Fibrinolytic Factors Research Articles

Altered fibrinolytic system in rat models of depression and patients with first-episode depression

Tissue plasminogen activator (tPA) is a serine protease involved in cleavage of neurotrophic factors. In addition, tPA and neuroserpin can also directly bind to low density lipoprotein receptor-related protein 1 (LRP1), promoting neurogenesis and neurite outgrowth. Given both the cleavage and non-cleavage actions of the fibrinolytic system are crucial in neurological functions, the present study, for the first time, systematically detected the changes of fibrinolytic system factors in rats exposed to chronic unpredictable mild stress (CUMS) or lipopolysaccharide (LPS) and patients with depression. In general, our data demonstrated that both CUMS and LPS reduced tPA but elevated plasminogen activator inhibitor-1 (PAI-1; SERPINE1) mRNA expression. Intriguingly, decreased expression of neuroserpin and LRP1 was also observed in rats exposed to CUMS or LPS. The down-regulated neuroserpin and LRP1 signaling were confirmed by western blotting and immunoflurence data. Likewise, elevated PAI-1 but a significant reduction of neuroserpin and LRP1 mRNA expression were observed in the peripheral blood mononuclear cells (PBMCs) of patients with first-episode depression, and the mRNA levels of PAI-1, neuroserpin and LRP1 were correlated with the Beck Depression inventory (BDI) scores, further strengthening the clinical significance and involvement of the fibrinolytic system in depression. Collectively, the present study demonstrated the alterations of fibrinolytic system in stressed and inflamed brain and in patients with first-episode depression, firstly showing that not only the cleavage actions, but also the non-cleavage actions of the system may play an essential role in the development of depression.

The Effect of Immunosuppression on Coagulation After Liver Transplantation.

Everolimus (EVR) is a mammalian target of rapamycin (mTOR) inhibitor commonly used for immunosuppression (IS) after liver transplantation (LT). However, there are concerns about whether mTOR inhibitors may move the hemostatic balance toward a higher likelihood of thrombosis. The present study aimed to investigate potential coagulation disorders after the administration of EVR. We evaluated 54 patients after conversion to an EVR-based IS regimen (n=26) and compared those patients with patients who were switched to extended-release tacrolimus (TAC) but had never received EVR (n=28). At baseline and again at 1 month and 6 months after conversion, we measured international normalized ratio, activated partial thromboplastin time, and anticoagulation and fibrinolysis factors, and we performed rotational thromboelastometry (ROTEM). Data were analyzed with a Mann-Whitney U test, a repeated-measure analysis of variance, and a Fisher's exact test. Statistical significance was set at the level of P ≤ 0.05. Plasma levels of von Willebrand factor, fibrinogen, and factor VIII were significantly higher than baseline levels at 1 month and 6 months after conversion of IS to EVR (P<0.001); plasma levels of protein C, protein S, and plasminogen also increased significantly (P<0.001). ROTEM confirmed a significant increase in maximum clot firmness in EXTEM, INTEM, and FIBTEM assays (P<0.001). In all assays, maximum lysis was significantly lower than baseline levels at 1 month and 6 months after conversion to EVR. Patients converted to IS with extended-release TAC exhibited no significant changes in coagulation variables. Retrospective analysis showed a significantly higher incidence of thromboembolic complications among patients treated with EVR-based IS than among those treated with extended-release TAC (P<0.01). In conclusion, the administration of EVR after LT seems to modify hemostasis to a procoagulant state. Thrombophilia screening before conversion may determine which patients will benefit from conversion to EVR-based IS.

Antithrombotic Effect of Oral Administration of Mozuku (Cladosiphon okamuranus, Brown Seaweed) Extract in Rat.

Dysfunction of vascular endothelial cells causes the risk of thrombosis. Aim of this study is to evaluate the antithrombotic effect of Okinawa mozuku (brown seaweed, Cladosiphon okamuranus) extract by using cultured vascular endothelial cells and rat carotid arterial thrombosis model induced by ferric chloride (FeCl3). The cell line (TKM-33) established from human umbilical vein endothelial cells were cultured with or without Okinawa mozuku extract. After incubation for 24 h, the conditioned medium was collected to evaluate urokinase-type plasminogen activator (u-PA) activity. Next, rats were fed with water or water containing 5% of Okinawa mozuku extract for 8 wk. After 8 wk of treatments, the rats were provided for the carotid arterial thrombosis model, and fibrinolytic factor and coagulation factor in blood were measured. Okinawa mozuku extract significantly augmented u-PA activity in the conditioned medium. The decrease of carotid artery blood flow induced by 40% FeCl3 injury in rats fed with Okinawa mozuku extract was less than that in control rats. Thus, oral administration of Okinawa mozuku extract prevented thrombus formation in this model. Oral administration of Okinawa mozuku extract significantly increased u-PA activity in euglobulin fraction, compared with control group. On the other hand, platelet aggregation activity, activated partial thromboplastin time, and active PAI-1 level in plasma exhibited no significant differences between control and Okinawa mozuku groups. These results indicate that oral administration of Okinawa mozuku enhances fibrinolytic activity in plasma and prevents thrombus formation which is induced by injury of vascular endothelial cells.

