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.

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.

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.

Contact system and intrinsic pathway activation in patients with advanced pancreatic cancer: a prospective cohort study.

BACKGROUND: Despite high risk of venous thromboembolism (VTE) in patients with pancreatic cancer, there are little data on contact system activation in these patients. OBJECTIVES: To quantify contact system and intrinsic pathway activation and subsequent VTE risk in patients with pancreatic cancer. METHODS: Patients with advanced pancreatic cancer were compared with controls. Blood was drawn at baseline and patients were followed for 6 months. Complexes of proteases with their natural inhibitors, C1-esterase inhibitor (C1-INH), antithrombin (AT), or alpha-1 antitrypsin (α1at), were measured for complexes containing kallikrein (PKa:C1-INH), factor (F)XIIa (FXIIa:C1-INH), and FXIa (FXIa:C1-INH, FXIa:AT, FXIa:α1at). The association of cancer with complex levels was assessed in a linear regression model, adjusted for age, sex, and body mass index. In a competing risk regression model, we assessed associations between complex levels and VTE. RESULTS: One hundred nine patients with pancreatic cancer and 22 controls were included. The mean age was 66 years (SD, 8.4) in the cancer cohort and 52 years (SD, 10.1) in controls. In the cancer cohort, 18 (16.7%) patients developed VTE during follow-up. In the multivariable regression model, pancreatic cancer was associated with increased complexes of PKa:C1-INH (P < .001), FXIa:C1-INH (P < .001), and FXIa:AT (P < .001). High FXIa:α1at (subdistribution hazard ratio, 1.48 per log increase; 95% CI, 1.02-2.16) and FXIa:AT (subdistribution hazard ratio, 2.78 highest vs lower quartiles; 95% CI, 1.10-7.00) were associated with VTE. CONCLUSION: Complexes of proteases with their natural inhibitors were elevated in patients with cancer. These data suggest that the contact system and intrinsic pathway activation are increased in patients with pancreatic cancer.

Severity of systemic inflammation is the main predictor of ACLF and bleeding in individuals with acutely decompensated cirrhosis.

BACKGROUND & AIMS: Hypercoagulability and hypofibrinolysis in acutely decompensated cirrhosis (AD) may be implicated in disease progression and haemostatic complications. We conducted a prospective study to: (1) characterise haemostatic alterations in AD; (2) evaluate whether such alterations can predict acute-on-chronic liver failure (ACLF) and bleeding/thrombosis. METHODS: Hospitalised individuals with AD were prospectively recruited and underwent an extensive haemostatic profiling including coagulation factors, thrombomodulin-modified thrombin generation assay with evaluation of endogenous thrombin potential (ETP; marker for plasmatic hypercoagulability), fibrinolytic factors, and plasmin-antiplasmin complex (fibrinolysis activation marker). Inflammation severity was assessed by C-reactive protein (CRP). In part 1 of the study, we compared haemostasis in AD vs. controls (stable decompensated and compensated cirrhosis). In part 2 of the study, we prospectively followed individuals with AD for 1 year and investigated predictors of ACLF and bleeding/thrombosis. RESULTS: A total of 169 individuals with AD were recruited (median model for end-stage liver disease score 20; CLIF-C AD 54). Compared with controls, AD was associated with more pronounced hypercoagulability (ETP: 871 vs. 750 vs. 605 nmol/L per min; p <0.0001), without differences in fibrinolysis activation. During follow-up, 55 individuals developed ACLF. CLIF-C AD, CRP, and Child-Pugh were independently associated with ACLF. A predictive model combining these variables (Padua model) accurately identified individuals at higher risk of ACLF (AUROC 0.857; 95% CI 0.798-0.915; sensitivity 74.5%, specificity 83.3%). Notably, CRP and progression to ACLF, but not baseline coagulopathy, were associated with bleeding (n = 11); CRP and antifibrinolytic factor PAI-1 >50 ng/ml were associated with thrombosis (n = 14). The prognostic value of the Padua model was validated in an independent, bicentric European cohort (N = 301). CONCLUSIONS: Inflammation severity, and not coagulopathy, is the most important predictor of ACLF and bleeding in AD. The Padua model can be used to identify individuals with AD at risk of ACLF. IMPACT AND IMPLICATIONS: A better understanding of haemostasis in individuals with acutely decompensated cirrhosis may help to identify those at higher risk of progression and complications. In this prospective study, we found no significant association between alterations of haemostasis and cirrhosis progression, indicating that the assessment of haemostatic alterations is not useful to identify those at risk. However, we found that C-reactive protein (a simple blood test that reflects severity of inflammation) and severity of chronic liver disease itself (as assessed by specific scores) were associated with cirrhosis progression and development of bleeding complications. Therefore, we developed a simple predictive model - based on C-reactive protein and liver disease scores - that, if validated by independent studies, could be used in clinical practice to assist physicians in identifying individuals with decompensated cirrhosis at higher risk of disease progression and death (i.e. in whom to consider an expedited evaluation for liver transplantation).

