Plasma Fibrinolytic Potential Research Articles

Exploring Coagulation and Fibrinolysis Activation As Mechanisms of Sepsis-Related Tissue Damage in Patients with Severe Neutropenia and Thrombocytopenia Secondary to Hematological Malignancies

Sepsis is a life-threatening end-organ dysfunction resulting from dysregulated host responses to infection with physiopathology not fully understood. Yet, it has been long recognized that platelets and neutrophil extracellular traps collaborate to promote secondary tissue damage. Interestingly, patients with hematological malignancies and febrile neutropenia (FN) secondary to chemotherapy, with neutrophils and platelet counts frequently around zero, develop sepsis and septic shock, raising questions about which mechanisms are involved. Here, we show that extracellular vesicles (EVs) and activation of the fibrinolytic system are two potential mediators of tissue damage in sepsis among patients with very low platelets and neutrophil counts. The study population consisted of patients with hematologic malignancies who underwent antineoplastic treatment and that presented neutrophil counts < 500/μl and temperature ≥ 37.5 °C. Sepsis was defined as an increase of at least 2 points in the Sequential Organ Failure Assessment (SOFA) score and septic shock was defined by the need for vasoactive drugs after adequate volume infusion. Samples were obtained after FN diagnosis, at sepsis onset, and 72 h thereafter. A control group of patients with hematological malignancies and chemotherapy-induced neutropenia without fever was also included. Forty-two subjects, both genders, aged from 21 to 71 years were included and categorized into 4 groups, referred to: FN (n=18), FN + sepsis (n=10), FN + sepsis 72h (n=9), and the control group (n=18). The clinical and laboratory characteristics of these groups are described in Table 1. We first evaluated whether the release of EVs could be associated with the development of sepsis in these patients. EVs were characterized and enumerated from platelet-poor plasma (PPP) obtained from all groups and then separated through high-speed centrifugation (30 min, 20,000 g, 4ºC). EVs were marked with CD146 and 144 for endothelial cells; CD66b for granulocytes; CD14 and CD68 for monocytes, CD41 for platelets, and CD142 for tissue factor (TF) expression. Lactadherin and calcein were used to identify EVs according to size and integrity. Samples were acquired in a flow cytometer, Cytoflex S, with 30 μL/min flow rate and calibration microbeads were used to identify EV of specific sizes (Rosetta). The main findings are shown in Table 2. The main results were an increase in monocyte-derived EVs in patients with FN after sepsis onset (FN + sepsis 72h), compared to all other groups (p=0.01). Moreover, the expression of TF + was higher in these EVs (FN + sepsis 72h) when compared to EVs obtained at sepsis onset (FN + sepsis): 0.50 (IQR 0.29 - 1.10) events/μL versus 0.28 (IQR 0.12 - 0.56) in FN + Sepsis (p=0.03). Additionally, an increase in endothelial (CD146 +) and granulocyte-derived EVs were observed at the same time-point compared to FN and the control group. Of note, monocyte-derived EVs counts were not correlated with monocyte cell counts (Rs= 0.03; p=0.94). Given that coagulation activation plays a role in the immunothrombotic cascade known to mediate tissue damage in sepsis, we also explored whether thrombin and plasmin generation were increased in these patients. Briefly, the assay used PPP and two independent fluorometric substrates for thrombin (Boc-Val-Pro-Arg-MCA) and plasmin (Boc-Glu-Lys-Lys-MCA) in a 96-well plate, lately initiated by the addition of TF (10 pM). The plate was read in a fluorometric spectrophotometer for 4 h. Results are shown in Table 2, and no differences were observed in thrombin generation. However, the area under the curve of plasmin generation, which could be interpreted as a proxy of the plasma fibrinolytic potential, was lower in both FN + sepsis and FN + sepsis 72 h when compared to FN and the control group. Our findings reveal that EVs might present a role in the pathogenesis of sepsis in a scenario where cells known to be critical to this process are virtually absent. The increase of TF + EVs from monocytes, which has been demonstrated in classical sepsis, seems to be relevant even in this context, warranting additional studies using functional assays to further explore this mechanism of tissue damage. Our results also demonstrate that hypofibrinolysis is a characteristic of FN-associated sepsis, suggesting that deregulation of the fibrinolytic system is at least in part independent of the participation of granulocytes and platelets in these patients.

