Inhibitor Of Urokinase Plasminogen Activator Research Articles

Diastereoselective synthesis and biological evaluation of new fluorine-containing α-aminophosphonates as anticancer agents and scaffold to human urokinase plasminogen activator inhibitors

Phosphonate analogs of α-amino acids are increasingly valued for their significant potential in medicinal chemistry. Fluorine is a “magic” element that plays a huge role in modulating the properties of organic compounds. In this work, we combined the two pharmacophores in the synthesis of three series of new α-aminophosphonates. These compounds were obtained by diastereoselective hydrophosphonylation of imines prepared by an environmentally friendly mechanochemical approach.Results of computational SwissADME analysis suggested favorable drug-like properties of the α-aminophosphonates and indicated their potential for interaction with diverse biological targets including proteases, showing promising pharmacokinetic profiles compared to 5-fluoro-2'-deoxyuridine (FdU) used as a standard anticancer drug.Screening against ten cancer cell lines from seven types of cancer showed that five of the twenty compounds tested (1c, 2a, 2h, 3e, and 3f) exhibited superior activity against the HeLa cell line and lower cytotoxicity against normal MRC-5 cells than FdU. Compound 3e showed notable inhibitory effect on the MDA-MB-231 cell line, while 3a, 3h, and 3g demonstrated significant cytotoxic activity against U-87 MG and U-251 MG lines.Molecular docking highlighted the strong binding of compound 2a to the urokinase-type plasminogen activator (uPA) protein, with a binding affinity of −6.41 kcal/mol, suggesting the anti-metastatic potential of the compound. These findings enable to position the newly synthesized α-aminophosphonates as promising scaffolds for developing targeted anticancer therapies for metastatic cancers characterized by elevated uPA expression.

Alterations of Hemostatic Molecular Markers During Acute Large Vessel Occlusion Stroke.

This study aimed to investigate regional levels of TAT (thrombin-antithrombin complex), PIC (plasmin-α2 plasmin inhibitor complex), t-PAIC (tissue plasminogen activator-plasminogen activator inhibitor complex), sTM (soluble thrombomodulin), and D-dimer, along with their associations with clinical and procedural characteristics in patients with acute ischemic stroke undergoing endovascular thrombectomy. We retrospectively analyzed 166 consecutive patients with acute ischemic stroke (62±11.54 years of age, 34.3% women) using prospectively maintained clinical databases and blood samples from local ischemic (proximal to thrombus) and systemic (femoral artery, self-control) arterial compartments. Levels of TAT, PIC, t-PAIC, and D-dimer were significantly elevated, whereas sTM was significantly reduced, in local ischemic regions compared with their systemic levels. Each 1-unit increase in ischemic TAT (adjusted odds ratio [aOR], 1.086 [95% CI, 1.03-1.145]; P=0.002; area under the curve [AUC], 0.833) and PIC (aOR, 1.337 [95% CI, 1.087-1.644]; P=0.006; AUC, 0.771) correlated significantly with higher symptomatic intracranial hemorrhage risk. Additionally, each 1-unit increase in ischemic TAT (aOR, 1.076 [95% CI, 1.016-1.139]; P=0.013; AUC, 0.797), PIC (aOR, 1.554 [95% CI, 1.194-2.022]; P=0.001; AUC, 0.798), and sTM (aOR, 0.769 [95% CI, 0.615-0.961]; P=0.021; AUC, 0.756) was significantly associated with an increased risk of an unfavorable 90-day outcome (modified Rankin scale of 3-6). These hemostatic molecules, individually or combined, significantly improved the predictive power of conventional risk factors, as evidenced by significant increases in net reclassification improvement and integrated discrimination improvement (all P<0.01). We observed a hyperactive state of the coagulation-fibrinolysis system within the local ischemic region during hyperacute stroke. Rapid automated measurement of hemostatic molecular markers, particularly TAT, PIC, and sTM, during intra-arterial procedures may provide additional information for stroke risk stratification and therapeutic decision-making, and warrants further investigation.

