Role of fibrinogen concentrates for treatment of critical perioperative hemorrhage.

Acquired hypofibrinogenemia is a frequent cause of maintained bleeding in perioperative high-risk settings. Loss, consumption and dilution under resuscitation fluid therapy are the principal causes for fibrinogen depletion. Severe hypofibrinogenemia is frequently associated with an early bleeding complication that cannot be reliably avoided with high-ratio plasma transfusion strategies. Real-time monitoring with viscoelastic hemostatic assays is a useful tool for timely diagnosis and treatment of detected coagulopathies. Replenishment of fibrinogen in uncontrolled bleeding events is currently recommended by most published guidelines, suggesting treatment thresholds to maintain a minimum of 1.5 g/L plasma fibrinogen concentration for nonobstetrical hemorrhage. Fibrinogen concentrates, originally licensed for treatment of bleeding episodes in patients with congenital hypo-, dys- or afibrinogenemia disorders, are used in many clinical situations as supplementary therapy for the treatment of acquired hypofibrinogenemia. This review seeks to provide an overview of the most relevant topics associated to fibrinogen replacement therapy for critical perioperative hemorrhage highlighting currently available evidence on the risk/benefit profile of purified fibrinogen concentrates for this extended clinical indication.

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.

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.

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.

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.

The role of the endothelium in the short-term complications of hematopoietic SCT.

In this review, we analyse the role of the endothelium in the development of several complications that appear soon after haematopoietic SCT (HSCT). Once it had been demonstrated that sinusoidal damage is the initiating event of the sinusoidal obstruction syndrome, it was considered that other short-term complications with overlapping clinical manifestations, such as capillary leak syndrome, engraftment syndrome, transplant-associated microangiopathy, diffuse alveolar haemorrhage and idiopathic pneumonia syndrome, could have an endothelial origin. During HSCT, endothelial cells (ECs) are activated and damaged by several factors, including conditioning, cytokines released by damaged tissues, endotoxins translocated through damaged mucosa, drugs used in the procedure, the engraftment, and--in the allogeneic setting--immunological reactions. The different clinical syndromes that occur could be determined by the predominant phenotypic change in the ECs and the location of this change (organ dependant or systemic). Several translational studies have provided evidence of this endothelial dysfunction on the basis of analysis of soluble markers, soluble forms of adhesion molecules, the enumeration of circulating ECs and microparticles, and morphologic and functional changes induced in cultured ECs. This increased knowledge has opened up a wide range of potential pharmacologic interventions to prevent or treat endothelial damage and, consequently, to improve the outcome of patients receiving HSCT.

The secretory mechanisms in equine platelets are independent of cytoskeletal polymerization and occur through membrane fusion.

Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cytoskeletal assembly, by polyacrylamide gel electrophoresis, and iii) specific molecules involved in activation and secretion, by western blot. In activated human platelets, centralization of granules, cytoskeletal assembly and fusion of granules with the open canalicular system were observed. In activated equine platelets, granules fused together forming an organelle chain that fused with the surface membrane and released its content directly outside the platelets. Human platelets responded to activation with actin polymerization and the assembly of other contractile proteins to the cytoskeleton. These events were almost undetectable in equine platelets. When exploring the involvement of the synaptosomal-associated protein-23 (SNAP-23), a known regulator of secretory granule/plasma membrane fusion events, it was present in both human and equine platelets. SNAP-23 was shown to be more activated in equine platelets than human platelets in response to activation, especially with collagen. Thus, there are significant differences in the secretion mechanisms between human and equine platelets. While in human platelets, activation and secretion of granules depend on mechanisms of internal contraction and membrane fusion, in equine platelets the fusion mechanisms seem to be predominant.

Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway.

