Normalization of blood clotting characteristics using prothrombin complex concentrate, fibrinogen and FXIII in an albumin based fluid : experimental studies in thromboelastometry

Colloid fluids supplemented with adequate combinations of coagulation factor concentrates with the capability to restore coagulation could be a desirable future treatment component in massive transfusion. Starting from a coagulation factor and blood cell-free albumin solution we added Prothrombin Complex Concentrate, Fibrinogen Concentrate and Factor XIII in different combinations and concentrations to analyze their properties to restore thromboelastometry parameters without the use of plasma. Further analysis under the presence of platelets was performed for comparability to whole blood conditions. Albumin solutions enriched with Fibrinogen Concentrate, Factor XIII and Prothrombin Complex Concentrate at optimized concentrations show restoring coagulation potential. Prothrombin Complex Concentrate showed sufficient thrombin formation for inducing fibrinogen polymerization. The combination of Prothrombin Complex Concentrate and Fibrinogen Concentrate led to the formation of a stable in vitro fibrin clot. Fibrinogen and Factor XIII showed excellent capacity to improve fibrin clot firmness expressed as Amplitude at 10 min and Maximal Clot Firmness. Fibrinogen alone, or in combination with Factor XIII, was able to restore normal Amplitude at 10 min and Maximal Clot Firmness values. In the presence of platelets, the thromboelastometry surrogate parameter for thrombin generation (Clotting Time) improves and normalizes when compared to whole blood. Combinations of coagulation factor concentrates suspended in albumin solutions can restore thromboelastometry parameters in the absence of plasma. This kind of artificial colloid fluids with coagulation-restoring characteristics might offer new treatment alternatives for massive transfusion. Study registered at the institutional ethic committee "Institut de Recerca, Hospital Santa Creu i Sant Pau, with protocol number IIBSP-CFC-2013-165

Integrated GWAS and Gene Expression Suggest ORM1 as a Potential Regulator of Plasma Levels of Cell-Free DNA and Thrombosis Risk

Plasma cell-free DNA (cfDNA) is a surrogate marker of neutrophil extracellular traps (NETs) that contribute to immunothrombosis. There is growing interest about the mechanisms underlying NET formation and elevated cfDNA, but little is known about the factors involved. We aimed to identify genes involved in the regulation of cfDNA levels using data from the Genetic Analysis of Idiopathic Thrombophilia (GAIT-2) Project. Imputed genotypes, whole blood RNA-Seq data, and plasma cfDNA quantification were available for 935 of the GAIT-2 participants from 35 families with idiopathic thrombophilia. We performed heritability and GWAS analysis for cfDNA. The heritability of cfDNA was 0.26 ( p  = 3.7 × 10 (−6) ), while the GWAS identified a significant association (rs1687391, p  = 3.55 × 10 (−10) ) near the ORM1 gene, on chromosome 9. An eQTL (expression quantitative trait loci) analysis revealed a significant association between the lead GWAS variant and the expression of ORM1 in whole blood ( p  = 6.14 × 10 (−9) ). Additionally, ORM1 expression correlated with levels of cfDNA ( p  = 4.38 × 10 (−4) ). Finally, genetic correlation analysis between cfDNA and thrombosis identified a suggestive association ( ρ (g)  = 0.43, p  = 0.089). All in all, we show evidence of the role of ORM1 in regulating cfDNA levels in plasma, which might contribute to the susceptibility to thrombosis through mechanisms of immunothrombosis

Nous fenotips intermediaris basats en el model cel·lular de la coagulació com a factors de risc de trombosi venosa