Early identification of lung cancer patients with venous thromboembolism: development and validation of a risk prediction model

IntroductionVenous thromboembolism(VTE) is a leading cause of death in patients with lung cancer. Furthermore, hospitalization of patients with advanced lung cancer for VTE treatment represents a major economic burden on the national public health resources. Therefore, we performed this prospective study to identify clinical biomarkers for the early identification of VTE in lung cancer patients.MethodsThis prospective study enrolled 158 patients with confirmed lung cancer, including 27 who were diagnosed with VTE within six months of the follow-up after lung cancer diagnosis. Multivariate logistic regression analysis was used to evaluate the diagnostic performancese of all the relevant clinical features and laboratory indicators in identifying lung cancer patients with a higher risk of VTE. A novel risk prediction model was constructed consisting of five clinical variables with the best diagnostic performances and was validated using the receiver operation characteristic(ROC) curves. The diagnostic performances of the new risk prediction model was also compared with the Khorana risk score (KRS) and the Padua risk score (PRS).ResultsThe VTE group of lung cancer patients (n = 27) showed significantly higher serum levels of fibrin degradation products (FDP), D-dimer, thrombomodulin (TM), thrombin-antithrombin-complex (TAT), α2-plasmin inhibitor-plasmin Complex (PIC), and tissue plasminogen activator-plasminogen activator inhibitor complex (t-PAIC) compared to those in the non-VTE group (n = 131). ROC curve analyses showed that the diagnostic efficacy of the new VTE risk prediction model with TM ≥ 9.75 TU/ml, TAT ≥ 2.25ng/ml, t-PAIC ≥ 7.35ng/ml, history of VTE, and ECOG PS score ≥ 2 was superior than the KRS and the PRS in the early identification of lung cancer patients with a higher risk of VTE.ConclusionsThe new risk prediction model showed significantly high diagnostic efficacy in the early identification of lung cancer patients with a high risk of VTE. The diagnostic efficacy of the new risk prediction model was higher than the KRS and the PRS in this cohort of lung cancer patients.

Clinical study on the feasibility of new thrombus markers in predicting massive cerebral infarction.

This study investigated the diagnostic performance of the thrombin-antithrombin complex (TAT), plasmin-α2 plasmin inhibitor complex (PIC), tissue plasminogen activator-plasminogen activator inhibitor complex (t-PAIC), and thrombomodulin (TM) in the early identification of massive cerebral infarction. A total of 423 patients with cerebral infarction confirmed by imaging examination were divided into the massive cerebral infarction (MCI) group and the non-massive cerebral infarction (NMCI) group. TAT, PIC, t-PAIC, and TM were measured immediately after admission. The diagnostic performance was analyzed by the receiver characteristic operating curve (ROC). The median plasma concentrations of TAT, PIC, and t-PAIC in patients with MCI at early onset were 5.10 ng/ml, 1.11 μg/ml, and 8.80 ng/ml, respectively, which were higher than those in patients with NMCI (2.20 ng/ml, 0.59 μg/ml, and 7.35 ng/ml), and the difference was statistically significant (P < 0.001). TAT was shown to be an independent risk factor for the development of massive cerebral infarction by a multivariate logistic regression analysis (OR = 1.138). A ROC curve analysis showed that PIC had the best performance in identifying MCI at an early stage (AUC = 82.8%), with a sensitivity of 80.7% and a specificity of 76.2% when the PIC concentration was ≥0.8 μg/ml; TAT had the highest specificity in identifying MCI, with a specificity of 80.6% when the TAT concentration was ≥3.97 ng/ml. The detection of PIC, TAT, t-PAIC, and TM is a comprehensive assessment of vascular endothelial damage and activation of the coagulation and fibrinolytic systems and has diagnostic value for early identification of patients with MCI, which, together with its ease of detection, can be used as a plasma marker for early identification of large vessel occlusion.

Plasminogen Binding and Activation at the Mesothelial Cell Surface Promotes Invasion through a Collagen Matrix.