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548. Effects of flavonoids on platelet adhesion were assessed using arterial subendothelium with annular plate perfusion chambers, and global evaluation of apigenin on high-shear-dependent platelet function was determined by the PFA-100. To evaluate the ability of apigenin to potentiate the effect of aspirin, arachidonic acid-induced platelet aggregation was measured prior to and after consumption of subaggregatory doses of aspirin in the presence or absence of apigenin. Binding assays revealed that apigenin was an efficient competitor of [ (3)H]SQ29548 binding to PRP ( K i = 155.3 +/- 65.4 microM), and perfusion studies showed that apigenin, genistein, and catechin significantly diminished thrombus formation when compared to control (26.2 +/- 3.8, 33.1 +/- 5.2, and 26.2 +/- 5.2 vs 76.6 +/- 2.6%, respectively; p < 0.05). Apigenin, similarly to the TP antagonist SQ29548, significantly prolonged collagen epinephrine-induced PFA-100 closure time in comparison to the control and, when added to platelets that had been exposed in vivo to aspirin, potentiated its inhibitory effect on platelet aggregation. The inhibitory effect of some flavonoids in the presence of plasma, particularly apigenin, might in part rely on TxA(2) receptor antagonism. There is a clear increase in the ex vivo antiplatelet effect of aspirin in the presence of apigenin, which encourages the idea of the combined use of aspirin and certain flavonoids in patients in which aspirin fails to properly suppress the TxA(2) pathway.

Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species.

BACKGROUND: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. OBJECTIVE: To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. METHODS: Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber. RESULTS AND CONCLUSIONS: Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.

[Uremic media affects hemostatic properties of human endothelial cells in culture and increases the production of von Willebrand factor]. / El medio urémico altera las propiedades hemostáticas de células endoteliales humanas en cultivo y aumenta la producción de factor von Willebrand.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effect of the uremic media on the morphology of ECs, and their resistance to flow was analyzed. The reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic media resulted in abnormal morphology and signs of accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (22% vs 13%). Platelet deposition and formation of aggregates were significantly elevated on ECMs generated in the presence of uremic media (40.23 +/- 6.43% vs 25.42 +/- 2.69%, p < 0.05, n = 5). Immunocytochemical methods detected an enhanced expression of von Willebrand factor antigen on uremic ECMs (uremic 17.1 +/- 4.2% vs control 13.57 +/- 3.98%, p < 0.05) and its mRNA expression in endothelial cells (uremic 213.24 +/- 6.13 vs control 200.77 +/- 7.52, p < 0.05). These results suggest that uremic medium alters endothelial function and impairs the antithrombotic functions of cultured endothelial cells. This effect may contribute to the increased cardiovascular and thrombotic risk reported in ESRD patients.

El medio urémico altera las propiedades hemostáticas de células endoteliales humanas en cultivo y aumenta la producción de factor von Willebrand / Uraemic medium alters endothelial function and increases the thrombogenicity and von Willebrand factor expression of the extracellular matrix

El objetivo de este estudio fue determinar la capacidad del suero urémico de modificar las propiedades hemostáticas del endotelio. Para ello se analizó el efecto del medio urémico sobre la morfología y resistencia al flujo de las células endoteliales, así como la trombogenicidad de la matriz subendotelial generada por las células endoteliales.La exposición de células endoteliales en cultivo a un suero urémico indujo alteraciones en su morfología y un crecimiento acelerado de las mismas.Cuando las células endoteliales en cultivo eran expuestas a sangre circulante el desprendimiento de las mismas era superior cuando fueron cultivadas en suero urémico (22 por ciento vs 13 por ciento). La adhesión de plaquetas y la formación de agregados eran superiores en las matrices subendoteliales generadas en presencia de medio urémico (40,23 ñ 6,43 por ciento vs 25,42 ñ 2,69 por ciento, p < 0,05, n = 5).Asimismo, se detectó un aumento en la expresión de antígeno del factor von Willebrand mediante métodos inmunocitoquímicos en las matrices subendoteliales urémicas (17,1 ñ 4,2 por ciento vs 13,57 ñ 3,98, p < 0,05) y la de la expresión de su ARNm en células endoteliales (213,24 ñ 6,13 vs 200,77 ñ 7,52, p < 0,05).Estos resultados indican que el medio urémico altera la función endotelial in vitro y aumenta la trombogenicidad del subendotelio. Estos cambios podrían estar implicados en el aumento del riesgo cardiovascular y de sufrir fenómenos trombóticos que presentan los pacientes con insuficiencia renal crónica. (AU)

Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.

We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.

State-of-the-art knowledge on thrombosis and hemostasis.

The XVIII Congress of the International Society on Thrombosis and Haemostasis, held July 6-12, 2001, in Paris, France, covered the latest advances in the field of thrombosis and hemostasis, including vascular biology. Plenary conferences, state-of-the-art lectures, symposia, and oral and poster presentations focused most of the attention on the current research on the pathogenesis and treatment of thrombotic and hemorrhagic disorders and provided updates on trials currently under way or already completed.