Publications by authors named "Ana Rosa Cid"

24 Publications

  • Page 1 of 1

The ISTH bleeding assessment tool as predictor of bleeding events in inherited platelet disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

J Thromb Haemost 2021 May;19(5):1364-1371

Department of Internal Medicine, IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy.

Background: The ISTH Bleeding Assessment Tool (ISTH-BAT) has been validated for clinical screening of suspected von Willebrand disease (VWD) and for bleeding prediction. Recently it has been validated for subjects with inherited platelet disorders (IPD) (BAT-VAL study).

Objectives: To determine whether the ISTH-BAT bleeding score (BS) predicts subsequent bleeding events requiring treatment in IPD patients.

Methods: Patients with IPD, type 1 VWD (VWD-1) and age- and sex-matched healthy controls enrolled in the BAT-VAL study were prospectively followed-up for 2 years and bleeding episodes requiring treatment were recorded.

Results: Of the 1098 subjects initially enrolled, 955 were followed-up and 124 suffered hemorrhages during follow-up, 60% of whom had inherited platelet function disorders (IPFD). Total number of events was significantly higher in IPFD (n = 235) than VWD-1 (n = 52) or inherited thrombocytopenia (IT; n = 20). Events requiring transfusions were 66% in IPFD, 5.7% in VWD-1, and 3% in IT. Baseline BS was significantly higher in IPFD patients with a bleeding event at follow-up than in those without (p < .01) and the percentage of subjects suffering a bleeding event increased proportionally to baseline BS quartile. A significant association between the BS and the chance of suffering severe bleeding was found in the overall, IPFD, and VWD-1 populations. Similar results were obtained for the pediatric population.

Conclusions: Inherited platelet function disorder patients with high BS at enrollment are more likely to suffer from bleeding events requiring treatment at follow-up. Moreover, the higher the baseline BS quartile the greater the incidence of subsequent events, suggesting that independently from diagnosis a high BS is associated with a greater risk of subsequent hemorrhage.
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http://dx.doi.org/10.1111/jth.15263DOI Listing
May 2021

Unraveling the Influence of Common von Willebrand factor variants on von Willebrand Disease Phenotype: An Exploratory Study on the Molecular and Clinical Profile of von Willebrand Disease in Spain Cohort.

Thromb Haemost 2020 Mar 5;120(3):437-448. Epub 2020 Mar 5.

Department of Hematology, Hospital Sta Creu i St Pau, Barcelona, Spain.

The clinical diagnosis of von Willebrand disease (VWD), particularly type 1, can be complex because several genetic and environmental factors affect von Willebrand factor (VWF) plasma levels. An estimated 60% of the phenotypic variation is attributable to hereditary factors, with the ABO blood group locus being the most influential. However, recent studies provide strong evidence that nonsynonymous single nucleotide variants (SNVs) contribute to VWF and factor VIII phenotypic variability in healthy individuals. This study aims to investigate the role of common VWF SNVs on VWD phenotype by analyzing data from 219 unrelated patients included in the "Molecular and Clinical Profile of von Willebrand Disease in Spain project." To that end, generalized linear mixed-effects regression models were fitted, and additive and epistatic analyses, and haplotype studies were performed, considering five VWD-related measures (bleeding score, VWF:Ag, VWF:RCo, factor VIII:C, and VWF:CB). According to these analyses, homozygotes: for p.Thr789Ala(C) would be expected to show 39% higher VWF:Ag levels; p.Thr1381Ala(C), 27% lower VWF:Ag levels; and p.Gln852Arg(C), 52% lower VWF:RCo levels. Homozygotes for both p.Thr789Ala(C) and p.Gln852Arg(T) were predicted to show 185% higher VWF:CB activity, and carriers of two copies of the p.Thr1381Ala(T)/p.Gln852Arg(T) haplotype would present a 100% increase in VWF:RCo activity. These results indicate a substantial effect of common variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on , these findings provide new evidence regarding the contribution of common variants to VWD, which should be taken into account to enhance the accuracy of the diagnosis and classification of this condition. ClinicalTrials.gov identifier: NCT02869074.
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http://dx.doi.org/10.1055/s-0040-1702227DOI Listing
March 2020

Factor XIII deficiency in two Spanish families with a novel variant in gene F13A1 detected by next-generation sequencing; symptoms and clinical management.

