Coagulation factor XI in cirrhosis does not predict thrombo-hemorrhagic complications and hepatic decompensation.

INTRODUCTION: Factor XI (FXI) is associated with thrombosis in patients without liver disease, but it alterations and prognostic value in cirrhosis are uncertain. PATIENTS AND METHODS: We studied a prospective cohort of cirrhosis patients determining FXI and its association with portal vein thrombosis (PVT), bleeding, and hepatic decompensation/ACLF during 1-year follow-up. Odds ratios (OR) and 95 % CIs were calculated using logistic regression. RESULTS: We included 183 patients (Child-Pugh [CP] A/B/C 57/59/57). FXI was reduced in cirrhosis, decreasing with CP stage (78 % [66-94] vs. 58 % [44-78] vs. 41 % [30-52] in CP A, B, and C, respectively; p < 0.001). FXI was correlated with MELD score (rho: -0.6, p < 0.001), INR (rho: -0.6, p < 0.001), and platelet count (rho: 0.4, p < 0.001). Sixteen patients (8.7 %) experienced PVT, which only predictor was baseline platelet count (OR: 0.94; CI95 %: 0.91-0.97, p < 0.001). Bleeding occurred in 7 patients (3.8 %). Cirrhosis severity, platelet count, fibrinogen, and FXI (60% vs. 78 %; p = 0.2) were comparable between bleeding and non-bleeding individuals. Finally, no association was found between FXI and hepatic decompensation/ACLF, which were predicted by lower albumin and platelet count, respectively. CONCLUSION: FXI seems not to be responsible for thrombosis and cirrhosis progression. The lack of association between low FXI and bleeding events, however, indirectly opens to future studies evaluating FXI inhibitors in cirrhosis.

Procoagulant phenotype of virus-infected pericytes is associated with portal thrombosis and intrapulmonary vascular dilations in fatal COVID-19.

BACKGROUND & AIMS: Mechanisms and clinical impact of portal microthrombosis featuring severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesized that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19. METHODS: Ninety-three patients who died from COVID-19, were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-ß) markers, tissue factor (TF), viral spike-protein and nucleoprotein (SP, NP), fibrinogen, platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy (TEM). Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells (HPMEC) was assessed upon vWF treatment. RESULTS: IPVD was present in 16/66 COVID-19 patients with both liver and lung histology, with a younger age (62 vs 78yo), longer illness (25 vs 14 days), worsening hypoxemia (PaO2/FiO2 from 209 to 89), and more ventilatory support (63 vs 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, were confirmed by chest-CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-ß+/ SP+/NP+ pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA+ area, whereas infected SP+/NP+ pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In-vitro SARS-CoV-2 infection of pericytes up-regulated TF and induced endothelial cells to overexpress vWF, which expanded HPMEC tubules. CONCLUSIONS: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19. IMPACT AND IMPLICATIONS: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a pro-coagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterized at the clinical level by development of hypoxemia and at the histological level, by phlebosclerosis and reduced caliber of the portal vein branches in the absence of cirrhosis. Moreover, our findings bring light to an as yet overlooked player of thrombosis pathophysiology, i.e. pericytes, which may provide novel therapeutic tools to halt prothrombotic mechanisms.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

Cryptogenic ischemic stroke in cardiac transthyretin amyloidosis and sinus rhythm: a case report.

