Diagnostic challenges in von Willebrand disease. Report of two cases with emphasis on multimeric and molecular analysis.

Identification of qualitative variants of von Willebrand disease (VWD) can be a diagnostic challenge because of discrepant results obtained in the multiple laboratory tests available for its appropriate classification. We report two cases of infrequent inherited variants of VWD with unclear preliminary results with the test panel available at the time of first consultation and that were finally diagnosed as a VWD type 2A/IID with a c.8318 G > C, p.Cys2773Ser mutation and a VWD type 2M with c.4225 T > G, p.Val1409Phe mutation, respectively. The description of these two cases highlights that despite the limited diagnostic panel for the evaluation of von Willebrand Factor (VWF) functionality, the multimeric analysis and genetic family studies were fundamental tools to achieve the final diagnosis.

Towards the understanding of the UV light, riboflavin and additive solution contributions to the in vitro lesions observed in Mirasol®-treated platelets.

OBJECTIVES: Pathogen reduction technologies are implemented to increase the safety of blood products. We previously showed that the UVB alone significantly contributes to the storage lesions observed in platelets treated with riboflavin/UVB using a home-made illuminator. The present study aims at confirming these observations using the commercial Mirasol® technology. METHODS: A three-arm study (untreated, UV-, Mirasol®-treated platelets) was conducted to investigate the platelet storage lesions throughout storage (n=4). A two-arm study was then designed to compare Intersol and T-PAS+ additive solutions (n=3). Phenotype and functional platelet characteristics were assessed using flow cytometry, aggregometry, antioxidant assays and metabolic parameters. RESULTS: Mirasol®-treated platelets exhibit enhanced storage lesions compared to controls (increase of activation markers and glycolysis rate, lower hypotonic shock and double-agonist activation responses, and decrease of total antioxidant capacity). Here, we also confirmed that the UV radiation alone is causing platelet lesions. Riboflavin tends to have an intracellular protective role while it decreases the extracellular antioxidant defenses. Furthermore, benefits of platelet additive solutions containing potassium and magnesium were confirmed as it reduces the extent of storage lesions. CONCLUSIONS: The photosensitizer, UV illumination and composition of the platelet additive solutions are key parameters influencing the platelet storage lesion. The clinical relevance of these findings is not fully understood and recent published clinical studies could not show increase in bleeding in patients receiving Mirasol-treated platelets. New developments in storage solutions might help to improve storage conditions of PRT-treated platelets and should be prioritised as research subject in the future.

Emicizumab for routine prophylaxis to prevent bleeding episodes in patients with hemophilia A.

Hemophilia A is an X-linked bleeding disorder caused by defects in the gene encoding factor VIII (FVIII). Routine prophylaxis with exogenous FVIII requires frequent intravenous injections. One of the most challenging issues in the treatment of hemophilia A is the development of alloantibodies against infused FVIII. Presence of inhibitors results in an ineffective factor replacement therapy and increases the risk of morbidity and mortality in these patients. Therefore, there is growing interest in the development of new strategies for the prophylaxis and prevention of bleeding in patients with hemophilia to circumvent these drawbacks. Emicizumab (ACE-910; Roche, Genentech and Chugai Pharmaceutical) is a recombinant humanized bispecific antibody that restores the function of missing FVIII by bridging activated FIX and FX, simulating the cofactor function of FVIII.

Betrixaban: a direct oral inhibitor of activated factor X for the prophylaxis of venous thromboembolism in patients hospitalized for acute medical illness.

Venous thromboembolism (VTE), comprising deep vein thrombosis and pulmonary embolism, is a serious clinical and public health concern. Hospitalization is a major risk factor for developing VTE. Hospital-associated events account for more than 50% of all cases of VTE. Heparins have demonstrated to be efficacious in the prevention of VTE in medically ill patients. Despite the demonstrated efficacy and safety of the available direct oral anticoagulants in the prevention and treatment of different thromboembolic conditions, their net benefit in the prevention of VTE in hospitalized medically ill patients has not been fully confirmed. Betrixaban is an oral, specific and direct inhibitor of human coagulation factor Xa with demonstrated efficacy and safety for the prevention of VTE in patients undergoing total knee replacement and in patients with nonvalvular atrial fibrillation. Recent studies have successfully evaluated betrixaban 80 mg once daily in the prevention of VTE in acute medically ill patients in a large phase III trial. This review will address preclinical pharmacology and main aspects of the clinical development of betrixaban as an antithrombotic agent, with specific attention to recent studies on the prophylaxis of VTE in a specific population of patients hospitalized for acute medical illnesses.

