Verkleij, Chantal J N; Stuijver, Danka J F; van Zaane, Bregje; Squizzato, Alessandro; Brandjes, Dees P M; Büller, Harry R; Meijers, Joost C M; Gerdes, Victor E A
Endocrine disorders affect both the coagulation and fibrinolytic systems, and have been associated with the development of cardiovascular diseases. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a link between coagulation and the fibrinolytic system. The aim of this study was to determine the effect of thyroid hormone excess and deficiency on TAFI levels and function. The effect of hyperthyroxinemia on TAFI was studied in healthy volunteers who were randomised to receive levothyroxine or no medication for 14 days in a crossover design. The effect of hypothyroidism on TAFI was studied in a multicentre observational cohort study. Blood was drawn before treatment of patients with newly diagnosed hypothyroidism and when euthyroidism was achieved. Plasma clot-lysis times, activated TAFI (TAFIa)-dependent prolongation of clot-lysis and TAFI levels were measured. Thyroid hormone excess resulted in a hypofibrinolytic condition and in an enhanced TAFIa-dependent prolongation of clot lysis. A trend towards decreased plasma TAFI levels was observed in healthy volunteers who used levothyroxine. Hypothyroidism resulted in hyperfibrinolysis and a reduced TAFIa-dependent prolongation of clot lysis. In conclusion, alterations of TAFIa-dependent prolongation of clot lysis in patients with thyroid disorders may cause an impaired haemostatic balance. The disturbed haemostatic balance in patients with hyperthyroidism might make them prone to thrombosis, while the risk for bleeding may increase in patients with hypothyroidism.
Yener, S; Akarsu, M; Demir, T; Akinci, B; Sagol, O; Bayraktar, F; Ozcan, M A; Tankurt, E; Yesil, S
This study was conducted to demonstrate the plasminogen activator inhibitor- 1 (PAI-1) and thrombin activatable fibrinolysis inhibitor antigen (TAFI-Ag) levels in non-alcoholic steatohepatitis (NASH). Twenty-seven patients with biopsy-proven NASH and 18 healthy controls (HC) were recruited for the study. Anthropometric data, liver histology (no.=20) and laboratory parameters including PAI-1 and TAFI-Ag assessments were recorded. When compared with HC, patients with NASH had higher body weight, higher waist circumference, elevated blood pressure, higher fasting plasma glucose (FPG) levels and higher homeostasis model assessment (HOMA) scores. The mean plasma PAI-1 levels of patients was found to be higher than HC (87.60 ng/ml vs 30.84 ng/ml p=0.000) and mean plasma TAFI-Ag levels of patients was found to be significantly lower (8.69 microg/ml vs 12.19 microg/ml p=0.000). PAI-1 levels were correlated with systolic blood pressure, age, body weight, transaminases, waist circumference, FPG, body mass index, and HOMA score. TAFI-Ag levels were found to be negatively correlated with transaminases, waist circumference, and body weight. In multiple regression analysis, BMI was the independent variable effecting PAI-1 levels. We did not show any association between PAI-1, TAFI-Ag, disease activity score and fibrosis score. HOMA was the independent variable effecting liver fibrosis in our patients. In this study we demonstrated that patients with biopsy-proven NASH had higher PAI-1 and lower TAFI-Ag expression than HC. Elevated levels of PAI-1 in NASH is the consequence of insulin resistance state. Lower TAFI-Ag levels may be related to the overactivation of TAFI pathway resulting in TAFI-Ag depletion. Furthermore, liver function disturbances may impair TAFI production in NASH. We also showed that NASH patients even with slight elevations of transaminases feature marked insulin resistance and components of metabolic syndrome.
Valls Serón, M; Haiko, J; DE Groot, P G; Korhonen, T K; Meijers, J C M
Pathogenic bacteria modulate the host coagulation system to evade immune responses or to facilitate dissemination through extravascular tissues. In particular, the important bacterial pathogens Salmonella enterica and Yersinia pestis intervene with the plasminogen/fibrinolytic system. Thrombin-activatable fibrinolysis inhibitor (TAFI) has anti-fibrinolytic properties as the active enzyme (TAFIa) removes C-terminal lysine residues from fibrin, thereby attenuating accelerated plasmin formation. Here, we demonstrate inactivation and cleavage of TAFI by homologous surface proteases, the omptins Pla of Y. pestis and PgtE of S. enterica. We show that omptin-expressing bacteria decrease TAFI activatability by thrombin-thrombomodulin and that the anti-fibrinolytic potential of TAFIa was reduced by recombinant Escherichia coli expressing Pla or PgtE. The functional impairment resulted from C-terminal cleavage of TAFI by the omptins. Our results indicate that TAFI is degraded directly by the omptins PgtE of S. enterica and Pla of Y. pestis. This may contribute to the ability of PgtE and Pla to damage tissue barriers, such as fibrin, and thereby to enhance spread of S. enterica and Y. pestis during infection. © 2010 International Society on Thrombosis and Haemostasis.
Bridge, Katherine I; Bollen, Lize; Zhong, Jim; Hesketh, Mark; Macrae, Fraser L; Johnson, Anne; Philippou, Helen; Scott, D Julian; Gils, Ann; Ariёns, Robert A S
Intra-luminal thrombosis is a key factor in Abdominal Aortic Aneurysms (AAA) growth. Patients with AAA form dense clots that are resistant to fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) has been shown to influence AAA development in murine models. The aim of this study is to characterise the role of TAFI in human AAA. Plasma levels of TAFI, TAFI activation peptide (TAFI-AP), activated/inactivated TAFI (TAFIa/ai) and plasmin-α2-antiplasmin complex were measured by ELISAs in patients with AAA (n=202) and controls (n=188). TAFIa/ai and TAFI-AP levels were higher in patients than controls (median (IQR): 20.3 (14.6-32.8) vs. 14.2 (11.2-19.3) and 355.0 (232.4-528.1) vs. 248.6 (197.1-328.1) ng/ml). TAFIa/ai was positively correlated with TAFI-AP (r=0.164). Intact TAFI levels were not different between patients and controls (13.4 (11.2-16.1) vs. 12.8 (10.6-15.4) μg/ml). Plasmin-α2-antiplasmin was higher in AAA patients than controls (690.0 (489.1-924.3) vs. 480.7 (392.6-555.3) ng/ml). The increase in TAFIa/ai and TAFI-AP suggest an increased TAFI activation in patients with AAA. Prospective studies are required to further elucidate the role of TAFI and fibrinolysis in AAA pathogenesis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
Pang, H; Zhang, C; Liu, F; Gong, X; Jin, X; Su, C
A study was made of the changes in the serum levels of thrombin activatable fibrinolysis inhibitor (TAFI), proinflammatory cytokines and acute phase proteins in the acute stage of acute coronary syndrome (ACS), in order to explore the possibility of using TAFI as a biomarker for ACS risk assessment. A total of 211 patients with ACS were enrolled, and healthy subjects were used as controls. Blood samples were taken within 24h after admission. Serum TAFI levels were determined by immunoturbidimetry. Serum levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA). Procalcitonin (PCT) and C-reactive protein (CRP) levels were measured by gold-immunochromatographic assay. Serum TAFI levels in ACS patients were significantly decreased versus the controls. The IL-1β, IL-6, TNF-α, PCT and CRP levels were markedly higher in the ACS patients than in the controls. Correlation analysis revealed a strong negative correlation between TAFI concentration and the IL-1β, IL-6, TNF-а, PCT and CRP levels in ACS patients and in controls. Multivariate logistic regression analysis suggested decreased serum TAFI to be an independent risk factor for ACS (OR 9.459; 95% CI 2.306-38.793; P=0.002). The area under the receiver operating characteristic (ROC) curve for TAFI was 0.872 (95% CI 0.787-0.909; P<0.001). The optimum TAFI cutoff point for the prediction of ACS was 24μg/ml, with a sensitivity of 75.83% and a specificity of 72.57%. These findings suggest that TAFI can be useful as a potential biomarker for ACS risk assessment. Copyright © 2016 Elsevier España, S.L.U. y SEMICYUC. All rights reserved.
Brzoska, Tomasz; Suzuki, Yuko; Sano, Hideto; Suzuki, Seiichirou; Tomczyk, Martyna; Tanaka, Hiroki; Urano, Tetsumei
Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).
Valnickova, Zuzana; Sanglas, Laura; Arolas, Joan L.; Petersen, Steen V.; Schar, Christine; Otzen, Daniel; Aviles, Francesc X.; Gomis-Rüth, F. Xavier; Enghild, Jan J.
We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo. PMID:20880845
Grosso, Giorgia; Vikerfors, Anna; Woodhams, Barry; Adam, Mariette; Bremme, Katarina; Holmström, Margareta; Ågren, Anna; Eelde, Anna; Bruzelius, Maria; Svenungsson, Elisabet; Antovic, Aleksandra
Thrombosis and complement activation are pathogenic features of antiphospholipid syndrome (APS). Their molecular link is Plasma carboxypeptidase-B, also known as thrombin activatable fibrinolysis inhibitor (TAFIa), which plays a dual role: anti-fibrinolytic, by cleaving carboxyl-terminal lysine residues from partially degraded fibrin, and anti-inflammatory, by downregulating complement anaphylatoxins C3a and C5a. To investigate the levels of TAFI (proenzyme) and TAFIa (active enzyme) in relation to complement activation, fibrin clot permeability and fibrinolytic function in clinical and immunological subsets of 52 APS patients and 15 controls. TAFI (p<0.001), TAFIa (p<0.05) and complement factor C5a (p<0.001) were increased, while fibrin permeability (p<0.01) was decreased and clot lysis time (CLT) was prolonged (p<0.05) in APS patients compared to controls. Furthermore, TAFIa was increased (p<0.01) in samples from APS patients affected by arterial thrombosis compared to other APS-phenotypes. Positive associations were found between TAFI and age, fibrinogen and C5a, and between TAFIa and age, fibrinogen and thrombomodulin. TAFI and TAFIa levels were increased in patients with APS as a potential response to complement activation. Interestingly, TAFI activation was associated with arterial thrombotic APS manifestations. Thus, TAFIa may be considered a novel biomarker for arterial thrombosis in APS. Copyright © 2016 Elsevier Ltd. All rights reserved.