J Thromb Thrombolysis 2020 Oct;50(3):686-688

Unidad de Hemostasia Y Trombosis, Servicio de Hematología, Hospital Universitario Y Politécnico La Fe, Valencia, Spain.

Coagulation factor XIII (FXIII) has a major role in coagulation stabilizing the haemostatic clot. FXIII deficiency is associated with an increased risk of bleeding. Severe phenotypes lead to spontaneous, traumatic and surgical bleeding. Umbilical cord bleeding is especially common, and intracranial bleeding may occur in up to one third of patients without prophylaxis. In this work, we used NGS for screening all the coding and intronic boundary regions of F13A1 and F13B genes in two families affected by severe FXIII deficiency. Outcome confirmation analysis and variant studies in related patients was done by Sanger sequencing. Two variants were found: c.34A > G (p.Arg12Gly; NM_00129.3) and c.514C > T (p.Arg172Ter; NM_00129.3), both located in the F13A1 gene. The variant p.Arg172Ter is already described in literature and was found in homozygosis in one family and in compound heterozygosis in the other family. The variant p.Arg12Gly variant has not been described previously. This variant is located in the activation peptide of the FXIII A-subunit which is highly conserved among FXIII homologs. Given the high risk of dangerous bleeding and early manifestation in severe FXIII-deficient patients, a prompt genetic confirmation is imperative. In this sense, NGS technology allows a rapid and simultaneous analysis of all regions of all the genes involved in the pathology.
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http://dx.doi.org/10.1007/s11239-020-02065-zDOI Listing
October 2020

Body Mass Index Best Predicts Recovery of Recombinant Factor VIII in Underweight to Obese Patients with Severe Haemophilia A.

Thromb Haemost 2020 Feb 30;120(2):277-288. Epub 2019 Dec 30.

Novo Nordisk A/S, Søborg, Denmark.

Background:  Factor VIII (FVIII) products are usually dosed according to body weight (BW). This may lead to under- or over-dosing in underweight or obese patients, respectively.

Objective:  This article evaluates the pharmacokinetics (PK) of recombinant FVIII concentrate, particularly recovery, in relation to body mass index (BMI) and other body composition descriptors.

Materials And Methods:  Thirty-five previously treated adults with severe haemophilia A from five BMI categories (underweight, normal, overweight, obese class I and II/III) were included. PK was evaluated after 50 IU per kilogram of BW single-dose recombinant FVIII (turoctocog alfa). The body composition variable was based on measurements of weight, height, bioimpedance analysis, and dual-energy X-ray absorptiometry. A dosing model was derived to achieve similar peak FVIII activity levels across BMI categories.

Results:  A statistically significant positive association between BMI and C, IR, and AUC was observed; CL and V showed a significant negative association with BMI; t was independent of BMI and other parameters. The dosing model introduced a correction factor 'M' for each BMI category, based on linear regression analysis of C against BMI, which ranged from 0.55 for underweight to 0.39 for obese class II/III. This model achieved similar peak FVIII activity levels across BMI categories, estimating an average dose adjustment of +243.3 IU (underweight) to -1,489.6 IU (obese class II/III) to achieve similar C.

Conclusion:  BMI appears to be the best predictor of recombinant FVIII recovery; however, PK endpoints were also dependent on other body composition variables. The model demonstrated that dosing can be adjusted for individual BMI to achieve better FVIII predictability across BMI categories.
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http://dx.doi.org/10.1055/s-0039-3400745DOI Listing
February 2020

Validation of the ISTH/SSC bleeding assessment tool for inherited platelet disorders: A communication from the Platelet Physiology SSC.

J Thromb Haemost 2020 03 16;18(3):732-739. Epub 2019 Dec 16.

Department of Internal Medicine, IRCCS Policlinico S. Matteo Foundation, University of Pavia, Pavia, Italy.

Background: Careful assessment of bleeding history is the first step in the evaluation of patients with mild/moderate bleeding disorders, and the use of a bleeding assessment tool (BAT) is strongly encouraged. Although a few studies have assessed the utility of the ISTH-BAT in patients with inherited platelet function disorders (IPFD) none of them was sufficiently large to draw conclusions and/or included appropriate control groups.

Objectives: The aim of the present study was to test the utility of the ISTH-BAT in a large cohort of patients with a well-defined diagnosis of inherited platelets disorder in comparison with two parallel cohorts, one of patients with type-1 von Willebrand disease (VWD-1) and one of healthy controls (HC).