Cardiac amyloidosis is a group of diseases characterized by the deposition of amyloid fibers in cardiac tissue. Two forms are mainly reported: light chain (AL) and transthyretin (ATTR) amyloidosis. Among the complications of transthyretin amyloidosis there are thrombotic events and, to a lesser extent, hemorrhagic events. The latter are likely caused by perivascular amyloid deposition resulting in capillary fragility, in addition to INR lability during anticoagulant therapy. The onset of thrombotic events may be caused by the high prevalence of atrial fibrillation (AF), mechanical cardiac dysfunction and atrial myopathy observed in patients with transthyretin amyloidosis. It remains unclear why thromboembolic events occur even in patients with sinus rhythm or adequate anticoagulation, though a hypercoagulable state or underlying inflammation may be involved. We report a case of cryptogenic ischemic stroke in an 86-year-old woman with transthyretin amyloidosis and sinus rhythm. Traditional coagulation tests, whole blood rotational thromboelastometry and impedance aggregometry did not show a hypercoagulable state. The thrombin generation assay did not reveal a prothrombotic state. However, the study of extracellular vesicles highlighted underlying immune-mediated endothelial damage likely responsible for the thrombotic diathesis. It could be hypothesized that inflammation plays a role in the hypercoagulability of patients with transthyretin amyloidosis. Larger prospective studies are needed to validate our hypothesis.

Anticoagulant Therapy in Pregnant Women with Mechanical Heart Valves: Italian Federation of Centers for Diagnosis and Surveillance of the Antithrombotic Therapies (FCSA) Position Paper.

The management of anticoagulant therapy in pregnant women with mechanical heart valves (MHVs) is difficult and often challenging even for clinicians experienced in the field. These pregnancies, indeed, are burdened with higher rates of complications for both the mother and the fetus, compared to those in women without MHVs. The maternal need for an optimal anticoagulation as provided by vitamin K antagonists is counterbalanced by their teratogen effect on the embryo and fetus. On the other hand, several concerns have been raised about the efficacy of heparins in pregnant women with MHVs, considering the high risk of thrombotic complications in these patients. Therefore, numerous clinical issues about the management of pregnant women with MHVs remain unanswered, such as the selection of the best anticoagulant agent, the optimal anticoagulation levels to be achieved and maintained, and the evaluation of long-term effects for both the mother and the fetus. Based on a comprehensive review of the current literature, the Italian Federation of the Centers for the Diagnosis and the Surveillance of the Antithrombotic Therapies (FCSA) proposes experience-based suggestions and expert opinions. Particularly, this consensus document aims at providing practical guidance for clinicians dealing with pregnant women with MHVs, to optimize maternal and fetal outcomes while guaranteeing adequate anticoagulation. Finally, FCSA highlights the need for the creation of multidisciplinary teams experienced in the management of pregnant women with MHVs during pregnancy, delivery, and postpartum, in order to better deal with such complex clinical issues and provide a comprehensive counseling to these patients.

DOACs for Older adults with Atrial Fibrillation and Falls: Results from the prospective single-centre DOAFF study.

INTRODUCTION: Falls are one of the most fearsome events in anticoagulated older adults. The evidence concerning safety of direct oral anticoagulants (DOACs) in falling elderly patients with atrial fibrillation (AF) is still limited. METHODS: We prospectively enrolled consecutive anticoagulant-naïve patients aged 65 years and older, starting anticoagulation with DOACs for AF. The study cohort was stratified in fallers vs. non-fallers, according to the occurrence of at least one fall during the 2-year follow-up and bleeding outcomes were evaluated. RESULTS: We enrolled 524 consecutive patients. Mean age was 80.8 years and they were mostly women (54.0%). Among the study cohort, 148 patients (28.2%) presented at least one fall episode during the study period. After the adjustment for potential confounders, no difference was found between fallers and non-fallers for all the study outcomes: major bleeding [HR: 1.04 (95%CI: 0.58-1.85)], intracranial haemorrhage [HR: 1.63 (95%CI: 0.69-3.80)], clinically relevant non-major bleeding [HR: 1.21 (95%CI: 0.83-1.76)], and all-cause death [HR: 1.51 (95%CI: 0.85-2.69)]. The presence of a prior cerebrovascular event [HR: 2.27 (95%CI: 1.12-4.62); p-value: 0.02] and polypharmacy [HR: 1.60 (95%CI: 1.08-2.39); p-value: 0.02] were the main drivers for major and clinically relevant non-major bleedings, respectively. CONCLUSIONS: Falls in an anticoagulant-naïve population aged 65 years and over starting a DOAC for AF do not increase the bleeding risk. Thus, the presence of falls should not discourage clinicians from prescribing DOACs also in this subset of patients.