Влияние антенатальной гипоксии на коагуляционные свойства крови у половозрелых крыс

The paper presents the results of studies on the effects of hypoxia in the antenatal period on a number of blood coagulability indicators in adult rats. It was revealed that the long-term effect of prenatal hypoxia contributes to the acceleration of certain parameters of blood clotting. The established hypercoagulable shift in the system. It was shown that at the same time the blood coagulation time and recalcification accelerated; thromboplastic activity, prothrombin and thrombin time increased, and at the same time plasma high tolerance to heparin was noted. These studies suggest that prenatal hypoxia alters the overall state of blood coagulation, where it forms a qualitatively new stereotype of the relationship between hemocoagulation and fibrinolysis factors.

Blood Transfusion: a Guide to Clinical Decision Making

Nowadays, management of hemotherapy is regulated in Germany by the transfusion act and several guidelines while the transfusing physician is responsible for correct implementation at the bedside. Indications for blood products have to be carefully adapted to the patient's current clinical situation and pre-existing diseases have to be considered as well. Today, for most perioperative elective surgeries, evidence-based transfusion thresholds for packed red blood cell concentrates (RBC) have been defined and should be considered. Platelet concentrates (PC) have to be transfused in bleeding patients with thrombocytopenia or -pathy. In addition, PC are indicated prophylactically in clinically stable, not bleeding patients with a platelet trough count of less than 10/nl. Therapeutic plasma or fresh frozen plasma (FFP) is indicated for balanced substitution of coagulation factors, coagulation inhibitors and fibrinolysis factors in massive transfusion settings, where dilution of coagulation factors takes place. Plasma exchange in adults with thrombotic-thrombocytopenic purpura (TTP) or adult hemolytic-uremic syndrome (HUS) also requires FFP. In addition, FFP might be indicated, if no coagulation factor concentrate is available (e.g. FV deficiency). Adverse transfusion reactions are rare nowadays; however, hemolytic and allergic reactions are not too uncommon. For severe hemolytic transfusion reactions, in almost all cases, wrong blood in tube (WBIT) at the diagnostic bedside blood withdrawal or a mix-up of blood components before transfusion is causative. Massive transfusion situations require a proactive management, which includes RBC, FFP and potentially also PC and coagulation factor concentrates. Prior to elective surgery, anemic patients should be diagnosed and treated for the cause of their anemia, if possible.

Potential Protective Role of Blood Pressure-Lowering Drugs on the Balance between Hemostasis and Fibrinolysis in Hypertensive Patients at Rest and During Exercise.

In patients with hypertension, the triad represented by endothelial dysfunction, platelet hyperactivity, and altered fibrinolytic function disturbs the equilibrium between hemostasis and fibrinolysis and translates into a hypercoagulable state, which underlies the risk of thrombotic complications. This article reviews the scientific evidence regarding some biological effects of antihypertensive drugs, which can protect patients from the adverse consequences of hypertensive disease, improving endothelial function, enhancing antioxidant activity, and restoring equilibrium between hemostatic and fibrinolytic factors. These protective effects appear not to be mediated through blood pressure reduction and are not shared by all molecules of the same pharmacological class.

The Role of Fibrinolytic Regulators in Vascular Dysfunction of Systemic Sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease of autoimmune origin characterized by vascular dysfunction and extensive fibrosis of the skin and visceral organs. Vascular dysfunction is caused by endothelial cell (EC) apoptosis, defective angiogenesis, defective vasculogenesis, endothelial-to-mesenchymal transition (EndoMT), and coagulation abnormalities, and exacerbates the disease. Fibrinolytic regulators, such as plasminogen (Plg), plasmin, α2-antiplasmin (α2AP), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) and its receptor (uPAR), plasminogen activator inhibitor 1 (PAI-1), and angiostatin, are considered to play an important role in the maintenance of endothelial homeostasis, and are associated with the endothelial dysfunction of SSc. This review considers the roles of fibrinolytic factors in vascular dysfunction of SSc.