Plasminogen (Plg) activation to the serine protease plasmin (Pla) plays a key role in regulating wound healing and fibrotic responses, particularly when bound to cell surface receptors. Our previous work suggested that mesothelial cells bind Plg at the cell surface, though no Plg receptors were described for these cells. Since mesothelial cells contribute to injury responses, including cellular differentiation to a mesenchymal-like phenotype and extracellular matrix remodeling, we hypothesized that Plg binding would promote these responses. Here, we confirm that Plg binds to both pleural and peritoneal mesothelial cells via the lysine-binding domain present in Plg, and we demonstrate the presence of three Plg receptors on the mesothelial cell surface: α-Enolase, Annexin A2, and Plg-RKT. We further show that bound-Plg is activated to Pla on the cell surface and that activation is blocked by an inhibitor of urokinase plasminogen activator or by the presence of animal-derived FBS. Lastly, we demonstrate that Plg promotes mesothelial cell invasion through a type I collagen matrix but does not promote cellular differentiation or proliferation. These data demonstrate for the first time that mesothelial cells bind and activate Plg at the cell surface and that active Pla is involved in mesothelial cell invasion without cell differentiation.

In Vitro and In Vivo Effects of the Urokinase Plasminogen Activator Inhibitor WX-340 on Anaplastic Thyroid Cancer Cell Lines.

Increased expression of the urokinase-type plasminogen activator (uPA) system is associated with tumor invasion, neo-angiogenesis, and metastatic spread, and has been shown to positively correlate with a poor prognosis in several cancer types, including thyroid carcinomas. In recent years, several uPA inhibitors were found to have anticancer effects in preclinical studies and in some phase II clinical trials, which prompted us to evaluate uPA as a potential therapeutic target for the treatment of patients affected by the most aggressive form of thyroid cancer, the anaplastic thyroid carcinoma (ATC). In this study, we evaluated the in vitro and in vivo effects of WX-340, a highly specific and selective uPA inhibitor, on two ATC-derived cell lines, CAL-62 and BHT-101. The results obtained indicated that WX-340 was able to reduce cell adhesion and invasiveness in a dose-dependent manner in both cell lines. In addition, WX-340 increased uPA receptor (uPAR) protein levels without affecting its plasma membrane concentration. However, this compound was unable to significantly reduce ATC growth in a xenograft model, indicating that uPA inhibition alone may not have the expected therapeutic effects.

Efficacy and Safety of Postoperative Intravenous Tranexamic Acid in Total Knee Arthroplasty: A Prospective Randomized Controlled Study

ObjectiveTo assess the efficacy and safety of postoperative intravenous tranexamic acid (TXA) in patients undergoing total knee arthroplasty (TKA).MethodsFrom March 2020 to August 2020, all patients undergoing primary unilateral TKA in our hospital were considered in prospective randomized controlled study. Included patients were randomized into three groups to receive either two doses of 15 mg/kg intravenous TXA postoperatively, at 2 and 24 h after closing the incision (group A), or a single dose of 15 mg/kg intravenous TXA 2 h postoperatively (group B), or placebo (group C). The calculated total blood loss (TBL) and hidden blood loss (HBL), incidence of venous thromboembolism (VTE), and transfusion rate were compared among groups. The levels of prothrombotic state parameters including thrombomodulin (TM), thrombin‐anti‐thrombin complex (TAT), plasmin‐anti‐plasmin complex (PIC), and tissue‐type plasminogen activator‐plasminogen activator inhibitor complex (t‐PAI·C) in plasma were measured during the perioperative period. Patients were compared depending on the Kellgren‐Lawrence classification (K‐L types III and IV).ResultsAll patients were followed up for at least 4 weeks. The mean TBL and HBL in group C (1,182.45 ± 160.50; and 965.47 ± 139.61 mL, respectively) were significantly higher than those in groups A (944.34 ± 130.88 mL, P < 0.05; and 712.45 ± 129.82mL, P < 0.05, respectively) or B (995.20 ± 154.00 mL, P < 0.05; and 757.20 ± 134.39 mL, P < 0.05, respectively), but no significant differences were found between groups A and B (P > 0.05 and P > 0.05, respectively). None of the patients of three groups received blood transfusion, so there were no significant differences in blood transfusion rate among groups. Similar results were obtained with subgroups of patients who had the K‐L types III and IV. The DVT frequencies were four, three, and three in groups A, B, and C, respectively, with no significant differences after comparison (P > 0.05). There were no significant differences in the levels of prothrombotic state parameters (TM, TAT, PIC, t‐PAI·C) or incidence of VTE among groups (P > 0.05). Wound leakage was observed in five patients during the hospital stay (two patients in group A, one patient in group B, and two patients in group C), and no statistical difference was found in wound leakage or other complications among groups (P > 0.05).ConclusionsShort‐term application of postoperative intravenous TXA in TKAs resulted in reduced HBL without a measured increase in the actual incidence of VTE or the potential risk of thrombosis, but administration of TXA after the first 24 h had no significant effect.