Frère, Corinne; Tregouet, David-Alexandre; Morange, Pierre-Emmanuel; Saut, Noémie; Kouassi, Dinar; Juhan-Vague, Irène; Tiret, Laurence; Alessi, Marie-Christine
Recent studies revisiting the association between plasma thrombin-activatable fibrinolysis inhibitor (TAFI) Ag levels and polymorphisms of the CPB2 gene (coding for TAFI) suggested that TAFI Ag levels were influenced by 2 major quantitative trait nucleotides (QTNs) in European whites. However, the strong linkage disequilibrium (LD) between CPB2 polymorphisms in European whites did not allow one to distinguish which polymorphisms could be the putative QTNs. To get a better insight into the identification of QTNs, a transethnic haplotype analysis contrasting 2 populations of African and European subjects was performed using 13 CPB2 polymorphisms. Results of the haplotype analyses suggested that 3 QTNs had independent effects and explained about 15% of the TAFI variability, consistently in the 2 populations. The lower LD observed in the African population enabled us to identify the 1583T>A SNP located in 3'UTR as one of these QTNs, whereas the -2599C>G and -2345--2344insG SNPs located in the 5' region might be the 2 other QTNs. A phylogenetic study suggested that these 3 polymorphisms occurred before the period of migration "out of Africa." Although this transethnic comparison contributed to better map the putative CPB2 QTNs, further studies are required to clarify the role of the promoter region.
Zhou, J; Kochan, J; Yin, O; Warren, V; Zamora, C; Atiee, G; Pav, J; Orihashi, Y; Vashi, V; Dishy, V
Essentials DS-1040 inhibits the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa). Infusion of DS-1040 was safe and well tolerated in healthy young and elderly subjects. DS-1040 substantially decreased TAFIa activity but had no impact on bleeding time. DS-1040 may provide an option of safer thrombolytic therapy. Background Current treatments for acute ischemic stroke and venous thromboembolism, such as recombinant tissue-type plasminogen activator and thrombectomy, are limited by a narrow time window and the risk of bleeding. DS-1040 is a novel low molecular weight compound that inhibits the activated form of thrombin-activatable fibrinolysis inhibitor (TAFIa), and was developed as a fibrinolysis enhancer for the treatment of thromboembolic diseases. Objectives This first-in-human, randomized, placebo-controlled, three-part, phase 1 study was conducted to evaluate the safety, pharmacokinetics and pharmacodynamics of DS-1040 in healthy subjects. Subjects/Methods Young (18-45 years) or elderly (65-75 years) subjects (N = 103) were randomized to receive single ascending doses of DS-1040 ranging from 0.1 mg to 40 mg, or placebo, administered either as a 0.5-h intravenous infusion or as a 24-h continuous infusion. Results All doses of DS-1040 were tolerated, and no serious adverse events (AEs) or discontinuations resulting from AEs occurred during the study. Bleeding time remained within the normal range for all doses tested in all subjects. Plasma exposure of DS-1040 increased proportionally with increase in dose. Elderly subjects had higher exposures to DS-1040 and prolonged elimination times, probably because of decreased renal clearance. DS-1040 caused a substantial dose-dependent and time-dependent decrease in TAFIa activity and in 50% clot lysis time. The levels of D-dimer, indicative of endogenous fibrinolysis, increased in some individuals following DS-1040 treatment. No effects of DS-1040 on coagulation parameters or platelet
Bavbek, Nuket; Ceri, Mevlut; Akdeniz, Derya; Kargili, Ayse; Duranay, Murat; Erdemli, Kemal; Akcay, Ali; Guz, Galip
Coagulation abnormalities have been reported in familial Mediterranean fever (FMF) patients with amyloidosis and nephrotic syndrome; but there is not enough data about the continuity of the thrombogenic activity in FMF patients in clinical remission. The purpose of this study was to assess thrombin activatable fibrinolysis inhibitor (TAFI) levels and its relationship with fibrinolytic activity and also evaluate relationships between mutations and clinical signs in attack-free patients without amyloidosis. Seventy-nine FMF patients and 40 healthy adults were included. The study group was divided into five groups as follows: first group, homozygote M694V; second group, homozygote M680I; third group, M694V in one allele, the other allele have other mutations or not; fourth group, other mutations; and fifth group, no mutation. Serum TAFI levels were significantly increased in patients compared with healthy individuals (116.64 ± 21.8 vs. 78.48 ± 19.7 μg/mL, p < 0.001) and a positive correlation was detected between TAFI antigen level and erythrocyte sedimentation rate and C-reactive protein levels (r = 0.247, p = 0.029 and r = 0.252, p = 0.032, respectively). Mean fibrinogen and TAFI levels were significantly higher in Group 1 than the other groups (p = 0.04 and p = 0.001, respectively) and in Group 3 it was higher than Groups 2, 4 and 5 (p = 0.04 and p = 0.001, respectively). High level of TAFI antigen in attack-free period of FMF disease shows ongoing subclinical inflammation and hypercoagulability. Clinicians should be careful about thrombosis even in patients at clinical remission. Also, genetic tests must be considered to predict clinical outcome and to reduce complications of FMF disease.
Harmanci, Ayla; Kandemir, Nurgun; Dagdelen, Selcuk; Gonc, Nazli; Buyukasik, Yahya; Alikasifoglu, Ayfer; Kirazli, Serafettin; Ozon, Alev; Gurlek, Alper
Hypofibrinolysis is a state that is commonly observed in type 2 diabetic patients, a finding also possibly related to obesity and insulin resistance. There is little information, however, regarding the status of fibrinolytic system in Type 1 diabetes, in particular as reflected by thrombin-activatable fibrinolysis inhibitor (TAFI) activity and global fibrinolytic capacity (GFC). To provide information in this respect, 30 Type 1 diabetic patients (median age=16) and 28 healthy controls (median age=14) were enrolled in this study. The median duration of diabetes was 7 years, and median HbA(1c) was 8.85% (range: 5.5-11.9%) in the diabetic group. None of the patients had macrovascular complications. Microvascular complications were present in a total of eight patients (nephropathy: n=5; retinopathy: n=3). A comparison of the TAFI activity between the patient (median 84.9, range: 71.5-103.3%) and the control groups (median=83.3, range: 63.7-97.4%) yielded no statistically significant difference (P=.950). Similarly, GFC was comparable between the two groups (median=8.22, range: 0.72-22.38 microg/ml, and median=13.32, range: 3.0-23.22 microg/ml, respectively, in the diabetic and control groups, P=.086). TAFI activity did not significantly correlate with age, albumin excretion, fasting plasma glucose, HbA(1c), D-dimer, and fibrinogen by Spearman rank correlation test. There was as a significant inverse correlation between GFC and TAFI activity (r=-.414, P=.006). Contrary to the previous observations in Type 2 diabetes, our data suggest that fibrinolytic activity is not adversely affected by Type 1 diabetes, and it has no relationship with the degree of metabolic control.
Ozkan, Gulsum; Ulusoy, Sukru; Sönmez, Mehmet; Karahan, S Caner; Menteşe, Ahmet; Kaynar, Kubra; Bektaş, Ozlen
Hypertension is associated with fibrinolysis abnormality. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a novel molecule-linking coagulation and fibrinolysis. The aim of this study was to investigate the levels of TAFI in primary hypertensive patients and to compare the effects of amlodipine and ramipril on TAFI levels. The study was performed with 58 hypertensive subjects and 27 healthy volunteers. Biochemical and hematological parameters and TAFI levels were measured at baseline and after 1-month follow-up. TAFI concentrations increased in hypertensive patients compared with the controls (P = .030). Additionally, TAFI levels decreased with blood pressure control at 1-month follow-up (P = .026). There was no significant difference between TAFI levels in the amlodipine and ramipril groups at baseline. However, after 1-month follow-up, TAFI levels were decreased in the amlodipine group (P = .037) but not in the ramipril group. Our study is the first in the literature to determine increased TAFI levels in primary hypertension patients. In addition, we determined a decrease in TAFI levels in the amlodipine group after 1 month, but none in the ramipril group.
Schreuder, Herman; Liesum, Alexander; Lönze, Petra; Stump, Heike; Hoffmann, Holger; Schiell, Matthias; Kurz, Michael; Toti, Luigi; Bauer, Armin; Kallus, Christopher; Klemke-Jahn, Christine; Czech, Jörg; Kramer, Dan; Enke, Heike; Niedermeyer, Timo H. J.; Morrison, Vincent; Kumar, Vasant; Brönstrup, Mark
Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1’ binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site. PMID:27604544
Higuchi, Toshiyuki; Nakamura, Takashi; Kakutani, Hideki; Ishii, Hidemi
Cell malignancy is negatively correlated with the expression of thrombomodulin (TM), a thrombin receptor expressed on the surface of various cells, including tumor cells. TM accelerates thrombin-activatable fibrinolysis inhibitor (TAFI) activation catalyzed by thrombin. The active form of TAFI (TAFIa) contributes to inhibition of plasmin formation through its carboxypeptidase B (CPB)-like activity. Here, we examined whether TM- and tumor cell-dependent TAFI activation participates in controlling pericellular fibrinolysis and cell invasion. Human fibrosarcoma HT1080 cells retained the ability to activate both prothrombin and plasminogen, but did not express TM. HT1080 cells mediated activation of TAFI in plasma in the presence of soluble-type TM (sTM) through cell-dependent prothrombin activation. HT1080 cells stably expressing TM (TM-HT1080) mediated plasma TAFI activation without added sTM, but HT1080 (wild-type) and Mock-transfected HT1080 cells (Mock) did not. Production of TAFIa suppressed cell-mediated plasminogen activation. Matrigel invasion by wild-type and Mock cells was enhanced two-fold by diluted plasma (10%), whereas the plasma-induced invasion of TM-HT1080 cells (65% of wild-type invasion) was lower than those of wild-type and Mock cells. Cell invasion by TM-HT1080 was partially enhanced by addition of a TAFIa/CPB inhibitor. These results suggest that TM suppresses pericellular fibrinolysis and plasma-induced tumor cell invasion, and that it is mediated, at least in part, by plasma TAFI activation.