Formation of denser plasma fibrin clots is associated with coronary artery ectasias and aneurysms - preliminary results from the CARE-ANEURYSM study

Abstract Background Coronary artery ectasias and aneurysms (CAEAs) are defined as diffused or localized coronary artery dilations, that exceed diameter of the adjacent segment by at least 50%. In the Caucasian population, CAEAs are present in 2-5% of all-comer patients undergoing coronary angiography and are associated with increased risk of myocardial infarction and death. Purpose To determine whether CAEAs are associated with increased thrombin generation potential and unfavorably modified plasma fibrin clot properties. Methods In this prospective study 19 patients with CAEAs, 19 sex-, age-, fibrinogen- and diabetes mellitus-matched patients with coronary artery disease (CAD) but without CAEAs, and 20 healthy control subjects were assessed. Plasma fibrin clot permeability (Ks) reflecting an average pore size within fibrin network, plasma fibrinolytic potential expressed as clot lysis time (CLT), and thrombin generation capacity defined as endogenous thrombin potential (ETP) were assessed. The CARE-ANEURYSM study, founded by the Polish National Science Centre (no. UMO-2020/39/O/NZ5/02863), was approved by the Ethical Committee and conducted in accordance with the Declaration of Helsinki. Patients provided written informed consent. Results As many as 84.2% of CAEA patients were men, mean age was 69.6 years, and 73.7% individuals had CAD. On average, CAEA affected 3 coronary segments and exceeded vessels referral diameter by 2.15-fold. Patients with CAEA had 46.5% reduced Ks (median, 3.98 [3.30-4.63] vs 7.43 [6.15-8.88] x10-9cm2, p<0.001), a tendency to prolonged CLT (112 [103-116.5] vs 99 [89-114] min, p=0.07), and 51% higher ETP (1685 [1502-1786] vs 1119 [1025-1189] nM×min, p<0.001) compared to healthy control (Fig. 1A-C). Moreover, individuals with CAEA compared to CAD patients had 19.5% lower Ks (median 3.98 [3.30-4.63] vs 5.33 [4.86-6.78] x10-9cm2, p=0.016) and 21% longer CLT (112 [103-116.50] vs 92.52 [81.50-97.50] min, p=0.015) and (1685 [1502-1786] vs 1119 [1693-1966] nM×min, p<0.001) (Fig. 1A-C). In the CAEA group, Ks was associated with the CAEA maximal diameter (R=-0.595, p=0.007) and the ratio of CAEA maximal diameter to referral vessel diameter (R=0.743, p<0.001; Figu.1D). There were no associations between the number of coronary artery segments affected by the lesion and fibrin clot properties or ETP. However, CAEA patients in whom the ratio of the CAEA maximal diameter to the referral vessel diameter exceed 2 (n=9) had 24.5% lower Ks (3.45 [1.88-3.68] vs 4.57 [4.08-5.35] x10-9cm2, p=0.004) and 14.7% higher ETP (1779 [1685-1812] vs 1515 [1431-1674] nM×min, p=0.028) with no difference in CLT (p>0.05). Conclusion CAEAs are associated with denser fibrin clot structure and enhanced thrombin generation, which indicate that CAEA patients are characterized by prothrombotic state. Further studies are needed to elucidate whether prothrombotic clot phenotype may be associated with increased risk of thromboembolic events in CAEA patients.Figure 1.

Atorvastatin downregulates plasma procarboxypeptidase U concentrations and improves fibrinolytic potential dose-dependently in hyperlipidemic individuals

BackgroundStatins efficiently lower cholesterol and also exert pleiotropic effects that extend beyond lipid lowering. In a recent pilot study, valuable information on the carboxypeptidase U (CPU) system in hyperlipidemia and the effect of statin therapy was collected. It was shown that proCPU levels are increased in hyperlipidemic patients. Statins significantly decreased proCPU levels and improved plasma fibrinolysis. Furthermore, it was suggested that patients with high baseline proCPU levels are most likely to benefit from statin therapy. ObjectivesWe aimed to further substantiate the effect of hyperlipidemia and statin therapy on CPU-related parameters in a larger cohort of hyperlipidemic and statin-treated individuals. MethodsBlood was collected from 141 individuals treated with different dosages of atorvastatin (10-80 mg), 38 normolipidemic, and 37 hyperlipidemic controls. Lipid parameters and markers of fibrinolysis (proCPU and clot lysis time) were determined and compared between the groups. ResultsPilot study results of high proCPU concentrations in hyperlipidemic patients and the proCPU-reducing effect of atorvastatin were confirmed. Accordingly, an improvement in plasma fibrinolytic potential was seen under the influence of atorvastatin. High interindividual variation in proCPU concentrations was observed in the hyperlipidemic cohort, with up to 80% higher proCPU levels compared with normolipidemic controls. Furthermore, proCPU concentration and the dosage of atorvastatin were inversely correlated. ConclusionsThis study clearly shows that plasma proCPU concentrations and its expected effect on the fibrinolytic rate (as measured by clot lysis time) are increased in hyperlipidemic patients and that these effects can be normalized (and even further reduced compared with normolipidemic patients) by atorvastatin treatment.