Role of Substance P–Dependent Chemotactic Signaling in Postoperative Adhesion Formation

Postoperative adhesions are a potentially life-threatening complication of abdominal surgery. We previously showed that substance P (SP), acting through the neurokinin-1 receptor (NK-1R), is an important early mediator of adhesiogenesis through its regulation of the tissue plasminogen activator/plasminogen activator inhibitor-1 (PAI-1) fibrinolytic system. SP also mediates neurogenic inflammation by recruiting inflammatory leukocytes, such as neutrophils and macrophages. Our objective was to determine the role of SP-dependent chemotactic recruitment of these inflammatory cells through the CXCR2 in postsurgical adhesion formation. A mouse cecal cauterization model was used to generate intra-abdominal adhesions. Protein and mRNA levels of the chemokines CXCL1 and CXCL2 and their receptor CXCR2 were measured at 3 h and 6 h after surgery in peritoneal tissue and in peritoneal lavages in response to antagonists for the SP receptor and CXCR2, and in IFN-γ knockout mice. Postsurgical adhesion formation was inhibited by both an antagonist to NK-1R and an antagonist to CXCR2. Expression levels of neutrophil chemokines and CXCR2 in peritoneal tissue peaked 3-6 h after surgery and partially depended on SP and IFN-γ, one of its downstream mediators. An NK-1R antagonist inhibited SP-mediated increases in the expression of the PAI-1 inhibitory component of the fibrinolytic system, but the CXCR2 antagonist had no effect. Postsurgical adhesiogenesis involves upregulation of chemokine signaling that is partially SP- and IFN-γ-dependent. However, the adhesiogenic properties of chemokine signaling are not mediated through the inhibition of fibrinolysis with PAI-1, as was previously shown for SP.

A Pharmacokinetic and Pharmacodynamic Investigation of an ε-Aminocaproic Acid Regimen Designed for Cardiac Surgery With Cardiopulmonary Bypass

To investigate the pharmacokinetics and pharmacodynamics of an ε-aminocaproic acid (EACA) regimen designed for cardiac surgery with cardiopulmonary bypass (CPB). Prospective observational study requiring blood sampling to measure EACA concentrations and fibrinolysis markers (fibrinogen, D-dimer, α2-antiplasmin, and tissue plasminogen activator-plasminogen activator inhibitor [tPA-PAI-1] complex). Single-center, tertiary medical center. Patients who underwent cardiac surgery with CPB between 2018 and 2019 for aortic or mitral valve replacement/repair or coronary artery bypass grafting. Previous sternotomy patients were included. None. The pharmacokinetics of EACA, during CPB, were described by a 3-compartment disposition model. EACA concentrations were greater than 130 mg/L in all patients after CPB and in most patients during CPB. The D-dimer level trended up and reached a peak median level of 1.35 mg/L of fibrinogen equivalence units (FEU) at 15 minutes after protamine administration. The median change in D-dimer (ΔD-dimer) from baseline to 15 minutes after protamine was 0.34 (-0.48 to 3.81) mg/L FEU. ΔD-dimer did not correlate with EACA concentration intraoperatively, urine output, body weight, glomerular filtration rate, cell salvage volume, and ultrafiltration volume. The median 24-hour chest tube output was 445 (180-1,011) mL. This regimen provided maximum EACA concentrations near the time of protamine administration, with a total perioperative dose of 15 g. Most patients had EACA concentrations greater than the target during CPB. ΔD-dimer did not correlate with EACA concentration. The median 24-hour chest tube output compared well to similar studies that used higher doses of EACA.