Portal Vein Thrombosis: State-of-the-Art Review.

Background: Portal vein thrombosis (PVT) is a rare disease with an estimated incidence of 2 to 4 cases per 100,000 inhabitants. The most common predisposing conditions for PVT are chronic liver diseases (cirrhosis), primary or secondary hepatobiliary malignancy, major infectious or inflammatory abdominal disease, or myeloproliferative disorders. Methods: PVT can be classified on the basis of the anatomical site, the degree of venous occlusion, and the timing and type of presentation. The main differential diagnosis of PVT, both acute and chronic, is malignant portal vein invasion, most frequently by hepatocarcinoma, or constriction (typically by pancreatic cancer or cholangiocarcinoma). Results: The management of PVT is based on anticoagulation and the treatment of predisposing conditions. The aim of anticoagulation in acute thrombosis is to prevent the extension of the clot and enable the recanalization of the vein to avoid the development of complications, such as intestinal infarction and portal hypertension. Conclusions: The treatment with anticoagulant therapy favors the reduction of portal hypertension, and this allows for a decrease in the risk of bleeding, especially in patients with esophageal varices. The anticoagulant treatment is generally recommended for at least three to six months. Prosecution of anticoagulation is advised until recanalization or lifelong if the patient has an underlying permanent pro-coagulant condition that cannot be corrected or if there is thrombosis extending to the mesenteric veins.

Von Willebrand factor for outcome prediction within different clinical stages of advanced chronic liver disease.

BACKGROUND AND AIMS: The prognostic performance of von Willebrand factor (VWF) may vary across clinical stages of advanced chronic liver disease (ACLD). Therefore, we investigated the evolution of VWF and other biomarkers throughout the full ACLD spectrum and evaluated their stage-specific prognostic utility. METHODS: We retrospectively included Viennese ACLD patients with available information on hepatic venous pressure gradient (HVPG), C-reactive protein (CRP)/VWF levels and outcomes. ACLD stages were defined according to D'Amico et al. We included an external validation cohort from Padua. RESULTS: We observed gradual increases in VWF throughout ACLD stages. In contrast, HVPG levelled off in decompensated ACLD (dACLD), whereas MELD showed only minor changes in the early stages and CRP did not increase until stage 3. VWF was associated with hepatic decompensation/liver-related death in compensated ACLD (cACLD) in a fully adjusted model, while it was not independently predictive of ACLF/liver-related death in dACLD. After backward selection, HVPG/CRP/VWF remained the main predictors of hepatic decompensation/liver-related death in cACLD. Notably, the performance of the non-invasive CRP/VWF-based model was comparable to invasive HVPG-based models (C-index:0.765 ± 0.034 vs. 0.756 ± 0.040). The discriminative ability of the CRP/VWF-based model was confirmed in an external validation cohort using another VWF assay which yielded systematically lower values. CONCLUSION: VWF is the only biomarker that gradually increases across all ACLD stages. It is of particular prognostic value in cACLD, where a CRP/VWF-based model is equivalent to an invasive HVPG-based model. Systematic differences in VWF underline the importance of interlaboratory surveys. Moreover, our findings reinforce the notion that, already in cACLD, inflammation is a key disease-driving mechanism.

Profiling plasma alterations of extracellular vesicles in patients with acutely decompensated cirrhosis and bacterial infection.