Patients/methods: We enrolled 1098 subjects, 482 of whom had inherited platelet disorders (196 IPFD and 286 inherited platelet number disorders [IT]) from 17 countries.

Results: IPFD patients had significantly higher bleeding score (BS; median 9) than VWD-1 patients (median 5), a higher number of hemorrhagic symptoms (4 versus 3), and higher percentage of patients with clinically relevant symptoms (score > 2). The ISTH-BAT showed excellent discrimination power between IPFD and HC (0.9 < area under the curve [AUC] < 1), moderate (0.7 < AUC < 0.9) between IPFD and VWD-1 and between IPFD and inherited thrombocytopenia (IT), while it was inaccurate (AUC ≤ 0.7) in discriminating IT from HC.

Conclusions: The ISTH-BAT allows to efficiently discriminate IPFD from HC, while it has lower accuracy in distinguishing IPFD from VWD-1. Therefore, the ISTH-BAT appears useful for identifying subjects requiring laboratory evaluation for a suspected IPFD once VWD is preliminarily excluded.
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http://dx.doi.org/10.1111/jth.14683DOI Listing
March 2020

Antithrombotic prophylaxis for surgery-associated venous thromboembolism risk in patients with inherited platelet disorders. The SPATA-DVT Study.

Haematologica 2020 07 26;105(7):1948-1956. Epub 2019 Sep 26.

Department of Transfusion and Cell Transplantation Kitasato University School of Medicine, Sagamihara, Japan.

Major surgery is associated with an increased risk of venous thromboembolism (VTE), thus the application of mechanical or pharmacologic prophylaxis is recommended. The incidence of VTE in patients with inherited platelet disorders (IPD) undergoing surgical procedures is unknown and no information on the current use and safety of thromboprophylaxis, particularly of low-molecular-weight-heparin in these patients is available. Here we explored the approach to thromboprophylaxis and thrombotic outcomes in IPD patients undergoing surgery at VTE-risk participating in the multicenter SPATA study. We evaluated 210 surgical procedures carried out in 155 patients with well-defined forms of IPD (VTE-risk: 31% high, 28.6% intermediate, 25.2% low, 15.2% very low). The use of thromboprophylaxis was low (23.3% of procedures), with higher prevalence in orthopedic and gynecological surgeries, and was related to VTE-risk. The most frequently employed thromboprophylaxis was mechanical and appeared to be effective, as no patients developed thrombosis, including patients belonging to the highest VTE-risk classes. Low-molecular-weight-heparin use was low (10.5%) and it did not influence the incidence of post-surgical bleeding or of antihemorrhagic prohemostatic interventions use. Two thromboembolic events were registered, both occurring after high VTE-risk procedures in patients who did not receive thromboprophylaxis (4.7%). Our findings suggest that VTE incidence is low in patients with IPD undergoing surgery at VTE-risk and that it is predicted by the Caprini score. Mechanical thromboprophylaxis may be of benefit in patients with IPD undergoing invasive procedures at VTE-risk and low-molecular-weight-heparin should be considered for major surgery.
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http://dx.doi.org/10.3324/haematol.2019.227876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327644PMC
July 2020

Clinical and molecular characterization by next generation sequencing of Spanish patients affected by congenital deficiencies of fibrinogen.

Thromb Res 2019 Aug 25;180:115-117. Epub 2019 Jun 25.

Unidad de Hemostasia y Trombosis, Servicio de Hematología, Hospital Universitario y Politécnico La Fe, Valencia, Spain.

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http://dx.doi.org/10.1016/j.thromres.2019.06.015DOI Listing
August 2019

Unraveling the effect of silent, intronic and missense mutations on splicing: contribution of next generation sequencing in the study of mRNA.

Haematologica 2019 03 25;104(3):587-598. Epub 2018 Oct 25.

Hospital San Pedro de Alcántara, Cáceres, Spain.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on mRNA. This study aimed to elucidate the true effects of 18 mutations on mRNA processing, investigate the contribution of next-generation sequencing to mRNA study in von Willebrand disease, and compare the findings with prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. .
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http://dx.doi.org/10.3324/haematol.2018.203166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395343PMC
March 2019

Role of multimeric analysis of von Willebrand factor (VWF) in von Willebrand disease (VWD) diagnosis: Lessons from the PCM-EVW-ES Spanish project.