Specific inhibitors of urokinase plasminogen activator for treatment of cancers; In silico approach

Invasion, metastasis and angiogenesis are fundamental processes in the development of solid cancers. All these depend on the proteolysis where plasminogen activation system (PAS) is the prominent component. Plasmin of PAS, with a broad spectrum of proteins lysed, is secreted as a pro-enzyme and then activated by urokinase (uPA) or tissue plasminogen activator (tPA). Urokinase is in control of pericellular proteolysis while tPA mediates intravascular fibrinolysis. The most effective way to reduce excessive activity of plasmin in cancers is by inactivation of its activators (PAs). Inhibition of PAs reduces tumor size of cancers in vivo. However, for successful targeted anticancer therapy it is essential to find specific uPA inhibitors and to protect normal function of tPA. Unfortunately, most known inhibitors are unspecific, acting on both PAs. PAs are highly homologous enzymes with extreme similarities in their active sites so large numbers of chemicals need to be tested to find novel specific uPA inhibitors. Methods: As the availability of 3D protein structures determined experimentally by X-ray crystallography is growing, computational methods are ever more used in targeted drug discovery. AutoDock Vina molecular docking, exploring binding of small molecules to target protein using a Monte Carlo technique and scoring function, is gaining popularity due to its excellent prediction accuracy. Results: Using AutoDock we have found many inhibitors of PA from our 3D database of 6170 compounds which bind in silico to X-ray structures of the specificity pocket (B187-B197, B212-B229) of both PAs preventing activation of plasminogen. However we were successful in identifying a few molecules which are specific for uPA. For example: both amiloride and chrysin bind to the specificity pocket of uPA but not to tPA. We have found that they bind to other parts of tPA, distant from the specificity pocket, thus preserving the tPA enzymatic activity while being effective toward uPA. Conclusion: In silico search yield specific inhibitors of uPA which, when verified for safety and efficiency by in vivo testing, could be used as novel therapeutics to limit metastasis and angiogenesis in anticancer therapy.

SerpinB2 inhibits migration and promotes a resolution phase signature in large peritoneal macrophages

SerpinB2 (plasminogen activator inhibitor type 2) has been called the “undecided serpin” with no clear consensus on its physiological role, although it is well described as an inhibitor of urokinase plasminogen activator (uPA). In macrophages, pro-inflammatory stimuli usually induce SerpinB2; however, expression is constitutive in Gata6+ large peritoneal macrophages (LPM). Interrogation of expression data from human macrophages treated with a range of stimuli using a new bioinformatics tool, CEMiTool, suggested that SerpinB2 is most tightly co- and counter-regulated with genes associated with cell movement. Using LPM from SerpinB2−/− and SerpinB2R380A (active site mutant) mice, we show that migration on Matrigel was faster than for their wild-type controls. Confocal microscopy illustrated that SerpinB2 and F-actin staining overlapped in focal adhesions and lamellipodia. Genes associated with migration and extracellular matrix interactions were also identified by RNA-Seq analysis of migrating RPM from wild-type and SerpinB2R380A mice. Subsequent gene set enrichment analyses (GSEA) suggested SerpinB2 counter-regulates many Gata6-regulated genes associated with migration. These data argue that the role of SerpinB2 in macrophages is inhibition of uPA-mediated plasmin generation during cell migration. GSEA also suggested that SerpinB2 expression (likely via ensuing modulation of uPA-receptor/integrin signaling) promotes the adoption of a resolution phase signature.

Tranexamic acid is an active site inhibitor of urokinase plasminogen activator

Key PointsTXA is an active-site inhibitor of uPA. TXA attenuates MDA-MB-231 BAG cell migration and inhibits endogenous uPA activity.

Rapid identification of urokinase plasminogen activator inhibitors from Traditional Chinese Medicines based on ultrafiltration, LC–MS and in silico docking

The urokinase plasminogen activator (uPA) is regarded as the crucial trigger for plasmin generation, which is involved in several diseases especially for neoplasm metastasis. In this study, an efficient approach integrating ultrafiltration, LC/MS, bioassay and in silico docking, was proposed for rapidly detecting uPA ligands from Traditional Chinese Medicines (TCMs). Forty-two TCMs were initially assessed, and as illustrative case studies, Galla Chinensis and Sanguisorbae Radix, which appeared significant inhibitory activities on uPA, were chosen to develpe and verify the strategy. A total of seven uPA ligands were successfully detected and identified. Two of them, pentagalloylglucose and 28-O-β-d-glucopyranosyl pomolic acid, were demonstrated to be potential inhibitors, with IC50 at 1.639 μM and 37.82 μM repectively. Furthermore, a combinatorial compound library screening combined with in silico docking assay, was revealed that ursolic acid (IC50 = 2.623 μM) was also speculated to be a potent parent structure for inhibition of uPA. This approach offers a multidimensional perspective to discover uPA-binding leading compounds from TCMs or other complex mixtures, which would provide an efficient route for drug discovery.