BACKGROUND: Extracellular vesicles (EVs) modulate inflammation, coagulation and vascular homeostasis in decompensated cirrhosis. AIM: To characterize the profile of plasmatic EVs in patients with decompensated cirrhosis and bacterial infections and evaluate the association between EVs and the development of hemostatic complications. METHODS: We measured the levels of EVs using high-sensitivity flow cytometry and phospholipid-dependent clotting time (PPL) in a prospective cohort of hospitalized patients with acutely decompensated cirrhosis with versus without bacterial infections. A separate cohort of patients with bacterial infections without cirrhosis was also enrolled. We measured endothelium-, tissue factor (TF)-bearing, platelet- and leukocyte-derived EVs. In patients with infections, EVs were reassessed upon resolution of infection. Bleeding and thrombotic complications were recorded during 1-year follow-up. RESULTS: Eighty patients with decompensated cirrhosis were recruited (40 each with and without bacterial infections). Electron microscopy confirmed the presence of plasma EVs. Despite no difference in total EVs and PPL, patients with cirrhosis and infection had significantly higher TF+ EVs, P-Selectin+ EVs (activated platelet-derived), CD14+ EVs (monocyte/macrophages derived) and CD14+ TF+ EVs versus those with cirrhosis without infection. Upon infection resolution, levels of these EVs returned to those without infection. Patients with infections showed a significant association between reduced P-Selectin+ EVs and bleeding complications (HR 8.0 [95%CI 1.3-48.1]), whereas high levels of leukocyte-derived EVs (CD45+) and CD14+ EVs were significantly associated with thrombotic complications (HR 16.4 [95%CI 1.7-160] and 10.9 [95%CI 1.13-106], respectively). Results were confirmed in a validation cohort. CONCLUSION: Bacterial infections are associated with particular alterations of plasma EVs profile in decompensated cirrhosis. Bacterial infections trigger the release of EVs originating from various cell types, which may tip the precarious hemostatic balance of patients with acutely decompensated cirrhosis towards hyper- or hypocoagulability.

Reticulated platelets are increased and hyper-activated in patients with cirrhosis, especially those with poor outcome.

BACKGROUND: Reticulated platelets (RePLT) are emergency circulating platelets released to contrast peripheral platelet destruction. AIM: We conducted a prospective study to [a] characterize RePLT in cirrhosis; [b] evaluate the association between RePLT and hepatic decompensation/death. METHODS: Cirrhosis patients without hepatocellular carcinoma were prospectively recruited and underwent assessment of RePLT and thrombopoietin (TPO). RePLT were evaluated by cytofluorimetry and immuno-fluorescence microscopy. Twenty healthy subjects were included as controls. Patients were followed for 6 months for hepatic decompensation and further decompensation/ACLF. RESULTS: Forty-five patients were included (Child-Pugh [CP] A/B/C 18/11/16). Compared to controls, RePLT in cirrhosis were significantly increased (0.82% vs. 0.05%; p < 0.001) and hyperactivated (4.35% vs. 0.17%; p = 0.004). No correlation was observed between RePLT and CP, platelet count, TPO, MELD score, and C-reactive protein. TPO was lower in cirrhosis than controls (28 pg/mL vs. 52 pg/mL; p = 0.005), decreasing significantly with CP stage. In CP B/C patients (n = 27), RePLT were significantly higher in those who progressed towards further decompensation/ACLF (2.11 [0.56-2.95] vs. 0.69 [0.02-1.22]; p < 0.01). A proportion of RePLT >2% accurately identified high-risk patients (AUROC 0.818; 95%CI: 0.639-0.997; sensitivity 94%, specificity 73%). CONCLUSION: RePLT in cirrhosis are increased and hyper-activated. In decompensated patients, higher RePLT appear to be associated with worse outcomes.

Current Gaps in the Provision of Safe and Effective Anticoagulation in Atrial Fibrillation and the Potential for Factor XI-Directed Therapeutics.