Congruent identification of imbalanced fibrinolysis by 2 distinct clot lysis time assays

BackgroundA plasma-based clot lysis time (CLT) assay is an established research test to assess plasma fibrinolytic potential, with application in hyperfibrinolytic or hypofibrinolytic conditions. Interprotocol variations make comparisons between laboratories challenging. The aim of this study was to compare the results of 2 different CLT assays performed by 2 distinct research laboratories by using their own protocol. MethodsWe evaluated fibrinolysis in the plasma of 60 patients undergoing hepatobiliary surgery and in plasma from a healthy donor that was spiked with commonly used anticoagulant drugs (enoxaparin, dabigatran, and rivaroxaban) in 2 distinct laboratories (Aarhus and Groningen) by using 2 different assays that differ, among others, in tissue plasminogen activator (tPA) concentration. ResultsOverall conclusions on fibrinolytic potential in patients undergoing hepatobiliary surgery were similar between the 2 CLT assays, with hyperfibrinolytic and hypofibrinolytic profiles identified at the same time points during and after surgery. Severe hypofibrinolysis was less commonly reported in the Aarhus assay (36/319 samples; 11%) than in the Groningen assay (55/319 samples; 17%). No clot formation was observed in 31 of 319 samples in the Aarhus assay vs 0 of 319 samples in the Groningen assay. Clotting times increased much more profoundly on the addition of all 3 anticoagulants in the Aarhus assay. ConclusionsDespite the differences in laboratory, protocol, reagents, operator, data processing, and analysis, overall conclusions on fibrinolytic capacity are similar between the 2 laboratories. With a higher concentration of tPA in the Aarhus assay, the test becomes less sensitive for the detection of hypofibrinolysis and is more sensitive to the addition of anticoagulants.

Chronic prothrombotic tendency in patients with granulomatosis with polyangiitis.

Patients with granulomatosis with polyangiitis (GPA) show increased tendency toward thromboembolic phenomena in exacerbation of their disease. The aim of the study was to evaluate thrombin generation potential and fibrinolytic plasma activity in patients with GPA, both in exacerbation and in remission. We included 38 patients with GPA: 18 with exacerbated GPA and 20 in remission. The control group included 39 healthy participants matched for age and sex. Plasma thrombogenic potential was assessed using calibrated automated thrombography. Plasma fibrinolytic potential was assessed using clot lysis time (CLT). We also measured levels of inflammatory markers, thrombomodulin, and fibrinolysis proteins in all participants. In the whole group of patients with GPA, endogenous thrombin potential was higher by about 25% (P <0.001), while CLT was lower by about 20% (P = 0.02) when compared with controls. The endogenous thrombin potential was higher, the CLT lower, and the levels of thrombomodulin and inflammation markers (C‑reactive protein, fibrinogen, factor VIII) higher both in patients with exacerbation and in remission than in the control group; no such differences were noted when comparing those with exacerbation and those in remission, however. The only parameter that differentiated patients with GPA exacerbation from those in remission was the D‑dimer level (median [interquartile range], 1151 [597.2-2468.7] ng/ml vs 340.4 [255.1-500.7] ng/ml; P <0.001), a marker of lysis of intravascularly formed fibrin. Patients with GPA show an increased prothrombotic state, regardless of the disease phase. This is probably related to ongoing low-grade inflammation and endothelial injury. Large clinical studies are required to address the need for, and appropriate type of, antithrombotic prophylaxis during the course of GPA.

Effect of Statin Therapy on the Carboxypeptidase U (CPU, TAFIa, CPB2) System in Patients With Hyperlipidemia: A Proof-of-concept Observational Study

Statins are commonly used in patients with hypercholesterolemia to lower their cholesterol levels and to reduce their cardiovascular risk. There is also considerable evidence that statins possess a range of cholesterol-independent effects, including profibrinolytic properties. This pilot study aimed to explore the influence of statins on procarboxypeptidase U (proCPU) biology and to search for possible effects and associations that can be followed up in a larger study. Blood was collected from 16 patients with hyperlipidemia, before and after 3 months of statin therapy (simvastatin 20 mg or atorvastatin 20 mg). Fifteen age-matched normolipemic persons served as control subjects. Lipid parameters and markers of inflammation and fibrinolysis (proCPU levels and clot lysis times) were determined in all samples. Mean (SD) proCPU levels were significantly higher in patients with hypercholesterolemia compared to control subjects (1186 [189] U/L vs 1061 [60] U/L). Treatment of these patients with a statin led to a significant average decrease of 11.6% in proCPU levels and brought the proCPU concentrations to the same level as in the control subjects. On a functional level, enhancement in plasma fibrinolytic potential was observed in the statin group, with the largest improvement in fibrinolysis seen in patients with the highest baseline proCPU levels and largest proCPU decrease upon statin treatment. Increased proCPU levels are present in patients with hyperlipidemia. Statin treatment significantly decreased proCPU levels and improved plasma fibrinolysis in these patients. Moreover, our study indicates that patients with high baseline proCPU levels are most likely to benefit from statin therapy. The latter should be examined further in a large cohort.