Andexanet alfa: a recombinant mimetic of human factor Xa for the reversal of anticoagulant therapies.

Activated coagulation factor X (FXa) is a common target for classic and newer anticoagulants. Parenteral anticoagulants with an indirect inhibitory action on FXa (low-molecular-weight heparins) have a well-established clinical efficacy in the prophylaxis and therapy of thromboembolic conditions. More recently developed direct oral anticoagulants (DOACs) have emerged as a new class of antithrombotic drugs. Rivaroxaban, apixaban and edoxaban are direct inhibitors of FXa approved for the management of venous thromboembolism and stroke prevention in atrial fibrillation. Although these DOACs are associated with fewer hemorrhagic side effects than classic vitamin K antagonists, bleeding is still a main complication. FXa antagonists had no specific agents that could reverse their antihemostatic effects. Andexanet alfa is a modified, recombinant human FXa molecule with an enhanced ability to bind to both direct and indirect FXa inhibitors, but unable to contribute to blood coagulation mechanisms. Andexanet alfa is designed to reverse the anticoagulant effects of FXa inhibitors. This review will address the preclinical pharmacology and the main aspects of the clinical development of andexanet alfa for the reversal of anticoagulant therapies with an inhibitory action on FXa. It will also summarize additional completed or ongoing studies on andexanet alfa available to the scientific community until present.

Endothelial damage is aggravated in acute GvHD and could predict its development.

The aim of the present study was to explore whether there is enhanced endothelial dysfunction in patients developing acute GvHD (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT) and to identify biomarkers with predictive and/or diagnostic value. In in vitro experiments, endothelial cells (ECs) were exposed to serum from patients with (aGvHD, n=31) and without (NoGvHD, n=13) aGvHD, to evaluate changes in surface adhesion receptors, the reactivity of the extracellular matrix by measuring the presence of Von Willebrand factor (VWF) and platelet adhesion, and the activation of intracellular signaling proteins. Plasma levels of VWF, ADAMTS-13, TNF receptor 1 (TNFR1), soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1 were also measured. In vitro results showed a more marked proinflammatory and prothrombotic phenotype in ECs in association with aGvHD. Regarding circulating biomarkers, levels of VWF and TNFR1 above an optimal cutoff score, taken independently or combined, at day 7 after allo-HCT, would be able to positively predict that around 90% of patients will develop aGvHD. Our results demonstrate that endothelial damage is aggravated in those allo-HCT recipients developing aGvHD, and that VWF and TNFR1 are promising predictive aGvHD biomarkers. These findings could contribute to improve the understanding of the pathophysiology of aGvHD.

Endothelial damage in major depression patients is modulated by SSRI treatment, as demonstrated by circulating biomarkers and an in vitro cell model.

There is a link between depression, cardiovascular events and inflammation. We have explored this connection through endothelial dysfunction, using in vivo and in vitro approaches. We evaluated circulating biomarkers of endothelial dysfunction in patients with major depression at their diagnosis (MD-0) and during antidepressant treatment with the selective serotonin reuptake inhibitor escitalopram, for 8 and 24 weeks (MD-8 and MD-24). Results were always compared with matched healthy controls (CON). We measured in vivo circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in blood samples, and assessed plasma levels of soluble von Willebrand factor (VWF) and vascular cell adhesion molecule-1 (VCAM-1). CEC counts, soluble VWF and VCAM-1 were statistically elevated in MD-0 (P<0.01 versus CON) and gradually decreased during treatment. Conversely, EPC levels were lower in MD-0, tending to increase throughout treatment. In vitro studies were performed in human endothelial cells cultured in the presence of sera from each study group. Elevated expression of the inflammation marker intercellular adhesion molecule-1 and oxidative stress, with lower presence of endothelial nitric oxide synthase and higher reactive oxygen species production, were found in cells exposed to MD-0 sera (P<0.05 versus CON). These results were normalized in cells exposed to MD-24 sera. Thrombogenicity of extracellular matrices generated by these cells, measured as expression of VWF, tissue factor and platelet reactivity, showed non-significant differences. We provide a model of cultured endothelial cells reproducing endothelial dysfunction in naive patients with major depression, demonstrating endothelial damage and inflammation at diagnosis, and recovering with selective serotonin reuptake inhibitor treatment for 24 weeks.