Repeat dose exposure of PM2.5 triggers the disseminated intravascular coagulation (DIC) in SD rats

Epidemiological evidence suggests that fine particulate matter (PM2.5) in air pollution promotes the formation of deep venous thrombosis. However, no evidence is available on the effects of PM2.5 lead to disseminated intravascular coagulation (DIC). For the first time, this study explored the effects of PM2.5 on DIC via coagulation disorders in vivo. SD rats received intratracheal instillation of PM2.5 once every three days for one month. Doppler ultrasound showed that the pulmonary valve (PV) and aortic valve (AV) peak flow were decreased after exposure to PM2.5. Fibrin deposition and bleeding were observed in lung tissue and vascular endothelial injury was found after exposure to PM2.5. Expression of thrombomodulin (TM) in vessel was downregulated after PM2.5-treated, whereas the levels of proinflammatory factors and adhesion molecules (IL-6, IL-1β, CRP, ICAM-1 and VCAM-1) were markedly elevated after exposure to PM2.5. Tissue factor (TF) and the coagulation factor of FXa were increased, while vWF was significantly lowered induced by PM2.5. Thrombin-antithrombin complex (TAT) and fibrinolytic factor (t-PA) were elevated, while there was no significantly change in the expression of anticoagulant factors (TFPI and AT-III). To clarify the relationship between PM2.5 and DIC, we examined the general diagnostic indices of DIC: PM2.5 prolonged PT and increased the expression of D-dimer but decreased platelet count and fibrinogen. In addition, the gene levels of JAK1 and STAT3 showed an upward trend, whereas there was little effect on JAK2 expression. And inflammatory factors (IL-6, IL-1β and TNF) in blood vessels of were up-reglated in PM2.5-treated rats. In summary, our results found that PM2.5 could induce inflammatory response, vascular endothelial injury and prothrombotic state, eventually resulted in DIC. It will provide new evidence for a link between PM2.5 and cardiovascular disease.

Deficiency of Bmal1 disrupts the diurnal rhythm of haemostasis.

Mice deficient in the circadian clock gene BMAL1 (Brain and Muscle ARNT-like protein-1) exhibit a hypercoagulable state and an enhanced arterial and venous thrombogenicity, which aggravates with age. We investigated the effect of BMAL1 deficiency in mice at a different age on the diurnal rhythm of factors involved in coagulation and fibrinolysis. Hepatic, cardiac and brain tissues were isolated from 10- and 25-weeks-old Bmal1-deficient (BMAL1-/-) and wild-type (BMAL1+/+) mice at ZT2 and at ZT14 to analyze the mRNA expression level of genes involved in coagulation and fibrinolysis. Body weight and brain weight were significantly lower in all BMAL1-/- versus BMAL1+/+ mice. Bmal1 deficiency disturbed the diurnal rhythm of plasminogen activator inhibitor-1 (PAI-1) in liver and plasma, but not in cardiac or brain tissues. BMAL1+/+ livers showed diurnal fluctuations in factor (F)VII, FVII, protein S and anti-thrombin gene expression, which were not observed in BMAL1-/- tissues. Interestingly, tissue plasminogen activator (t-PA) expression was significantly upregulated in all BMAL1-/- versus BMAL1+/+ brains at both time points. Plasma t-PA-PAI-1 complex levels were however similar for all groups. Bmal1 deficiency affected the biphasic rhythm of coagulation and fibrinolysis factors predominantly in the liver. In the brain, Bmal1-dependent control of t-PA gene expression was documented for the first time.

Thrombocytopenia in pregnant women with thrombophilia treated with low-molecular-weight heparin

Thrombophilia is a pathology introduced into the clinical practice, with risk of venous thromboembolism (VTE) in pregnancy. Objectives. The potential risks for the mother and fetus should be considered when using anticoagulants in pregnancy. Materials and method. In this study, we followed-up the evolution under prophylaxis with Fraxiparine® of 81 pregnant patients with inherited thrombophilia, of which 63 gave birth until the end of the study. Laboratory and adjacent tests were performed to determine the occurrence of complications related to thrombophilia, but also the adverse effects of the anticoagulant treatment. Results. Fraxiparine® was found not to be associated with dangerous adverse effects. Discussion. Procoagulant and fibrinolytic factors are involved in hemostasis. In addition, besides the coagulation cascade, the complement cascade, vascular factors, and quinine also play a role. Platelet aggregation can be diminished, being involved in pregnancy bleeding, especially at birth. The retractability of the clot may also be diminished by the occurrence of thrombasthenia, which should be monitored especially under anticoagulant treatment when a paradoxical thrombocytopenia syndrome might occur. Conclusions. We did not find any dangerous side effects, not even HIT syndrome (heparin-induced thrombocytopenia), which is common in other anticoagulant treatments.

Homocysteine-Enhanced Proteolytic and Fibrinolytic Processes in Thin Intraluminal Thrombus and Adjacent Wall of Abdominal Aortic Aneurysm: Study In Vitro.