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

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 hyperfibrinolysis, increased blood loss, and higher transfusion requirements upon graft reperfusion, compared with livers donated after brain death (DBD). A retrospective single-center analysis of all adult recipients of primary liver transplantation between 2000 and 2019 was performed (total cohort n = 628). Propensity score matching was used to balance baseline characteristics for DCD and DBD liver recipients (propensity score matching cohort n = 218). Intraoperative 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. No significant differences in median (interquartile range) postreperfusion blood loss (1.2 L [0.5-2.2] versus 1.3 L [0.6-2.2]; P = 0.62), red blood cell transfusion (2 units [0-4] versus 1.1 units [0-3]; P = 0.21), or fresh frozen plasma transfusion requirements (0 unit [0-2.2] versus 0 unit [0-0.9]; P = 0.11) were seen in DCD compared with DBD recipients, respectively. Furthermore, plasma fibrinolytic potential was similar in both groups. Transplantation of controlled DCD liver grafts does not result in higher intraoperative blood loss or more transfusion requirements, compared with DBD liver transplantation. In accordance with this, no evidence for increased hyperfibrinolysis upon reperfusion in DCD compared with DBD liver grafts was found.

Rosuvastatin use increases plasma fibrinolytic potential: a randomised clinical trial.

We conducted a study to assess the effect of rosuvastatin use on fibrinolysis in patients with previous venous thromboembolism (VTE). This was a post hoc analysis within the STAtins Reduce Thrombophilia (START) study (NCT01613794). Plasma fibrinolytic potential, fibrinogen, plasmin inhibitor, plasminogen activator inhibitor‐1 (PAI‐1) and thrombin‐activatable fibrinolysis inhibitor (TAFI) were measured before and after four weeks of rosuvastatin or no treatment in participants with prior confirmed VTE, after ending anticoagulant therapy. In the non‐rosuvastatin group (n = 121), plasma fibrinolytic potential and individual fibrinolysis parameters did not change at the end of the study versus the baseline, whereas in the rosuvastatin group (n = 126), plasma fibrinolytic potential increased: the mean clot lysis time decreased by 8·75 min (95% CI −13·8 to −3·72), and plasmin inhibitor levels and TAFI activity were lower at the end of the study (−0·05 U/ml; 95% CI −0·07 to −0·02 and −4·77%; 95% CI −6·81 to −2·73, respectively). PAI‐1 levels did not change and fibrinogen levels were 0·17 g/l (95% CI 0·04–0·29) higher. In participants with prior VTE, rosuvastatin use led to an increased fibrinolytic potential compared with non‐statin use. Our findings support the need for further studies on the possible role for statins in the secondary prevention of VTE.

Global Fibrinolysis Analysed By Clofal Method Is Not Associated with Post-Thrombotic Syndrome or Presence of Residual Vein Thrombosis in Patients with DVT in the Chronic Phase

Introduction: Deep venous thrombosis (DVT) is a common disease and post-thrombotic syndrome (PTS) is a complication present in about 20-50% of patients after the thrombotic episode (Kahn, 2016 - Hematology Am Soc Hematol Educ Program). The presence of residual venous thrombosis (RVT) can contribute to DVT recurrence and its role in SPT is controversial (Pradoni et al., 2009 - Annals of Internal Medicine; Cosmi et al., 2010 - Eur J Vasc Endovasc Surg). Fibrinolysis is the mechanism responsible for blood clot dissolution and decreased plasma fibrinolytic potential (DPFP), leads to an increased risk of DVT and appears to be associated with PTS, but not with recurrence(Lisman, 2017 - Semin Thromb Hemost; Meltzer et al., 2008 - PLoS Med). Furthermore, hypercoagulability and hypofibrinolysis synergistically enhance the risk of a first venous event, driven probably by elevated plasma levels of TAFI and PAI-1(Bombardier et al., 2012 - Thromb Res; Lisman, 2017 - Semin Thromb Hemost). Relationship between fibrinolysis and DVT has been focused on plasma levels of proteins or protease-inhibitor complexes, and not on plasma fibrinolytic potential (PFP). CloFAL is a turbidimetric assay that calculates the coagulation index (CI) and fibrinolytic index (FI), and allows the evaluation of global fibrinolysis based on clot lysis, with good analytical sensitivity for hypercoagulability and hypofibrinolysis. Preliminary studies of our group demonstrated decreased median fibrinolytic index in patients with lower limb DVT when compared to controls (98% vs 146%, p = 0.003). With the objective to increase scientific knowledge, we evaluated PFP in DVT patients and its association with PTS, RVT, and PAI-1 levels. Methods: PFP was determined by CloFAL assay and PAI-1 by ELISA method. The reference values of CI and FI were calculated using the median values obtained in plasma samples of 20 healthy subjects, along with the percentiles 25th and 75th (for CI, median was 104%, interquartile range (IQR) = 70-140%; for F, median = 97%; IQR = 80-180%). PTS and RVT were evaluated by Villalta scale and doppler ultrasound (US), respectively. Outpatients with DVT of lower limbs up to two years after the acute episode attended at Hemostasis Clinic of Hemocentro (University of Campinas - UNICAMP, SP Brazil) from 2014 to 2019 were included. Healthy controls were workers from Hemocentro. Exclusion criteria were cancer, anticoagulation, renal or hepatic disease, and infection. Continuous variables among groups were compared with Mann-Whitney test. A multiple regression analysis was performed by to evaluate the effect of age, sex, type of thrombosis (spontaneous or provoked), PAI-1, RVT, time between DVT and blood collection and CI on the observed values of FI. Results: Seventy-eight patients and 42 controls were included. The samples were collected 16 months (range = 1-27) after acute DVT. Forty-three (55%) patients presented PTS and 41 (71%) showed RVT on US. Our results corroborate our previous study, as patients demonstrated a significant increase of CI (p = 0.0009) and decrease of FI (p = 0.0009) (Table 1). However, when patients were analyzed according to the presence of SPT or RVT, no difference was observed. Median PAI-1 levels corrected by BMI were higher in patients compared to controls (8.5 vs 4.8, p&lt; 0.0001). No difference was observed in PAI-1 levels when we compared patients according to SPT and RVT status. When multiple variables were analyzed with a multiple regression analysis, only CI statistically significantly predicted FI (R2= 0.278; p=0.003). Conclusions: Patients with DVT, even in the chronic phase, presented hypofibrinolysis, when compared to controls. We initially found higher PAI-1 levels in patients when compared to controls but with a multivariate analysis, CI was the only variable that predicted FI. Therefore, other factors not included in this study may contribute to the lower FI values observed in our cohort. The presence of RVT or PTS did not induce modifications of global fibrinolysis. Disclosures No relevant conflicts of interest to declare.