El tromboembolisme venós (TEV) és una malaltia complexa. És conseqüència de la interacció de factors ambientals i genètics que molts cops es reflecteixen en els anomenats fenotips intermediaris. Aquests, s’associen per definició al risc de trombosi i per això tenen una gran utilitat tant diagnòstica com en la busca de factors causals. La forma d’estudiar l’hemostàsia ha variat i, avui en dia, es parla del model cel·lular. El model cel·lular proporciona una visió més àmplia dels processos de l’hemostàsia projectant les múltiples interaccions entre els elements que hi intervenen. En aquest model, les cèl·lules no actuen només com una superfície fosfolipídica que serveix de suport als diferents factors, sinó que tenen un paper actiu. Això fa que les cèl·lules sanguínies siguin objectius on focalitzar la busca de nous fenotips implicats en les malalties de l’hemostàsia. El principal objectiu d’aquesta tesi ha estat la caracterització de nous fenotips eritrocitaris, plaquetaris i leucocitaris per descriure la seva associació amb el risc de TEV. Els estudis realitzats s’han desenvolupat en l’àmbit de dos projectes: Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) un estudi de 35 famílies, i el projecte Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), un estudi de casos i controls no emparentats amb 400 casos amb TEV i 400 controls. L’estudi de la sèrie eritrocitària a la població GAIT-2 ha conduit cap a la identificació de 5 fenotips intermediaris genèticament correlacionats amb la trombosi: Hematòcrit, l’amplitud de distribució eritrocitària (RDW), índex de fluorescència reticulocitària (IRF), el receptor soluble de la transferrina i el ferro sèric. Aquests genotips poden utilitzar-se per a la recerca de gens relacionats amb la trombosi. L’estudi de la funció plaquetària en pacients amb trombosi (projecte RETROVE) mitjançant dispositius habitualment utilitzats per a l’estudi de malalties hemorràgiques, com el PFA-100®, ens ha permès demostrar que el risc de TEV és superior en aquells pacients amb temps d’obturació curts. Aquest fet podria indicar que l’adhesió plaquetària tindria un paper en la formació i probablement en la propagació del trombus. I finalment l’estudi de la població leucocitària fet dins el projecte RETROVE, ens ha permès identificar la xifra de monòcits superior a 0,7x109/L com un factor de risc de TEV, fet que concorda amb estudis previs. I per altra banda, s’ha observat que els neutròfils dels pacients amb antecedents de TEV, tenen major capacitat per a fer NETosi, fet que s’observa amb un increment dels marcadors plasmàtics de trampes de neutròfils (NETs). Aquests marcadors poden ser utilitzats com a fenotips intermediaris de trombosi, però, a més, en un futur, poden arribar a ser dianes terapèutiques antitrombòtiques.Venous thromboembolism (VTE) is a complex disease. It is the result of the interaction between the environment and genes. This interaction is manifested by “intermediary phenotypes”. These intermediary phenotypes are useful for estimating the risk of developing the disease. The prevailing view of hemostasis has changed and nowadays, there is another point of view based on a model in which coagulation is regulated by properties of cell surfaces: The Cell-based Model of Hemostasis. This model provides a wide vision of the hemostatic process and reflects the multiple pathways of hemostasis in vivo. The clearer understanding of the role of cells will allow us to answer some unanswered questions in hemostasis. The main objective of this thesis has been the characterization of new erythrocyte, platelet and leucocyte phenotypes that will allow us to describe the association between blood cells and VTE risk. The studies included in this thesis were performed in the context of two projects: the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) project, a study of 35 families and 935 individuals, and the project Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), a case-control study of 400 unrelated cases with VTE and 400 controls. Using the extended pedigrees of GAIT-2 project, we have identified 5 intermediate erythrocyte-related phenotypes: Hematocrit, red cell distribution width (RDW), index reticulocyte fluorescence (IRF), soluble transferrin receptor and serum iron, that are genetically correlated with VTE. They can be used in the research for thrombosis –related genes. The study of platelet function in patients with thrombosis (in the RETROVE project) using devices that are frequently used for the study of hemorrhagic diseases, like PFA-100®, has allowed us to demonstrate that the risk of VTE is higher in patients with shortest closure times. This fact, supports the relevance of platelet adhesion in thrombus formation and probably, in their perpetuation. And finally, the study of leucocyte counts in the RETROVE project, have allowed us, in one hand, to identify that monocyte counts over 0,7x109/L are statistically associated with previous VTE. Our findings are in line with previous studies that reported the same association. In the other hand, we have observed that circulating neutrophils in patients with VTE are prone to undergo NETosis, which is associated with enhanced expression of neutrophil extracellular traps (NETs) biomarkers. These biomarkers can be used as intermediary phenotypes for VTE, and, in the future, they can be therapeutic targets

Nous fenotips intermediaris basats en el model cel·lular de la coagulació com a factors de risc de trombosi venosa