Homocysteine (Hcy) may affect the pathogenesis of abdominal aortic aneurysms (AAAs) through enhancement of proteolysis and an impaired coagulation/fibrinolysis system. Intensified haemostatic capacity may promote local proteolytic degradation of the aortic wall. This study aimed to examine the effects of Hcy on haemostatic and proteolytic processes in samples of thick and thin fragments of the ILT and underlying walls. Subjects and Methods. Thirty-six patients who underwent AAA surgery were enrolled. Aneurysm tissue sections were incubated with DL-Hcy (100 and 500 μmol/L) in a series of experiments and analyzed for concentration/activity of proteolytic and haemostatic markers by enzyme-linked immunosorbent assay. Results. Incubation of wall underlying thin ILT segments (B) with DL-Hcy resulted in an increase of active MMP-2 levels compared to control tissue (9.54 ± 5.88 versus 7.44 ± 4.48, p=0.011). DL-Hcy also induced t-PA and plasminogen concentration increases in thin thrombus sections (B1) compared to control tissue (respectively: 1.39 ± 1.65 versus 0.84 ± 0.74, p=0.024; 11.64 ± 5.05 versus 10.34 ± 5.52, p=0.018). In contrast, wall adjacent to thick thrombus segments (A) showed decreases in MMP-2 and TF activities compared to control (respectively, 5.89 ± 3.39 versus 7.26 ± 5.49, p=0.046; 67.13 ± 72.59 versus 114.46 ± 106.29, p=0.007). In thick ILT sections (A1), DL-Hcy decreased MMP-2 activity and t-PA and plasminogen concentrations compared to control tissue (respectively, 2.53 ± 2.02 versus 3.28 ± 2.65, p=0.006; 0.67 ± 0.57 versus 0.96 ± 0.91, p=0.021; 9.25 ± 4.59 versus 12.63 ± 9.56, p=0.017). In addition, analysis revealed positive correlations at all sites between activities/concentrations of MMP-2, TF, and PAI-1 measured in control tissues and after incubation with DL-Hcy. Conclusions. These data indicate the potential for excess Hcy to enhance damage of arterial wall in thinner AAA segments as a result of the increased activity of MMP-2 and fibrinolytic factors.

Assessment and determinants of whole blood and plasma fibrinolysis in patients with mild bleeding symptoms

Enhanced clot lysis is associated with bleeding, but assessment of lysis capacity remains difficult. The plasma turbidity lysis and whole blood tissue Plasminogen Activator-Rotational Thromboelastometry (tPA-ROTEM) assays estimate fibrinolysis under more physiological conditions than clinically used assays. We hypothesized that these assays could find signs of enhanced lysis capacity in patients who report bleeding symptoms, but are not diagnosed with bleeding disorders. We also aimed to gain insight in determinants of the results of these lysis assays. Data from 240 patients with and 95 patients without self-reported bleeding symptoms were obtained, who were included in a study that primarily aimed to assess prevalence of haemostatic abnormalities in preoperative patients. ROTEM and turbidity assays were performed with rtPA. Blood counts, fibrinolysis and coagulation factor activities were determined. Data were analysed using multivariable linear regression models. Remarkably, patients reporting bleeding symptoms showed signs of significantly impaired lysis capacity in the tPA-ROTEM, but not in the turbidity lysis assay. In these patients, the tPA-ROTEM results depended on FII, FXII, plasminogen, α2-antiplasmin, PAI-1 and TAFI levels. The turbidity lysis results were significantly influenced by fibrinogen, α2-antiplasmin, PAI-1 and TAFI. In conclusion, the tPA-ROTEM and the turbidity lysis assay could not detect enhanced fibrinolytic capacity in patients with bleeding symptoms. This suggests that these symptoms are not caused by enhanced fibrinolytic activity. As both assays were sensitive to important determinants of fibrinolysis they may be able to detect a fibrinolytic imbalance, but this needs to be validated in patients with known hypo- or hyperfibrinolytic disorders.

Coagulation and Fibrinolysis Imbalance Demonstrated By Novel Simultaneous Thrombin and Plasmin Generation Assay during L-Asparaginase Treatment Phase of Induction Therapy in Pediatric Acute Lymphoblastic Leukemia