Global hemostatic profiling in patients with decompensated cirrhosis and bacterial infections.

Background & Aims: Bacterial infections in cirrhosis are associated with increased bleeding risk. To assess the factors responsible for bleeding tendency in patients with bacterial infections, we conducted a prospective study comparing all 3 aspects of hemostasis (platelets, coagulation, and fibrinolysis) in hospitalized patients with decompensated cirrhosis with vs. without bacterial infections. Methods: Primary hemostasis assessment included whole blood platelet aggregation and von Willebrand factor (VWF). Coagulation assessment included procoagulant factors (fibrinogen, factor II, V, VII, VIII, IX, X, XI, XII, XIII), natural anticoagulants (protein C, protein S, antithrombin) and thrombomodulin-modified thrombin generation test. Fibrinolysis assessment included fibrinolytic factors (plasminogen, t-PA, PAI-1, α2-AP, TAFIa/ai) and plasmin-antiplasmin complex (PAP). Results: Eighty patients with decompensated cirrhosis were included (40 with and 40 without bacterial infections). Severity of cirrhosis and platelet count were comparable between groups. At baseline, patients with cirrhosis and bacterial infections had significantly lower whole blood platelet aggregation, without significant differences in VWF. Regarding coagulation, bacterial infections were associated with reduced procoagulant factors VII and XII, and a significant reduction of all natural anticoagulants. However, thrombomodulin-modified thrombin generation was comparable between the study groups. Finally, although mixed potentially hypo-fibrinolytic (lower plasminogen) and hyper-fibrinolytic (higher t-PA) changes were present in bacterial infections, a comparable level of PAP was detected in both groups. Upon resolution of infection (n = 29/40), platelet aggregation further deteriorated whereas coagulation and fibrinolysis factors returned to levels observed in patients without bacterial infections. Conclusion: In hospitalized patients with decompensated cirrhosis, bacterial infections are associated with reduced whole blood platelet aggregation and a significant decrease of all natural anticoagulants, which may unbalance hemostasis and potentially increase the risk of both bleeding and thrombosis. Lay summary: Bacterial infections are a common issue in hospitalized patients with decompensated cirrhosis (i.e. patients hospitalized due to severe complications of advanced chronic liver disease). Patients with decompensated cirrhosis who acquire infections may be at increased risk of bleeding complications following invasive procedures (that is a procedure in which the body is penetrated or entered, for instance by a needle or a tube). As bleeding complications in decompensated cirrhosis are associated with a high risk of further decompensation and death, there is an urgent need to understand the factors responsible for such increased bleeding tendency. Herein, we investigated the alterations of hemostasis (that is the physiological process responsible for clot formation and stability) in patients with decompensated cirrhosis and bacterial infections. We found that development of bacterial infections in these patients is associated with alterations of hemostasis (particularly of platelets and clotting cascade) that may increase the risk of both bleeding and thrombotic complications.

