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Introduction: Adherence is a cornerstone of factor VIII prophylactic treatment. Information regarding the factors with potential influence on adherence is limited, particularly in adult patients., Aim: To assess adherence in adult patients with severe haemophilia A receiving prophylactic treatment in a real-life setting, and investigate the factors influencing adherence., Methods: Observational, prospective study including adult patients receiving factor VIII therapy in 15 Spanish centres. Patients recorded infusion doses on a logbook and answered various questionnaires to assess their health beliefs. Adherence rate was the percentage of infused doses over the prescribed ones. Self-perceived adherence was assessed using the VERITAS-Pro questionnaire, the psychometric properties of which were validated in the Spanish population. The relationship between adherence rate and treatment, clinical and demographic characteristics, health beliefs and perceived self-efficacy was investigated., Results: A total of 66 patients were followed up for 12 months. Mean adherence rate at the end of follow-up was 82.5%. Most of the study patients (n = 53, 80.3%) showed a moderate-to-high adherence rate (>70%). The VERITAS-Pro revealed a high perception of adherence. Multivariate analyses to predict treatment adherence identified the knee as a target joint and longer treatment duration as variables with significant (negative) influence on adherence. Adherence rate was not influenced by the patient's health beliefs or perceived self-efficacy., Conclusion: Most adult patients receiving factor VIII prophylactic treatment in Spain have moderate-to-high treatment adherence. Treatment duration and the knee as a target joint are factors with a moderate negative influence on treatment adherence., (© 2020 John Wiley & Sons Ltd.)

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 VWF variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on VWF , 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., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

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.)

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.

Treatment adherence in adolescents with chronic diseases is around 50%, and failure is more common in preventive therapy. In haemophilia, contradictory results are reported by the published studies. The objective of this study was to evaluate adherence with factor VIII (FVIII) prophylaxis in Spanish patients with severe haemophilia A between age 6 and 20 years. Data were collected retrosp-ectively in the previous 2 years. The primary endpoint was the absolute adherence index (AAI), and the endpoints were related to clinical status, age, prophylaxis regimen, responsibility for factor administration and quality of life (QoL), assessed by the Haemo-QoL questionnaires. A total of 78 patients from 14 Spanish hospitals were recruited. Adherence ranged between -64.4 and 66.7 (mean -3.08). No differences were observed between children and adolescents (7.11 vs. 6.39; P = 0.809). A statistically significant association (P < 0.010) between infra adherent group and target joint was found, as was a statistically significant difference (P < 0.010) between the number of bleeding episodes experienced by the adherent group (mean 1.4) and by infra adherents (mean 4.5). There was no significant difference between AAI and prophylactic regimen (6.35 vs. 6.96, P = 0.848), neither between AAI and the person responsible for factor administration (5.57 vs. 8.79, P = 0.326). The Haemo-QoL scores (8-12 years) were related to adherence level (P < 0.05). Adherence was approximately ideal and patients perceived a high QoL. Because of the repercussions for compliance, it is essential to work during puberty on emotional and self-acceptance aspects of the disease, as well as coping, and the patient's family, school and health team relationships., (© 2015 John Wiley & Sons Ltd.)

Hypercoagulable states can be detected by measuring activation peptides, enzyme-inhibitor complexes, and fibrin/fibrinogen degradation products, which are markers of hemostatic activation. A series of these prethrombotic markers has been evaluated in the elderly, pregnancy, diabetes and acute myocardial infarction patients (n=30 in each group) as well as in hematologic malignancies (n=42). The parameters assayed were: prothrombin fragment 1+2 (F1+2), thrombin-antithrombin III complexes (TAT), fibrinopeptide A (FPA), plasmin-alpha2 antiplasmin complexes (PAP) and D-Dimer. Results were compared with those obtained in a group of 30 healthy subjects. We found a significant increase of F1+2, TAT and FPA in elderly (p<0.05), acute myocardial infarction (AMI) (p<0.01), hematologic malignancies (p<0.01), and pregnancy (p<0.0001), indicating a marked clotting activation. Diabetic patients under strict metabolic control only presented a moderate increase of TAT (p<0.05), suggesting a slight activation. We also observed a highly significant elevation of PAP and D-Dimer in elderly (p<0.001), AMI (p<0.0001), and malignancy (p<0.0001), indicating an activation of the fibrinolytic system. The combination of selected fibrinolytic and coagulation measurements is useful for the detection of a hypercoagulable state in conditions characterized by a risk of thrombosis.

We have evaluated the effect of plasminogen activators (t-PA and urokinase) on an experimental model of disseminated intravascular coagulation (DIC) in rabbits by injection of 20 micrograms/kg/h of E. coli lipopolysaccharide during 6 h t-PA (0.2 mg/kg and 0.7 mg/kg), urokinase (3000 U/kg/h) and saline (control) were given simultaneously with endotoxin. Results indicated that urokinase and low dose of t-PA significantly reduced the increase of plasminogen activator inhibitor (PAI) activity observed 2 h after endotoxin (p < 0.001). High t-PA dose also diminished the PAI levels at 6 h (p < 0.001). A significant reduction of fibrin deposits in kidneys was observed din both t-PA treated groups as compared with findings in the group of rabbits infused with saline solution (p < 0.005), whereas urokinase had no significant effect on the extent of fibrin deposition. Finally, the mortality rate in the control group (70%) was reduced to 50% in rabbits receiving high doses of t-PA. In conclusion, treatment with t-PA resulted in reduced PAI generation, fibrin deposits and mortality in endotoxin-treated rabbits.