Kozian, Detlef H; Lorenz, Martin; März, Winfried; Cousin, Emmanuelle; Mace, Sandrine; Deleuze, Jean-Francois
The thrombin-activatable fibrinolysis inhibitor (TAFI) is a key mediator in the regulation of endogenous fibrinolysis, down-regulating clot lysis by degrading the C-terminal lysine residues from fibrin, which are important for binding and activating plasminogen. Elevated TAFI antigen levels have been suggested to be associated with promoter variants and the Ala147Thr polymorphism; increased TAFI stability and antifibrinolytic potential instead have been associated with the Thr325Ile polymorphism. We investigated the influence of these two polymorphisms on cardiovascular and thrombotic events in patients of the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. The LURIC study is a prospective cohort study comprising more than 3,300 patients aimed at identifying biochemical and genetic markers for metabolic and cardiovascular diseases. We demonstrate that the Ile/Ile genotype at position 325 of TAFI associates with the incidence of stroke and the age at onset of first stroke in patients of the LURIC cohort. Both the incidence of stroke and the risk of a premature event are higher in TAFI Ile325Ile patients with predisposing risk factors for thrombotic events such as diabetes mellitus, myocardial infarction or hypertension, alone or in combination. In contrast, no significant association was identified for the TAFI Ala147Thr polymorphism. The robust association of the TAFI Thr325Ile polymorphism with the incidence and the age at onset of first stroke strongly suggests a key role for TAFI in the pathogenetic mechanism of stroke.
Soluble CD40 ligand, plasminogen activator inhibitor-1 and thrombin-activatable fibrinolysis inhibitor-1-antigen in normotensive type 2 diabetic subjects without diabetic complications. Effects of metformin and rosiglitazone.
Yener, Serkan; Comlekci, Abdurrahman; Akinci, Baris; Demir, Tevfik; Yuksel, Faize; Ozcan, Mehmet Ali; Bayraktar, Firat; Yesil, Sena
To evaluate subclinical inflammation and fibrinolysis in low-risk type 2 diabetic subjects and to assess the efficacy of metformin and rosiglitazone in this group. Sixty-one normotensive, normoalbuminuric type 2 diabetic subjects without diabetes-related complications were included in a 4-week standardization period with glimepiride. After the standardization period, 21 subjects were excluded and the remaining 40 were randomly divided into two groups matched for age, gender, body mass index and disease duration. The first group (n = 20) received metformin (1,700 mg/day), the second group (n = 20) rosiglitazone (4 mg/day) for 12 weeks. Patients with low-density lipoprotein-cholesterol higher than 130 mg/dl at the beginning of the randomization period were treated with simvastatin (maximum dose 20 mg/day). Twenty-three healthy controls were also recruited. Cytokine measurements were performed with ELISA kits. Baseline plasma plasminogen activator inhibitor-1 (PAI-1) level of type 2 diabetic subjects was significantly elevated (p = 0.038), but baseline levels of soluble CD40 ligand (sCD40L) and thrombin-activatable fibrinolysis inhibitor-1 (TAFI) antigen did not differ from healthy controls. Twelve weeks of metformin or rosiglitazone therapy did not cause significant changes in sCD40L, PAI-1 and TAFI antigen levels. In simvastatin-treated subjects (n = 9) significant reductions of PAI-1 were achieved (p = 0.028), while sCD40L and TAFI-Ag did not differ from baseline values. Our results showed that nonobese diabetic patients at low cardiovascular risk had similar levels of subclinical markers of inflammation and fibrinolysis as matched healthy controls. Neither metformin nor rosiglitazone caused marked changes in sCD40L, PAI-1 and TAFI antigen levels. A subset of patients who received simvastatin showed a modest decrease in PAI-1 level and could contribute to beneficial vasculoprotective effect of the drug in type 2 diabetics. Copyright (c) 2009 S. Karger AG, Basel.
Özcan, Mehmet Ali; Akarsu, Mesut; Demirkan, Fatih; Akpınar, Hale; Yüksel, Faize; Özsan, Güner Hayri; Ündar, Bülent; Pişkin, Özden; Alacacıoğlu, İnci
GİRİŞ: İnflamatuar barsak hastalığının (IBH) seyri, arteriyel ya da venöz sistemde olabilen tromboembolik olaylar ile sık olarak komplike olmaktadır. Tümü olmasa da çalışmaların çoğu IBH olan hastalarda koagülasyon ve fibrinoliz göstergelerindeki değişiklikleri ortaya koymaktadır. YÖNTEM: Çalışmaya IBH olan 45 hasta (31 UC, 14 CD) dahil edildi. Yaşı ve cinsiyeti uyumlu 16 gönüllü kontrol grubu olarak alındı. TAFI antijeninin kantitatif olarak saptanması için VisuLiseTM ELISA kiti kullanıldı. SONUÇLAR: Beyaz küre sayısı, trombosit sayısı, eritrosit sedimentasyon hızı ve C-reaktif protein gibi inflamasyon belirteçleri aktif hastalığı olanlarda belirgin yüksek bulundu. Aktif ya da inaktif IBH olanların protrombin zamanı, aktive parsiyel tromboplastin zamanı ve d-dimer düzeyleri gibi koagülasyon ölçekleri arasında anlamlı fark bulunmadı. Hastalığı aktif olanların fibrinojen düzeyleri belirgin olarak daha yüksekti. Aktif ya da inaktif hastalığı olanlar ile sağlıklı kontrollerin plazma TAFI düzeyleri arasında anlamlı bir fark gösterilemedi. Aktif ve inaktif hastalığı olanların β-TG ve PF-4 düzeyleri arasında da anlamlı bir değişiklik gözlenmedi. SONUÇ: IBH’da TAFI düzeylerini araştırdık. Literatürdeki bilgilerle çelişecek şekilde TAFI düzeyleri IBH’nın aktivasyon göstergesi olarak kullanılabilir gözükmemektedir. Hastalığın farklı aşamalarında ve aktivasyon düzeylerinde olan daha fazla hastayı kapsayan çalışmaların yapılması konunun daha iyi aydınlamasına yardımcı olacaktır.
Panigada, Mauro; Zacchetti, Lucia; L’Acqua, Camilla; Cressoni, Massimo; Anzoletti, Massimo Boscolo; Bader, Rossella; Protti, Alessandro; Consonni, Dario; D’Angelo, Armando; Gattinoni, Luciano
Introduction Impairment of fibrinolysis during sepsis is associated with worse outcome. Early identification of this condition could be of interest. The aim of this study was to evaluate whether a modified point-of-care viscoelastic hemostatic assay can detect sepsis-induced impairment of fibrinolysis and to correlate impaired fibrinolysis with morbidity and mortality. Methods This single center observational prospective pilot study was performed in an adult Intensive Care Unit (ICU) of a tertiary academic hospital. Forty consecutive patients admitted to the ICU with severe sepsis or septic shock were included. Forty healthy individuals served as controls. We modified conventional kaolin activated thromboelastography (TEG) adding urokinase to improve assessment of fibrinolysis in real time (UK-TEG). TEG, UK-TEG, plasminogen activator inhibitor (PAI)-1, thrombin-activatable fibrinolysis inhibitor (TAFI), d-dimer, DIC scores and morbidity (rated with the SOFA score) were measured upon ICU admission. Logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (95% CIs) of mortality at ICU discharge. Results UK-TEG revealed a greater impairment of fibrinolysis in sepsis patients compared to healthy individuals confirmed by PAI-1. TAFI was not different between sepsis patients and healthy individuals. 18/40 sepsis patients had fibrinolysis impaired according to UK-TEG and showed higher SOFA score (8 (6–13) vs 5 (4–7), p = 0.03), higher mortality (39% vs 5%, p = 0.01) and greater markers of cellular damage (lactate levels, LDH and bilirubin). Mortality at ICU discharge was predicted by the degree of fibrinolysis impairment measured by UK-TEG Ly30 (%) parameter (OR 0.95, 95% CI 0.93–0.98, p = 0.003). Conclusions Sepsis-induced impairment of fibrinolysis detected at UK-TEG was associated with increased markers of cellular damage, morbidity and mortality. PMID:26308340
Podorol'skaia, L V; Andreenko, G V; Poliantseva, L R; Bumblite, I D
Functional activities of plasminogen activators (FPAA) and their inhibitors and plasminogen activators's (PA), antigen level were determined in 31 patients with chronic glomerulonephritis, 23 patients with amyloidosis and 15 healthy persons. High FPAA correlated with favourable prognosis of diseases, elevated PA antigen level and diminished alpha 1-antitrypsin, alpha 2-macroglobulin and antiactivator activities. There were decreased PA antigen level and increased inhibitor's activities in group with zero FPAA. Protein loaded functional probe demonstrated the presence of PA reserves in high FPAA patients and "pathological proteolysis" in zero FPAA patients. The last phenomenon was likely connected to nonspecific proteases differed from PA.
Krishnan, Lissy K; Vijayan Lal, Arthur; Uma Shankar, P R; Mohanty, Mira
Experiments have been carried out to determine if aprotinin and epsilon -amino caproic acid increases the quality of Fibrin glue. A rat model was used for tissues such as liver and skin while rabbits were used for application of glue in dura mater. Apposition of all the tissues, glued with fibrin was found to be good and remnants of the polymerized fibrin were seen even on the seventh day of application, though inhibitors were not incorporated with the glue. In skin, excessive amounts of fibrin remained as a result of addition of aprotinin and epsilon -amino caproic acid, as compared to the glue applied without any inhibitor. After dural sealing, the wound repair and new bone formation at craniotomy site progressed well in the fibrin glue applied area as compared to the commercially available glue that contained aprotinin. The adhesive strength of the glue without or with fibrinolysis inhibitors was found to be similar, after 1h grafts on rat back. The observations from this study suggests that the use of aprotinin with fibrin glue may not be required because, even liver tissue that is known to have high fibrinolytic activity was sealed and repaired well in the absence of plasminogen inhibitors. On the other hand, it was found that if inhibitors were added, nondegraded matrix remained in the tissue even after 15 days and affected migration of repair cells. Thus, the inhibition of fibrinolysis after fibrin glue application is found detrimental to wound healing.
Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul
Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870
van Geffen, Mark; Loof, Arnoud; Lap, Paul; Boezeman, Jan; Laros-van Gorkom, Britta A P; Brons, Paul; Verbruggen, Bert; van Kraaij, Marian; van Heerde, Waander L
Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis. A novel hemostasis assay (NHA) was developed to measure thrombin and plasmin generation in a single well by a fluorimeter. The NHA uses two fluorescent substrates with non-interfering fluorescent excitation and emission spectra. The assay was tested in vitro using modulators like heparin, hirudin, epsilon-aminocaproic acid, gly-pro-arg-pro peptide and reptilase and validated by measurement of prothrombin fragment 1+2 and plasmin-alpha2-antiplasmin levels. Intra- and inter-assay coefficients of variation were < 9% and 6-25%, respectively. Interplay between coagulation and fibrinolysis was demonstrated by the effect of tissue-type plasminogen activator on thrombin generation and by the different responses of activated protein C and thrombomodulin on fibrinolysis. The last responses showed the linkage between coagulation and fibrinolysis by thrombin activatable fibrinolysis inhibitor. In conclusion, this strategy allows detection of coagulation, fibrinolysis and their interplay in a single assay.
Cesari, Matteo; Pahor, Marco; Incalzi, Raffaele Antonelli
The close relationship existing between aging and thrombosis has growingly been studied in this last decade. The age-related development of a pro-thrombotic imbalance in the fibrinolysis homeostasis has been hypothesized at the basis of this increased cardiovascular and cerebrovascular risk. Fibrinolysis is the resulting of the interactions among multiple plasminogen activators and inhibitors constituing the enzymatic cascade, and ultimately leading to the degradation of fibrin. The plasminogen activator system plays a key role in a wide range of physiological and pathological processes. Plasminogen activator inhibitor-1 (PAI-1) is a member of the superfamily of serine-protease inhibitors (or serpins), and the principal inhibitor of both the tissue-type and the urinary-type plasminogen activator, the two plasminogen activators able to activate plasminogen. In this review, current evidence describing the central role played by PAI-1 in a number of age-related subclinical (i.e., inflammation, atherosclerosis, insulin resistance) and clinical (i.e., obesity, comorbidities, Werner syndrome) conditions is presented. Despite some controversial and unclear issues, PAI-1 represents an extremely promising marker which may become a biological parameter to be growingly considered in the prognostic evaluation, in the disease monitoring, and as treatment target of age-related conditions in the next future. PMID:20626406
Mimuro, J; Kimura, S; Aoki, N
When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis. Images PMID:2419360
Kuleš, Josipa; Gotić, Jelena; Mrljak, Vladimir; Barić Rafaj, Renata
Canine babesiosis is a tick-borne disease caused by hemoprotozoan parasites of the genus Babesia. The disease can be clinically classified into uncomplicated and complicated forms. The aim of this study was to assess the level of endothelial activation and alterations in the fibrinolytic pathway during canine babesiosis. Blood samples were collected on the day of admission and on the 6th day after treatment with imidocarb propionate, from 30 dogs of various breeds and of both sexes with naturally occurring babesiosis caused by B. canis. In this prospective study, plasminogen activity was assessed using a chromogenic assay, and concentrations of high mobility group box-1 protein (HMGB-1), intercellular adhesive molecule-1 (ICAM-1), vascular adhesive molecule-1 (VCAM-1), soluble urokinase receptor of plasminogen activator (suPAR), thrombin activatable fibrinolysis inhibitor (TAFI), soluble thrombomodulin (TM) and plasminogen activator inhibitor-1 (PAI-1) were determined using a canine specific ELISA. Concentrations of TM, HMGB-1, VCAM-1 and suPAR were increased in dogs with babesiosis at admission compared to healthy dogs. After treatment, concentrations of TM were lower in infected dogs compared to healthy dogs. Dogs with babesiosis also had increased concentrations of TM, ICAM-1 and HMGB-1 and decreased plasminogen and PAI-1 at presentation compared to day 6 after treatment. Dogs with complicated babesiosis had higher concentrations of TM, HMGB1 and TAFI at admission compared to the 6th day. Biomarkers of endothelial activation and fibrinolysis were altered in dogs with babesiosis. Further studies into their usefulness as biomarkers of disease severity or prognosis is warranted.
Nishimura, Toshihiko; Myles, Timothy; Piliposky, Adrian M.; Kao, Peter N.; Berry, Gerald J.
Plasma procarboxypeptidase B (proCPB) is activated by the endothelial thrombin-prothrombomodulin complex. Activated (CPB) functions as a fibrinolysis inhibitor, but it may play a broader role by inactivating inflammatory mediators. To test this hypothesis, C5a-induced alveolitis was studied in wild-type (WT) and proCPB-deficient mice (proCPB−/−). C5a-induced alveolitis, as measured by cell counts and total protein contents in bronchoalveolar lavage fluids, was markedly enhanced in the proCPB−/− mice. E229K thrombin, a thrombin mutant with minimal clotting activity but retaining its ability to activate protein C and proCPB, attenuated C5a-induced alveolitis in WT but not in proCPB−/− mice, indicating that its beneficial effect is mediated primarily by its activation of proCPB. Lung tissue histology confirmed these cellular inflammatory responses. Delayed administration of E229K thrombin after the C5a instillation was ineffective in reducing alveolitis in WT mice, suggesting that the beneficial effect of E229K thrombin is due to the direct inhibition of C5a by CPB. Our studies show that thrombin-activatable proCPB, in addition to its role in fibrinolysis, has intrinsic anti-inflammatory functions. Its activation, along with protein C, by the endothelial thrombin-TM complex represents a homeostatic response to counteract the inflammatory mediators generated at the site of vascular injury. PMID:17105819
Tomkiewicz-Pajak, Lidia; Hoffman, Piotr; Trojnarska, Olga; Lipczyńska, Magdalena; Podolec, Piotr; Undas, Anetta
Thrombosis occurs in up to 30% of patients with various forms of congenital single ventricle after the Fontan procedure. We investigated hemostatic abnormalities in adult Fontan patients. Forty-eight Fontan patients between ages 18 and 40 years, including 10 (21%) patients with previous thromboembolism 5 to 15 years after surgery, and 35 control subjects matched for age and sex were studied. Coagulation factors and inhibitors, together with markers of fibrinolysis, platelets, and endothelial activation, were determined in peripheral venous blood plasma. Compared with control subjects, Fontan patients showed lower, although mostly within normal ranges, values of all coagulation factors, as well as reduced free protein S, in association with higher antithrombin and free tissue factor pathway inhibitor levels. Thrombin generation, reflected by prothrombin fragment 1.2, and platelet activation markers were increased in Fontan patients. The plasma clot lysis time was prolonged in Fontan patients, which was associated with an increased activity of thrombin-activatable fibrinolysis inhibitor. Fontan patients with previous thromboembolism had lower oxygen saturation, coagulation factors V and VIII, and free protein S, and increased von Willebrand factor, soluble CD40 ligand, and P-selectin. Other laboratory or clinical parameters were not associated with prior thrombotic episodes. Adult Fontan patients are characterized by enhanced platelet activation and endothelial injury, heightened thrombin formation, and impaired fibrinolysis. Patients showed reduced free protein S levels, increased platelet activation, and endothelial damage after thromboembolic events observed late after Fontan surgery. Our findings indicate novel prothrombotic mechanisms in adult Fontan patients, which might help to optimize thromboprophylaxis. Copyright © 2014 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.
Colucci, Mario; Pentimone, Anna; Binetti, Bianca M; Cramarossa, Marialisa; Piro, Donatella; Semeraro, Nicola
Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 micrograms/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through "localized" TAFIa generation.
Koncz, Zsuzsa; Bagoly, Zsuzsa; Haramura, Gizella; Mezei, Zoltán A; Muszbek, László
It has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FV(Leiden)) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α(2)-plasmin inhibitor (α(2)-PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FV(Leiden) carriers. In the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FV(Leiden) coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α(2)-PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method. rhTM significantly delayed the formation of α(2)-PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FV(Leiden). rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FV(Leiden). When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α(2)-PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FV(Leiden). FV(Leiden) abrogates the delaying effect of rhTM on α(2)-PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FV(Leiden) carriers. Copyright © 2012 Elsevier Ltd. All rights reserved.
Chapin, John C.; Hajjar, Katherine A.
Fibrin plays an essential role in hemostasis as both the primary product of the coagulation cascade and the ultimate substrate for fibrinolysis. Fibrinolysis efficiency is greatly influenced by clot structure, fibrinogen isoforms and polymorphisms, the rate of thrombin generation, the reactivity of thrombus-associated cells such as platelets, and the overall biochemical environment. Regulation of the fibrinolytic system, like that of the coagulation cascade, is accomplished by a wide array of cofactors, receptors, and inhibitors. Fibrinolytic activity can be generated either on the surface of a fibrin-containing thrombus, or on cells that express profibrinolytic receptors. In a widening spectrum of clinical disorders, acquired and congenital defects in fibrinolysis contribute to disease morbidity, and new assays of global fibrinolysis now have potential predictive value in multiple clinical settings. Here, we summarize the basic elements of the fibrinolytic system, points of interaction with the coagulation pathway, and some recent clinical advances. PMID:25294122
Chapin, John C; Hajjar, Katherine A
Fibrin plays an essential role in hemostasis as both the primary product of the coagulation cascade and the ultimate substrate for fibrinolysis. Fibrinolysis efficiency is greatly influenced by clot structure, fibrinogen isoforms and polymorphisms, the rate of thrombin generation, the reactivity of thrombus-associated cells such as platelets, and the overall biochemical environment. Regulation of the fibrinolytic system, like that of the coagulation cascade, is accomplished by a wide array of cofactors, receptors, and inhibitors. Fibrinolytic activity can be generated either on the surface of a fibrin-containing thrombus, or on cells that express profibrinolytic receptors. In a widening spectrum of clinical disorders, acquired and congenital defects in fibrinolysis contribute to disease morbidity, and new assays of global fibrinolysis now have potential predictive value in multiple clinical settings. Here, we summarize the basic elements of the fibrinolytic system, points of interaction with the coagulation pathway, and some recent clinical advances.