Documento multidisciplinar de consenso sobre el manejo de la hemorragia masiva (documento HEMOMAS) / Multidisciplinary consensus document on the management of massive haemorrhage (HEMOMAS document)

La hemorragia masiva es una entidad frecuente que se asocia a una elevada morbimortalidad. Ante la necesidad de la implementación y estandarización de su manejo, se realizó una revisión sistemática de la literatura, con extracción de recomendaciones en base a las evidencias existentes. A partir de las mismas se redactó un documento de consenso multidisciplinar. Desde las definiciones de hemorragia masiva y transfusión masiva, se establecen recomendaciones de actuación estructuradas en las medidas generales de manejo de las mismas (valoración clínica de la hemorragia, manejo de la hipotermia, reposición de la volemia, reanimación hipotensiva y cirugía de contención de daños), monitorización de la volemia, administración de hemocomponentes (concentrado de hematíes, plasma fresco, plaquetas, y óptima relación de administración entre ellos), y de hemostáticos (complejo protrombínico, fibrinógeno, factor VIIa, antifibrinolíticos) (AU)

Massive haemorrhage is common and often associated with high morbidity and mortality. We perform a systematic review of the literature, with extraction of the recommendations from the existing evidences because of the need for its improvement and the management standardization. From the results we found, we wrote a multidisciplinary consensus document. We begin with the agreement in the definitions of massive haemorrhage and massive transfusion, and we do structured recommendations on their general management (clinical assessment of bleeding, hypothermia management, fluid therapy, hypotensive resuscitation and damage control surgery), blood volume monitoring, blood products transfusion (red blood cells, fresh frozen plasma, platelets and their best transfusion ratio), and administration of hemostatic components (prothrombin complex, fibrinogen, factor VIIa, antifibrinolytic agents) (AU)

[Multidisciplinary consensus document on the management of massive haemorrhage (HEMOMAS document)]. / Documento multidisciplinar de consenso sobre elmanejo de la hemorragia masiva (document HEMOMAS)

Massive haemorrhage is common and often associated with high morbidity and mortality. We perform a systematic review of the literature, with extraction of the recommendations from the existing evidences because of the need for its improvement and the management standardization. From the results we found, we wrote a multidisciplinary consensus document. We begin with the agreement in the definitions of massive haemorrhage and massive transfusion, and we do structured recommendations on their general management (clinical assessment of bleeding, hypothermia management, fluid therapy, hypotensive resuscitation and damage control surgery), blood volume monitoring, blood products transfusion (red blood cells, fresh frozen plasma, platelets and their best transfusion ratio), and administration of hemostatic components (prothrombin complex, fibrinogen, factor VIIa, antifibrinolytic agents).

Documento multidisciplinar de consenso sobre el manejo de la hemorragia masiva (documento HEMOMAS) / Multidisciplinary consensus document on the management of massive hemorrhage (HEMOMAS document)

La hemorragia masiva es una entidad frecuente que se asocia a una elevada morbimortalidad. Ante la necesidad de la implementación y estandarización de su manejo, se realizó una revisión sistemática de la literatura, con extracción de recomendaciones en base a las evidencias existentes. A partir de las mismas se redactó un documento de consenso multidisciplinar. Desde las definiciones de hemorragia masiva y transfusión masiva, se establecen recomendaciones de actuación estructuradas en las medidas generales de manejo de las mismas (valoración clínica de la hemorragia, manejo de la hipotermia, reposición de la volemia, reanimación hipotensiva y cirugía de contención de daños), monitorización de la volemia, administración de hemocomponentes (concentrado de hematíes, plasma fresco, plaquetas, y óptima relación de administración entre ellos), y de hemostáticos (complejo protrombínico, fibrinógeno, factor VIIa, antifibrinolíticos) (AU)

Massive haemorrhage is common and often associated with high morbidity and mortality. We perform a systematic review of the literature, with extraction of the recommendations from the existing evidences because of the need for its improvement and the management standardization. From the results we found, we wrote a multidisciplinary consensus document. We begin with the agreement in the definitions of massive haemorrhage and massive transfusion, and we do structured recommendations on their general management (clinical assessment of bleeding, hypothermia management, fluid therapy, hypotensive resuscitation and damage control surgery), blood volume monitoring, blood products transfusion (red blood cells, fresh frozen plasma, platelets and their best transfusion ratio), and administration of hemostatic components (prothrombin complex, fibrinogen, factor VIIa, antifibrinolytic agents) (AU)

Multidisciplinary consensus document on the management of massive haemorrhage (HEMOMAS document).

Massive haemorrhage is common and often associated with high morbidity and mortality. We perform a systematic review of the literature, with extraction of the recommendations from the existing evidences because of the need for its improvement and the management standardization. From the results we found, we wrote a multidisciplinary consensus document. We begin with the agreement in the definitions of massive haemorrhage and massive transfusion, and we do structured recommendations on their general management (clinical assessment of bleeding, hypothermia management, fluid therapy, hypotensive resuscitation and damage control surgery), blood volume monitoring, blood products transfusion (red blood cells, fresh frozen plasma, platelets and their best transfusion ratio), and administration of hemostatic components (prothrombin complex, fibrinogen, factor VIIa, antifibrinolytic agents).