PLoS One 2018 20;13(6):e0197876. Epub 2018 Jun 20.

Servicio Hematología, Hospital Universitario Fundación Alcorcón, Madrid, Spain.

The multimeric analysis (MA) of plasma von Willebrand factor (VWF) evaluates structural integrity and helps in the diagnosis of von Willebrand disease (VWD). This assay is a matter of controversy, being considered by some investigators cumbersome and only slightly informative. The centralised study 'Molecular and Clinical Profile of von Willebrand Disease in Spain (PCM-EVW-ES)' has been carried out by including the phenotypic assessment and the genetic analysis by next generation sequencing (NGS) of the VWF gene (VWF). The aim of the present study was to evaluate the role of MA to the diagnosis of these patients and their potential discrepancies. Two hundred and seventy out of 480 patients centrally diagnosed with VWD had normal multimers, 168 had abnormal multimers and 42 a total absence of multimers. VWF MA was of great significance in the diagnosis of 83 patients (17.3%), it was also of help in the diagnosis achieved in 365 additional patients (76%) and was not informative in 32 cases (6.7%). With regard to discrepancies, 110 out of 480 (23%) patients centrally diagnosed with VWD presented some kind of discordance between VWF:RCo/VWF:Ag and/or VWF:CB/VWF:Ag ratios, multimeric study and/or genetic results. The VWF MA was key in the presence of novel mutations as well as in cases with phenotypic discrepancies. A comparison between the contribution of MA and VWF:CB showed a clearly higher contribution of the former in the diagnostic process. These data seem to reinforce the relevance of the VWF MA in VWD diagnosis, despite all its limitations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197876PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010290PMC
December 2018

α2-Macroglobulin Is a Significant In Vivo Inhibitor of Activated Protein C and Low APC:α2M Levels Are Associated with Venous Thromboembolism.

Thromb Haemost 2018 04 15;118(4):630-638. Epub 2018 Feb 15.

Grupo de Investigación en Hemostasia, Trombosis, Arteriosclerosis y Biología Vascular, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Hospital Universitario y Politécnico La Fe, Valencia, Spain.

Background: Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α-antitrypsin (αAT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α-macroglobulin (αM) have been observed by immunoblotting. Here, we report an ELISA for APC:αM complexes in plasma.

Methods: Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:αM assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with αM, PCI and αAT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls.

Results: In all the in vivo experiments, αM was a significant APC inhibitor. The VTE case-control study showed that VTE patients had significantly lower APC:αM and APC levels than the controls ( < 0.001). Individuals in the lowest quartile of APC:αM or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:αM or of APC. The risk increased for individuals with low levels of both parameters.

Conclusion: The APC:αM assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:αM level is associated with increased VTE risk.
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http://dx.doi.org/10.1055/s-0038-1629902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002867PMC
April 2018

Risk Factors for the Progression from Low to High Titres in 260 Children with Severe Haemophilia A and Newly Developed Inhibitors.

Thromb Haemost 2017 12 6;117(12):2274-2282. Epub 2017 Dec 6.

Julius Center for Health Sciences and Primary Care, University Medical Center, Utrecht, The Netherlands.

In children with severe haemophilia A, inhibitors to factor VIII (FVIII) usually develop during the first 50 treatment exposure days and are classified as low or high titre depending on the peak inhibitor titre being greater or less than 5 Bethesda units/mL (BU/mL). Classification of the inhibitor may change with time, as some low-titre inhibitors progress to high titre following re-exposure to FVIII concentrate. The aim of this study was to investigate potential risk factors for such a progression in children with severe haemophilia A and newly diagnosed inhibitors. This study was a follow-up study of the PedNet Registry and included 260 children with severe haemophilia A and inhibitors born between 1990 and 2009 and recruited consecutively from 31 haemophilia centres. Clinical and laboratory data were collected from the date of each child's first positive inhibitor test for at least 3 years. At the time of first positive inhibitor test, 49% (n = 127) had low-titre inhibitors, with 50% of them progressing to high titre and only 25% maintaining low titres. The FVIII gene (F8) mutation type was known in 247 patients (95%), and included 202 (82%) null mutations. The progression to high-titre inhibitors was associated with null F8 mutations (odds ratio [OR]: 2.6; 95% confidence interval [CI]: 1.0–6.5), family history of inhibitors (OR: 7.2; 95% CI: 1.8–28.4) and the use of high-dose immune tolerance induction, defined as ≥100 IU FVIII concentrate/kg/d (OR: 3.9; 95% CI: 1.5–10.0). These results suggest that high-dose immune tolerance induction should be avoided as the initial strategy in patients who develop low-titre FVIII inhibitors.
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http://dx.doi.org/10.1160/TH17-01-0059DOI Listing
December 2017

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): comprehensive genetic analysis by next-generation sequencing of 480 patients.

