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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 VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico 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 VWF 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 VWF 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 VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074 ., (Copyright© 2019 Ferrata Storti Foundation.)

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 VWF , including intronic and promoter regions, was achieved in the 556 individuals recruited via 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 VWF , 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico 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., (Copyright© 2017 Ferrata Storti Foundation.)

Background and Objectives: We compared the efficacy and safety of low molecular weight heparins (LMWH) and unfractionated heparin (UFH) in the treatement of deep venous thrombosis (DVT). A comparison between two daily subcutaneous injections of LMWH against a single injection was also performed., Design and Methods: The study was performed by a meta-analysis. Clot improvement in venography, recurrency, total mortality and major hemorrhages were assessed in 4,472 patients with DVT from 21 studies treated with subcutaneous LMWH or UFH., Results: An improvement in clot reduction (odds ratio 0.73, 95% confidence interval 0.59 to 0.90, p = 0.004), a decrease in total mortality (0. 68, 0.50 to 0.91, p = 0.012) and a lower incidence of hemorrhage (0. 65, 0.43 to 0.98, p = 0.047) were observed in LMWH treated patients. There were no differences in recurrences (0.78, 0.59 to 1.04, p = 0. 10). A single dose of LMWH was better than two in reducing major bleeding (c2 = 4.99, p = 0.025); however, the two dose regimen was more effective in clot reduction (c2 = 8.56, p = 0.004)., Interpretation and Conclusions: LMWH is superior to UFH in terms of safety and efficacy. A single daily dose of LMWH dose is a suitable therapeutic regimen and could facilitate the outpatient treatment of venous thromboembolism.

Background and Objective: Vascular fibrinolysis, a major natural defense mechanism against thrombosis, is a highly regulated process. The aim of this study was to evaluate the effect of endotoxin, tumor necrosis factor-alpha (TNFalpha) and interleukin-1alpha (IL-1alpha), on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVEC)., Design and Methods: Samples of stimulated conditioned media were collected over a period of 24 hours to determine: plasminogen activator (PA) and plasminogen activator inhibitor (PAI) activity, PAI-1 mRNA, tissue-type plasminogen activator (t-PA) antigen and urokinase-type plasminogen activator (u-PA) antigen., Results: Similar changes were observed after endotoxin and cytokine stimulation: there was a significant increase of PAI activity (p<0.01), starting at 6 hours, which remained 24 hours after stimulation. PAI-1 mRNA also showed an important rise with these agents, although cytokines induced an earlier and more intense inhibitor response (up to 6-fold increase). PA activity increased significantly at 6 hours (p<0.01) to drop at 24 hours and was mainly related to the presence of u-PA., Interpretation and Conclusions: We conclude that endotoxin,+TNFalpha and IL-1alpha induce profound alterations in the fibrinolytic potential of HUVEC, characterized by an initial rise of activators (u-PA) followed by a strong increase of PAI-1. These changes may be of pathophysiologic significance for thrombosis and inflammatory reactions.

Background and Objective: Acute generalized, widespread bleeding is often related to disseminated intravascular coagulation (DIC), a pathologic process which complicates the clinical course of many diseases and is characterized by huge amounts of thrombin and plasmin within the circulation. The final result is the consumption of platelets, coagulation factors and inhibitors, as well as secondary hyperfibrinolysis, all leading to diffuse hemorrhage and microthromboses. This review article examines the present attitudes to the diagnosis and treatment of overt DIC in clinical practice, emphasizing the importance of an accurate differential diagnosis from some other processes characterized by acute generalized, widespread bleeding., Information Sources: The authors have been working in this field, both at experimental and clinical levels, contributing original papers for many years. In addition, material examined in this review includes articles published in journals covered by MedLine, recent reviews in journals with high impact factor and in relevant books on hemostasis and thrombosis., State of Art and Perspectives: DIC is an intermediary mechanism of disease which complicates the clinical course of many well-known disorders. Although the systemic hemorrhagic syndrome is the predominant clinical manifestation, massive intravascular thrombosis frequently occurs contributing to ischemia and associated organ damage, making the mortality rate of this condition high. Current concepts on the pathophysiology, laboratory diagnosis and management of DIC are presented. Complex pathophysiological interrelations make the diagnosis of the etiology of the DIC difficult in clinical practice, although simple tests are useful for identification of patients with the process. Laboratory diagnosis of DIC is mainly based on screening assays, which allow a rapid diagnosis, whereas some other highly sensitive but more complex assays are not always available to routine clinical laboratories. The management of DIC is based on the treatment of the underlying disease, supportive and replacement therapies and the control of the coagulation mechanisms. Although some advances have been achieved, management decisions are still controversial, so that therapy should be highly individualized depending on the nature of the DIC and severity of clinical symptoms. Many syndromes sharing common findings with DIC, such as primary hyperfibrinolysis or thrombotic thrombocytopenic purpura, should be excluded. Finally, new therapeutic approaches to the management of this potentially catastrophic syndrome are required.

Background and Objective: The control of well-known atherosclerotic risk factors represents the optimal strategy in the prevention of acute coronary syndromes. It was the aim of this work to analyze the effects of a long-term cardiac rehabilitation program on the changes of fibrinolysis parameters and plasma lipid profile in coronary patients., Design and Methods: The study was carried out in 30 (M/F:22/8, mean age 47 years) survivors of a first acute myocardial infarction (AMI) and in 30 healthy controls who underwent a cardiac rehabilitation program (9 months duration). Samples were taken before, at 3 and 9 months after the beginning of the program to measure: tissue-type plasminogen activator (t-PA) antigen and plasminogen activator inhibitor (PAI-1) activity and antigen. A lipid profile including cholesterol (both HDL and LDL) and lipoprotein(a) was also assessed. The Wilcoxon and Mann-Whitney tests were used for statistical comparisons., Results: There was a marked decrease of functional PAI-1 after 3 and 9 months as compared with baseline in AMI patients (p < 0.01). Results showed a significant increase of HDL-cholesterol (p < 0.01) and decrease of lipoprotein(a) levels after the exercise program (p < 0.01)., Interpretation and Conclusions: The cardiac rehabilitation program improved fibrinolysis, by reducing the functional levels of PAI-1, and ameliorated the lipid profile by decreasing lipoprotein(a) and increasing HDL-cholesterol in patients with AMI. A long-term cardiac rehabilitation has positive effects on some risk factors for coronary disease.