El tromboembolisme venós (TEV) és una malaltia complexa. És conseqüència de la interacció de factors ambientals i genètics que molts cops es reflecteixen en els anomenats fenotips intermediaris. Aquests, s’associen per definició al risc de trombosi i per això tenen una gran utilitat tant diagnòstica com en la busca de factors causals. La forma d’estudiar l’hemostàsia ha variat i, avui en dia, es parla del model cel·lular. El model cel·lular proporciona una visió més àmplia dels processos de l’hemostàsia projectant les múltiples interaccions entre els elements que hi intervenen. En aquest model, les cèl·lules no actuen només com una superfície fosfolipídica que serveix de suport als diferents factors, sinó que tenen un paper actiu. Això fa que les cèl·lules sanguínies siguin objectius on focalitzar la busca de nous fenotips implicats en les malalties de l’hemostàsia. El principal objectiu d’aquesta tesi ha estat la caracterització de nous fenotips eritrocitaris, plaquetaris i leucocitaris per descriure la seva associació amb el risc de TEV. Els estudis realitzats s’han desenvolupat en l’àmbit de dos projectes: Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) un estudi de 35 famílies, i el projecte Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), un estudi de casos i controls no emparentats amb 400 casos amb TEV i 400 controls. L’estudi de la sèrie eritrocitària a la població GAIT-2 ha conduit cap a la identificació de 5 fenotips intermediaris genèticament correlacionats amb la trombosi: Hematòcrit, l’amplitud de distribució eritrocitària (RDW), índex de fluorescència reticulocitària (IRF), el receptor soluble de la transferrina i el ferro sèric. Aquests genotips poden utilitzar-se per a la recerca de gens relacionats amb la trombosi. L’estudi de la funció plaquetària en pacients amb trombosi (projecte RETROVE) mitjançant dispositius habitualment utilitzats per a l’estudi de malalties hemorràgiques, com el PFA-100®, ens ha permès demostrar que el risc de TEV és superior en aquells pacients amb temps d’obturació curts. Aquest fet podria indicar que l’adhesió plaquetària tindria un paper en la formació i probablement en la propagació del trombus. I finalment l’estudi de la població leucocitària fet dins el projecte RETROVE, ens ha permès identificar la xifra de monòcits superior a 0,7x109/L com un factor de risc de TEV, fet que concorda amb estudis previs. I per altra banda, s’ha observat que els neutròfils dels pacients amb antecedents de TEV, tenen major capacitat per a fer NETosi, fet que s’observa amb un increment dels marcadors plasmàtics de trampes de neutròfils (NETs). Aquests marcadors poden ser utilitzats com a fenotips intermediaris de trombosi, però, a més, en un futur, poden arribar a ser dianes terapèutiques antitrombòtiques.Venous thromboembolism (VTE) is a complex disease. It is the result of the interaction between the environment and genes. This interaction is manifested by “intermediary phenotypes”. These intermediary phenotypes are useful for estimating the risk of developing the disease. The prevailing view of hemostasis has changed and nowadays, there is another point of view based on a model in which coagulation is regulated by properties of cell surfaces: The Cell-based Model of Hemostasis. This model provides a wide vision of the hemostatic process and reflects the multiple pathways of hemostasis in vivo. The clearer understanding of the role of cells will allow us to answer some unanswered questions in hemostasis. The main objective of this thesis has been the characterization of new erythrocyte, platelet and leucocyte phenotypes that will allow us to describe the association between blood cells and VTE risk. The studies included in this thesis were performed in the context of two projects: the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) project, a study of 35 families and 935 individuals, and the project Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), a case-control study of 400 unrelated cases with VTE and 400 controls. Using the extended pedigrees of GAIT-2 project, we have identified 5 intermediate erythrocyte-related phenotypes: Hematocrit, red cell distribution width (RDW), index reticulocyte fluorescence (IRF), soluble transferrin receptor and serum iron, that are genetically correlated with VTE. They can be used in the research for thrombosis –related genes. The study of platelet function in patients with thrombosis (in the RETROVE project) using devices that are frequently used for the study of hemorrhagic diseases, like PFA-100®, has allowed us to demonstrate that the risk of VTE is higher in patients with shortest closure times. This fact, supports the relevance of platelet adhesion in thrombus formation and probably, in their perpetuation. And finally, the study of leucocyte counts in the RETROVE project, have allowed us, in one hand, to identify that monocyte counts over 0,7x109/L are statistically associated with previous VTE. Our findings are in line with previous studies that reported the same association. In the other hand, we have observed that circulating neutrophils in patients with VTE are prone to undergo NETosis, which is associated with enhanced expression of neutrophil extracellular traps (NETs) biomarkers. These biomarkers can be used as intermediary phenotypes for VTE, and, in the future, they can be therapeutic targets

Nous fenotips intermediaris basats en el model cel·lular de la coagulació com a factors de risc de trombosi venosa