Mixed Fibrinolytic Phenotypes in Decompensated Cirrhosis and Acute-on-Chronic Liver Failure with Hypofibrinolysis in Those With Complications and Poor Survival.

Background and AimsPatients with liver disease acquire complex changes in their hemostatic system, which results in a fragile rebalanced status. The status of the fibrinolytic system is controversial, as is the role of fibrinolytic dysfunction in bleeding and thrombosis in patients with cirrhosis. Here, we aimed to determine fibrinolytic status and its relationship with outcome in acutely ill patients with cirrhosis.Approach and ResultsWe assessed plasma fibrinolytic potential in a large cohort of patients with acutely decompensated cirrhosis (AD, n = 52) or acute‐on‐chronic liver failure (ACLF, n = 57). Compared with 40 healthy volunteers, median clot lysis times (CLTs) were shorter in patients with AD but comparable to controls in patients with ACLF. However, the variability in CLTs in patients was much larger than in healthy controls, and in both patient groups, a proportion of patients had clearly prolonged or shortened CLTs. The variability in CLTs in patients was not readily explained by variations in plasma levels of key fibrinolytic proteins. However, CLTs were clearly related to clinical characteristics, with longer CLTs in patients with sepsis and patients with any organ failure (as defined by the European Foundation for the Study of Chronic Liver Disease organ failure scores). CLTs were not different between patients that did or did not experience bleeding or a thrombotic event during follow‐up. Baseline CLTs were substantially longer in patients that died within 30 days of admission.ConclusionsOur study demonstrates a mixed fibrinolytic phenotype in acutely ill patients with cirrhosis with baseline hypofibrinolysis associated with sepsis, organ failure, and short‐term mortality. These associations may be explained by defective clearance of intraorgan microthrombi that have been proposed to drive organ failure.

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

Preserved hemostatic status in patients with non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is associated with an increased risk of thrombosis. However, it remains unclear if hypercoagulability contributes to this risk. We, therefore, determined an in-depth hemostatic profile in a cohort of well-defined patients with NAFLD. We drew blood samples from 68 patients with biopsy-proven NAFLD (simple steatosis n=24, NASH n=22, and NASH cirrhosis n=22), 30 lean controls, 30 overweight controls (body mass index (BMI) >25kg/m2), and 15 patients with alcoholic (ASH) cirrhosis, and performed in-depth hemostatic profiling. Basal and agonist-induced platelet activation, plasma levels of markers of platelet activation, and plasma levels of the platelet adhesion regulators von Willebrand factor and ADAMTS13 were comparable between patients with non-cirrhotic NAFLD and controls. Agonist-induced platelet activation was decreased in patients with cirrhosis. Thrombomodulin-modified thrombin generation was comparable between all patients and controls, although patients with cirrhosis had a reduced anticoagulant response to thrombomodulin. Thromboelastography test results were comparable between controls and non-cirrhotic NAFLD patients, but revealed moderate hypocoagulability in cirrhosis. Plasma fibrinolytic potential was decreased in overweight controls and non-cirrhotic NAFLD, but accelerated fibrinolysis was observed in ASH cirrhosis. Clot permeability was decreased in overweight controls and patients with NAFLD. The overall hemostatic profile is comparable between patients with non-cirrhotic NAFLD and controls. Additionally, pro-thrombotic features (hypofibrinolysis and a pro-thrombotic structure of fibrin clot) in patients with NAFLD are likely driven by obesity. Our study suggests a limited role for hyperactive hemostasis in the increased thrombotic risk in NAFLD. The combined results of this study show that the overall hemostatic status is comparable between healthy individuals and patients with a fatty liver disease.