Introduction: L-asparaginase (L-Asp) is one of the risk factor of thromboembolism during ALL chemotherapy. Although the pathogenesis has been still unclarified, L-Asp may have profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In addition, recent studies demonstrated that the age of over 10-years might be a risk factor for this L-Asp associated coagulopathy. In this study, we hypothesized that change of balance between coagulation and fibrinolysis contributes to hyper-coagulation condition during chemotherapy including L-Asp. In order to clarify our hypothesis, we investigated the global coagulation and fibrinolytic function during the ALL induction therapy with simultaneous thrombin and plasmin generation assay (T/P-GA). Patients: Seventy-two pediatric patients aged 1.0 to 15.2 years (43 males and 29 females) with newly diagnosed ALL were enrolled from Aug. 2014 to Mar. 2018 at four hospitals in Japan. All patients and their families had no thrombotic predisposition. Fifty-five cases (76.4%) (BCP-ALL; n=47, T-ALL; n=7, MPAL; n=1) received BFM-95 oriented protocol consisting of a total of 8 doses of E.coli L-Asp 5,000 U/m2, whilst the others (BCP-ALL; n=13, T-ALL; n=2, MPAL; n=1, Ph-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS)-ALL02-protcol consisting of a total of 6 doses of E.coli L-Asp 6,000 U/m2. Fifty-two cases (72.2%) were categorized to younger group (1-10 years; median, 4.3 years) and the other cases to older group (>10 years; median, 12.6 years). The percentage of patients of each category were similar in the two treatment protocols (p=0.764). Methods: The global functions of coagulation and fibrinolysis were evaluated using T/P-GA [Matsumoto et al. TH 2013]. This clotting was initiated by the mixture of recombinant human tissue factor (Innovin®, f.c. 1 pM), phospholipid vesicles (f.c. 4 μM) and tissue-type plasminogen activator (f.c. 3.2 nM). Thrombin and plasmin generation were monitored simultaneously using thrombin- and plasmin-specific fluorogenic substrate (Z-Gly-Gly-Arg-AMC and BOC-Glu-Lys-Lys-MAC, respectively) in separate microtiter wells. The first derivatives (velocity) of thrombin and plasmin generation were utilized to derive the parameters, lag time (LT), endogenous potential (EP), peak levels (peak), and time to peak (ttPeak). EP of thrombin generation (T-EP) and plasmin-peak (P-peak) were selected as parameters for evaluation in this study. A ratio of T-EP and P-peak of patients' plasmas to those of control normal plasma were calculated. The conventional laboratory markers of coagulation and fibrinolysis were also monitored by fibrinogen (Fbg), fibrin-Fbg degradation products (FDP), antithrombin (AT), thrombin-AT complex and plasmin-α2 plasmin inhibitor complex. Plasmas were collected at T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post induction phase. Results: None of the cases developed L-Asp associated coagulopathy, and two cases (2.8%) developed induction failure. Thirteen cases (18.1%) received fresh frozen plasma transfusion for low Fbg level, whilst 52 (72.2%) cases received AT supplement for low AT level. Fbg showed a median of 165, 99.0, 97.0 and 316 mg/dl at T0, T1, T2 and T3, respectively, and AT showed a median of 143, 80.9, 80.0 and 105%, respectively, whilst the value of other conventional markers remained within reference value. T-EP revealed a median of 1,134, 1,135, 1,221, 1,277 and 1,102 nM, whilst P-peak showed a median of 5.33, 3.75, 3.63, 4.91 and 5.51 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-peak ratios (Fig. 1). T-EP ratios showed significantly higher at T2 and T3 than at T0 and T1 (p<0.05), whilst P-peak ratios showed significantly lower at T1 and T2 than at T0 and T3 (p<0.01) (median ratios of T-EP/P-peak; T0; 1.06/0.97, T1; 1.04/0.65, T2; 1.12/0.65, T3; 1.17/0.87, respectively). P-peak ratios of the older group showed significantly lower than the younger group at T1 (median; 0.58 vs 0.69, p=0.016). Conclusion: The results from T/P-GA showed that their hemostatic dynamics appear likely to shift to hyper-coagulation and hypo-fibrinolysis at L-Asp treatment phase. These results suggest that the imbalance of coagulation and fibrinolysis might cause thrombotic complication during the L-Asp treatment for ALL, especially in patients aged 10-years older. [Display omitted] DisclosuresNogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.