El tromboembolisme venós (TEV) és una malaltia complexa. És conseqüència de la interacció de factors ambientals i genètics que molts cops es reflecteixen en els anomenats fenotips intermediaris. Aquests, s'associen per definició al risc de trombosi i per això tenen una gran utilitat tant diagnòstica com en la busca de factors causals. La forma d'estudiar l'hemostàsia ha variat i, avui en dia, es parla del model cel·lular. El model cel·lular proporciona una visió més àmplia dels processos de l'hemostàsia projectant les múltiples interaccions entre els elements que hi intervenen. En aquest model, les cèl·lules no actuen només com una superfície fosfolipídica que serveix de suport als diferents factors, sinó que tenen un paper actiu. Això fa que les cèl·lules sanguínies siguin objectius on focalitzar la busca de nous fenotips implicats en les malalties de l'hemostàsia. El principal objectiu d'aquesta tesi ha estat la caracterització de nous fenotips eritrocitaris, plaquetaris i leucocitaris per descriure la seva associació amb el risc de TEV. Els estudis realitzats s'han desenvolupat en l'àmbit de dos projectes: Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) un estudi de 35 famílies, i el projecte Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), un estudi de casos i controls no emparentats amb 400 casos amb TEV i 400 controls. L'estudi de la sèrie eritrocitària a la població GAIT-2 ha conduit cap a la identificació de 5 fenotips intermediaris genèticament correlacionats amb la trombosi: Hematòcrit, l'amplitud de distribució eritrocitària (RDW), índex de fluorescència reticulocitària (IRF), el receptor soluble de la transferrina i el ferro sèric. Aquests genotips poden utilitzar-se per a la recerca de gens relacionats amb la trombosi. L'estudi de la funció plaquetària en pacients amb trombosi (projecte RETROVE) mitjançant dispositius habitualment utilitzats per a l'estudi de malalties hemorràgiques, com el PFA-100®, ens ha permès demostrar que el risc de TEV és superior en aquells pacients amb temps d'obturació curts. Aquest fet podria indicar que l'adhesió plaquetària tindria un paper en la formació i probablement en la propagació del trombus. I finalment l'estudi de la població leucocitària fet dins el projecte RETROVE, ens ha permès identificar la xifra de monòcits superior a 0,7x109/L com un factor de risc de TEV, fet que concorda amb estudis previs. I per altra banda, s'ha observat que els neutròfils dels pacients amb antecedents de TEV, tenen major capacitat per a fer NETosi, fet que s'observa amb un increment dels marcadors plasmàtics de trampes de neutròfils (NETs). Aquests marcadors poden ser utilitzats com a fenotips intermediaris de trombosi, però, a més, en un futur, poden arribar a ser dianes terapèutiques antitrombòtiques.Venous thromboembolism (VTE) is a complex disease. It is the result of the interaction between the environment and genes. This interaction is manifested by "intermediary phenotypes". These intermediary phenotypes are useful for estimating the risk of developing the disease. The prevailing view of hemostasis has changed and nowadays, there is another point of view based on a model in which coagulation is regulated by properties of cell surfaces: The Cell-based Model of Hemostasis. This model provides a wide vision of the hemostatic process and reflects the multiple pathways of hemostasis in vivo. The clearer understanding of the role of cells will allow us to answer some unanswered questions in hemostasis. The main objective of this thesis has been the characterization of new erythrocyte, platelet and leucocyte phenotypes that will allow us to describe the association between blood cells and VTE risk. The studies included in this thesis were performed in the context of two projects: the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) project, a study of 35 families and 935 individuals, and the project Riesgo de Enfermedad TROmboembólica Venosa (RETROVE), a case-control study of 400 unrelated cases with VTE and 400 controls. Using the extended pedigrees of GAIT-2 project, we have identified 5 intermediate erythrocyte-related phenotypes: Hematocrit, red cell distribution width (RDW), index reticulocyte fluorescence (IRF), soluble transferrin receptor and serum iron, that are genetically correlated with VTE. They can be used in the research for thrombosis -related genes. The study of platelet function in patients with thrombosis (in the RETROVE project) using devices that are frequently used for the study of hemorrhagic diseases, like PFA-100®, has allowed us to demonstrate that the risk of VTE is higher in patients with shortest closure times. This fact, supports the relevance of platelet adhesion in thrombus formation and probably, in their perpetuation. And finally, the study of leucocyte counts in the RETROVE project, have allowed us, in one hand, to identify that monocyte counts over 0,7x109/L are statistically associated with previous VTE. Our findings are in line with previous studies that reported the same association. In the other hand, we have observed that circulating neutrophils in patients with VTE are prone to undergo NETosis, which is associated with enhanced expression of neutrophil extracellular traps (NETs) biomarkers. These biomarkers can be used as intermediary phenotypes for VTE, and, in the future, they can be therapeutic targets

