Procoagulant changes in fibrin clot structure in patients with cirrhosis are associated with oxidative modifications of fibrinogen

Patients with cirrhosis have hemostatic changes, which may contribute to a risk of thrombosis. This in vitro study compares clot formation and structure between patients and healthy subjects. Clot formation is delayed in patients; ultimately, however, clot permeability is decreased. The thrombogenic structure of fibrin clots may contribute to the thrombotic risk in cirrhosis. Background and Objectives: Patients with cirrhosis can be at risk of thrombotic complications due to an imbalance between hemostatic components. However, little is known on how the disease affects clot generation or how alterations in the structure of fibrin clots may affect the hemostatic function of these patients. Methods: We investigated the formation and structure of clots generated with plasma and purified fibrinogen of 42 patients with cirrhosis. Clots generated with plasma and fibrinogen of 29 healthy volunteers were studied for comparison. Clot formation and structure were assessed by turbidity, permeation studies, confocal laser and scanning electron microscopy (SEM). The extent of fibrinogen oxidation was assessed by measuring the carbonyl content of purified fibrinogen samples. Results: Tissue factor and thrombin-induced clotting of plasma was delayed in patients. The clotting rate was also decreased, but change in turbidity, fibrin density and fiber thickness were largely comparable to healthy volunteers. Conversely, clot permeability was significantly decreased in patients. When clots were generated with purified fibrinogen, differences in clot formation and structure similar to those in plasma were found. The carbonyl content was increased in patient fibrinogen and correlated with disease severity and clot permeability. Conclusions: Delayed clot formation in cirrhosis ultimately results in decreased clot permeability. Similar alterations in clots generated with purified fibrinogen suggest that modifications of the molecule are (partly) responsible. Taken together, these findings are indicative of hypercoagulable features of clots of patients with cirrhosis, which may explain the increased risk of thrombosis associated with this condition

The international normalised ratio to monitor coagulation factor production during normothermic machine perfusion of human donor livers

Background: Normothermic machine perfusion (NMP) of donor livers allows for new diagnostic and therapeutic strategies. As the liver produces most of the haemostatic proteins, coagulation assays such as the International Normalised Ratio (INR) performed in perfusate may be useful to assess hepatocellular function of donor livers undergoing NMP. However, high concentrations of heparin and low levels of fibrinogen may affect coagulation assays.Methods: Thirty donor livers that underwent NMP were retrospectively included in this study, of which 18 were subsequently transplanted. We measured INRs in perfusate in presence or absence of exogenously added fibrinogen and/or polybrene. Additionally, we prospectively included 14 donor livers that underwent NMP (of which 11 were transplanted) and measured INR using both a laboratory coagulation analyser and a point-of-care device.Results: In untreated perfusate samples, the INR was above the detection limit in all donor livers. Addition of both fibrinogen and polybrene was required for adequate INR assessment. INRs decreased over time and detectable perfusate INR values were found in 17/18 donor livers at the end of NMP. INR results were similar between the coagulation analyser and the point-of-care device, but did not correlate with established hepatocellular viability criteria.Conclusions: Most of the donor livers that were transplanted showed a detectable perfusate INR at the end of NMP, but samples require processing to allow for INR measurements using laboratory coagulation analysers. Point-of-care devices bypass this need for processing. The INR does not correlate with established viability criteria and might therefore have additional predictive value.</p

Aggravation of fibrin deposition and microthrombus formation within the graft during kidney transplantation

In kidney transplantation, microthrombi and fibrin deposition may lead to local perfusion disorders and subsequently poor initial graft function. Microthrombi are often regarded as donor-derived. However, the incidence, time of development, and potential difference between living donor kidneys (LDK) and deceased donor kidneys(DDK), remains unclear. Two open-needle biopsies, taken at preimplantation and after reperfusion, were obtained from 17 LDK and 28 DDK transplanted between 2005 and 2008. Paraffin-embedded sections were immunohistochemically stained with anti-fibrinogen antibody. Fibrin deposition intensity in peritubular capillaries(PTC) and glomeruli was categorized as negative, weak, moderate or strong and the number of microthrombi/mm(2) was quantified. Reperfusion biopsies showed more fibrin deposition (20% to 100% moderate/strong, p < 0.001) and more microthrombi/mm(2) (0.97 ± 1.12 vs. 0.28 ± 0.53, p < 0.01) than preimplantation biopsies. In addition, more microthrombi/mm(2) (0.38 ± 0.61 vs. 0.09 ± 0.22, p = 0.02) and stronger fibrin intensity in glomeruli (28% vs. 0%, p < 0.01) and PTC (14% vs. 0%, p = 0.02) were observed in preimplantation DDK than LDK biopsies. After reperfusion, microthrombi/mm(2) were comparable (p = 0.23) for LDK (0.09 ± 0.22 to 0.76 ± 0.49, p = 0.03) and DDK (0.38 ± 0.61 to 0.90 ± 1.11, p = 0.07). Upon reperfusion, there is an aggravation of microthrombus formation and fibrin deposition within the graft. The prominent increase of microthrombi in LDK indicates that they are not merely donor-derived

