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.

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.

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.

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.

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.

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.

Perioperative coagulation assessment of patients undergoing major elective orthopedic surgery.

Traditional coagulative parameters are of limited use in identifying perioperative coagulopathy occurring in patients undergoing major elective orthopedic surgery (MEOS). The aim of our study was to evaluate the coagulation changes in patients undergoing MEOS and to facilitate an early detection of perioperative coagulopathy in patients experiencing major intraoperative bleeding. We enrolled 40 consecutive patients (M/F 10/30, age range 34-90 years) who underwent MEOS at the Orthopedic Unit of the Padua University Hospital, Italy, between January 2014 and January 2015. Blood samples were obtained at the following time points: T0-pre: 30 min before surgery; T0-post: 30 min after the end of the procedure; T1: morning of the first postoperative day; T2: 7 ± 2 days after surgery. Patients who experienced an intraoperative blood loss ≥250 mL/h were considered as cases. Routine coagulative parameters, thromboelastometry and thrombin generation (TG) profiles were evaluated. At baseline, a significantly lower platelet count and FIBTEM MCF/AUC were observed in patents with excessive bleeding (p < 0.05 and 0.02/0.01, respectively). At T0-post and T1 intervals, cases showed hypocoagulation characterized by a significantly low platelet count (p = 0.001), prolonged CFT INTEM/EXTEM, reduction of alpha-angle and MaxV INTEM/EXTEM, MCF and AUC INTEM/EXTEM/FIBTEM (p < 0.05 in all comparisons). The only TG parameter standing out between study groups was time to peak at T0-pre. A low platelet count and fibrinogen activity were associated with significant intraoperative bleeding in patients undergoing MEOS. Thromboelastometry performed by ROTEM(®) identifies patients with coagulopathy.

Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity.

Microparticles are now considered as critical effectors involved in numerous biological processes (coagulation, inflammation, and vascular biology). Microparticle can be measured using a quantitative and descriptive approach by flow cytometry (FCT) or by a functional approach assessing microparticle biological activities by a factor Xa-based clotting assay. FCT can be used to determine the cellular origin of the different microparticles, although there are concerns about the detection limit of this approach. Functional assays measure only the procoagulant activity of isolated microparticles and give no information on the cellular source or the physical properties of the microparticles. The advantage of the functional assays is that the assays use well defined reagents, and they are readily automated with high sensitivity and simplicity. In this study, we analyzed samples from 60 patients with active cancer of different type, 60 patients with a BMI more than 25 kg/m, and 49 carriers of Factor V Leiden with and without a prior venous thromboembolic episode. The study showed a significant correlation (P < 0.05) between microparticles determined by annexin V-microparticle-based FCT and a procoagulant activity-based clotting assay. This indicates that the procoagulant assay could be used as a routine screening test to screen out the normal samples so that only the abnormal samples need further testing by FCT.

Does decreased fibrinolysis have a role to play in the development of non-neoplastic portal vein thrombosis in patients with hepatic cirrhosis?

Hepatic cirrhosis is characterized by complex abnormalities of the fibrinolytic system. Little is known about the possible association between these alterations and thrombosis. The aim of this study was to evaluate the fibrinolytic profile in cirrhotic individuals with and without portal vein thrombosis (PVT). We measured thrombin activatable fibrinolysis inhibitor (TAFI), total amount of activated TAFI (TAFIa/ai), plasminogen activator inhibitor (PAI-1), plasminogen and fibrinogen plasma levels in 66 cirrhotic patients (33 with and 33 without PVT) and in 66 healthy volunteers. TAFI plasma levels (median [range]) were significantly lower in cirrhotic individuals (5.6 µg/ml [1.7-11.7]) than in controls (10.1 µg/ml [6.6-14.2], p < 0.0001), while TAFIa/ai levels were significantly higher in cases (18.3 ng/ml [0.3-35.4]) than in controls (15.9 ng/ml [7.4-41], p = 0.02). Cirrhotic patients with PVT had higher TAFI (6.6 µg/ml [2.9-10.1]), TAFIa/ai (19.2 ng/ml [11.6-35.4]) and PAI-1 (33.1 ng/ml [27.6-56.3]) plasma levels than those without PVT (3.9 µg/ml [1.7-11.7], p = 0.001; 15.6 ng/ml [10.3-33.9], p = 0.037; 15.9 ng/ml [2.5-29.1], p = 0.004. The fibrinolytic profile in cirrhotic individuals with PVT is characterized by higher levels of TAFI, TAFIa/ai and PAI-1 than in those without PVT. These alterations identify a hypofibrinolytic condition that may increase the risk of developing a thrombotic event.

Thromboelastographic evaluation of coagulative profiles in pig-to-monkey kidney xenotransplantation.