Innes, D.; Sevitt, S.
Serial changes in coagulation and fibrinolysis studied among 42 patients admitted to hospital with a wide variety of injuries are reported. The first hours after trauma are dominated by an acceleration of fibrinolysis (clot lysis) and clotting time which are often followed by an abrupt rebound to prolonged fibrinolysis and normal clotting. Evidence is presented that acceleration of fibrinolysis is due to flooding of the circulation by plasminogen activator and that prolongation is probably due to an inhibitor. A prolonged prothrombin time, increased prothrombin consumption index, an acceleration of the heparin-retarded clotting time, and a fall in the platelet count are also frequent during the first hours after injury. There is evidence also of an early deficiency in factor V and the onset of a fall in factor VII and prothrombin. The following days are characterized by continued prolongation of fibrinolysis, a lengthening of clotting time, and an increased prothrombin consumption index suggestive of a defect in thrombo-plastin generation. Subsequent periods of prolonged fibrinolysis may develop. Prothrombin time often continues prolonged for one to three weeks and may vary phasically; plasma prothrombin and factor VII are reduced but there is now little change in factor V. The platelet count continues to fall for one to three days, then a thrombocytosis develops, often with abnormally high platelet levels, a week or so later. Plasma fibrinogen rises within 24 hours to reach a plateau maximum a few days later and levels remain high for prolonged periods in the severely injured. Various changes are related to or influenced by the severity of trauma. Mechanisms are discussed, including thrombosis in vivo, and reference is made to homeostatic significance and its possible breakdown. PMID:14103515
Wardle, E. N.; Menon, I. S.; Rastogi, S. P.
Plasma and urine fibrinolysis were studied in 36 patients with glomerulonephritis and proteinuria. In 40% of these plasma fibrinolytic activator activity was moderately reduced and fibrinolytic inhibitors were increased. Globulins with antiplasmin effect were raised, particularly in the earlier months. Both the serum cholesterol and the plasma fibrinogen were related to the level of serum albumin, and those patients with high fibrinogen levels were also those with poor plasma fibrinolytic activator and those showing a steady deterioration. Urinary fibrinolysis was greatly reduced in most patients and bore no relation to plasma fibrinolysis levels. Hence urokinase is not derived from circulating plasminogen activator. PMID:4246192
Atkinson, J M; Pullen, N; Johnson, T S
Chronic kidney disease (CKD) is characterised by the pathological accumulation of extracellular matrix (ECM) proteins leading to progressive kidney scarring via glomerular and tubular basement membrane expansion. Increased ECM synthesis and deposition, coupled with reduced ECM breakdown contribute to the elevated ECM level in CKD. Previous pre-clinical studies have demonstrated that increased plasmin activity has a beneficial effect in the protein overload model of CKD. As plasmin activation is downregulated by the action of the thrombin activated fibrinolytic inhibitor (TAFI), we tested the hypothesis that inhibition of TAFI might increase plasmin activity and reduce ECM accumulation in an in vitro model of glucose induced ECM expansion. Treatment of NRK52E tubular epithelial cells with increasing concentrations of glucose resulted in a 40% increase in TAFI activity, a 38% reduction in plasmin activity and a subsequent increase in ECM accumulation. In this model system, application of the previously reported TAFI inhibitor UK-396082 [(2S)-5-amino-2-[(1-n-propyl-1H-imidazol-4-yl)methyl]pentanoic acid] caused a reduction in TAFI activity, increased plasmin activity and induced a parallel decrease in ECM levels. In contrast, RNAi knockdown of plasmin resulted in an increase in ECM levels. The data presented here indicate that high glucose induces TAFI activity, inhibiting plasmin activation which results in elevated ECM levels in tubular epithelial cells. The results support the hypothesis that UK-396082 is able to reduce TAFI activity, normalising plasmin activity and preventing excess ECM accumulation suggesting that TAFI inhibition may have potential as an anti-scarring strategy in CKD.
Minet, Valentine; Alpan, Lutfiye; Mullier, François; Toussaint, Olivier; Lucas, Stéphane; Dogné, Jean-Michel; Laloy, Julie
Nanoparticles (NPs) are developed for many applications in various fields, including nanomedicine. The NPs used in nanomedicine may disturb homeostasis in blood. Secondary hemostasis (blood coagulation) and fibrinolysis are complex physiological processes regulated by activators and inhibitors. An imbalance of this system can either lead to the development of hemorrhages or thrombosis. No data are currently available on the impact of NPs on fibrinolysis. The objectives of this study are (1) to select a screening test to study ex vivo the impact of NPs on fibrinolysis and (2) to test NPs with different physicochemical properties. Euglobulin clot lysis time test was selected to screen the impact of some NPs on fibrinolysis using normal pooled plasma. A dose-dependent decrease in the lysis time was observed with silicon dioxide and silver NPs without disturbing the fibrin network. Carbon black, silicon carbide, and copper oxide did not affect the lysis time at the tested concentrations.
Wardle, E. N.; Taylor, G.
In chronic renal failure and after acute renal failure, fibrinogen levels are raised and there is diminished fibrinolysis as the result of renal damage. A similar situation is found in nephrosis, possibly due to fibrinolytic inhibitors. Increased levels of cryofibrinogen were found in one quarter of cases of acute nephritis, nephrosis, and acute and chronic renal failure. In addition, after acute renal failure low platelet counts, prolonged thrombin times, and high levels of fibrin degradation products, yet with diminished fibrinolysis, indicate that intravascular coagulation has occurred. A positive result for fibrin degradation products was found in 17 of 20 cases of acute renal failure but in none of 10 cases of chronic uraemia. Intravascular coagulation is a process in which fibrin is deposited in the glomerular filters and may account for anuria, and, in the renal vasculature, where it may cause ischaemic tubular necrosis. Images PMID:5697045
Gabriel, D A; Muga, K; Boothroyd, E M
Fibrin structure contributes to the regulation of the fibrinolytic rate. As the fibrin fiber size is decreased, the fibrinolytic rate also decreases. Fibrin structure was altered by either changing the ratio of thrombin to fibrinogen, i.e. altering the assembly rate or by adding a fibrin assembly inhibitor, iopamidol. Changes in the fibrinolytic rate were followed by measuring the time dependence of the decrease in the fiber mass/length ratio during fibrinolysis. A measure of the overall fibrinolytic rate was determined from the decrease in the mass/length ratio versus time. An 8-fold reduction in the fibrinolytic rate was seen on decreasing the mass/length ratio from 2.7 x 10(12) daltons/cm to 0.5 x 10(12) daltons/cm. It is shown that thin fibrin fibers have a decreased rate of conversion of plasminogen to plasmin by tissue plasminogen activator and that thin fibrin fibers are lysed more slowly than thick fibrin fibers.
Ke, Zhang; Hao, Xu; Ning, Wei; Zu, Mao-heng; Fun, Yu-fei
Pathogenesis and clinical characteristics of the Budd-Chiari syndrome (BCS) in Asia are somewhat different from the ones observed in Western countries. Obstruction of the inferior vena cava (IVC) or of the hepatic veins is caused to a greater extent by membranous webs than by thrombosis. Impaired fibrinolysis has been found in European patients with BCS, but its status in Chinese patients with this condition is still unknown. To explore the characteristics of fibrinolysis in BCS patients in this country, we measured the euglobulin lysis time (ELT) for overall fibrinolysis and the plasma levels of five fibrinolytic components in 65 Chinese patients with BCS and 43 healthy controls. In patients, ELTs were slightly shorter than in controls (mean, 293 vs. 357 min, P < 0.02), tissue type plasminogen activator levels were higher than in controls (mean, 239 vs. 185 pg/ml, P < 0.01), and plasminogen activator inhibitor 1 levels were lower than in controls (mean, 1.43 vs. 1.73 ng/ml, P < 0.001). To explore BCS in more detail, we subgrouped the cases according to age, type of venous occlusion, Child-Pugh score, and thrombosis. As a result of this analysis, we found that young patients (age <30 years) had a longer ELT (mean, 440 min) than the older patient groups (30 ≤ age ≤ 44, 45 ≤ age ≤ 54, age>54 years; mean ELT = 242, 198, and 289 min, respectively, all P < 0.05). The independent hepatic vein occlusion subgroup showed a longer ELT (mean, 367 min) than the combined hepatic vein and IVC or the independent IVC occlusion subgroup (mean ELT = 233 and 260 min, both P < 0.05). ELT did not show significant differences between Child-Pugh class A and B subgroups (mean, 267 vs. 333 min, P > 0.05). ELT in the subgroup without thrombosis was shorter than in controls (mean, 288 vs. 358 min, P < 0.05), and in the subgroup with thrombosis, it was also slightly shorter than in controls, without reaching statistical significance (mean, 306 vs. 358 min, P > 0.05). By and large
Macaulay, William; Westrich, Geoffrey; Sharrock, Nigel; Sculco, Thomas P; Jhon, Peter H; Peterson, Margaret G E; Salvati, Eduardo A
The purpose of this prospective randomized clinical study was to investigate the enhanced systemic fibrinolysis mechanism of venous thrombosis prevention by pneumatic compression after total hip arthroplasty. Fifty patients were randomized into one of two groups (one with pneumatic compression [n=25] and one without [n=25]). Blood was drawn from a radial arterial line immediately preoperatively (baseline), at skin closure, and 8 hours and 22 hours after the baseline sample. Serum determinations of antigen of tissue plasminogen activator and plasminogen activator inhibitor-1 were done using enzyme-linked immunosorbent assays. These data do not support the enhancement of systemic fibrinolysis mechanism for lowering thromboembolic risk after total hip arthroplasty by pneumatic compression devices. The results of this study showed no differences that were statistically significant between the two groups. The greatest difference was observed 8 hours after surgery for the plasminogen activator inhibitor-1 marker, (28.12 with compression versus 22.07 ng/mL without); however, this result was not statistically significant. The beneficial effect of mechanical compression is more likely achieved through increased flow, local fibrinolytic effects, or both.