Increased platelet aggregation in patients with decompensated cirrhosis indicates higher risk of further decompensation and death.

BACKGROUND & AIMS: Studies on platelet aggregation in cirrhosis are controversial because interpretation of platelet function is challenged by thrombocytopenia. We conducted a prospective study to investigate whole blood platelet aggregation in cirrhosis and its association with liver-related outcomes. METHODS: Platelet aggregation was assessed by whole blood aggregometry (Multiplate®). To overcome the influence of platelet count and compare cirrhosis with thrombocytopenia vs. controls with normal platelet count, we calculated a ratio between platelet aggregation and platelet count (PLT ratio). Then, we prospectively followed patients with cirrhosis and ascertained predictors of decompensation, transplantation, and death. RESULTS: Two-hundred and three patients with cirrhosis were prospectively recruited (77% decompensated). PLT ratio was significantly higher in cirrhosis than in those with chronic hepatitis and healthy individuals (0.44 vs. 0.25 and 0.26, respectively; p <0.0001). In cirrhosis, the ratio increased with disease severity (Child-Pugh class C>B>A) and was particularly elevated in decompensated patients with severe thrombocytopenia. Among decompensated patients, 65 had further decompensation, underwent transplantation, or died during a 6-month follow-up. On multivariate analysis, PLT ratio (odds ratio 1.87; 95% CI 1.23-2.84; p = 0.003) and MELD score (odds ratio 1.05; 95% CI 1.01-1.08; p = 0.01) were independently associated with outcome. The relative risk of events was 7.5-fold higher in patients with PLT ratio >0.75 vs. patients with PLT ratio <0.25 (95% CI 2.5-21.9; p = 0.003). The increased PLT ratio, its discriminative ability for composite outcome, and the prognostic value of PLT ratio >0.75 were confirmed in an independent cohort of hospitalized patients with decompensated cirrhosis (n = 41). CONCLUSIONS: Patients with cirrhosis, particularly when decompensated, exhibit significantly increased whole blood platelet aggregation. Decompensated patients with a PLT ratio >0.75 have a >80% probability of further decompensation, transplantation, or liver-related death within 6 months. LAY SUMMARY: In patients with cirrhosis, previous studies have suggested that platelets (i.e. circulating blood cells that help form clots to stop bleeding) are dysfunctional. In particular, these studies suggested that platelet aggregation (the process by which platelets adhere to each other to form clots) is reduced. Since platelet aggregation is important for clot formation, it has been hypothesized that alterations of platelet aggregation may be responsible for the increased risk of bleeding observed in patients with cirrhosis. Our study demonstrates: i) that platelet aggregation in patients with cirrhosis is higher than in healthy individuals; ii) that platelet aggregation in patients with decompensated cirrhosis (i.e. those who have already experienced some complications of cirrhosis) is particularly elevated and associated with risk of further complications and death.

Assessing Clinically Meaningful Hypercoagulability after COVID-19 Vaccination: A Longitudinal Study.

A large number of daily requests to exclude possible prothrombotic risk factors for coronavirus disease 2019 (COVID-19) vaccines were received. Our aim was to longitudinally evaluate coagulation profiles in a series of healthy subjects who received COVID-19 vaccination and assess hypercoagulability thereafter. Volunteers awaiting a first or second dose of either the ChAdOx1 or BNT162b2 vaccine were enrolled. Venous samples were obtained at baseline (before the vaccine) and longitudinally 3 ± 2 days (T1) and 10 ± 2 days after the vaccine (T2). Global coagulation monitoring was assessed via platelet count, whole blood thromboelastometry and impedance aggregometry, plasma thrombin generation, and anti-platelet factor 4 (PF4)/heparin immunoglobulin G antibodies. One hundred and twenty-two subjects were enrolled (61 [50%] ChAdOx1 and 61 BNT162b2). The ChAdOx1 cohort showed a slight but transient increase in thrombin generation (mainly endogenous thrombin potential [ETP] with thrombomodulin and ETP ratio) at T1, which promptly decreased at T2. In addition, the second dose of either vaccine was associated with increased thrombin peak, ETP with thrombomodulin, and ETP ratio. At baseline, 3.2% of the ChAdOx1 cohort and 1.6% BNT162b2 cohort were positive for PF4/heparin antibodies with a stable titer through T1 and T2. No relevant differences were detected in platelet count and aggregation, or thromboelastometry parameters. No thrombotic or hemorrhagic events occurred. We can confirm that no clinically meaningful hypercoagulability occurred after either vaccine, albeit keeping in mind that thrombin generation may increase in the first days after the second dose of either vaccine and after the first dose of the ChAdOx1 vaccine.