Evidence for a rebalanced hemostatic system in pediatric liver transplantation:A prospective cohort study

In adults with end-stage liver disease concurrent changes in pro- and antihemostatic pathways result in a rebalanced hemostasis. Children though, have a developing hemostatic system, different disease etiologies, and increased risk of thrombosis. This study aimed to assess the hemostatic state of children during and after liver transplantation. Serial blood samples were obtained from 20 children (≤16 years) undergoing primary liver transplantation (September 2017-October 2018). Routine hemostasis tests, thrombomodulin-modified thrombin generation, clot lysis times, and hemostatic proteins were measured. Reference values were established using an age-matched control group of 30 children. Thrombocytopenia was present in study patients. Von Willebrand factors were doubled and ADAMTS13 levels decreased during and after transplantation up until day 30, when platelet count had normalized. Whereas prothrombin time and activated partial thromboplastin time were prolonged during transplantation, thrombin generation was within normal ranges, except during perioperative heparin administration. Fibrinogen, factor VIII levels, and clot lysis time were elevated up until day 30. In conclusion, children with end-stage liver disease are in tight hemostatic balance. During transplantation a temporary heparin-dependent hypocoagulable state is present, which rapidly converts to a hemostatic balance with distinct hypercoagulable features that persist until at least day 30. This hypercoagulable state may contribute to the risk of posttransplant thrombosis

Collagens are functional, high affinity ligands for the inhibitory immune receptor LAIR-1

Collagens are the most abundant proteins in the human body, important in maintenance of tissue structure and hemostasis. Here we report that collagens are high affinity ligands for the broadly expressed inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). The interaction is dependent on the conserved Gly-Pro-Hyp collagen repeats. Antibody cross-linking of LAIR-1 is known to inhibit immune cell function in vitro. We now show that collagens are functional ligands for LAIR-1 and directly inhibit immune cell activation in vitro. Thus far, all documented ligands for immune inhibitory receptors are membrane molecules, implying a regulatory role in cell–cell interaction. Our data reveal a novel mechanism of peripheral immune regulation by inhibitory immune receptors binding to extracellular matrix collagens

Extended hypothermic oxygenated machine perfusion enables ex situ preservation of porcine livers for up to 24 hours

Background & Aims: End-ischemic hypothermic oxygenated machine perfusion (HOPE) of the donor liver for 1-2 h mitigates ischemia-reperfusion injury during subsequent liver transplantation. Extended preservation time may be preferred to facilitate difficult recipient hepatectomy or to optimize logistics. We therefore investigated whether end-ischemic dual HOPE (DHOPE) could extend preservation time for up to 24 h using a porcine liver reperfusion model. Methods: Following 30 min warm ischemia, porcine livers were subjected to 2 h static cold storage (SCS), followed by 2 h, 6 h, or 24 h DHOPE (n = 6 per group). Subsequent normothermic reperfusion was performed for 4 h using autologous blood. Two livers preserved by 24 h SCS served as additional controls. A proof of principle confirmation was carried out in 2 discarded human livers subjected to extended DHOPE. Hepatocellular and cholangiocyte injury and function were assessed. Oxidative stress levels and histology were compared between groups. Results: Perfusion flows remained stable during DHOPE, regardless of duration. After normothermic reperfusion, livers perfused for 24 h by DHOPE had similar lactate clearance, blood pH, glucose, and alanine aminotransferase levels, and biliary pH, bicarbonate, and LDH levels, as livers perfused for 2 h and 6 h. Levels of malondialdehyde and high-mobility group box 1 in serum and liver parenchyma were similar for all groups. Histological analysis of bile ducts and liver parenchyma revealed no differences between the groups. Extended DHOPE in discarded human livers preserved hepatocellular and cholangiocyte function and histology after reperfusion. In contrast, livers preserved by 24 h SCS were non-functioning. Conclusion: Extended end-ischemic DHOPE enabled successful preservation of porcine and discarded human donor livers for up to 24 h. Extended DHOPE enables safe extension of preservation time, which may facilitate allocation and transplantation from a logistical perspective, and further expand the donor pool. Lay summary: It has been suggested that preserving liver grafts with a technique called (dual) hypothermic oxygenated machine perfusion ([D]HOPE) leads to better outcomes after transplantation than if livers are stored on ice, especially if an organ is of lesser quality. In this study, we showed that DHOPE could be used to preserve liver grafts for up to 24 h. This extended procedure could be used globally to facilitate transplantation and expand the donor pool

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

Nonmalignant portal vein thrombi in patients with cirrhosis consist of intimal fibrosis with or without a fibrin-rich thrombus