Haematologica 2017 12 29;102(12):2005-2014. Epub 2017 Sep 29.

Hospital Universitario Fundación de Alcorcón, Madrid, Spain.

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the , including intronic and promoter regions, was achieved in the 556 individuals recruited the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along , 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.
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http://dx.doi.org/10.3324/haematol.2017.168765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709099PMC
December 2017

Bleeding risk of surgery and its prevention in patients with inherited platelet disorders.

Haematologica 2017 07 6;102(7):1192-1203. Epub 2017 Apr 6.

Hematology Department, S. Bortolo Hospital, Vicenza, Italy.

Excessive bleeding at surgery is a feared complication in patients with inherited platelet disorders. However, very few studies have evaluated the frequency of surgical bleeding in these hemorrhagic disorders. We performed a worldwide, multicentric, retrospective study to assess the bleeding complications of surgery, the preventive and therapeutic approaches adopted, and their efficacy in patients with inherited platelet disorders: the Surgery in Platelet disorders And Therapeutic Approach (SPATA) study. We rated the outcome of 829 surgical procedures carried out in 423 patients with well-defined forms of inherited platelet disorders: 238 inherited platelet function disorders and 185 inherited platelet number disorders. Frequency of surgical bleeding was high in patients with inherited platelet disorders (19.7%), with a significantly higher bleeding incidence in inherited platelet function disorders (24.8%) than in inherited platelet number disorders (13.4%). The frequency of bleeding varied according to the type of inherited platelet disorder, with biallelic Bernard Soulier syndrome having the highest occurrence (44.4%). Frequency of bleeding was predicted by a pre-operative World Health Organization bleeding score of 2 or higher. Some types of surgery were associated with a higher bleeding incidence, like cardiovascular and urological surgery. The use of pre-operative pro-hemostatic treatments was associated with a lower bleeding frequency in patients with inherited platelet function disorders but not in inherited platelet number disorders. Desmopressin, alone or with antifibrinolytic agents, was the preventive treatment associated with the lowest bleedings. Platelet transfusions were used more frequently in patients at higher bleeding risk. Surgical bleeding risk in inherited platelet disorders is substantial, especially in inherited platelet function disorders, and bleeding history, type of disorder, type of surgery and female sex are associated with higher bleeding frequency. Prophylactic pre-operative pro-hemostatic treatments appear to be required and are associated with a lower bleeding incidence.
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http://dx.doi.org/10.3324/haematol.2016.160754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566025PMC
July 2017

A simplified assay for the quantification of circulating activated protein C.

Clin Chim Acta 2016 Aug 2;459:101-104. Epub 2016 Jun 2.

Grupo de Investigación en Hemostasia, Trombosis, Arteriosclerosis y Biología Vascular, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain. Electronic address:

Available assays for circulating levels of activated protein C (APC) are either time-consuming or difficult to use in a routine laboratory, or have a detection limit above normal levels. We have developed a simplified assay that measures both the in vivo free APC and the in vivo APC complexed to PC inhibitor (PCI). We measured APC levels, with both assays, in 339 plasma samples, 165 from patients with venous thromboembolism (VTE) and 174 from healthy individuals. The mean APC level in the 339 samples was 0.038±0.010 nM, using a previous assay that measures only the in vivo APC level, and 0.041±0.010 nM with the present new assay. The coefficient of correlation between assays was r=0.954 (P<0.001). The mean APC level in VTE patients was 0.034±0.009 nM (previous assay) and 0.037±0.009 nM (new assay), significantly lower than those in controls (P<0.001). In both groups there was a significant correlation between the levels obtained by the two assays (P<0.001). These results show that both assays are equivalent, and confirm that the APC level is lower in VTE patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories.
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http://dx.doi.org/10.1016/j.cca.2016.05.025DOI Listing
August 2016

Novel mutations in RASGRP2, which encodes CalDAG-GEFI, abrogate Rap1 activation, causing platelet dysfunction.