Reduced ADAMTS13 activity is associated with thrombotic risk in systemic lupus erythematosus.

BACKGROUND: Severe deficiency of ADAMTS13 activity leads to von Willebrand factor (VWF) ultralarge multimers with high affinity for platelets, causing thrombotic thrombocytopenic purpura. Other pathological conditions with moderate ADAMTS13 activity exhibit a thrombotic risk. We examined the ADAMTS13 activity in systemic lupus erythematosus (SLE) and its value as a thrombotic biomarker. METHODS: ADAMTS13 activity, VWF antigen and multimeric structure, and vascular cell adhesion molecule 1 (VCAM-1) were measured in plasma samples from 50 SLE patients and 50 healthy donors. Disease activity (systemic lupus erythematosus disease activity index; SLEDAI) and organ damage (systemic lupus international collaborating clinics) scores, thrombotic events, antiphospholipid syndrome (APS) and antiphospholipid antibodies (aPLs) were registered. RESULTS: SLE patients showed decreased ADAMTS13 activity and high VWF levels compared with controls (66 ± 27% vs. 101 ± 8%, P < 0.01, and 325 ± 151% vs. 81 ± 14%, P < 0.001). VCAM-1 levels were higher in SLE patients (P < 0.05). Considering three groups of SLE patients depending on ADAMTS13 activity (>60%, 60-40% and <40%), comparative analysis showed significant association between ADAMTS13 activity and SLEDAI (P < 0.05), presence of aPLs (P < 0.001), APS (P < 0.01) and thrombotic events (P < 0.01). Reduced ADAMTS13 activity together with increased VWF levels were especially notable in patients with active disease and with aPLs. CONCLUSION: ADAMTS13 activity, in combination with other laboratory parameters, could constitute a potential prognostic biomarker of thrombotic risk in SLE.

Vorapaxar for the reduction of atherothrombotic events.

Vorapaxar is a novel platelet inhibitor that potently and selectively inhibits thrombin-mediated platelet activation without interfering with thrombin-mediated cleavage of fibrinogen via antagonism of the platelet proteinase-activated receptor PAR1. Vorapaxar is a non-peptide himbacine analogue that has been developed for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or peripheral arterial disease.

Hemorragias en pacientes anticoagulados: utilidad real de los antídotos y modo de actuación con los nuevos anticoagulantes orales / Bleeding in patients on anticoagulant therapy: the real utility of antidotes and how to manage bleeding in patients on new-generation oral anticoagulants

Una de las complicaciones más temidas de los anticoagulantes son las hemorragias. Aunque en muchas ocasiones la suspensión de la anticoagulación y medidas de soporte es suficiente, en otras ocasiones exigen un tratamiento más específico. Clásicamente se han empleado los antagonistas de la vitamina K (AVK) en la prevención a largo plazo de las complicaciones tromboembólicas. Sin embargo, éstos presentan numerosas limitaciones que complican su manejo clínico. Los nuevos anticoagulantes orales, además de tener una eficacia al menos similar a los AVK con una menor incidencia de hemorragias intracraneales, no necesitan controles rutinarios de la coagulación y se pautan a dosis fijas. Sin embargo, no están exentos de presentar complicaciones hemorrágicas. Con la única diferencia de que los AVK tienen un antídoto específico, el manejo de las hemorragias con los nuevos anticoagulantes orales es muy similar al de la hemorragia asociada a los AVK (AU)

Bleeding is one of the most feared complications of anticoagulant therapy. Discontinuing the anticoagulant and establishing of support measures will often resolve the emergency, but some cases require more specific treatment. Vitamin K antagonists (VKAs) have traditionally been used for long-term prevention of thromboembolic complications, but these drugs have many limitations that complicate clinical management. The new-generation of oral anticoagulants are similar in efficacy to VKAs but are associated with a lower incidence of intracranial hemorrhage, do not require routine scheduling of laboratory tests, and can be prescribed at fixed dosages. However, these drugs are not complication-free. The management of acute bleeding is very similar for patients on either VKA or a new oralanticoagulant, with the single exception that a specific antidote is available to reverse the effect of a VKA (AU)

Apixaban in the prevention of stroke and systemic embolism in nonvalvular atrial fibrillation.