BACKGROUND: Activation of the clotting cascade is central in acute xenograft rejection (AHXR) that occurs when pig organs are transplanted into primates. The coagulopathy reported in this model is a very complex process that involves simultaneously coagulation factors, platelets and phospholipid-bearing cells (i.e., leukocytes, red blood cells, and endothelial cells). Choosing whole blood for coagulation analysis theoretically appears more favorable compared with plasma. Whole blood rotation thromboelastometry (ROTEM(®) ) is a point-of-care global coagulation analyzer able to evaluate the characteristics of clot formation and lysis by dynamic monitoring. The aim of this study was to record thromboelastographic profiles, performed by ROTEM(®) , in a series of immunosuppressed nephrectomized primates that received a life-supporting kidney. METHODS: Of the eight primates, n = 4 received a pig kidney transgenic for human decay-accelerating factor (hDAF/Gal+); n = 2, an α 1,3-galactosyltransferase gene-knockout (GT-KO) pig kidney transgenic for human CD39, CD55, CD59 and fucosyltransferase (HTF); and n = 2, a GT-KO pig kidney transgenic for hDAF. Blood samples were collected before and at least once per week after transplantation till euthanasia. Intrinsic (INTEM) and extrinsic (EXTEM) coagulation pathways and the function of fibrinogen (FIBTEM) were evaluated. Thromboelastographic parameters considered were clotting time (CT, seconds) and clot formation time (CFT, seconds) in INTEM and EXTEM and maximum clot firmness (MCF, mm) in FIBTEM. The correlations between CT in INTEM and activated partial thromboplastin time (aPTT), CT in EXTEM and PT, CFT in INTEM and EXTEM, and platelet counts and MCF in FIBTEM and fibrinogen plasma levels were also considered. RESULTS: In all animals, thromboelastographic profiles showed progressive prolongation of CT (activation of coagulative cascade) in INTEM. A close correspondence was observed between (i) the prolongation of the CFT values (propagation of clot formation), both in INTEM and EXTEM, and the decrease in platelet counts; (ii) the reduction in MCF values (clot firmness) ​​in FIBTEM and the decrease in fibrinogen plasma levels. No concordance between CT in INTEM and aPTT and between CT in EXTEM and PT was observed. CONCLUSIONS: Our study demonstrated that ROTEM(®) analyzer could be a useful and complementary tool to study the consumptive coagulopathy, either "compensated" or "non-compensated," that takes place when transgenic pig kidneys are transplanted into primates. Larger and prospective studies are needed to confirm our results and to evaluate the role of ROTEM(®) to guide the management of consumptive coagulopathy in order to prolong the survival of the transplanted organ.

Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism.

BACKGROUND: Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Microparticles (MPs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play a role in VTE, although evidence of this association is still limited. OBJECTIVES: To evaluate the hypothesis that elevated numbers of endothelial (EMPs), platelets (PMPs), and Tissue Factor-bearing MPs (TF(+)MPs) in plasma may contribute to cancer-associated thrombosis. PATIENTS/METHODS: EMPs, PMPs and TF(+)MPs plasma levels were measured in 90 consecutive patients (cases) referred to our Department (30 with a first episode of unprovoked VTE; 30 with active cancer; 30 with a diagnosis of acute VTE associated with active cancer), and in a group of 90 healthy subjects (controls). MPs analyses were performed by flow-cytometry (Cytomics FC500). RESULTS: Cases showed statistically significant higher (mean ± SD) circulating EMPs and PMPs plasma levels (920 ± 341 and 1221 ± 413 MP/µL, respectively) than controls (299 ± 102 and 495 ± 241 MP/µL; p<0.005). Moreover cancer patients (with and without VTE) showed higher (mean ± SD) TF(+)MPs (927 ± 415 MPs/µL) than controls (204 ± 112 MPs/µL; p<0.001). The subgroup of cancer patients plus VTE showed statistically significant higher TF(+)MPs plasma levels (1019 ± 656 MPs/µL) than cancer patients without VTE (755 ± 391 MPs/µL, p = 0.002). Multivariate analysis failed to show a significant association between elevated TF(+)MPs and VTE in cancer patients. CONCLUSIONS: Our results suggest that MPs might be an important intermediate in the cascade of cellular injury and vascular dysfunctions underlying the process of thrombosis, particularly in cancer. Further clinical investigations are needed to confirm the precise role of MPs in predicting hypercoagulable state in patients with cancer.

In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM-3, in the xenogeneic pig-to-primate context.

BACKGROUND: Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia-reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. METHODS: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 microm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-alpha) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v. to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. RESULTS: At concentrations <500 microm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. CONCLUSIONS: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs.