Mechanisms of enhanced thrombin generating capacity in patients with cirrhosis.

The liver is the site of synthesis of many proteins involved in hemostasis including pro- and anticoagulant proteins, pro- and antifibrinolytic proteins, and thrombopoietin. Consequently, patients with liver disease acquire complex changes within their hemostatic system[1]. Historically, these changes were thought to result in a bleeding diathesis. Although bleeding complications are common in patients with chronic liver disease, the most common bleeding complication, variceal bleeding, is a consequence of portal hypertension, rather than hemostatic failure

Haemostatic effects of recombinant coagulation factor VIIa

Recombinant coagulation factor VIIa (rFVIIa) has recently become available for treatment of patients with inhibitor-complicated haemophilia. It has been postulated that rFVIIa could become a universal haemostatic agent. Case reports and small studies confirm efficacy and safety of rFVIIa in a variety of haemostatic disorders including liver disease, platelet-related bleeding disorders, and in patients without prior coagulopathy who are bleeding excessively due to trauma or surgery. Although clinical evidence for efficacy and safety of rFVIIa in its various applications is rapidly accumulating, very little is know about the molecular mechanisms by which rFVIIa induces haemostasis. In this thesis, novel hypotheses on the mechanism of action of rFVIIa in different haemostatic disorders are described. In the major part of this thesis, the focus is on the involvement of thrombin activatable fibrinolysis inhibitor (TAFI)-mediated down-regulation of fibrinolysis in in vitro systems representing various haemostatic disorders, and the effect of rFVIIa. In chapter 2, it has been described that rFVIIa, next to its procoagulant properties, possesses antifibrinolyic properties in plasma from patients with severe haemophilia A. This antifibrinolytic effect was due to enhanced activation of TAFI. Chapter 3 describes the complex haemostatic disorder induced by liver disease, and in chapter 4 the role of TAFI-mediated downregulation of fibrinolysis in liver disease was investigated. In contrast to earlier reports, we did not find evidence of hyperfibrinolysis in liver disease, and also TAFI activation appeared normal, despite decreased plasma levels of TAFI antigen. In chapter 5, the effect of rFVIIa on coagulation and fibrinolysis in plasma from patients with liver disease are described. In contrast to the situation in haemophilia, rFVIIa did not display an antifibrinolytic effect in plasma from patients with liver disease. In contrast to the haemophilia situation, TAFI activation is already optimal in plasma from these patients. In chapter 6, the effect of anticoagulants targeting different steps in the coagulation cascade on coagulation and fibrinolysis were examined. It was found that anticoagulant drugs targeting factor Xa, but not those inhibiting thrombin or tissue factor accelerated fibrinolysis in vitro. In chapter 6, the effects of rFVIIa on coagulation and fibrinolysis of plasma anticoagulated with fondaparinux (a novel anticoagulant, specifically directed against factor Xa) were investigated. rFVIIa was able to completely reverse the effects of fondaparinux on coagulation, and was partially able to reverse the induction of hyperfibrinolysis by the drug. In chapter 8, the focus was on the effects of rFVIIa on platelet adhesion under flow conditions. More specifically, a model for the mechanism of action of rFVIIa in patients with Glanzmanns thrombasthenia, and patients treated with anti-aIIbB3 drugs is proposed based on results of in vitro perfusion studies. It was shown that tissue factor-indepdent thrombin generation via rFVIIa restored the defective platelet deposition induced by aIIbB3-deficiency. In chapter 9, the results presented in the previous chapters are discussed in a broader context. The advances in the understanding of the mechanism of action of rFVIIa in its different applications are discussed

Extracellular vesicles from amniotic fluid, milk, saliva, and urine expose complexes of tissue factor and activated factor VII

Background Tissue factor (TF) is expressed in the adventitia of the vessel wall and on extracellular vesicles (EVs) in body fluids. TF and activated coagulation factor (F) VII(a) together form the so-called extrinsic tenase complex, which initiates coagulation. Aim We investigated whether EVs in amniotic fluid, milk, saliva, and urine expose functional extrinsic tenase complexes that can trigger coagulation. Methods Milk, saliva, and urine were collected from healthy breastfeeding women (n = 6), and amniotic fluid was collected from healthy women undergoing routine amniocentesis (n = 7). EVs were isolated from body fluids by size exclusion chromatography (SEC) and clotting experiments were performed in the presence and absence of antibodies against TF and FVIIa in normal plasma and in FVII-deficient plasma. The ability of body fluids to generate FXa also was determined. Results Amniotic fluid, milk, saliva, and urine triggered clotting of normal plasma and of FVII-deficient plasma, which was almost completely inhibited by an anti-FVII antibody and to a lesser extent by an anti-TF antibody. Fractionation of body fluids by SEC showed that only the fractions containing EVs triggered clotting in normal plasma and FVII-deficient plasma and generated FXa, which again was almost completely inhibited by an anti-FVII antibody and partially by an anti-TF antibody. Conclusion Here we show that EVs from amniotic fluid, milk, saliva, and urine expose complexes of TF and FVIIa (i.e., extrinsic tenase complexes) that directly activate FX. Based on our present findings we propose that these EVs from normal body fluids provide hemostatic protection

The impact of ABO blood type on the prevalence of portal vein thrombosis in patients with advanced chronic liver disease

Background and aimsNon-O blood type (BT) is a risk factor for thromboses, which has been attributed to its effects on von Willebrand factor (VWF)/factor VIII (FVIII) levels. Although high VWF/FVIII may be risk factors for portal vein thrombosis (PVT) in patients with advanced chronic liver disease (ACLD), the impact of BT on PVT is unknown. We aimed to assess (I) whether non-O-BT is a risk factor for PVT and (II) whether non-O-BT impacts VWF/factor VIII in patients with ACLD. MethodsRetrospective analysis comprising two cohorts: (I) "US" including all adult liver transplantations in the US in the MELD era and (II) "Vienna" comprising patients with a hepatic venous pressure gradient (HVPG) >= 6 mmHg. Results(I) The "US cohort" included 84 947 patients (non-O: 55.43%). The prevalence of PVT at the time of listing (4.37% vs 4.56%; P = .1762) and at liver transplantation (9.56% vs 9.33%; P = .2546) was similar in patients with O- and non-O-BT. (II) 411 patients were included in the "Vienna cohort" (non-O: 64%). Mean HVPG was 18(9) mmHg and 90% had an HVPG >= 10 mmHg. Patients with non-O-BT had slightly increased VWF levels (318(164)% vs 309(176)%; P = .048; increase of 23.8%-23.9% in adjusted analyses), but this difference was driven by patients with less advanced disease. However, non-O-BT explained only 1% of the variation in VWF and had no effect on FVIII. ConclusionsAlthough non-O-BT impacts VWF in patients with early stage ACLD, its contribution to VWF variation is considerably smaller than in the general population. Moreover, non-O-BT had no impact on FVIII. These findings may explain the absence of an association between non-O-BT and PVT in patients with advanced cirrhosis

Intraperitoneal Activation of Coagulation and Fibrinolysis in Patients with Cirrhosis and Ascites

Development of ascites is the most common form of decompensation of cirrhosis. We aimed to investigate the coagulation system in ascitic fluid and plasma of patients with cirrhosis. We determined coagulation parameters and performed clotting and fibrinolysis experiments in ascitic fluid and plasma of thoroughly characterized patients with cirrhosis and ascites ( n  = 25) and in plasma of patients with cirrhosis but without ascites ( n  = 25), matched for severity of portal hypertension. We also investigated plasma D-dimer levels in an independent cohort of patients ( n  = 317) with clinically significant portal hypertension (HVPG ≥ 10 mmHg), grouped according to ascites severity. Ascitic fluid was procoagulant in a clotting assay. The procoagulant potential of ascitic fluid was abolished by depletion of extracellular vesicles from ascitic fluid by filtration or by addition of a tissue factor-neutralizing antibody. Compared with plasma, extracellular vesicle-associated tissue factor activity was high in ascitic fluid, while activities of other coagulation factors were low. The extracellular vesicle-depleted fraction of ascitic fluid induced fibrinolysis, which was prevented by aprotinin, indicating the presence of plasmin in ascitic fluid. Plasma peak thrombin generation and parameters reflecting fibrinolysis were independently associated with the presence of ascites. Finally, plasma D-dimer levels were independently linked to ascites severity in our second cohort comprising 317 patients. In conclusion, coagulation and fibrinolysis become activated in ascites of patients with cirrhosis. While tissue factor-exposing extracellular vesicles in ascitic fluid seem unable to pass the peritoneal membrane, fibrinolytic enzymes get activated in ascitic fluid and may re-enter the systemic circulation and induce systemic fibrinolysis

Controlled DCD Liver Transplantation Is Not Associated With Increased Hyperfibrinolysis and Blood Loss After Graft Reperfusion

BACKGROUND: The specific effect of donation after circulatory death (DCD) liver grafts on fibrinolysis, blood loss, and transfusion requirements after graft reperfusion is not well known. The aim of this study was to determine whether transplantation of controlled DCD livers is associated with an elevated risk of hyper-fibrinolysis, increased blood loss and higher transfusion requirements upon graft reperfusion, compared to livers donated after brain death (DBD). METHODS: A retrospective single-center analysis of all adult recipients of a primary liver transplantation between 2000 and 2019 was performed (total cohort n= 628). Propensity score matching (PSM) was used to balance baseline characteristics for DCD and DBD liver recipients (PSM cohort n= 218). Intra- and postoperative hemostatic variables between DCD and DBD liver recipients were subsequently compared. Additionally, in vitro plasma analyses were performed to compare the intraoperative fibrinolytic state upon reperfusion. RESULTS: No significant differences in median (interquartile range) postreperfusion blood loss (1.2 L [0.5-2.2] vs 1.3 L (0.6-2.2); P= 0.62), RBC transfusion (2 units [0-4) vs 1.1 units [0-3], P= 0.21), or FFP transfusion requirements (0 units [0-2.2] vs 0 units (0-0.9); P= 0.11) were seen in DCD compared to DBD recipients, respectively. Furthermore, plasma fibrinolytic potential was similar in both groups. CONCLUSIONS: Transplantation of controlled DCD liver grafts does not result in higher intraoperative blood loss or more transfusion requirements, compared to DBD liver transplantation. In accordance to this, no evidence for increased hyper-fibrinolysis upon reperfusion in DCD compared to DBD liver grafts, was found