Conventional anticoagulant therapies can significantly reduce the risk of stroke and related complications in patients with atrial fibrillation (AF). Classic oral anticoagulants based on vitamin K antagonism have shown effectiveness in the prevention of thromboembolic complications in this clinical setting. Unfortunately, vitamin K antagonists that have shown effectiveness in the prevention of thromboembolic complications in patients with nonvalvular AF hold inherent limitations including delayed onset of action, narrow therapeutic index, variability of their response, need for repeated control and numerous interactions with food and other drugs. Since the frequency of stroke related to AF increases with age, guidelines from different scientific societies advise that the risk of bleeding for a patient should be quantified before exposure to anticoagulation and balanced against the risk of stroke with and without anticoagulation. A consequence of assessing this risk/benefit balance is that not all patients with AF at thromboembolic risk receive adequate anticoagulant treatment. Apixaban is a new oral anticoagulant with a direct, specific and reversible inhibitory action on coagulation factor Xa and with demonstrated safety and efficacy in the prophylaxis and treatment of venous thromboembolism in several clinical studies involving thousands of patients subjected to major orthopedic surgery. Results of two large phase III trials have demonstrated the efficacy and safety of apixaban compared with aspirin or warfarin, in the prevention of stroke in patients with AF. Apixaban demonstrated superiority over classic vitamin K antagonists on the previously specified outcomes of stroke, systemic embolism, major bleeding and death. For those patients unsuitable for treatment with vitamin K antagonists because of an excessive bleeding risk, apixaban showed more efficacy than aspirin in stroke prevention with a not statistically significant modest increase of major bleeding.

Therapeutic efficacy of platelet components treated with amotosalen and ultraviolet A pathogen inactivation method: results of a meta-analysis of randomized controlled trials.

BACKGROUND AND OBJECTIVES: There are conflicting data regarding the therapeutic efficacy of platelets inactivated using amotosalen and ultraviolet A light. We have performed a meta-analysis to summarize the results of different randomized controlled trials (RCT). MATERIALS AND METHODS: Five RCTs reported through March 2011 met the criteria for meta-analysis. Weighted mean difference (WMD) in corrected count increment (CCI) at 1 h, CCI-24 h, and transfusion interval (days) and summary odds ratio (OR) of bleeding in inactivated platelet (I-P) group vs. noninactivated platelet (C-P) group were calculated across studies. RESULTS: Randomized controlled trials were statistically homogeneous when we analysed CCI-24 h, and the transfusion of C-P was associated with a higher CCI-24 h when compared with the transfusion of I-P (WMD, 3×10(3); 95% CI, 2·32×10(3)-3·69×10(3); P<0·00001). RCTs were statistically heterogeneous when we analysed CCI-1 h, transfusion interval and OR of bleeding. Regarding the OR of bleeding in the I-P and C-P groups, it varied by as much as a multiple of four among the trials, from 0·66 to 2·66. When we combined double-blinded and high methodologic quality score RCTs, the use of I-P was not statistically associated with an increase in the OR of bleeding when compared with the use of C-P (OR, 0·97; 95% CI, 0·75-1·27; P=0·84). CONCLUSION: Although the transfusion of I-P was associated with lower CCI-24 h when compared with the transfusion of C-P, this was not associated with differences in the OR of bleeding between I-P and C-P.

Translational evidence of endothelial damage in obese individuals: inflammatory and prothrombotic responses.

BACKGROUND: Obesity is associated with an increased atherothrombotic morbidity/mortality risk. However, there is no direct evidence of subclinical activation of the endothelium in obese subjects without other major cardiometabolic risk factors. OBJECTIVES: We applied a translational approach to investigate endothelial activation occurring in response to the components secreted by visceral and subcutaneous adipose tissue and their corresponding cell fractions obtained from obese subjects without other major cardiometabolic risk factors, as compared with non-obese controls. METHODS: Fat pads and cell fractions were incubated with serum-free medium to obtain their secretomes, which were analyzed by protein arrays. Endothelial cells (ECs) were exposed to the different secretomes to evaluate changes in gene expression, composition and reactivity of the extracellular matrix (ECM), and cell growth and viability. RESULTS: ECs incubated in the presence of obese secretomes displayed increased proliferation, altered cell morphology, augmented expression of VCAM-1, ICAM-1, and von Willebrand factor, and higher ECM reactivity towards circulating platelets. The visceral secretomes, especially the stromal one, induced the strongest expression of these markers, together with a more reactive ECM. These changes occurred through nuclear factor-κB (NF-κB) activation. CONCLUSION: This is the first translational study demonstrating that the cytokines secreted by the adipose tissue from obese individuals without other major cardiometabolic complications have a hazardous effect on the endothelium, through activation of the NF-κB pathway.