A sustained decrease in plasma fibrinolytic potential following partial liver resection or pancreas resection

BackgroundPatients undergoing partial hepatectomy have a substantial risk for postoperative venous thrombosis even in the presence of optimal thromboprophylaxis. Recently we demonstrated a hypercoagulable state following a partial hepatectomy which was related to decreased plasma levels of natural anticoagulants and elevated levels of FVIII. The fibrinolytic status following partial hepatectomy has not been studied, but may display unique features as a result of temporarily decreased synthesis of fibrinolytic proteins. MethodsWe included 17 patients undergoing a partial hepatectomy and determined plasma fibrinolytic potential and measured plasma levels of individual fibrinolytic proteins in serial samples taken perioperatively. Results were compared to ten patients undergoing pancreas resection and twenty-four healthy volunteers. Results and conclusionFollowing both partial hepatectomy and pancreas resection plasma fibrinolytic potential decreased at the end of surgery, normalized on post-operative day 1, and decreased again on post-operative day 3 after which the hypofibrinolytic state gradually resolved. The hypofibrinolytic state on day 1 associated with increased plasma levels of PAI-1 in both groups. Plasma levels of plasminogen, α2-antiplasmin and TAFI all decreased following partial hepatectomy and pancreas resection and levels recovered over time. The kinetics of recovery were different for the different proteins and were slower in the liver resection group, resulting in a unique ratio of pro-to-anti-fibrinolytic proteins at each time point. This may explain the hypofibrinolytic status from day 3 onwards. A sustained plasma hypofibrinolytic state in combination with the hypercoagulable state we previously identified may contribute to the increased risk of thrombotic complications after partial liver resection.

Epicatechin Impact on Primary Hemostasis, Coagulation and Fibrinolysis

IntroductionCardiovascular diseases are the leading causes of death worldwide. Among cardiovascular risk factors, some are linked to the stages of hemostasis : primary haemostasis, coagulation and fibrinolysis. There is a growing interest on the potential beneficial effects of some nutritional components on cardiovascular health, in particular in the field of thrombosis. Dark chocolate is associated with a one-third reduction in the risk of cardiovascular disease. Cocoa is particularly rich in a flavonoid subclass called flavan-3-ol which includes epicatechin. Epicatechin is a micronutrient found mainly in chocolate, green tea and grape. It has already been proven that epicatechin impacts platelet functions but there are only poor data on the role of epicatechin on coagulation and fibrinolysis. The aim of this study was to evaluate the impact of epicatechin in vitro on the different stages of hemostasis.Patients and methodsThe study was performed on 10 major healthy subjects. Exclusion criteria were antiplatelet or anticoagulant therapy intake, clinical history of thrombosis or bleeding, thrombocytopenia (platelet count < 150 G/L) and abnormal coagulation profile (fibrinogen < 1.8 g/L, Prothrombin time > 13 s, and activated partial thromboplastin time > 39 s).Platelets agregometry were performed according the ISTH recommendations with (final concentrations) : ADP (2 µmol/L), epinephrin (5 mmol/L), collagen (2 µg/ml), arachidonic acid (1 mmol/L) (Helena Laboratories, Beaumont, USA), thromboxan A2 analog, U46619 (1 mmol/L) (Merck, Darmstadt, Germany), Thrombin Receptor Activating peptide (TRAP, 10 µmol/L) (Roche Rotkreuz, Switzerland). Area under curve at 4 minutes and maximal agregation were the main parametres analysed.Thrombin generation assays (TGA) were performed with PPP reagent low (phospholipids (4 µmol/L) and tissue factor (TF, 1 pmol/L); Stago, Asnières sur Seine, France) on platelet free plasma (PFP). The platelets procoagulant activity was evaluated with PRP reagent (TF 1 pmol/L) (Stago) on platelets rich plasma (PRP) and microparticles procoagulant activity with PRP reagent on plasma rich in microparticles (without platelets). After activation by Ca ionophore ??Fibrinolysis was evaluated by a clot lysis assay determined by measurements of optical densitometry at 405 nm overtime. Coagulation was initiated by PPP reagent (Stago) sensibilised by an activator of plasminogen (t-PA, actylise®, Boehringer, France ; 200 ng/mL, final concentration). The plasma fibrinolytic potential was assessed by the clot lysis time (CTL), defined as the time from the midpoint of the baseline to maximum turbid transition, to the final plateau phase.In each of the experiments plasmas were used pure or supplemented with a range of (-)-epicatechin (Extrasynthese; Lyon, France) concentration (final concentrations of 0, 1, 10 and 100 mM).All ethical considerations were obtained.Results and discussion5 women and 5 men were included in this study, with a median age of 33 years (Q1 - Q3) (28-38).Epicatechin 100 µmol/L inhibits platelet aggregation induced by ADP and collagen with maximal agregation of 45 % versus 73 % in control (p< 0.001) and 67 % versus 79 % in control (p < 0.05) respectively. Epicatechin 10 µmol/L impact coagulation and decrease the TGA parameters such as peak or endogenous thrombin potential with 1345 (1161 - 1563) nM.min versus 1503 (1262 - 1728) nM.min (p<0.05) in control. Epicatechin did not seem to interact significantly on the platelet procoagulant activity. No impact of epicatechin was found in microparticles procoagulant activity assessed by TGA.Epicatechin play a profibrinolytic role as evaluated by a clot lysis assay : the CLT with epicatechin 100 µmol/L was significantly lower than without epicatechin with 11,0 (8,0 - 16,0) min versus 15,0 (9,5 - 20,8) min in control (p < 0.01).ConclusionEpicatechin seems to play a role in each stage of haemostasis. The impact on platelets functions is highlighted by lower maximal agregation induced by ADP and collagen. The coagulation is slightly decreased as shown by TGA on PFP. Epicatechin also impairs fibrinolysis evaluated with a clot lysis assay. Further in vivo studies are needed to confirm these results. DisclosuresNo relevant conflicts of interest to declare.