The study on the association between tissue plasminogen activator-plasminogen activator inhibitor-1 complex and risk of coronary disease

Objective The aim of this study was to discuss the effect and clinical value of plasma Tissue Plasminogen Activator-Plasminogen Activator Inhibitor-1 Complex (t-PAIC) in the coronary heart disease (CAD). Methods This is a retrospective study. From January 2017 to March 2018, 203 patients with CAD were recruited from Renmin Hospital of Wuhan University. 130 cases were selected for control group at the same period. Plasma concentrations of t-PAIC, blood RBC, Hb, WBC and other routine biochemical markers were quantified. Results The plasma t-PAIC level in CAD group 12.75(9.0-17.93) ng/ml was significantly higher than that in healthy control group 7.70(6.10-9.41) ng/ml (Z=9.208, P<0.001). Logistic regression analysis showed that the TG, LPa, t-PAIC were independent risk factors for CAD[OR(95%CI)], [1.762(1.076-2.884); 1.002(1.000-1.003); 1.338 (1.171-1.529)]. The t-PAIC level was positively correlated with the severity of CAD. Compared with control group, the level of t-PAIC was higher in mild group(11.64±0.59)ng/ml and severe group(15.84±0.72)ng/ml, and the plasma t-PAIC level was positively correlated with Gensini score(r=0.471, P<0.001). Multiple linear regression analysis showed that there was a positive correlation among the plasma t-PAIC and LDL-C, Glu, WBC (β coefficient=0.673, 0.305, 0.263, P=0.037, 0.002, 0.006). In addition, there was abnormal increase of plasma t-PAIC in CAD patients with poor prognosis (H=82.210, P<0.001). Conclusion Plasma t-PAIC is an independent risk factor of CAD. The significantly increase of plasma t-PAIC might be an important predictor of incidence of CAD and unfavourable prognosis. (Chin J Lab Med, 2018, 41: 420-424) Key words: Coronary disease; Tissue plasminogen activator; Plasminogen activator inhibitor 1; Risk factors

Effect of a synthetic inhibitor of urokinase plasminogen activator on the migration and invasion of human cervical cancer cells in vitro.

As a notable feature of malignant tumors, invasion and metastasis are important events in the process of tumor progression. Amiloride, a synthetic inhibitor of urokinase plasminogen activator (uPA), is involved in these events. To evaluate the therapeutic value of amiloride in cervical cancer, HeLa cells were used as in vitro cellular models. The migration and invasion abilities of HeLa cells, in addition to the mRNA expression of matriptase, uPA, uPA receptor and 72 kDa type IV collagenase (MMP-2), were detected using scratch assays, Transwell chamber assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of RT-qPCR demonstrated that the mRNA expression of uPA and MMP-2 in HeLa cells was downregulated significantly in a dose-dependent manner when incubated with various concentrations of amiloride for 24 h. The migration distance of HeLa cells was significantly shorter at 6, 12 and 24 h following incubation with amiloride (P<0.01), and there was a positive correlation between cell migratory ability and cellular uPA protein expression level (r=0.955, P<0.01). The number of HeLa cells that penetrated the Matrigel following incubation for 24 h with different concentrations of amiloride decreased significantly compared with the control group, indicating that cell invasiveness was positively correlated with the protein expression level of uPA in the cells (r=0.993, P<0.01). The present study demonstrated that amiloride was able to specifically inhibit the mRNA expression levels of uPA in HeLa cells, and sequentially downregulate the mRNA expression of downstream MMP-2 in the uPA system, thereby suppressing the migratory and invasive ability of HeLa cells. Therefore, amiloride may be a promising therapeutic target for the treatment of cervical cancer.