The global prevalence of atrial fibrillation is rapidly increasing, in large part due to the aging of the population. Atrial fibrillation is known to increase the risk of thromboembolic stroke by 5 times, but it has been evident for decades that well-managed anticoagulation therapy can greatly attenuate this risk. Despite advances in pharmacology (such as the shift from vitamin K antagonists to direct oral anticoagulants) that have increased the safety and convenience of chronic oral anticoagulation in atrial fibrillation, a preponderance of recent observational data indicates that protection from stroke is poorly achieved on a population basis. This outcomes deficit is multifactorial in origin, stemming from a combination of underprescribing of anticoagulants (often as a result of bleeding concerns by prescribers), limitations of the drugs themselves (drug-drug interactions, bioaccumulation in renal insufficiency, short half-lives that result in lapses in therapeutic effect, etc), and suboptimal patient adherence that results from lack of understanding/education, polypharmacy, fear of bleeding, forgetfulness, and socioeconomic barriers, among other obstacles. Often this adherence is not reported to treating clinicians, further subverting efforts to optimize care. A multidisciplinary, interprofessional panel of clinicians met during the 2023 International Society of Thrombosis and Haemostasis Congress to discuss these gaps in therapy, how they can be more readily recognized, and the potential for factor XI-directed anticoagulants to improve the safety and efficacy of stroke prevention. A full appreciation of this potential requires a reevaluation of traditional teaching about the "coagulation cascade" and decoupling the processes that result in (physiologic) hemostasis and (pathologic) thrombosis. The panel discussion is summarized and presented here.

Venous Thromboembolism in Children: The Rivaroxaban Experience.

The incidence of venous thromboembolism (VTE) in the pediatric population has increased more than 10-fold in the last 20 years, as a consequence of the advancement of resuscitation and surgical techniques and the global increase in life expectancy of children suffering from chronic pathologies. Monitoring anticoagulant therapy to achieve outcomes within the target range in childhood VTE, parenteral administration of medications, and frequent blood tests in children are often cumbersome. Availability of safe and effective oral agents with pediatric data to support use would be of clear benefit. A physiologically based pharmacokinetic model was developed to estimate the appropriate dosing schedule for rivaroxaban in children. This incorporated growth/maturation and variability in anthropometrics (e.g., body height, weight, and body mass index), anatomy (e.g., organ weight), physiology (e.g., blood flow rates), metabolism and excretion. Rivaroxaban use in pediatric population underwent a complete investigational program, consisting mainly of one phase I pharmacokinetics/pharmacodynamics trial, three phase II trials, one phase III trial. The phase III trial enrolled 500 patients from birth to <18 years and documented the efficacy and safety of rivaroxaban regimens at dose equivalent to the adult 20 mg dose for the prevention of fatal or symptomatic nonfatal recurrent VTE and major bleeding versus heparin or vitamin K antagonists. Results were similar to those in rivaroxaban studies in adults. The efficacy and safety of rivaroxaban in children reported in the EINSTEIN JUNIOR trial provide further support to previous trials in adults (EINSTEIN Program), which demonstrate a favorable profile for the use of rivaroxaban for the management of VTE in challenging patient populations. Other clinical evidence contributing to the use of rivaroxaban among different risk groups in pediatric VTE population confirms the consistency with principal trial. Our review aims to describe the rationale for using rivaroxaban oral suspension in clinical practice and to summarize its multiple indications in each vascular bed (e.g., cerebral venous thrombosis, symptomatic or asymptomatic central venous catheter-associated thrombosis), etiology, and patients setting.

The evolving knowledge on primary hemostasis in patients with cirrhosis: A comprehensive review.

Patients with cirrhosis develop complex alterations in primary hemostasis that include both hypocoagulable and hypercoagulable features. This includes thrombocytopenia, multiple alterations of platelet function, and increased plasma levels of von Willebrand factor. Contrary to the historical view that platelet dysfunction in cirrhosis might be responsible for an increased bleeding tendency, the current theory posits a rebalanced hemostasis in patients with cirrhosis. Severe thrombocytopenia is not indicative of the bleeding risk in patients undergoing invasive procedures and does not dictate per se the need for pre-procedural prophylaxis. A more comprehensive and individualized risk assessment should combine hemostatic impairment, the severity of decompensation and systemic inflammation, and the presence of additional factors that may impair platelet function, such as acute kidney injury and bacterial infections. Although there are multiple, complex alterations of platelet function in cirrhosis, their net effect is not yet fully understood. More investigations evaluating the association between alterations of platelet function and bleeding/thrombosis may improve risk stratification in patients with decompensated cirrhosis. Besides hemostasis, the assessment of von Willebrand factor Ag and ADP-induced, whole-blood platelet aggregation normalized by platelet count (VITRO score and PLT ratio) are promising biomarkers to predict the risk of hepatic decompensation and survival in both compensated and decompensated patients. Further investigations into the in vivo interplay between platelets, circulating blood elements, and endothelial cells may help advance our understanding of cirrhotic coagulopathy. Here, we review the complex changes in platelets and primary hemostasis in cirrhosis and their potential clinical implications.