Abnormal hemostatic function one year after orthotopic liver transplantation can be fully attributed to endothelial cell activation

The long-term risk of thrombotic and vascular complications is elevated in liver transplant recipients compared to the general population. Patients with cirrhosis are in a hypercoagulable status during and directly after orthotopic liver transplantation, but it is unclear whether this hypercoagulability persists over time. We aimed to investigate the hemostatic status of liver transplant recipients one year after transplantation. We prospectively collected blood samples of 15 patients with a functioning graft one year after orthotopic liver transplantation and compared the hemostatic status of these patients with that of 30 healthy individuals. Patients one year after liver transplantation had significantly elevated plasma levels of von Willebrand factor (VWF). Thrombin generation, as assessed by the endogenous thrombin potential, was decreased in patients, which was associated with increased plasma levels of the natural anticoagulants antithrombin and tissue factor pathway inhibitor. Plasma fibrinolytic potential was significantly decreased in patients and correlated inversely with levels of plasminogen activator inhibitor-1. One year after liver transplantation, liver graft recipients have a dysregulated hemostatic system characterised by elevation of plasma levels of endothelial-derived proteins. Increased levels of von Willebrand factor and decreased fibrinolytic potential may (in part) be responsible for the increased risk for vascular disease seen in liver transplant recipients.

Abnormal hemostatic function one year after orthotopic liver transplantation can be fully attributed to endothelial cell activation

Background: The long-term risk of thrombotic and vascular complications is elevated in liver transplant recipients compared to the general population. Patients with cirrhosis are in a hypercoagulable status during and directly after orthotopic liver transplantation, but it is unclear whether this hypercoagulability persists over time. Aim: We aimed to investigate the hemostatic status of liver transplant recipients one year after transplantation. Methods: We prospectively collected blood samples of 15 patients with a functioning graft one year after orthotopic liver transplantation and compared the hemostatic status of these patients with that of 30 healthy individuals. Results: Patients one year after liver transplantation had significantly elevated plasma levels of von Willebrand factor (VWF). Thrombin generation, as assessed by the endogenous thrombin potential, was decreased in patients, which was associated with increased plasma levels of the natural anticoagulants antithrombin and tissue factor pathway inhibitor. Plasma fibrinolytic potential was significantly decreased in patients and correlated inversely with levels of plasminogen activator inhibitor-1. Conclusion: One year after liver transplantation, liver graft recipients have a dysregulated hemostatic system characterised by elevation of plasma levels of endothelial-derived proteins. Increased levels of von Willebrand factor and decreased fibrinolytic potential may (in part) be responsible for the increased risk for vascular disease seen in liver transplant recipients.

Abnormal hemostatic function one year after orthotopic liver transplantation can be fully attributed to endothelial cell activation

Background: The long-term risk of thrombotic and vascular complications is elevated in liver transplant recipients compared to the general population. Patients with cirrhosis are in a hypercoagulable status during and directly after orthotopic liver transplantation, but it is unclear whether this hypercoagulability persists over time. Aim: We aimed to investigate the hemostatic status of liver transplant recipients one year after transplantation. Methods: We prospectively collected blood samples of 15 patients with a functioning graft one year after orthotopic liver transplantation and compared the hemostatic status of these patients with that of 30 healthy individuals. Results: Patients one year after liver transplantation had significantly elevated plasma levels of von Willebrand factor (VWF). Thrombin generation, as assessed by the endogenous thrombin potential, was decreased in patients, which was associated with increased plasma levels of the natural anticoagulants antithrombin and tissue factor pathway inhibitor. &nbsp;Plasma fibrinolytic potential was significantly decreased in patients and correlated inversely with levels of plasminogen activator inhibitor-1. Conclusion: One year after liver transplantation, liver graft recipients have a dysregulated hemostatic system characterised by elevation of plasma levels of endothelial-derived proteins. Increased levels of von Willebrand factor and decreased fibrinolytic potential may (in part) be responsible for the increased risk for vascular disease seen in liver transplant recipients.