Blood 2016 09 27;128(9):1282-9. Epub 2016 May 27.

Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, Instituto Murciano de Investigaciones Biomédicas-Arrixaca, Murcia, Spain;

In addition to mutations in ITG2B or ITGB3 genes that cause defective αIIbβ3 expression and/or function in Glanzmann's thrombasthenia patients, platelet dysfunction can be a result of genetic variability in proteins that mediate inside-out activation of αIIbβ3 The RASGRP2 gene is strongly expressed in platelets and neutrophils, where its encoded protein CalDAG-GEFI facilitates the activation of Rap1 and subsequent activation of integrins. We used next-generation sequencing (NGS) and whole-exome sequencing (WES) to identify 2 novel function-disrupting mutations in RASGRP2 that account for bleeding diathesis and platelet dysfunction in 2 unrelated families. By using a panel of 71 genes, we identified a homozygous change (c.1142C>T) in exon 10 of RASGRP2 in a 9-year-old child of Chinese origin (family 1). This variant led to a p.Ser381Phe substitution in the CDC25 catalytic domain of CalDAG-GEFI. In 2 Spanish siblings from family 2, WES identified a nonsense homozygous variation (c.337C>T) (p.Arg113X) in exon 5 of RASGRP2 CalDAG-GEFI expression was markedly reduced in platelets from all patients, and by using a novel in vitro assay, we found that the nucleotide exchange activity was dramatically reduced in CalDAG-GEFI p.Ser381Phe. Platelets from homozygous patients exhibited agonist-specific defects in αIIbβ3 integrin activation and aggregation. In contrast, α- and δ-granule secretion, platelet spreading, and clot retraction were not markedly affected. Integrin activation in the patients' neutrophils was also impaired. These patients are the first cases of a CalDAG-GEFI deficiency due to homozygous RASGRP2 mutations that are linked to defects in both leukocyte and platelet integrin activation.
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http://dx.doi.org/10.1182/blood-2015-11-683102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009515PMC
September 2016

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): Proposal for a new diagnostic paradigm.

Thromb Haemost 2016 Jan 6;115(1):40-50. Epub 2015 Aug 6.

Francisco Javier Batlle Fonrodona, M. D., Servicio de Hematología y Hemoterapia. INIBIC., Complexo Hospitalario Universitario A Coruña, Edificio Hospital Materno Infantil, Carretera del Pasaje s/n, 15006 - A Coruña, Spain, Tel.: +34 981 178000 Ext. 292113, Fax: +34 981 178392, E-mail:

The diagnosis of von Willebrand disease (VWD) remains difficult in a significant proportion of patients. A Spanish multicentre study investigated a cohort of 556 patients from 330 families who were analysed centrally. VWD was confirmed in 480. Next generation sequencing (NGS) of the whole coding VWF was carried out in all recruited patients, compared with the phenotype, and a final diagnosis established. A total of 238 different VWF mutations were found, 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 463 were found to have VWF mutation/s. A good phenotypic/genotypic association was estimated in 96.5% of the patients. One hundred seventy-four patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred sixteen patients presented with mutations that had previously been associated with increased von Willebrand factor (VWF) clearance. RIPA unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 70 patients. The NGS study facilitated an appropriate classification in 63 of them. The remaining seven patients presented with a VWF novel mutation pending further investigation. In five patients with a type 3 and two with a type 2A or 2B phenotype with no mutation, an acquired von Willebrand syndrome (AVWS) was suspected/confirmed. These data seem to support NGS as a first line efficient and faster paradigm in VWD diagnosis.
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http://dx.doi.org/10.1160/TH15-04-0282DOI Listing
January 2016

Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative study.

Orphanet J Rare Dis 2014 Dec 24;9:213. Epub 2014 Dec 24.

Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, Murcia, 30003, Spain.

Background: The diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project "Functional and Molecular Characterization of Patients with Inherited Platelet Disorders" under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis.

Patients/methods: Subjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest.

Results: Of the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed.

Conclusions: Our study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.
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http://dx.doi.org/10.1186/s13023-014-0213-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4302577PMC
December 2014

Novel missense mutation c.2685G>C (p.Q895H) in VWF gene associated with very low levels of VWF mRNA.

Ann Hematol 2009 Mar 20;88(3):245-7. Epub 2008 Aug 20.