More Pronounced Hypercoagulable State and Hypofibrinolysis in Patients With Cirrhosis With Versus Without HCC.

In patients with cirrhosis, particularly those with hepatocellular carcinoma (HCC), hypercoagulability may be associated with purported increased risks of portal vein thrombosis and cirrhosis progression. In this study, we extensively investigated hemostatic alterations potentially responsible for the thrombotic tendency in HCC, and evaluated whether such alterations were predictive of hepatic decompensation. Patients with cirrhosis at all stages were prospectively recruited and underwent an extensive hemostatic assessment, including all procoagulant factors and inhibitors, thrombin generation with and without thrombomodulin (TG), profibrinolytic and antifibrinolytic factors, and plasmin-antiplasmin complex. In study part 1 (case control), we compared alterations of coagulation and fibrinolysis in patients with cirrhosis with versus without HCC. In study part 2 (prospective), the subgroup of patients with decompensated cirrhosis was followed for development of further decompensation, and predictors of outcome were assessed by multivariate analysis. One-hundred patients were recruited (50 each with and without HCC). Severity of cirrhosis was comparable between groups. Median HCC volume was 9 cm3 (range: 5-16). Compared with controls, patients with HCC demonstrated a significantly more prothrombotic hemostatic profile due to increased TG and reduced activation of fibrinolysis, independent of cirrhosis stage. During a median follow-up of 175 days, 20 patients with decompensated cirrhosis developed further episodes of decompensation that were predicted by low FVII and high plasminogen activator inhibitor-1 levels, independent of Model for End-Stage Liver Disease score. Conclusion: Patients with cirrhosis with HCC have profound hyper-coagulable changes that can account for their increased thrombotic tendency. In contrast, hypercoagulability in patients with decompensated cirrhosis is more likely a consequence of chronic liver disease rather than a driver for cirrhosis progression.

Coagulopathy is not predictive of bleeding in patients with acute decompensation of cirrhosis and acute-on-chronic liver failure.

BACKGROUND & AIMS: Understanding factors responsible for the increased bleeding tendency in acute-on-chronic liver failure (ACLF) would improve the management of these complications. We investigated coagulation alterations in ACLF and assessed whether they were predictive of bleeding. METHODS: Cirrhosis patients with ACLF (cases) and acute decompensation (AD, controls) were prospectively recruited and underwent an extensive haemostatic assessment including standard tests, pro and anticoagulant factors, thrombomodulin-modified thrombin generation (TG) and thromboelastometry (ROTEM® ). In study part 1 (case-control), we compared coagulation in ACLF vs AD. In study part 2 (prospective), all patients were followed for bleeding, and predictors of outcome were assessed. RESULTS: Ninety-one patients were included (51 with ACLF, 40 with AD). Infections and ascites/renal dysfunction were the most common precipitating and decompensating events. Platelet count was lower while INR and activated partial thrombin time were longer in ACLF cohort vs AD. Regarding clotting factors, fibrinogen and factor VIII were comparable between groups while protein C and antithrombin were significantly reduced in ACLF. Endogenous thrombin potential by TG was comparable between groups. Clotting formation time and clot stability by ROTEM® were significantly lower in ACLF, indicative of a more hypocoagulable state. No haemostasis alteration could discriminate between patients who had bleeding complications during hospitalization and those who did not. CONCLUSION: We found coagulation changes in ACLF to largely overlap with that of AD and evidence of preserved coagulation capacity in both groups. ROTEM alterations were indicative of a more pronounced hypocoagulable state in ACLF; however, no correlation was found between such alterations and bleeding.