Abnormal hemostatic function one year after orthotopic liver transplantation can be fully attributed to endothelial cell activation

Background: The long-term risk of thrombotic and vascular complications is elevated in liver transplant recipients compared to the general population. Patients with cirrhosis are in a hypercoagulable status during and directly after orthotopic liver transplantation, but it is unclear whether this hypercoagulability persists over time.Aim: We aimed to investigate the hemostatic status of liver transplant recipients one year after transplantation.Methods: We prospectively collected blood samples of 15 patients with a functioning graft one year after orthotopic liver transplantation and compared the hemostatic status of these patients with that of 30 healthy individuals.Results: Patients one year after liver transplantation had significantly elevated plasma levels of von Willebrand factor (VWF). Thrombin generation, as assessed by the endogenous thrombin potential, was decreased in patients, which was associated with increased plasma levels of the natural anticoagulants antithrombin and tissue factor pathway inhibitor. Plasma fibrinolytic potential was significantly decreased in patients and correlated inversely with levels of plasminogen activator inhibitor-1.Conclusion: One year after liver transplantation, liver graft recipients have a dysregulated hemostatic system characterised by elevation of plasma levels of endothelial-derived proteins. Increased levels of von Willebrand factor and decreased fibrinolytic potential may (in part) be responsible for the increased risk for vascular disease seen in liver transplant recipients.

Hyperhomocysteinemia in patients with pulmonary embolism is associated with impaired plasma fibrinolytic capacity

Hyperhomocysteinemia (HHcy) affects haemostasis and shifts its balance in favour of thrombosis. In vitro and in vivo studies suggested that HHcy may impair fibrinolysis either by influencing the plasma levels of fibrinolytic factors or by altering the fibrinogen structure. We investigated the influence of mild HHcy levels on plasma fibrinolytic potential by using clot lysis time (CLT) and fibrin susceptibility to plasmin-induced lysis in 94 patients with previous pulmonary embolism and no pulmonary hypertension. CLT was measured as lysis time of tissue factor induced clots exposed to exogenous tissue plasminogen activator (t-PA). The rate of in vitro plasmin-mediated cleavage of fibrin β-chain was assessed over a 6-h period on fibrin clots, which were obtained by exposition to thrombin of purified fibrinogen. Homocysteine plasma levels were measured by Abbott Imx immunoassay and we considered as altered the values above 15 μmol/L according to the literature. In 68 patients homocysteine levels were below 15 μmol/L (NHcy) and in 26 they were above (HHcy). Significant differences were observed between the two groups regarding plasma fibrinolytic potential (p = 0.016), TAFIact (expressed as clot lysis ratio) (p = 0.02), t-PA (0.008) and PLG (0.037), but not for the other assessed components. The HHcy-patients had a threefold higher risk to have an impaired fibrinolysis. Instead, a multivariate logistic regression analysis adjusted for significances of univariate showed that HHcy (OR 5.2 95% CI 1.7-15.9; p = 0.003) and BMI (OR 5.0 95% CI 1.6-15.9; p = 0.006) resulted independently associated with impaired fibrinolytic activity. HHcy affects TAFI-mediated hypofibrinolysis but not fibrin(ogen) structure or function as documented by fibrin degradation analysis.

Effect of fibrinolysis on bleeding phenotype in moderate and severe von Willebrand disease

Patients with von Willebrand disease (VWD), the most common inherited bleeding disorder, display large variation in bleeding tendency, which is not completely related to VWF levels. The cause of variability in clinical expression is largely unknown. The effect of plasma fibrinolytic capacity on bleeding tendency in VWD patients has not been investigated. We hypothesized that enhanced fibrinolysis may result in a more severe bleeding phenotype. Therefore, we measured the fibrinolytic potential in patients with moderate or severe VWD to investigate the contribution of fibrinolysis to the bleeding tendency. Fibrinolytic potential was measured as plasma clot lysis time (CLT) with and without addition of potato carboxypeptidase inhibitor (PCI) in 638 patients with moderate or severe VWD who participated in a nationwide multicentre cross-sectional study. Bleeding severity was measured using the Bleeding Score (BS).The CLTs were significantly longer, indicative of hypofibrinolysis, in men compared to women with VWD [106.2 (IQR 95.7-118.1) vs. 101.9 (IQR 92.8-114.0) min]. The CLTs prolonged with increasing age. No association was found between VWF or FVIII levels and CLT, or between VWF or FVIII levels and CLT(+PCI) . No association was observed for BS in a model with 10log-transformed CLT, adjusted for age, gender, VWF:Act and FVIII [b = 6.5 (95%CI -0.3 to 13.4)]. Our study showed that the plasma fibrinolytic potential does not influence bleeding tendency in VWD patients and therefore does not explain the variability in bleeding phenotype in VWD.