Kristiansen, Jacobina; Grove, Erik L; Rise, Nina; Neergaard-Petersen, Søs; Würtz, Morten; Kristensen, Steen Dalby; Hvas, Anne-Mette
Remote ischaemic conditioning (RIC) reduces infarct size and may improve prognosis in patients with acute myocardial infarction. To explore the potential mechanisms, we investigated the effects of RIC on coagulation and fibrinolysis. Interventional crossover study including 30 healthy drug-naïve males. Participants were exposed to a sham intervention (visit 1) and to RIC (visit 2 and 3) induced by intermittent arm ischaemia through four cycles of 5-min inflation of a blood pressure cuff followed by 5-min deflation. Prior to visit 3, all participants received aspirin 75mg daily for seven days. Blood samples were obtained at baseline as well as 5 and 45min after intervention. Whole blood coagulation was assessed by thromboelastometry (ROTEM®) and thrombin generation. Fibrinolysis was evaluated by clot turbidity-lysis, tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1). No differences were found in clot initiation, clot propagation or clot strength evaluated by ROTEM® (all p-values≥0.98). During aspirin treatment, clot initiation and clot propagation decreased after RIC evaluated by ROTEM® EXTEM® clotting time (p=0.04) and ROTEM® EXTEM® maximum velocity (p=0.03). After sham and RIC, thrombin generation declined as evaluated by reduced endogenous thrombin potential (RIC: p=0.001) and peak (RIC: p=0.01). After RIC during aspirin treatment, changes in thrombin generation were inconsistent; increased peak (p=0.04) and time to peak (p<0.001) and a decrease in lag-time (p<0.001). Clot lysis time was prolonged both after sham and after RIC (p<0.001). After RIC, PAI-1 levels declined (p=0.03), but t-PA levels also declined after all interventions (p-values≤0.04). RIC did not have substantial effects on coagulation or fibrinolysis compared to sham. Overall, aspirin did not influence the results. Copyright © 2016. Published by Elsevier Ltd.
Barlow, N; Nasser, Y; Zhao, P; Sharma, N; Guerrero-Alba, R; Edgington-Mitchell, L E; Lieu, T; Veldhuis, N A; Poole, D P; Conner, J W; Lindström, E; Craig, A W; Graham, B; Vanner, S J; Bunnett, N W
Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders. © 2015 John Wiley & Sons Ltd.
Tasci, Tonguc O; Disharoon, Dante; Schoeman, Rogier M; Rana, Kuldeepsinh; Herson, Paco S; Marr, David W M; Neeves, Keith B
Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, the polymer that forms between and around platelets to provide mechanical stability. Fibrinolysis rates however are often constrained by transport-limited delivery to and penetration of fibrinolytics into the thrombus. Here, these limitations are overcome with colloidal microwheel (µwheel) assemblies functionalized with the fibrinolytic tissue-type plasminogen activator (tPA) that assemble, rotate, translate, and eventually disassemble via applied magnetic fields. These microwheels lead to rapid fibrinolysis by delivering a high local concentration of tPA to induce surface lysis and, by taking advantage of corkscrew motion, mechanically penetrating into fibrin gels and platelet-rich thrombi to initiate bulk degradation. Fibrinolysis of plasma-derived fibrin gels by tPA-microwheels is fivefold faster than with 1 µg mL(-1) tPA. µWheels following corkscrew trajectories can also penetrate through 100 µm sized platelet-rich thrombi formed in a microfluidic model of hemostasis in ≈5 min. This unique combination of surface and bulk dissolution mechanisms with mechanical action yields a targeted fibrinolysis strategy that could be significantly faster than approaches relying on diffusion alone, making it well-suited for occlusions in small or penetrating vessels not accessible to catheter-based removal. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Chu, Arthur J; Mathews, Suresh T
The elevated level of histidine-rich glycoprotein was considered a risk factor of inherited thrombophilia. However, the mode of action remains largely unclear. In the current study, we employ poly-l-histidine (PLH) mimicking the histidine-rich region and determine whether PLH modulates urokinase (uPA)-dependent fibrinolysis. In an in vitro model, turbidity appearance and clearance monitored fibrin polymer formation and lysis, respectively. Fibrin polymer formed upon fibrinogen incubation with thrombin. In the presence of uPA or plasmin, fibrin polymer lysis took place in a dose-dependent manner as a function of time. We demonstrated that PLH significantly downregulated uPA-dependent fibrinolysis. PLH had no effect on plasminogen activation, as evidenced by no inhibitions on either uPA amidolytic activity or plasmin formation derived from its zymogen. Nor did PLH show any inhibition on plasmin amidolytic activity. PLH caused a profound delay of plasmin-dependent fibrinolysis upon pre-incubation of either plasmin or fibrinogen with PLH. The observations taken together suggest that the complex [plasmin-PLH-fibrin] formation significantly delayed plasmin-dependent fibrinolysis.
Zhao, Jing; Jin, Guorui; Weng, Guojun; Li, Jianjun; Zhu, Jian; Zhao, Junwu
Fluorescence imaging is superior in sensitivity and resolution compared with other imaging modalities; however, its application is hindered by high background noise. Tissue-selective strategies, such as passive, active, and activatable targeting, hold great promise in accelerating clinical translation by significantly improving the tumor:background ratio (TBR) and, in turn, the sensitivity and contrast of fluorescence imaging. Compared with the 'always on' contrast agents, activatable probes, which remain nonfluorescent until being activated by tumor-specific molecular targets, further enhance TBR and at the same time provide additional molecular information that can be related to tumor staging and therapy response. In this review, we summarize recent advances in the development of activatable fluorescence probes and provide insights into their advantages and limitations when used for tumor imaging. Copyright © 2017. Published by Elsevier Ltd.
Drake, Christopher R.; Miller, David C.; Jones, Ella F.
The early detection of many human diseases is crucial if they are to be treated successfully. Therefore, the development of imaging techniques that can facilitate early detection of disease is of high importance. Changes in the levels of enzyme expression are known to occur in many diseases, making their accurate detection at low concentrations an area of considerable active research. Activatable fluorescent probes show immense promise in this area. If properly designed they should exhibit no signal until they interact with their target enzyme, reducing the level of background fluorescence and potentially endowing them with greater sensitivity. The mechanisms of fluorescence changes in activatable probes vary. This review aims to survey the field of activatable probes, focusing on their mechanisms of action as well as illustrating some of the in vitro and in vivo settings in which they have been employed. PMID:23519774
Marzano, A V; Tedeschi, A; Polloni, I; Crosti, C; Cugno, M
Bullous pemphigoid (BP) is a potentially life-threatening autoimmune blistering disease that is burdened with an increased risk of cardiovascular events. In BP, there is an interplay between inflammation and coagulation both locally, which contributes to skin damage, and systemically, which leads to a prothrombotic state. Fibrinolysis is an important defence mechanism against thrombosis, but has only been studied locally in BP and no systemic data are available. The aim of this observational study was to evaluate systemic fibrinolysis and coagulation activation in patients with BP. We measured parameters of fibrinolysis and coagulation by immunoenzymatic methods in plasma from 20 patients with BP in an active phase and during remission after corticosteroid treatment. The controls were 20 age- and sex-matched healthy subjects. Plasma levels of plasminogen activator inhibitor type 1 (PAI-1) antigen, PAI-1 activity and tissue plasminogen activator (t-PA) antigen were significantly higher in the BP patients with active disease than in healthy controls (P = 0·0001 for all), as were the plasma levels of the fibrin fragment d-dimer and prothrombin fragment F1+2 (P = 0·0001 for both). During remission after treatment, levels of PAI-1 antigen and PAI-1 activity decreased significantly (P = 0·008 and P = 0·006, respectively), and there was also a significant decrease in plasma levels of d-dimer (P = 0·0001) and F1+2 (P = 0·0001). Fibrinolysis is inhibited in patients with active BP, due mainly to an increase in plasma levels of PAI-1. Corticosteroids not only induce the regression of BP lesions, but also reduce the inhibition of fibrinolysis, which may contribute to decreasing thrombotic risk. PMID:23199326
Matsumoto, Tomoko; Nogami, Keiji; Shima, Midori
Normal haemostasis is maintained by a controlled balance between coagulation and fibrinolysis, involving thrombin and plasmin the respective key enzymes. Simultaneous evaluation of both enzymes facilitates, therefore, an overall understanding of normal and pathological haemostasis. Combined thrombin and plasmin generation (T/P-G) assays have been recently described, and we have adapted the technique to investigate the interplay between coagulation and fibrinolysis in patients with various haemostatic disorders. Our modified T/P-G was initiated by the addition of a mixture of optimised lower concentrations of tissue factor and tissue-type plasminogen activator. Thrombin generation (TG) and plasmin generation (PG) were monitored simultaneously using individual fluorescent substrates in separate microtitre wells. The relationship between coagulation and fibrinolysis was demonstrated by analysing the effects of thrombin inhibitors, activated protein C and thrombomodulin. The most evident impairments in TG were observed with plasma samples deficient of coagulation factors participating in the prothrombinase complex. Defects in PG were observed with deficiencies of factor (F)V, FX, fibrinogen, and plasminogen. TG appeared to be a prerequisite for the initiation of PG, and overall PG was governed by fibrinogen concentration. TG in patients with haemophilia A correlated with levels of FVIII activity, but there was no significant relationship between PG and FVIII:C, confirming that the abnormal haemostasis in haemophilia A results in a severe imbalance between coagulation and fibrinolysis. The findings demonstrate that global haemostasis depends on a sensitive balance between coagulation and fibrinolysis, and that the modified T/P-G assay could provide an enhanced understanding of haemorrhage and thrombosis in clinical practice.