A Novel Plasma Panel for the Assessment of Chronic Liver Disease Severity

Background: Hepatocytes produce a variety of clotting and fibrinolytic factors. In chronic liver disease (CLD), biosynthesis of these factors is often altered leading to hemostatic aberrations. Recently, the usefulness of PT/INR - one of the five parameters used in the Child-Pugh (CP) scoring system - has been questioned. In this retrospective study, we explored the patterns of plasma alterations relative to CP scores to establish a plasma-based panel that can help evaluate the extent of CLD severity without the use of PT/INR and/or such parameters as the extent of ascites and hepatic encephalopathy.Methods: Blood was collected via venipuncture (0.129 mol/l sodium citrate) from CLD patients suffering from cirrhosis due to untreated HCV and/or excessive alcohol consumption, that were previously assigned a CP score: CP-A, n=12; CP-B, n=19; CP-C, n=11. Healthy control subjects (n=30) were age and sex matched; all subjects gave informed consent. Within 2 h post-collection, specimens were centrifuged for 15 min at 1500 x g, and then 13000 x g for 2 min to obtain plasma that was immediately frozen at -80°C until used. The following was measured: pro-coagulant phospholipid, fXa, D-dimer, fVIII, sP-selectin, thrombomodulin, protein C, protein S, alternatively spliced Tissue Factor (asTF), free TFPI, TF/TFPI ratio, and TFPI/protein S ratio. Thrombin generation tests (TGT) with and without thrombomodulin were performed. To interrogate the biology of CLD, patient plasma was also analyzed for the levels of microvesicles (MVs) of various origin: endothelium (CD31+/CD41-), monocytes (CD14+), leukocytes (CD11b+), neutrophils (CD16+), platelets (CD41a+, CD41a+/CD62P+, CD62P+), and exosomes (CD63+). Platelet counts and the levels of Annexin V+ MVs were also evaluated. Results were analyzed using GraphPad Prism v.6.03, and the parameters that were significantly different between control and CLD plasma were further evaluated. A system was developed with specified cutoffs for each parameter, derived using clinically normal ranges as well as the data generated in this study, to compile a composite score for CLD severity.Results: CLD plasma had endogenous thrombin potential comparable to that of control plasma; however, the rate of thrombin generation and peak thrombin generation diminished with increased CLD severity (both p < 0.0001), prolonging the time until peak (p < 0.01) as assessed by TGT. Of the 14 remaining parameters investigated in CLD and control plasma, the levels of fVIII, D-dimer, and asTF increased with CLD severity while protein C, fV, protein S, and sP-selectin levels decreased (Table 1) and in combination, these 7 parameters were able to predict the previously assigned CP scores with high accuracy. Our scoring system assigns a numeric value (0, 1, or 2) for each parameter; a cumulative score of <2 is normal, 2-3 CP-A, 4-7 CP-B, and ≥8 CP-C (Table 2). The system was 97.2% accurate at predicting the CP score: of the 72 plasma samples analyzed, there was only one false positive (a control scored as a CP-A), and one CP-C sample scored as CP-B. Omission of any of the 7 components markedly lowered the system's accuracy. Levels of CD31+/CD41-, CD62+, CD63+, and CD16+ MVs rose with disease severity (p < 0.0001 for all) while CD41a+ MVs decreased (p < 0.0001). Of the panel's 7 components, asTF is relatively novel and so asTF's association with various markers was further examined. There was a positive correlation between asTF and D-dimer (r = 0.305, p = 0.05) and asTF and CD63 (r = 0.346, p = 0.025); asTF negatively correlated with CD14+ MVs (r = -0.348, p = 0.024). There was a strong and highly significant positive correlation between asTF and CD31+/CD41- MVs (r = 0.630, p = 7.81x10-6).Conclusions: Here we report a novel plasma panel with potential utility in assessing CLD severity in a point-of-care setting. Our scoring system exhibits 97.2% accuracy and does not require clinical assessment beyond plasma analysis. asTF levels positively correlate with those of CD31+/CD41- MVs and both increase with CLD severity; this is in agreement with earlier reports of inflammatory cytokines eliciting asTF secretion from endothelial cells. Larger-scale prospective studies are warranted to validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Indoxyl Sulfate Promotes Arterial Thrombosis in Rat Model via Increased Levels of Complex TF/VII, PAI-1, Platelet Activation as Well as Decreased Contents of SIRT1 and SIRT3.

Patients suffering from chronic kidney disease (CKD) are at a 20-fold higher risk of dying due to cardiovascular diseases (CVDs), primarily thrombosis following vascular injury. CKD is connected with retention of uremic toxins, especially indoxyl sulfate (IS), which are currently considered as a non-classical CKD-specific risk factor for CVDs. The present study aimed to examine the effect of chronic exposure to IS on the hemostatic system and arterial thrombosis in a model without greater interferences from the uremic milieu consisting of additional uremic toxins. Forty-eight male Wistar Crl:WI (cmdb) rats were divided into three groups: one control group and two experimental groups, which were exposed to 100 or 200 mg/kg of b.w./day of IS in drinking water for a period of 28 days. The control group received water without IS. At the end of the experiment, the induction of arterial thrombosis was performed. We investigated the impact of IS on thrombosis incidence, kinetics and strength of clot formation, platelet activity, aortic contents of sirtuin (SIRT) 1 and sirtuin 3 (SIRT3), hemostatic system, cardiorespiratory parameters, biochemistry of plasma and urine as well as histology of the thrombus, kidney, and liver. Obtained data revealed that chronic exposure to IS promotes arterial thrombosis via increased levels of complex tissue factor/factor VII, plasminogen activator inhibitor-1 (PAI-1), platelet activation, as well as decreased aortic levels of SIRT1 and SIRT3. Therefore, we hypothesize that IS enhances primary hemostasis leading to augmented formation of platelet plug with increased amounts of fibrin and affects secondary hemostasis through the influence on plasma coagulation and fibrinolysis factors, which results in the increased kinetics and strength of clot formation. The findings described may contribute to a better understanding of the mechanisms leading to increased thrombotic events in patients with CKD with elevated levels of IS.

The fibrinolytic factor tPA drives LRP1-mediated melanoma growth and metastasis.