BACKGROUND AND AIM: Portal vein thrombosis (PVT) is a common complication of cirrhosis. The exact pathophysiology remains largely unknown, and treatment with anticoagulants does not lead to recanalization of the portal vein in all patients. A better insight into the structure and composition of portal vein thrombi may assist in developing strategies for the prevention and treatment of PVT. APPROACH AND RESULTS: Sixteen prospectively and 63 retrospectively collected nonmalignant portal vein thrombi from patients with cirrhosis who underwent liver transplantation were included. Histology, immunohistochemistry, and scanning electron microscopy were used to assess structure and composition of the thrombi. Most recent CT scans were reanalyzed for thrombus characteristics. Clinical characteristics were related to histological and radiological findings. All samples showed a thickened, fibrotic tunica intima. Fibrin-rich thrombi were present on top of the fibrotic intima in 9/16 prospective cases and in 21/63 retrospective cases. A minority of the fibrotic areas stained focally positive for fibrin/fibrinogen (16% of cases), von Willebrand factor (VWF; 10%), and CD61 (platelets, 21%), while most of the fibrin-rich areas stained positive for those markers (fibrin/fibrinogen, 100%; VWF, 77%; CD61, 100%). No associations were found between clinical characteristics including estimated thrombus age and use of anticoagulants and presence of fibrin-rich thrombi. CONCLUSION: We demonstrate that PVT in patients with cirrhosis consists of intimal fibrosis with an additional fibrin-rich thrombus in only one-third of cases. We hypothesize that our observations may explain why not all portal vein thrombi in patients with cirrhosis recanalize by anticoagulant therapy

In vitro effects of proteases in human pancreatic juice on stability of liquid and carrier-bound fibrin sealants

Background: Fibrin sealants are used in pancreatic surgery to prevent leakage of pancreatic fluid and reduce associated complications. The efficacy of this approach is unclear. Methods: Fibrin clots were generated in vitro from two commercially available liquid fibrin sealants (Tissucol Duo (R) and Evicel (R)) and the carrier-bound fibrin sealant Tachosil (R), and exposed to normal saline or human pancreatic fluid. Stability of the sealants was assessed by release of the fibrin and collagen degradation products, D-dimer and hydroxyproline. The effect of protease inhibitors on sealant breakdown was assessed. Results: Clots generated from liquid fibrin sealants degraded rapidly in pancreatic fluid, but not in normal saline. D-dimer release from fibrin clots by pancreatic fluid was approximately 1700 mu g/ml after 24 h and less than 20 mu g/ml by saline. Pancreatic fluid, but not normal saline, degraded both the fibrin and collagen component of Tachosil (R). After 6 h, mean(s.e.m.) D-dimer levels in pancreatic fluid exposed to Tachosil (R) were 850(183) ng/ml, compared with 60(6) ng/ml in normal saline. The mean(s.e.m.) hydroxyproline concentration in pancreatic fluid was 497(17) mu g/ml after a 24-h exposure to Tachosil (R), compared with 26(12) mu g/ml in normal saline. Protease inhibitors significantly inhibited breakdown of liquid sealants (D-dimer levels less than 50 mu g/ml after 24 h) and Tachosil (R) (D-dimer release 179(12) ng/ml at 6 h; hydroxyproline release 181(29) mu g/ml at 24 h). Conclusion: Proteases in pancreatic juice effectively degrade both liquid and carrier-bound fibrin sealants in vitro. The use of these products in pancreatic surgery with the aim of preventing leakage of pancreatic fluid is not supported by this experimental study

Normothermic Machine-perfused Human Donor Livers Produce Functional Hemostatic Proteins

BACKGROUND: Normothermic machine perfusion (NMP) is used for the viability assessment of high-risk donor livers before transplantation. The production of hemostatic proteins is one of the major synthetic functions of the liver. The objective of this study was to measure the concentration and functionality of hemostatic proteins concentration in the NMP perfusate of human donor livers.METHODS: Thirty-six livers that underwent NMP for viability assessment were included in this study. Perfusate samples taken during NMP (start, 150 min, and 300 min) were used for the measurement of antigen and activity levels of hemostatic proteins (factors II, VII, and X; fibrinogen; plasminogen; antithrombin; tissue plasminogen activator; von Willebrand factor; and proteins induced by vitamin K absence). The antigen levels were correlated with hepatocellular function according to previously proposed individual hepatocellular viability criteria: lactate clearance and perfusate pH.RESULTS: Antigen levels of hemostatic proteins reached subphysiological levels in the NMP perfusate. Hemostatic proteins that were produced during NMP were at least partially active. All livers produced all hemostatic proteins tested within 150 min of NMP. Hemostatic protein concentrations did not significantly correlate with perfusate lactate and perfusate pH after 150 min of NMP.CONCLUSIONS: All livers produce functional hemostatic proteins during NMP. The generation of a functional hemostatic system in NMP perfusate confirms the need for adequate anticoagulation of the perfusate to avoid generation of (micro)thrombi that may harm the graft.</p