Absence of hypercoagulability after nCoV-19 vaccination: An observational pilot study.

BACKGROUND: It is still unknown whether COVID-19 vaccines induce a prothrombotic state or increase the hypercoagulable condition in subjects with a predisposition to thrombosis. OBJECTIVES: We evaluated the coagulation profile in a series of healthy subjects who received the first dose of the BNT162b2 or the ChAdOx1 vaccines and assessed whether hypercoagulability developed. PATIENTS/METHODS: Volunteers among the staff of the University of Padua or health care professionals in the Padua University Hospital who had received either the ChAdOx1 or BNT162b2 vaccine in the previous 10 ± 2 days were eligible. A cohort of unvaccinated volunteers among family members of the University staff acted as control group. Global coagulation monitoring was assessed by whole blood rotational thromboelastometry, whole blood impedance aggregometry and thrombin generation. Platelet count was also obtained. RESULTS: One hundred and ninety subjects were enrolled: 101 (53.2%) received the ChAdOx1 vaccine and 89 (46.8%) the BNT162b2 vaccine. Twenty-eight non-vaccinated subjects acted as controls. Thromboelastometry parameters were all comparable among groups. Thrombin receptor activating peptide (TRAP)-, ADP- and ASPI-induced platelet aggregation were similar among groups, as well as platelet count. Endogenous thrombin potential (ETP) was comparable among groups. The results were confirmed after controlling for age, gender and hormonal. Considering women taking combined oral contraceptives or thrombophilia carriers, no differences were detected in thromboelastometry or thrombin generation parameters between subjects who received ChAdOx1 vs. BNT162b2 vaccines. CONCLUSIONS: No significant activation of fibrinogen-driven coagulation, plasma thrombin generation or clinically meaningful platelet aggregation after ChAdOx1 or BNT162b2 vaccination was observed.

Influence of Hepatocellular Carcinoma on Platelet Aggregation in Cirrhosis.

Hyper-functional platelets are being proposed as a potential therapeutic target in multiple cancers. Whether this can be considered in patients with cirrhosis and hepatocellular carcinoma (HCC) is unknown as their platelet function has not yet been investigated. We evaluated platelet function in cirrhosis patients with HCC. Patients with cirrhosis with and without HCC were prospectively recruited. Platelet aggregation, a marker of platelet function, was assessed by impedance aggregometry with adenosine diphosphate (ADP), arachidonic acid (ASPI), and thrombin (TRAP) stimulation. Plasmatic levels of Von Willebrand factor antigen (VWF) were also determined. One-hundred patients were recruited (50 cirrhotics with and 50 without HCC). Cirrhosis severity by Child class and platelet count were comparable between cirrhotics with and without HCC. Cirrhotics with HCC had higher ADP- (45 vs. 28; p < 0.001), ASPI- (47 vs. 28; p < 0.001), and TRAP- (85 vs. 75; p = 0.01) induced platelet aggregation than cirrhotics without HCC, all indicative of platelet hyper-function. The relatively increased platelet aggregation in patients with HCC was confirmed after adjusting the analysis for platelet count/severity of thrombocytopenia. Levels of VWF were higher in patients with vs. without HCC (348 vs. 267; p = 0.006), particularly in compensated cirrhosis. In patients with cirrhosis, HCC is associated with increased platelet aggregation and higher VWF. The clinical implications of these findings deserve further investigation.

The risk of arterial thrombosis in carriers of natural coagulation inhibitors: a prospective family cohort study.