We present a patient diagnosed of polycythemia vera who developed a multiple myeloma 13 years after the initial diagnosis of polycythemia vera. Although an unusual finding, another 17 patients with polycythemia and myeloma have been described. In some patients the diagnosis of both diseases was simultaneous but in most cases myeloma developed years after polycythemia vera. Some patients received only venipuncture for treatment of polycythemia vera so the appearance of multiple myeloma could not be explained on the grounds of chemotherapy induced second neoplasm. An explanation for this association is suggested.

Purpose: To assess the therapeutic efficacy of agents capable of stimulating the fibrinolytic system, such as tissue plasminogen activator (t-PA) and urokinase (UK) on endotoxin-induced disseminated intravascular coagulation (DIC) in the rabbit., Material and Methods: DIC was induced by intravenous administration of endotoxin, 20 micrograms/kg/hr during 6 hr. Four different groups were established: a) control group, receiving only saline solution; b) t-PA group receiving 0.2 mg/kg; c) t-PA group receiving 0.7 mg/kg, and d) UK group, which was given 3,000 IU/kg/hr for 6 hr. Blood samples were drawn before and after 2 hr and 6 hr of endotoxin administration. Platelet count, and fibrinogen, factor XII and antithrombin III concentrations, were assessed in each sample. Mean, standard deviation and percentage of increase or decrease with respect to the basal value, this considered 100%, were used to evaluate the findings. For comparison of values, Student's t and Mann Whitney's U were used; the Fisher test was used for mortality studies., Results: No statistical differences appeared for any of the values in the rabbits under basal conditions. The rabbits in the control group developed DIC. No doses of t-PA modified the changes appearing in blood coagulation. UK reduced the fibrinogen and factor XII consumption induced by endotoxin. The mortality rate in the control group reached 70%. High-dose t-PA decreased such figure to 50%, while low-dose t-PA or UK failed to reduce mortality., Conclusions: High-dose t-PA has beneficial effects on endotoxin-induced DIC in rabbits. UK failed to achieve such effect at the doses given in this experimental DIC model.

We have evaluated the effect of 1-Deamino-8D-arginine vasopressin (DDAVP) on an experimental model of intravascular coagulation (DIC) induced in rabbits by injection of 20 micrograms kg-1 h-1 during 6 h of E. coli lipopolysaccharide. DDAVP significantly ameliorated the platelet drop and fibrinogen decrease (p less than 0.01) induced by endotoxin in control animals. A significant reduction in factor XII consumption (p less than 0.01) and a decrease in the generation of endotoxin induced PAI-1 activity in rabbits circulation was also observed (p less than 0.005). Moreover, fibrin deposition in kidneys of rabbits receiving DDAVP was significantly reduced as compared to control animals. Finally, the mortality rate in the control group was significantly higher than in DDAVP-treated rabbits (p less than 0.01). The hemostatic changes induced by DDAVP correlated with lower fibrin deposition and reduction in mortality rates.

The influence of cardiopulmonary bypass (CPB) on fibrinolytic activity was assessed in 100 patients with valvular heart disease or atrial septal defects. Euglobulin fibrinolytic activity (EFA), tissue type plasminogen activator (t-PA) activity, plasminogen activator inhibitor 1 (PAI-1) activity, plasminogen, alpha 2-antiplasmin (alpha 2-AP), fibrinogen degradation products (FDP), and D-dimer were measured pre-, intra-, and postoperatively. There were significant increases in EFA and t-PA activity (p less than 0.002), and decreases in plasminogen and alpha 2-AP (p less than 0.0001) intraoperatively with respect to baseline values. t-PA activity decreased significantly after surgery (p less than 0.002), whereas PAI-1 activity showed a marked increase shortly after operation and on postoperative day 1 (p less than 0.0001). FDP and D-dimer levels were significantly increased both intra- and postoperatively, the latter showing higher values (p less than 0.01 and p less than 0.0001, respectively). This study shows that there is an activation of the fibrinolytic system, as a result of the increased activation of plasminogen and decreased levels of plasmin inhibitors, during CPB surgery followed by a postoperative fibrinolytic shutdown.

Mean plasma concentrations of the main inhibitors of blood coagulation antithrombin III (AT III), protein C and protein S were determined in 149 patients with different local and disseminated malignancies. Results were compared with findings in 44 healthy subjects. No statistical significant differences for AT III, protein C and free protein S were found between patients and controls. However, total protein S showed a significant increase in the patient group (p less than 0.0003). None of the parameters analyzed showed differences according to the degree of tumoral activity nor to the tumour localization. We conclude that the main inhibitors of the blood coagulation seem not to play an important role in the pathogenesis of the hypercoagulable state in malignancy. The increased levels in total protein S would indicate endothelial synthesis in response to thrombogenic stimuli.

Different coagulation and fibrinolysis parameters were investigated in 149 patients with metastatic and non-metastatic tumours and results were compared with those obtained in a healthy population. Results showed a significant increase of thrombin-antithrombin complexes, fibrinopeptide A (FPA) and fibrin monomers in the group of patients (p less than 0.001). There was also a significant prolongation of euglobulin lysis time (p less than 0.005) and an increase of plasminogen activator inhibitor activity (p less than 0.0001), fibrinogen degradation products (p less than 0.001), and D-dimer (p less than 0.05) in the group of patients as compared to controls; FPA levels were also increased in patients with metastases (p less than 0.005). This study demonstrates clotting activation, at the level of fibrinogen to fibrin conversion, and impairment of fibrinolysis in patients with malignancy.