Cellular Components Contributing to the Development of Venous Thrombosis in Patients with Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer and has a poor prognosis. Patients with PDAC are at high risk of developing thromboembolic events, which is a leading cause of morbidity and mortality following cancer progression. Plasma-derived coagulation is the most studied process in cancer-associated thrombosis. Other blood components, such as platelets, red blood cells, and white blood cells, have been gaining less attention. This narrative review addresses the literature on the role of cellular components in the development of venous thromboembolism (VTE) in patients with PDAC. Blood cells seem to play an important role in the development of VTE. Altered blood cell counts, i.e., leukocytosis, thrombocytosis, and anemia, have been found to associate with VTE risk. Tumor-related activation of leukocytes leads to the release of tissue factor-expressing microvesicles and the formation of neutrophil extracellular traps, initiating coagulation and forming a scaffold for thrombi. Tissue factor-expressing microvesicles are also thought to be released by PDAC cells. PDAC cells have been shown to stimulate platelet activation and aggregation, proposedly via the secretion of podoplanin and mucins. Hypofibrinolysis, partially explained by increased plasminogen activator inhibitor-1 activity, is observed in PDAC. In short, PDAC-associated hypercoagulability is a complex and multifactorial process. A better understanding of cellular contributions to hypercoagulability might lead to the improvement of diagnostic tests to identify PDAC patients at highest risk of VTE.

In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency.

BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

Whole blood thrombin generation shows a significant hypocoagulable state in patients with decompensated cirrhosis.

BACKGROUND: Patients with cirrhosis have a normal to increased thrombin generation (TG) capacity in platelet-poor plasma (PPP). By reflecting the contribution of all circulating blood cells, whole blood (WB) TG may allow a more physiological assessment of coagulation. OBJECTIVES: We compared WB-TG vs PPP-TG in patients with cirrhosis. METHODS: Assessment of coagulation included routine tests, factor VIII, natural anticoagulants, PPP-TG, and WB-TG. TG assays were performed with and without thrombomodulin. Twenty-five healthy subjects were included as controls. RESULTS: We included 108 patients (Child-Pugh A/B/C, 44/24/40). Compared with controls, patients had significantly lower platelet count, longer international normalized ratio, higher FVIII, and lower levels of protein C/S and antithrombin. Regarding thrombomodulin-modified TG assays, in compensated cirrhosis, both PPP-TG and WB-TG indicated an increased TG capacity, as reflected by an endogenous thrombin potential (ETP) significantly higher than controls. In contrast, in decompensated cirrhosis, PPP-TG indicated a hypercoagulable state with increased ETP, higher peak height, and shorter time-to-peak than controls, whereas WB-TG revealed a progressive impairment of TG kinetics and total capacity, ultimately resulting in a profound hypocoagulable state in patients with Child-Pugh C cirrhosis (ie, significant prolongation of lag time and time-to-peak with reduction of both ETP and peak height). In decompensated patients, bacterial infections and severity of anemia were associated with a further reduction of both ETP and peak height. CONCLUSION: Compensated cirrhosis is associated with an increased TG capacity. In decompensated cirrhosis, contrary to PPP-TG, which indicates hypercoagulability, WB-TG shows a significant hypocoagulable state. The clinical value of these findings deserves further investigation.