresa; Martinez Perez, Angel; Isabel Anton, Ana; Martinez, Constantino; Panes, Olga; Vicente, Vicente; Mezzano, Diego; Soria, Jose Manuel; Corral, Javier
Background
Platelet activation by charged ligands and nanoparticles: platelet glycoprotein receptors as pattern recognition receptors
(2021) Montague, Samantha J.; Patel, Pushpa; Martin, Eleyna M.; Slater, Alexandre; Quintanilla, Lourdes Garcia; Perrella, Gina; Kardeby, Caroline; Nagy, Magdolna; Mezzano, Diego; Mendes, Paula M.; Watson, Steve P.
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.
Platelet activation in chronic cocaine users: Effect of short term abstinence
(TAYLOR & FRANCIS INC, 2011) Pereira, Jaime; Saez, Claudia G.; Pallavicini, Julio; Panes, Olga; Pereira Flores, Karla; Cabreras, Manuel J.; Massardo, Teresa; Mezzano, Diego
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.
Platelet tissue factor activity and membrane cholesterol are increased in hypercholesterolemia and normalized by rosuvastatin, but not by atorvastatin.
(2017) Panes Becerra, Olga Teresa; González, César; Hidalgo, Patricia; Valderas Igor, Juan Patricio; Acevedo B., Mónica; Contreras, Susana; Sánchez, Ximena; Pereira Garcés, Jaime Ignacio; Rigotti Rivera, Attilio; Mezzano, Diego
Procarboxypeptidase U (TAFI) and the Thr325Ile proCPU polymorphism in patients with hereditary mucocutaneous hemorrhages
(ELSEVIER, 2009) Matus, Valeria; Willemse, Johan; Quiroga, Teresa; Goycoolea, Manuela; Aranda, Eduardo; Panes, Olga; Pereira, Jaime; Hendriks, Dirk; Mezzano, Diego
Background: Patients with hereditary mucocutaneous bleeding are difficult to diagnose and many of them fulfill the category of bleeders of unknown cause (BUC). The pathogenic role of hyperfibrinolysis has received little attention, despite the successful use of antifibrinolytic drugs in treating many of these patients. Theoretically, decreased plasma procarboxypeptidase U (proCPU) levels or lower carboxypeptidase U (CPU) stability would result in higher fibrinolytic activity and bleeding tendency.
Rare homozygous status of P43 beta 1-tubulin polymorphism causes alterations in platelet ultrastructure
(GEORG THIEME VERLAG KG, 2011) Navarro Nunez, Leyre; Teruel, Raul; Isabel Anton, Ana; Nurden, Paquita; Martinez Martinez, Irene; Luisa Lozano, Maria; Rivera, Jose; Corral, Javier; Mezzano, Diego; Vicente, Vicente; Martinez, Constantino
beta 1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased beta 1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals. Our study included 19 subjects genotyped for TUBB1 Q43P polymorphism (4 QQ, 4 QP, and 2 PP). The two PP individuals were recruited after genotyping of 2073 individuals. Biochemical, microscopy, and molecular studies were performed. Real-time PCR showed a similar to 40% decrease in TUBB1 mRNA in the two PP individuals compared to four QQ subjects. Western blot analysis confirmed this reduction. Electron microscopy revealed a majority of normal discoid platelets in PP individuals, although platelets with loose, re-orientated or invaginated protofilaments, and an over-developed open canalicular system were observed. Such abnormalities were not observed in QQ subjects. Morphometric analyses showed no differences between PP and QQ individuals. Immunofluorescence confirmed the presence of a normal marginal band in a majority of platelets from PP subjects. Interestingly, both PP subjects had a 40% lower platelet count than QP and QQ. TUBB1 Q43P polymorphism in homozygosity mildly affects platelet ultrastructure and our data further suggest that high levels of beta 1-tubulin might not be critical to sustain platelet discoid shape.
Serotonin secretion by blood platelets: accuracy of high-performance liquid chromatography-electrochemical technique compared with the isotopic test and use in a clinical laboratory
(2023) Aranda, Eduardo; Iha, Seiki; Solari, Sandra; Rodriguez, David; Romero, Viviana; Villarroel, Luis; Pereira, Jaime; Panes, Olga; Mezzano, Diego
Background: Mild secretion defects are the most frequent and challenging blood platelet disorders to diagnose. Most d-granule secretion tests lack validation, are not quantitative, or have unreliable response to weak platelet agonists.

Browsing by Author "Mezzano, Diego"

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