Li, Jingjing; Cheng, Fangfang; Huang, Haiping; Li, Lingling; Zhu, Jun-Jie
Activatable imaging probes as alternatives to "always on" imaging probes have attracted more and more attention due to their improved sensitivity and specificity. They are commonly designed to amplify or boost imaging signals only in response to specific biomolecular recognition or interaction. Thus, the design strategies play a vital role in the fabrication of activatable imaging probes. In this review, we focus on the design mechanisms and biological applications of those nanomaterial-based activatable imaging probes reported in the past five years, benefitting greatly from the good development of nanotechnology. These probes not only include the most studied activatable fluorescence imaging probes, but also cover more activatable MR imaging probes based on nanoparticle contrast agents and activatable photoacoustic imaging probes, providing more bases for clinical translation.
Bjerregaard, Nils; Dupont, Daniel M; Andreasen, Peter A
Uncontrolled bleeding is a major cause of mortality. Lysine analogues are routinely used in the management of bleeding, but several studies indicate a risk of serious detrimental effects upon their administration. In this study, we report a bivalent conjugate "3218" of two RNA aptamers selected for binding to the serine protease tissue-type plasminogen activator (tPA), the principal initiator of fibrinolysis in mammals. The constituent monomeric aptamers, K32v2 and K18v2, were previously demonstrated to weakly inhibit fibrinolysis. We now show that K32v2 and K18v2 recognize distinct binding sites, presumably in the A- and B-chain of tPA, respectively. Both aptamers bind tPA with low nanomolar affinity and inhibit tPA-mediated activities in a way that is consistent with the proposed localization of their binding sites. The 3218 conjugate possesses the inhibitory activities of both K32v2 and K18v2 and additionally exhibits increased inhibitory efficiency relative to the monomeric aptamers. The 3218 conjugate proved an efficient inhibitor of fibrinolysis and may find application in the management of bleeding as a substitute for, or in combination with, currently used lysine analogues.
Couturier, Roland; Rubatti, Marina; Credico, Carmen; Louvain-Quintard, Virginie; Anerkian, Vregina; Doubine, Sylvie; Vasse, Marc; Grassin-Delyle, Stanislas
Tranexamic acid is given continuously or discontinuously as an anti-fibrinolytic therapy during cardiac surgery, but the effects on fibrinolysis parameters remain poorly investigated. We sought to assess the effects of continuous and discontinuous tranexamic acid on fibrinolysis parameters in children undergoing cardiac surgery with cardiopulmonary bypass (CPB). Children requiring cardiac surgery or repeat surgery by sternotomy with CPB for congenital heart disease were randomized to receive either continuous or discontinuous tranexamic acid. Blood tranexamic acid, D-dimers, tissue plasminogen activator (tPA), tPA-plasminogen activator inhibitor 1 (tPA-PAI1) complexes, fibrinogen and fibrin monomers were measured and compared to values obtained from children who did not receive tranexamic acid. Tranexamic acid inhibited the CPB-induced increase in D-dimers, with a similar potency between continuous and discontinuous regimens. Time courses for tPA, fibrin monomers, and fibrinogen were also similar for both regimen, and there was a significant difference in tPA-PAI1 complex concentrations at the end of surgery, which may be related to a significantly higher tranexamic acid concentration. Continuous and discontinuous regimen are suitable for an effective inhibition of fibrinolysis in children undergoing cardiac surgery with CPB, but the continuous regimen was previously shown to be more effective to maintain stable tranexamic acid concentrations.
Erlinger, T P; Conlin, P R; Macko, R F; Bohannon, A D; Miller, E R; Moore, T J; Svetkey, L P; Appel, L J
Hypertension is associated with impaired fibrinolysis. Both angiotensin receptor blockers (ARB) and the DASH (Dietary Approaches to Stop Hypertension) diet effectively lower blood pressure in hypertensive patients. Some evidence suggests that treatment with ARBs could increase fibrinolysis, however, data is conflicting. The impact of the DASH diet on fibrinolytic parameters is not known. Fifty-five hypertensive participants (35 African-American, 20 white) were randomly assigned to receive 8 weeks of either a control diet or the DASH diet. The diets did not differ in sodium content (approximately 3 g/day). Within each diet, individuals were randomly assigned to receive losartan or placebo for 4 weeks in double-blind, cross-over fashion. Tissue plasminogen activator (t-PA) antigen, t-PA activity, plasminogen activator inhibitor-1 (PAI-1) activity and plasma renin activity (PRA) were measured at the end of a 2-week run-in period on the control diet and after each treatment period. The DASH diet did not affect markers of fibrinolysis. Losartan significantly lowered t-PA antigen levels (-1.8 ng/mL, P = 0.045), but had no effect on t-PA or PAI-1 activities. This effect was more pronounced in whites (-4.1 ng/mL (P = 0.003)) compared with African-Americans (-0.3 ng/mL (P = 0.7), P-interaction = 0.03). Results were not materially affected by adjustment for basline values or changes in blood pressure. This study demonstrates that losartan reduces t-PA antigen levels in white, but not African-American hypertensive individuals. In contrast, the DASH diet had no significant effect on markers of fibrinolysis in whites or African-Americans.
Abdelhamid, Mohamed F; Davies, Robert S M; Vohra, Rajiv K; Adam, Donald J; Bradbury, Andrew W
Abdominal aortic aneurysm (AAA) is associated with a prothrombotic diathesis that may increase the risk of cardiovascular events. This diathesis is exacerbated in the short term by open aneurysm repair (OAR) and endovascular aneurysm repair (EVAR). However, the effect of EVAR and OAR on coagulation and fibrinolysis in the medium and long term is poorly understood. The purpose of this study was to investigate the medium-term effects of EVAR and OAR on thrombin generation, neutralization, and fibrinolysis. Prothrombin fragment (PF)1+2, thrombin antithrombin (TAT) complex, plasminogen activator inhibitor (PAI) activity, and tissue-plasminogen activator (t-PA) antigen were measured in eight age-matched controls (AMCs), 29 patients with AAA immediately before (preoperatively) and 12 months after EVAR (post-EVAR), and in 11 patients at a mean of 16 months after OAR (post-OAR). Preoperatively, PF1+2 levels were significantly higher in patients with AAAs than in AMC. PF1+2 levels post-EVAR and post-OAR were significantly lower than preoperative values and similar to AMC. There was no significant difference in TAT, PAI, or t-PA between AMC, AAA preoperatively, and post-EVAR. Post-OAR, PAI activity was significantly higher than in preoperative patients. AAA is associated with increased thrombin generation without upregulation of fibrinolysis. The prothrombotic, hypofibrinolytic diathesis observed in patients with AAA returns toward normal in the medium term after EVAR and OAR, although there is a trend toward decreased fibrinolysis post-OAR. Copyright © 2013 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.
Walsh, Mark; Shreve, Jacob; Thomas, Scott; Moore, Ernest; Moore, Hunter; Hake, Daniel; Pohlman, Tim; Davis, Patrick; Ploplis, Victoria; Piscoya, Andres; Wegner, Julie; Bryant, John; Crepinsek, Anton; Lantry, James; Sheppard, Forest; Castellino, Francis
The emphasis on fibrinolysis as an important contributor to trauma-induced coagulopathy (TIC) has led to a debate regarding the relative clinical significance of fibrinolysis in the setting of trauma. The debate has centered on two camps. The one camp defines fibrinolysis in trauma by standard coagulation tests as well as fibrin split products, D-dimers, and plasmin/antiplasmin levels. This camp favors a more liberal use of tranexamic acid and attributes more significance to hyperfibrinolysis in TIC. The other camp favors a definition of fibrinolysis based on the viscoelastic tests (VET), rotational thromboelastometry (ROTEM), and thromboelastography (TEG). These whole blood assays define hyperfibrinolysis at a higher threshold than plasma-based tests. Therefore, this VET camp reserves antifibrinolytic treatment for patients who demonstrate severe coagulopathy associated with hyperfibrinolysis. This bimodal attribution of the clinical relevance of fibrinolysis in trauma suggests that there may be an underlying "Myth" of the concept of TIC that was historically defined by plasma-based tests and a future "Reality" of the concept of TIC that is grounded on an understanding of TIC based on a VET-defined "fibrinolytic spectrum" of TIC. This narrative review explores this "Myth" and "Reality" of fibrinolysis in TIC and proposes a direction that will allow a "Future" interpretation of TIC that incorporates both the past "Myth" and present "Reality" of fibrinolysis TIC. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.
Gordon, P. A.; Breeze, G. R.; Mann, J. R.; Stuart, J.
A study of fibrinolytic activity in sickle-cell patients during asymptomatic periods has shown a normal fibrinolytic response to exercise and local heat to the arm. During vasoocclusive crises there was no significant decrease in fibrinolytic activity. These results contrast with earlier reports of decreased fibrinolysis during crisis and a suggestion that fibrinolytic activators might be of value in preventing vasoocclusive episodes. Patients in painful crisis showed a significant rise in fibrinogen concentration and fall in platelet count. The former may contribute to localized vascular sludging by increasing whole-blood viscosity, while the latter probably results from local trapping of platelets in areas of sickling or from subsequent splenic sequestration of damaged platelets. There was no evidence of disseminated, as opposed to localized, intravascular coagulation during crisis. PMID:4412492
Okon, Aniekan; Matos de Souza, Marcos Romário; Shah, Rachit; Amorim, Raquel; da Costa, Luciana Jesus; Wagner, Carston R
This work describes the synthesis and biological evaluation of an anchimerically activated proTide of 2'-C-β-methylguanosine as an inhibitor of dengue virus 2 (DENV-2). The proTide incorporates a chemically cleavable 2-(methylthio)ethyl moiety and a HINT1 hydrolyzable tryptamine phosphoramidate. Inhibition of DENV-2 replication by proTide 6 was 5-fold greater than the parent nucleoside while displaying no apparent cytotoxicity. Furthermore, we demonstrate with a HINT1 inhibitor that the anti DENV-2 activity of the proTide correlates with the activity of HINT1. Taken together, these results demonstrate that a phosphoramidate based pronucleotide that undergoes an initial nonenzymatic activation step based on anchimeric assistance followed by P-N bond cleavage by HINT1 can be prepared.