The multifunctional endocytic receptor low-density lipoprotein receptor-related protein (LRP)1 has recently been identified as a hub within a biomarker network for multicancer clinical outcome prediction. The mechanism how LRP1 modulates cancer progression is poorly understood. In this study we found that LRP1 and one of its ligands, tissue plasminogen activator (tPA), are expressed in melanoma cells and control melanoma growth and lung metastasis in vivo. Mechanistic studies were performed on 2 melanoma cancer cell lines, B16F10 and the B16F1 cells, both of which form primary melanoma tumors, but only B16F10 cells metastasize to the lungs. Tumor-, but not niche cell-derived tPA, enhanced melanoma cell proliferation in tPA-/- mice. Gain-of-function experiments revealed that melanoma LRP1 is critical for tumor growth, recruitment of mesenchymal stem cells into the tumor bed, and metastasis. Melanoma LRP1 was found to enhance ERK activation, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity, a well-known modulator of melanoma metastasis. Restoration of LRP1 and tPA in the less aggressive, poorly metastatic B16F1 tumor cells enhanced tumor cell proliferation and led to massive lung metastasis in murine tumor models. Antimelanoma drug treatment induced tPA and LRP1 expression. tPA or LRP1 knockdown enhanced chemosensitivity in melanoma cells. Our results identify the tPA-LRP1 pathway as a key switch that drives melanoma progression, in part by modulating the cellular composition and proteolytic makeup of the tumor niche. Targeting this pathway may be a novel treatment strategy in combination treatments for melanoma.-Salama, Y., Lin, S.-Y., Dhahri, D., Hattori, K., Heissig, B. The fibrinolytic factor tPA drives LRP1-mediated melanoma growth and metastasis.

Co-exposure subacute toxicity of silica nanoparticles and lead acetate on cardiovascular system.

BackgroundThe harmful effects following the release of nanomaterials into environment are of great concern today.PurposeIn this study, subacute effect due to co-exposure to low-dose silica nanoparticles (SiNPs) and lead acetate (Pb) on cardiovascular system was detected in Sprague Dawley male rats.Materials and MethodsHistopathological and ultrastructural changes of heart, aortic arch and abdominal aorta were detected. Blood routine and blood biochemistry examinations were used to show the changes of blood components. The fibrinolytic and plasmin factors, inflammation-related factors and myocardial-related enzyme in serum were analysised by ELISA and Western blot assay.ResultsHistopathological and ultrastructural examination of heart, aortic arch, and abdominal aorta showed that serious damage occurred in co-exposure group (n=6/group). Blood routine examination showed that leukocytosis and thrombocytopenia increased markedly, while changes in the erythrocyte count were not obvious in the co-exposure group. The expression of alanine transaminase (ALT) decreased obviously in co-exposure group, while no significant changes were noted in the expression of aspartate aminotransferase (AST), cholesterol (CHO), triglyceride (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) in the co-exposure group on blood biochemistry analysis. In addition, data from ELISA analysis showed that the levels of fibrinolytic and plasmin factors, including thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), tissue-type plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), and antithrombin III (AT III), were decreased, while those of human fibrinogen (FIB) and D-dimer (D2D) increased significantly in the co-exposure group. Moreover, the myocardial-related enzyme in serum, tested by ELISA, and cardiovascular-related protein expression of atrial natriuretic peptide and brain natriuretic peptide, tested by Western blot assay, was increased in the heart. Furthermore, the expression of inflammation factors such as C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) was increased in heart tissue subjected to combined exposure, which was manifested by Western blot assay, while the protein levels of angiotensin II (ANG II) and endothelin 1 were (ET-1) elevated in blood vessels in the co-exposure group.ConclusionIn conclusion, the major interactions involved in subacute toxicity due to co-exposure to low doses of SiNPs and Pb on cardiovascular system were expected to be additive and synergistic in nature. Co-exposure to SiNPs and Pb could aggravate the cardiovascular toxicity via endothelial damage, hypercoagulation, and cardiac injury in vivo.

Preoperative screening for bleeding disorders: A comprehensive laboratory assessment of clinical practice

Unlabelled Box BackgroundPatients with mild bleeding disorders are at risk of perioperative bleeding, but screening for these disorders remains challenging. ObjectivesWe aimed to assess the prevalence of hemostatic abnormalities in patients with and without reported bleeding symptoms on a preoperative questionnaire, consisting of guideline‐proposed questions, and appraised the diagnostic value of several screening modalities for the identification of patients with hemostatic abnormalities. MethodsIn this observational study, 240 patients with and 95 patients without bleeding symptoms on the preoperative questionnaire were included. Patients with known bleeding disorders, antithrombotic drugs, thrombocytopenia, and anemia were excluded. Preoperatively, all patients underwent elaborate hemostatic testing. Hemostatic abnormalities were defined as coagulation, vWF, or fibrinolysis factor levels below reference range and platelet function defects. Screening modalities included the ISTH Bleeding Assessment Tool (ISTH‐BAT), PT, aPTT, TT, Euglobulin Lysis Time (ELT), and Platelet Function Analyser (PFA). ResultsIn 21 of 240 (8.8%) patients reporting bleeding symptoms, hemostatic abnormalities were found, including 7 reduced coagulation factor levels, 10 platelet function abnormalities, and 4 reduced vWF levels. In comparison, 10 of 95 (10.5%) patients not reporting bleeding symptoms had abnormalities. The ISTH‐BAT could not identify patients with abnormalities, while PT, aPTT, TT, ELT, and PFA had high specificity but low sensitivity to detect abnormalities. ConclusionsThe prevalence of hemostatic abnormalities in both patients with and without reported bleeding symptoms was 9%‐10%. This suggests that the guideline‐based questionnaire cannot differentiate between patients with and without abnormalities, while the discriminative power of the screening modalities is also limited.