Unidad de Coagulopatías Congénitas, Hospital Universitario La Fe., Av. Campanar 21, 46009 Valencia, Spain.

Homozygous patients for null alleles in VWF gene show a severe von Willebrand phenotype, whereas compound heterozygous patients only show the phenotype of the expressed allele. Five members of the same family were studied. The two patients showed borderline VWF levels, a mild factor VIII (FVIII) deficiency and a decrease of the binding of VWF to exogenous FVIII. The genetic analyses of the VWF gene confirmed that the patients were compound heterozygous for c.2561G>A (R854Q) and c.2685G>C (p.Q895H) mutations. The latter, is located in the 3' extreme of exon 20, and it has not been previously described. Studies of the cDNA from platelet mRNA were performed to investigate the expression of p.H895 allele. The loss of heterozygosity at the cDNA level suggests a lack of expression of the p.H895 allele. The overall studies can explain the type 2N phenotype of the two patients, since the allele carrying the new missense mutation p.Q895H has shown a low expression of VWF gene.
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http://dx.doi.org/10.1007/s00277-008-0576-7DOI Listing
March 2009

Severe and moderate hemophilia A: identification of 38 new genetic alterations.

Haematologica 2008 Jul 9;93(7):1091-4. Epub 2008 Apr 9.

Unidad de Coagulopatías Congénitas, Hospital Universitari La Fe, Av. Campanar, 21, 46009 Valencia, Spain.

Hemophilia A is an X-linked recessive disorder caused by a lack or decrease of factor VIII activity. Its socio-economic impact is high given its high bleeding expression and treatment cost. Our aim was to establish the mutation of each patient to improve family management. A total of 116 unrelated families with severe and moderate hemophilia A were involved. Non-carriers of intron 22 and intron 1 rearrangements were included in F8 gene screening. Intron 1 and 22 inversion frequencies were 3% and 52.5% respectively. Putative mutations were identified in all the families; 38 were new. The cumulative inhibitor incidence was 22%. Approximately half the families carry non-recurrent mutations, which were unique in around one third. Harmful effects for mutations predicting null alleles are expected. Missense mutation consequences are not easily predictable, despite the help of some bio-informatics tools.
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http://dx.doi.org/10.3324/haematol.12344DOI Listing
July 2008

Von Willebrand's disease: a novel mutation, P1824H and the incidence of R1205H defect among families with dominant quantitative von Willebrand factor deficiency.

Haematologica 2006 Aug 25;91(8):1130-3. Epub 2006 Jul 25.

Unidad de Coagulopatías Congénitas, Hospital Universitari La Fe, Av. Campanar, 21, 46009 Valencia, Spain.

To date, few mutations associated with a dominant quantitative deficiency of von Willebrand factor (VWF) and a high penetrance have been reported. This phenotype was confirmed in seven unrelated families of several patients diagnosed with von Willebrand's disease out of 70 who requested genetic studies of the VWF gene. The mutations linked to this type were identified: R1205H in five families; T1156M in one family; and the new P1824H alteration in one other family. The R1205H mutation linked to the different haplotypes might well be frequent among this variant. The P1824H in the A3 domain is associated with very low VWF levels and with a moderate-to-severe bleeding tendency, unlike the other mutations reported in this domain.
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August 2006

Treatment of intra-abdominal bleeding with recombinant activated factor VII in a patient with disseminated intravascular coagulation secondary to septic shock.

Blood Coagul Fibrinolysis 2005 Jun;16(4):297-9

Department of Haematology, Hospital Universitario La Fe, Av. Campanar 21, 46009 Valencia, Spain.

Although mainly indicated for treatment of bleeding in haemophilia patients with inhibitors, recombinant activated factor VII (rFVIIa) has also been successfully used in other situations. However, no data are available on its use in the treatment of disseminated intravascular coagulation (DIC) secondary to septic shock. We report a man with DIC and septic shock due to retrocaecal appendicitis and severe intra-abdominal bleeding after surgery. Despite conventional treatment, the bleeding persisted, and treatment with rFVIIa controlled the haemorrhage. No side-effects related to rFVIIa were noted. This case suggests a potential role of rFVIIa in the treatment of severe bleeding associated with DIC.
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http://dx.doi.org/10.1097/01.mbc.0000169224.23550.3fDOI Listing
June 2005