Correlation between plateletworks® and pfa-100® for measuring platelet function before urgent surgery in patients with chronic antiplatelet therapy

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Biometa: Tecnologías y Sistemas SAL.Hemostasis is crucial for reducing bleeding during surgical procedures. The points-of-care based on the platelet function test could be useful to minimize the complications related to chronic antiplatelet therapy during surgery. The present study is aimed at comparing two point-of-care platelet function devices-Platelet Function Analyzer PFA-100® (Siemens Canada, Mississauga, ON, Canada) and Plateletworks® (Helena Laboratories, Beaumont, TX, USA). Our objective is to evaluate if they provide comparable and useful information to manage anti-aggregate patients before surgery. We included patients with a femoral fracture receiving chronic antiplatelet therapy and a median age of 89 years (range from 70 to 98). A platelet function evaluation was performed on all patients before surgery using both devices-Plateletworks® and PFA-100®. The correlation between Plateletworks® and PFA-100® was performed using Cohen's Kappa coefficient. Twenty consecutive patients par-ticipated in the trial; 16 patients were under treatment with 75 mg/day of clopidogrel, three with >300 mg/day of acetylsalicylic acid (ASA), and only one was in treatment with both antiplatelet agents. Cohen's Kappa coefficient was 0.327 comparing PFA-100®-ADP (adenosine diphosphate) and Plateletworks® and, 0.200 comparing PFA-100®-EPI (epinephrine) and Plateletworks®. In conclusion, we found a weak concordance comparing PFA-100® and Plateletworks®. This could partially be due to the advanced age of the included patients. However, given the limited sample size, more studies are necessary to confirm these results

Assessing the potential of sputtered gold nanolayers in mass spectrometry imaging for metabolomics applications.

Mass spectrometry imaging (MSI) is a molecular imaging technique that maps the distribution of molecules in biological tissues with high spatial resolution. The most widely used MSI modality is matrix-assisted laser desorption/ionization (MALDI), mainly due to the large variety of analyte classes amenable for MALDI analysis. However, the organic matrices used in classical MALDI may impact the quality of the molecular images due to limited lateral resolution and strong background noise in the low mass range, hindering its use in metabolomics. Here we present a matrix-free laser desorption/ionization (LDI) technique based on the deposition of gold nanolayers on tissue sections by means of sputter-coating. This gold coating method is quick, fully automated, reproducible, and allows growing highly controlled gold nanolayers, necessary for high quality and high resolution MS image acquisition. The performance of the developed method has been tested through the acquisition of MS images of brain tissues. The obtained spectra showed a high number of MS peaks in the low mass region (m/z below 1000 Da) with few background peaks, demonstrating the ability of the sputtered gold nanolayers of promoting the desorption/ionization of a wide range of metabolites. These results, together with the reliable MS spectrum calibration using gold peaks, make the developed method a valuable alternative for MSI applications

Genetic Determinants of Thrombin Generation and Their Relation to Venous Thrombosis: Results from the GAIT-2 Project.

BACKGROUND:Venous thromboembolism (VTE) is a common disease where known genetic risk factors explain only a small portion of the genetic variance. Then, the analysis of intermediate phenotypes, such as thrombin generation assay, can be used to identify novel genetic risk factors that contribute to VTE. OBJECTIVES:To investigate the genetic basis of distinct quantitative phenotypes of thrombin generation and its relationship to the risk of VTE. PATIENTS/METHODS:Lag time, thrombin peak and endogenous thrombin potential (ETP) were measured in the families of the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project. This sample consisted of 935 individuals in 35 extended families selected through a proband with idiopathic thrombophilia. We performed also genome wide association studies (GWAS) with thrombin generation phenotypes. RESULTS:The results showed that 67% of the variation in the risk of VTE is attributable to genetic factors. The heritabilities of lag time, thrombin peak and ETP were 49%, 54% and 52%, respectively. More importantly, we demonstrated also the existence of positive genetic correlations between thrombin peak or ETP and the risk of VTE. Moreover, the major genetic determinant of thrombin generation was the F2 gene. However, other suggestive signals were observed. CONCLUSIONS:The thrombin generation phenotypes are strongly genetically determined. The thrombin peak and ETP are significantly genetically correlated with the risk of VTE. In addition, F2 was identified as a major determinant of thrombin generation. We reported suggestive signals that might increase our knowledge to explain the variability of this important phenotype. Validation and functional studies are required to confirm GWAS results