Impaired plasminogen binding in patients with venous thromboembolism: Association with protein carbonylation

IntroductionVenous thromboembolism (VTE) is associated with hypofibrinolysis. Its mechanisms are poorly understood. We investigated plasminogen-fibrin interaction and its association with fibrinolytic capacity and protein oxidation/carbonylation in VTE patients. Materials and methodsPlasma-purified plasminogen conversion to plasmin and surface plasmon resonance employed for plasminogen-fibrin interactions were individually evaluated in all healthy controls and non-anticoagulated patients following VTE, 10–23months since the event. We also assessed plasma fibrin clot permeability (Ks), clot lysis time (LT), activators and inhibitors of fibrinolysis together with oxidation/carbonylation markers. ResultsVTE patients had impaired plasminogen binding to fibrin (apparent Kd, +290%, p=0.002), reduced rate of plasmin generation (−4.7%, p=0.001), and longer LT (+18.6%, p<0.001) compared with controls. Fibrinogen and Ks were similar in both groups. Apparent Kd correlated with LT (r=0.43, p=0.037), tissue plasminogen activator-plasminogen activator inhibitor 1 (tPA-PAI-1) complexes (r=0.63, p=0.012), and active PAI-1 (r=0.49, p=0.03). Compared with controls, VTE patients had higher thiobarbituric acid reactive substances (TBARS), total protein carbonyl content (PC), and lower total antioxidant capacity (all p<0.001), that all were associated with LT (r=0.61, r=0.56, and r=−0.47, respectively, all p<0.05). Impaired plasminogen binding to fibrin reflected by apparent Kd positively correlated with TBARS (r=0.48, p=0.032) and PC (r=0.54, p=0.013) in the whole group. ConclusionsPlasminogen-fibrin interactions are altered in young and middle-aged VTE patients, without known thrombophilias, except increased factor VIII. The mechanisms underlying these phenomena remain to be established.

Abolishment of proximal tubule albumin endocytosis does not affect plasma albumin during nephrotic syndrome in mice

The megalin/cubilin receptor complex is required for proximal tubular endocytosis and degradation of filtered albumin. An additional high-capacity retrieval pathway of intact albumin for the recovery of large amounts of filtered albumin has been proposed, possibly involving cooperation between megalin/cubilin and the neonatal Fc receptor. To clarify the potential role of such a pathway, we examined the effects of megalin/cubilin gene inactivation on tubular albumin uptake and plasma albumin levels in nephrotic, podocin knockout mice. Immunofluorescence microscopy of megalin/cubilin/podocin knockout mouse kidneys demonstrated abolishment of proximal tubule albumin uptake, in contrast to the excessive albumin accumulation observed in podocin knockout mice compared to controls. Correspondingly, urinary albumin excretion was increased 1.4 fold in megalin/cubilin/podocin compared to podocin knockout mice (albumin/creatinine: 226 vs. 157 mg/mg). However, no difference in plasma albumin levels was observed between megalin/cubilin/podocin and podocin knockout mice, as both were reduced to approximately 40% of controls. There were no differences in liver albumin synthesis by mRNA levels and protein abundance. Thus, megalin/cubilin knockout efficiently blocks proximal tubular albumin uptake in nephrotic mice but plasma albumin levels did not differ as a result of megalin/cubilin-deficiency, suggesting no significance of the megalin/cubilin-pathway for albumin homeostasis by retrieval of intact albumin.

The serine protease inhibitor serpinB2 binds and stabilizes p21 in senescent cells

SerpinB2 is a serine protease inhibitor also known as plasminogen activator inhibitor type 2 (PAI-2). It has been well documented that serpinB2 is an inhibitor of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). Interestingly, serpinB2 levels are increased in senescent cells and serpinB2 is thus considered a senescence biomarker. In this study, by mimicking the elevated levels of serpinB2 in senescent cells, proliferating human fibroblasts were induced into senescence. Senescence induced by serpinB2 did not relate to its extracellular function, as inhibition of serpinB2 secretion, exogenous introduced serpinB2, or a serpinB2 mutant that failed to bind to its extracellular target uPA did not affect senescence. We also showed that serpinB2 is a direct downstream target of p53 that is activated by the DNA damage response pathway. Significantly, serpinB2 bound to and stabilized p21 to mediate senescence in a proteasome-independent manner, indicating that serpinB2 has a direct role in senescence. Thus, this study reveals a unique mechanism by which serpinB2 maintains senescence through stabilization of p21 protein levels.