BACKGROUND: Whether the carriership of inherited antithrombin (AT), protein C (PC), and protein S (PS) deficiency increases the risk of arterial thromboembolic events (ATE) is controversial. This information has the potential to inform the management of family members of probands with inherited deficiency of natural anticoagulants. PATIENTS/METHODS: We conducted a large prospective family cohort study in 640 subjects (of whom 341 carriers and 299 non-carriers) belonging to 86 families with inherited deficiency of AT, PC, or PS. RESULTS: A total of 4240 and 3810 patient-years were available for carriers and non-carriers, respectively. Risk factors for atherosclerosis were similarly distributed in the two groups. Of the 26 ATE that were recorded, 19 occurred in carriers (5.6%), as compared to 7 in non-carriers (2.3%) [p = 0.07]. After adjusting for confounders, the hazard ratio (HR) for ATE was 4.9 (95% CI 1.5-16.3) in carriers as compared to non-carriers. CONCLUSIONS: Among family members of probands with an inherited deficiency of natural anticoagulants, carriers exhibit a risk of ATE that is almost five times higher than in non-carriers.

Thrombin generation in patients with COVID-19 with and without thromboprophylaxis.

OBJECTIVES: Thrombin generation (TG) with and without thrombomodulin (TM) was evaluated in COVID-19 patients with different disease severity and thromboprophylaxis regimen, in order to understand the prothrombotic profile. METHODS: We enrolled consecutive patients with confirmed diagnosis of COVID-19 admitted to Medical Departments (MD) or Intensive Care Units (ICU), and 54 healthy controls. RESULTS: Eighty-nine patients were included (mean age 60.4±16.1 years, 68.5% male); 33.7% admitted to ICU. Twenty-four patients (26.9%) were enrolled before thromboprophylaxis administration; 45 patients (50.6%) received standard and 20 (22.5%) intermediate sub-therapeutic dose thromboprophylaxis. Overall, patients with COVID-19 showed a TG profile comparable to that of healthy subjects (i.e. comparable peak height, endogenous thrombin potential [ETP] with and without TM). The only exception was lag time and time to peak, prolonged in COVID-19 patients vs. controls. MD patients showed a similar TG profile to healthy controls, and ICU patients showed significantly decrease ETP (p=0.030) compared to MD. As for thromboprophylaxis, TG profile was significantly increased in COVID-19 patients without thromboprophylaxis vs. controls and vs. those with thromboprophylaxis. In this latter group, ETP inhibition was significantly decreased (p=0.0003) and positively correlated with anti-Xa activity (r=0.49, p=0.0017). However, patients with thromboprophylaxis had similar TG profile vs. controls. Intermediate dose thromboprophylaxis more effectively inhibited TG in severe COVID-19 patients by increasing ETP inhibition via ETP with TM reduction vs. standard dose. CONCLUSIONS: COVID-19 patients showed increased TG at diagnosis. Standard thromboprophylaxis reduced TG to levels of healthy controls. Intermediate sub-therapeutic thromboprophylaxis more effectively inhibited TG by decreasing ETP with TM.

Longitudinal Trend of Plasma Concentrations of Extracellular Vesicles in Patients Hospitalized for COVID-19.