Patients with Stevens - Johnson syndrome and Toxic Epidermal Necrolysis Develop Coagulopathies Similar to Those Seen in Burn Patients: A Pilot Study

Background: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are a continuum of a life-threatening skin loss condition due to an immune or hypersensitivity reaction. Patients are frequently treated in burn centers. Objective: This study was undertaken to determine if patients with SJS and TEN have a coagulopathy with comparable hemostatic perturbations to those seen in patients with burn injury. Materials &amp; Methods: Blood plasma parameters studied were factors of coagulation, fibrinolysis, Interleukin-6 (IL-6) and Endothelin-1. Results were compared to historical hemostatic and cytokine data from burn patients treated at the same center. Results: Sixteen patients with SJS-TEN (6 males/10 females) with ≥20 % total body surface area (TBSA) sloughed skin were studied. The majority had received phenytoin or an antibiotic as the precipitating medication for the SJS-TEN. There was a significant increase in Thrombin-Antithrombin Complex (TAT) p&lt;0.0004, tissue Plasminogen Activator (tPA), p&lt;0.02, Plasminogen Activator Inhibitor-1 (PAI-1), p&lt;0.02, and D-dimer p&lt;0.007 plasma levels on admission. Antithrombin (AT), p&lt;0.04 and plasminogen (PLG) p&lt;0.02 plasma levels were significantly decreased. Conventional global coagulation tests (prothrombin and partial thromboplastin times) were not abnormal in patients with ≤7 days duration of the rash on admission. Patients with delayed admission at &gt;7 days after the start of the rash had a significantly increased chance of demise, p&lt;0.01. These patients also had a significantly decreased AT levels (p&lt;0.01) compared to normal controls and to patients admitted at ≤7 days of the disease process, (p&lt;0.01). The pattern of hemostatic aberrations of TAT, tPA, PAI1, D-dimer, Interleukin -6, AT, and PLG was similar to that seen in burn patients during the acute phase of injury and resuscitation. The mortality rate was 37.5 %. Conclusions: Patients with ≥20% TBSA SJS-TEN had hemostatic perturbations consistent with those observed in ≥20% TBSA burn injuries coagulopathies.

Changes in graft gene expression contribute to coagulopathy in uDCD liver transplantation

Uncontrolled DCD donors suffer cardiac arrest followed by unsuccessful resuscitation. Their livers have been successfully transplanted, though strict maintenance and selection criteria are necessary to avoid adverse outcomes, namely ischemic type biliary lesions. We demonstrated that uDCD liver recipients develop severe coagulopathy and fibrinolysis (Blasi 2016). Given the liver is the major source of clotting and fibrinolytic factors, we hypothesized that changes in gene expression may be detected in uDCD livers and provide a basis for abnormalities observed. Methods: 32 samples were taken from 16 uDCD livers: “A” before cold preservation and “B” after reperfusion. Total RNA was extracted and analyzed using microarrays; results were confirmed using RT-PCR. Finally, 61 biopsies from 38 uDCD livers were evaluated by light microscopy to rule out fibrin microthrombi. Results: Both before preservation and after reperfusion, uDCD livers had significantly upregulated expression of genes that provoke fibrinolysis and inhibit coagulation, including TPA, urokinase plasminogen activator, urokinase plasminogen activator receptor, and thrombomodulin. There was also decreased expression of genes implicated in hemostasis (vWF) and inhibition of fibrinolysis (alpha-2-macroglobulin):TableGene"A"FCFDR"B"FCFDRA2M−2.20.006−2.60.0009PLAT1.90.0022.70.0002PLAU2.00.0052.20.0008PLAUR2.50.00023.22E-5TM----1.5<0.01VWF−2.40.0004−2.10.001 Open table in a new tab On histology, no fibrin microthrombi were detected, even in cases that developed ITBL (13%). Conclusion: Changes in gene expression in uDCD livers help explain severe coagulopathy and fibrinolysis in recipients. Endogenous fibrinolysis is upregulated in these livers most at risk for ITBL, and clinical DCD protocols using TPA to help prevent ITBL should undergo critical appraisal.