Blood coagulation biomarkers for postoperative venous thromboembolism diagnosis in orthopedic traumatic patients: a case control study

Objective To evaluate the value of blood coagulation biomarkers in orthopaedic traumatic patients after surgery and analyze its diagnostic values for venous thrombosis embolism. Methods In thiscase control study, we consecutive enrolled 108 traumatic patients after surgery. 54 patients have thrombosis and other 54 patients have no thrombosis. Blood was taken 3-4 days after surgery. Routine coagulation screening test , FDP(fibrinogen/fibrin degradation products) , D dimer and new item such as TM(thrombomodulin), TAT(thrombin-anti-thrombin complex), t-PAIC(tissue- type plasminogen activator-plasminogen activator inhibitor complex), PIC(plasmin-anti-plasmin complex) were tested. The difference between groups of these biomarkers was compared, and then the receiver operation curve (ROC) was drew to determine the diagnostic cut-off point and diagnostic performance. Results ALL blood coagulation biomarkers in orthopaedic traumatic patients after surgery were significantly increased. The group of patients with thrombosis have higher TM(9.04±2.06)IU/ml, t-PAIC(10.15±4.23)ng/ml, PIC(1.15±0.70)μg/ml, D dimer(5.31±5.10)ng/ml than group without thrombosis TM(7.50±1.70)IU/ml, t-PAIC (6.97±2.56)ng/ml, PIC(0.93±0.84)μg/ml, D dimer(2.35±2.12)ng/ml, and P=0.000 2, <0.000 1, <0.000 1, <0.000 1, respectively. However, TAT(4.79±4.32)ng/ml, (6.51±5.92)ng/ml, FDP(8.87±7.68)μg/ml, (4.91±4.67)μg/ml showed no difference between thrombosis groupand no thrombosis group, (P=0.212 3, 0.050 8; respectively). The area under the ROC curve of TM, t-PAIC, PIC and D-dimer were 0.718 5, 0.741 6, 0.648 0, 0.670 0, respectively; P values were <0.000 1, <0.000 1, 0.009 3, 0.004 1, respectively; cut-off values were 11.15 IU/ml, 10.65 ng/ml, 1.36 μg/ml, 7.69 ng/ml, respectively; positive likelihood ratios were 9.00, 11.29, 3.66, 14.60, respectively; specificity were 98.15%, 96.23%, 90.20%, 97.96%, respectively; the diagnostic rates were 20.3%, 46.3%, 35.8%, 25.9%, respectively. Conclusions There were coagulation and fibrinolysis system activated in orthopaedic traumatic patients after surgery.TM, t-PAIC, PIC, D dimer were good biomarkers for the diagnosis of thrombosis after trauma surgery. TAT was not fit for screening thrombosis after surgery because of influence of anti-coagulation.(Chin J Lab Med, 2016, 39: 751-755) Key words: Postoperative complications; Venous thrombosis; Biological markers; Thrombomodulin; Tissue plasminogen activator; Fibrin fibrinogen degradation products

Discovery and SAR of Novel and Selective Inhibitors of Urokinase Plasminogen Activator (uPA) with an Imidazo[1,2-a]pyridine Scaffold.

Urokinase plasminogen activator (uPA) is a biomarker and therapeutic target for several cancer types. Its inhibition is regarded as a promising, noncytotoxic approach in cancer therapy by blocking growth and/or metastasis of solid tumors. Earlier, we reported the modified substrate activity screening (MSAS) approach and applied it for the identification of fragments with affinity for uPA's S1 pocket. Here, these fragments are transformed into a novel class of uPA inhibitors with an imidazo[1,2-a]pyridine scaffold. The SAR for uPA inhibition around this scaffold is explored, and the best compounds in the series have nanomolar uPA affinity and selectivity with respect to the related trypsin-like serine proteases (thrombin, tPA, FXa, plasmin, plasma kallikrein, trypsin, FVIIa). Finally, the approach followed for translating fragments into small molecules with a decorated scaffold architecture is conceptually straightforward and can be expected to be broadly applicable in fragment-based drug design.