Plasma concentrations of extracellular vesicles (EVs) originating from cells involved in COVID-19-associated coagulopathy (CAC), their longitudinal trend and association with clinical outcomes were evaluated. Blood samples of consecutive COVID-19 patients admitted to a medical Unit were longitudinally collected within 48 h of admission, at discharge and 30 days post-discharge. EVs were analyzed using high sensitivity flow cytometry and phospholipid-dependent clotting time (PPL). The following EVs were measured: endothelium-, platelet-, leukocyte-derived, bearing tissue factor (TF)+, angiotensin-converting enzyme (ACE2)+, platelet-derived growth factor receptor-ß (PDGF-ß)+ and SARS-CoV-2-nucleoprotein (NP)+. 91 patients were recruited for baseline EV analysis (mean age 67 ± 14 years, 50.5% male) and 48 underwent the longitudinal evaluation. From baseline to 30-days post-discharge, we observed significantly decreased plasma concentrations of endothelium-derived EVs (E-Selectin+), endothelium-derived bearing TF (E-Selectin+ TF+), endothelium-derived bearing ACE2 (E-Selectin+ACE2+) and leukocyte-EVs bearing TF (CD45+TF+), p < 0.001, p = 0.03, p = 0.001, p = 0.001, respectively. Conversely, platelet-derived (P-Selectin+) and leukocyte-derived EVs (CD45+) increased from baseline to 30-days post-discharge (p = 0.038 and 0.032, respectively). EVs TF+, ACE2+, PDGF-ß+, and SARS-CoV-2-NP+ did not significantly change during the monitoring. PPL increased from baseline to 30-days post-discharge (+ 6.3 s, p = 0.006). P-Selectin + EVs >1,054/µL were associated with thrombosis (p = 0.024), E-Selectin + EVs ≤531/µL with worsening/death (p 0.026) and 30-days P-Selectin+ and CD45 + EVs with persistent symptoms (p < 0.0001). We confirmed increased EVs originating from cells involved in CAC at admission and discharge. EVs derived from activated pericytes and expressing SARS-CoV-2-NP were also detected. 30-days post-discharge, endothelium-EVs decreased, while platelet- and leukocyte-EVs further increased, indicating that cellular activation persists long after the acute phase.

Noninvasive Risk Stratification After HCV Eradication in Patients With Advanced Chronic Liver Disease.

BACKGROUND AND AIMS: Risk stratification after cure from hepatitis C virus (HCV) infection remains a clinical challenge. We investigated the predictive value of noninvasive surrogates of portal hypertension (liver stiffness measurement [LSM] by vibration-controlled transient elastography and von Willebrand factor/platelet count ratio [VITRO]) for development of hepatic decompensation and hepatocellular carcinoma in patients with pretreatment advanced chronic liver disease (ACLD) who achieved HCV cure. APPROACH AND RESULTS: A total of 276 patients with pretreatment ACLD and information on pretreatment and posttreatment follow-up (FU)-LSM and FU-VITRO were followed for a median of 36.6 months after the end of interferon-free therapy. FU-LSM (area under the receiver operating characteristic curve [AUROC]: 0.875 [95% confidence interval [CI]: 0.796-0.954]) and FU-VITRO (AUROC: 0.925 [95% CI: 0.874-0.977]) showed an excellent predictive performance for hepatic decompensation. Both parameters provided incremental information and were significantly associated with hepatic decompensation in adjusted models. A previously proposed combined approach (FU-LSM < 12.4 kPa and/or FU-VITRO < 0.95) to rule out clinically significant portal hypertension (CSPH, hepatic venous pressure gradient ≥10 mm Hg) at FU assigned most (57.3%) of the patients to the low-risk group; none of these patients developed hepatic decompensation. In contrast, in patients in whom FU-CSPH was ruled in (FU-LSM > 25.3 kPa and/or FU-VITRO > 3.3; 25.0% of patients), the risk of hepatic decompensation at 3 years following treatment was high (17.4%). Patients within the diagnostic gray-zone for FU-CSPH (17.8% of patients) had a very low risk of hepatic decompensation during FU (2.6%). The prognostic value of this algorithm was validated in an internal (n = 86) and external (n = 162) cohort. CONCLUSION: FU-LSM/FU-VITRO are strongly and independently predictive of posttreatment hepatic decompensation in HCV-induced ACLD. An algorithm combining these noninvasive markers not only rules in or rules out FU-CSPH, but also identifies populations at negligible versus high risk for hepatic decompensation. FU-LSM/FU-VITRO are readily accessible and enable risk stratification after sustained virological response, and thus facilitate personalized management.

Partial F8 gene duplication (factor VIII Padua) associated with high factor VIII levels and familial thrombophilia.

High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.