Use of Laboratory Markers in Addition to Symptoms for Diagnosis of Inflammatory Bowel Disease in Children:A Meta-analysis of Individual Patient Data

IMPORTANCE: Blood markers and fecal calprotectin are used in the diagnostic workup for inflammatory bowel disease (IBD) in pediatric patients. Any added diagnostic value of these laboratory markers remains unclear.OBJECTIVE: To determine whether adding laboratory markers to evaluation of signs and symptoms improves accuracy when diagnosing pediatric IBD.DATA SOURCES: A literature search of MEDLINE and EMBASE from inception through September 26, 2016. Studies were identified using indexing terms and free-text words related to child, target condition IBD, and diagnostic accuracy.STUDY SELECTION: Two reviewers independently selected studies evaluating the diagnostic accuracy of more than 1 blood marker or fecal calprotectin for IBD, confirmed by endoscopy and histopathology or clinical follow-up, in pediatric patients with chronic gastrointestinal symptoms. Studies that included healthy controls and/or patients with known IBD were excluded.DATA EXTRACTION AND SYNTHESIS: Individual patient data from each eligible study were requested from the authors. In addition, 2 reviewers independently assessed quality with Quality Assessment of Diagnostic Accuracy Studies-2.MEAN OUTCOMES AND MEASURES: Laboratory markers were added as a single test to a basic prediction model based on symptoms. Outcome measures were improvement of discrimination by adding markers as a single test and improvement of risk classification of pediatric patients by adding the best marker.RESULTS: Of the 16 eligible studies, authors of 8 studies (n = 1120 patients) provided their data sets. All blood markers and fecal calprotectin individually significantly improved the discrimination between pediatric patients with and those without IBD, when added to evaluation of symptoms. The best marker-fecal calprotectin-improved the area under the curve of symptoms by 0.26 (95% CI, 0.21-0.31). The second best marker-erythrocyte sedimentation rate-improved the area under the curve of symptoms by 0.16 (95% CI, 0.11-0.21). When fecal calprotectin was added to the model, the proportion of patients without IBD correctly classified as low risk of IBD increased from 33% to 91%. The proportion of patients with IBD incorrectly classified as low risk of IBD decreased from 16% to 9%. The proportion of the total number of patients assigned to the intermediate-risk category decreased from 55% to 6%.CONCLUSIONS AND RELEVANCE: In a hospital setting, fecal calprotectin added the most diagnostic value to symptoms compared with blood markers. Adding fecal calprotectin to the diagnostic workup of pediatric patients with symptoms suggestive of IBD considerably decreased the number of patients in the group in whom challenges in clinical decision making are most prevalent.</p

Detection and localization of early- and late-stage cancers using platelet RNA

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I–IV cancer patients and in half of 352 stage I–III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening

Hemostatic and Non-hemostatic Functions of Platelets in Patients with Liver Disease

Chronic and acute liver diseases may be accompanied by substantial changes in primary hemostasis. Thrombocytopenia develops as a consequence of defective platelet production, increased consumption, and/or increased splenic sequestration. In addition, ill-defined platelet function defects may further impair primary hemostasis. However, defects in platelet number and function appear (partly) compensated for by increased levels of von Willebrand factor (VWF) and decreased circulating ADAMTS13. Consequently, patients with liver diseases may have a relatively well-functioning primary hemostatic system, which does not necessarily necessitate prophylactic transfusion of platelet concentrates, for example, prior to invasive procedures. Thrombopoietin receptor agonists are able to increase the platelet count, but normalization of the platelet count in patients with cirrhosis may lead to a thrombotic risk due to the high plasma levels of von Willebrand factor. The platelet count is a reasonable diagnostic and prognostic tool in patients with liver diseases. Platelets also have functions unrelated to cessation of bleeding in the context of liver disease. By incompletely understood mechanisms, platelets contribute to regeneration of the liver, for example, following a partial hepatectomy. In animal models of chronic or acute liver failure, platelets have been shown to exert both detrimental and beneficial effects. Whether platelets can both stimulate and inhibit progression of liver disease depending on the exact clinical context requires further study