To review: a) the role of extravascular fibrin deposition in the pathogenesis of acute lung injury; b) the abnormalities in the coagulation and fibrinolysis pathways that promote fibrin deposition in the acutely injured lung; and c) the pathways that contribute to the regulation of the fibrinolytic system via the lung epithelium, including newly recognized posttranscriptional and urokinase-dependent pathways. Another objective was to determine how novel anticoagulant or fibrinolytic strategies may be used to protect against acute inflammation or accelerated fibrosis in acute lung injury. Published medical literature. Alveolar fibrin deposition is characteristic of diverse forms of acute lung injury. Intravascular thrombosis or disseminated intravascular coagulation can also occur in the acutely injured lung. Extravascular fibrin deposition promotes lung dysfunction and the acute inflammatory response. In addition, transitional fibrin in the alveolar compartment undergoes remodeling leading to accelerated pulmonary fibrosis similar to the events associated with wound healing, or desmoplasia associated with solid neoplasms. In acute lung injury, alveolar fibrin deposition is potentiated by consistent changes in endogenous coagulation and fibrinolytic pathways. Procoagulant activity is increased in conjunction with depression of fibrinolytic activity in the alveolar compartment. Initiation of the procoagulant response occurs as a result of local overexpression of tissue factor associated with factor VII. Depression of fibrinolytic activity occurs as a result of inhibition of urokinase plasminogen activator (uPA) by plasminogen activators, or series inhibition of plasmin by antiplasmins. Locally increased amplification of plasminogen activator inhibitor-1 (PAI-1) is largely responsible for this fibrinolytic defect. Newly described pathways by which lung epithelial cells regulate expression of uPA, its receptor uPAR, and PAI-1 at the posttranscriptional level have been
Natorska, J; Undas, A
Aortic valve stenosis (AS) increasingly afflicts our aging population. However, the pathobiology of the disease is still poorly understood and there is no effective pharmacotherapy for treating those at risk for clinical progression. The progression of AS involves complex inflammatory and fibroproliferative processes that resemble to some extent atherosclerosis. Accumulating evidence indicates that several coagulation proteins and its inhibitors, including tissue factor, tissue factor pathway inhibitor, prothrombin, factor XIII, von Willebrand factor, display increased expression within aortic stenotic valves, predominantly on macrophages and myofibroblasts around calcified areas. Systemic impaired fibrinolysis, along with increased plasma and valvular expression of plasminogen activator inhibitor-1, has also been observed in patients with AS in association with the severity of the disease. There is an extensive cross-talk between inflammation and coagulation in stenotic valve tissue which contributes to the calcification and mineralisation of the aortic valve leaflets. This review summarises the available data on blood coagulation and fibrinolysis in AS with the emphasis on their interactions with inflammation and calcification.
Sumann, Günther; Fries, Dietmar; Griesmacher, Andrea; Falkensammer, Gerda; Klingler, Anton; Koller, Arnold; Streif, Werner; Greie, Sven; Schobersberger, Beatrix; Schobersberger, Wolfgang
Prolonged physical exercise is associated with multiple changes in blood hemostasis. Eccentric muscle activation induces microtrauma of skeletal muscles, inducing an inflammatory response. Since there is a link between inflammation and coagulation we speculated that downhill running strongly activates the coagulation system. Thirteen volunteers participated in the Tyrolean Speed Marathon (42,195 m downhill race, 795 m vertical distance). Venous blood was collected 3 days (T1) and 3 h (T2) before the run, within 30 min after finishing (T3) and 1 day thereafter (T4). We measured the following key parameters: creatine kinase, myoglobin, thrombin-antithrombin complex, prothrombin fragment F1 + 2, D-dimer, plasmin-alpha(2)-antiplasmin complexes, tissue-type plasminogen activator antigen, plasminogen-activator-inhibitor-1 antigen and thrombelastography with ROTEM [intrinsic pathway (InTEM) clotting time, clot formation time, maximum clot firmness, alpha angle]. Thrombin generation was evaluated by the Thrombin Dynamic Test and the Technothrombin TGA test. Creatine kinase and myoglobin were elevated at T3 and further increased at T4. Thrombin-antithrombin complex, prothrombin fragment F1 + 2, D-dimer, plasmin-alpha(2)-antiplasmin complexes, tissue-type plasminogen activator antigen and plasminogen-activator-inhibitor-1 antigen were significantly increased at T3. ROTEM analysis exhibited a shortening of InTEM clotting time and clot formation time after the marathon, and an increase in InTEM maximum clot firmness and alpha angle. Changes in TGA were indicative for thrombin generation after the marathon. We demonstrated that a downhill marathon induces an activation of coagulation, as measured by specific parameters for coagulation, ROTEM and thrombin generation assays. These changes were paralleled by an activation of fibrinolysis indicating a preserved hemostatic balance.
Geiser, Franziska; Gessler, Katharina; Conrad, Rupert; Imbierowicz, Katrin; Albus, Christian; Harbrecht, Ursula
Anxiety and depression are associated with an activation of coagulation and impairment of fibrinolysis. This study addresses the question whether these findings are reversed after psychotherapy and improvement of psychiatric symptoms. Three factors of coagulation and fibrinolysis as well as level of anxiety and depression were reassessed in 12 patients 1 to 3 years after intensive inpatient psychotherapy. The patients showed a substantial improvement of their severe anxiety disorder and comorbid depressive disorder. Simultaneously, we found a significant decrease in factor VII and plasminogen activator inhibitor. We conclude that reduction of severe anxiety and depression may be associated with a reversal of the procoagulant effect (activation of coagulation and impairment of fibrinolysis) of these psychological states. Because of the small sample size of this pilot study, further research is needed.
Schindler, Suzanne E; McCall, Jordan G; Yan, Ping; Hyrc, Krzystof L; Li, Mingjie; Tucker, Chandra L; Lee, Jin-Moo; Bruchas, Michael R; Diamond, Marc I
Techniques allowing precise spatial and temporal control of gene expression in the brain are needed. Herein we describe optogenetic approaches using a photo-activatable Cre recombinase (PA-Cre) to stably modify gene expression in the mouse brain. Blue light illumination for 12 hours via optical fibers activated PA-Cre in the hippocampus, a deep brain structure. Two-photon illumination through a thinned skull window for 100 minutes activated PA-Cre within a sub-millimeter region of cortex. Light activation of PA-Cre may allow permanent gene modification with improved spatiotemporal precision compared to standard methods.
Reddick, Keisha L B; Smrtka, Michael P; Grotegut, Chad A; James, Andra H; Brancazio, Leo R; Swamy, Geeta K
Pregnancy is associated with increased risk for thromboembolic events. Intermittent pneumatic compression (IPC) devices are the method of thromboprophylaxis in a nonpregnant population. The aim of this study was to examine the effects of IPC on markers of fibrinolysis during cesarean delivery. We conducted a randomized controlled trial from April 2009 to March 2010 of women undergoing scheduled elective cesarean delivery. Forty-nine women were randomized to IPCs or usual care. All participants had three blood samples obtained: (1) baseline, (2) 1 hour after randomization, and (3) 30 minutes after cesarean delivery. Tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), thrombin-antithrombin complex (TAT), plasminogen activator inhibitor-1 (PAI-1), and plasminogen activator inhibitor-2 (PAI-2) levels were analyzed in each sample using an enzyme-linked immunosorbent assay. Statistical analysis was performed using repeated measures two-way analysis of variance with α = 0.05. There was a time-dependent change in tPA, uPA, and PAI-1 levels following delivery but no difference in TAT and PAI-2 levels with time. There were no differences between women randomized to IPCs or usual care. Markers of fibrinolysis were not significantly altered by IPCs in this study of low-risk pregnant women. Further research regarding the mechanism and efficacy of IPCs in pregnant women is warranted. Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.
Oudijk, E J; Nieuwenhuis, H K; Bos, R; Fijnheer, R
The bleeding syndrome of acute promyelocytic leukemia (APL) is complex and consists of disseminated intravascular coagulation (DIC) and hyperfibrinolysis. Elastase, derived from malignant promyelocytes, is believed to mediate the fibrinogeno- and fibrinolysis by aspecific proteolysis. In this study we measured the role of elastase in fifteen patients with APL by using an assay for elastase degraded fibrin(ogen) and the results were compared with those obtained in patients with sepsis induced DIC. High levels of elastase were observed in sepsis and APL. The levels of fibrinogen and fibrin degradation products were significantly higher in APL patients compared to patients with sepsis induced DIC. Nevertheless, the level of elastase degraded fibrin(ogen) was higher in the sepsis group (635.3 ng/ml, compared to 144.3 ng/ml in APL; p <0.0001). So, the enormous increase in fibrin and fibrinogen degradation products in APL cannot be explained by elastase activity. This study suggests a minor role for elastase mediated proteolysis in the hemorrhagic diathesis in APL patients.
Rood, Marcus T.M.; Raspe, Marcel; ten Hove, Jan Bart; Jalink, Kees; Velders, Aldrik H.; van Leeuwen, Fijs W.B.
Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents. PMID:25985157
Kim, Junghyun; Jay, Michael
Neutron-activation is a promising method of generating radiotherapeutics with minimal handling of radioactive materials. Graphene oxide nanoplatelets (GONs) were examined as a carrier for neutron-activatable holmium with the purpose of exploiting inherent characteristics for theranostic application. GONs were hypothesized to be an ideal candidate for this application owing to their desirable characteristics such as a rigid structure, high metal loading capacity, low density, heat resistance, and the ability to withstand harsh environments associated with the neutron-activation process. Non-covalently PEGylated GONs (GONs-PEG) offered enhanced dispersibility and biocompatibility, and also exhibited increased holmium loading capacity nearly two-fold greater than GONs. Holmium leaching was investigated over a wide pH range, including conditions that mimic the tumor microenvironment, following neutron irradiation. The in vitro cell-based cytotoxicity analysis of GONs-based formulations with non-radioactive holmium confirmed their safety profile within cells. The results demonstrate the potential of GONs as a carrier of neutron-activatable radiotherapeutic agents. Copyright © 2017 Elsevier Inc. All rights reserved.
Rood, Marcus T M; Raspe, Marcel; ten Hove, Jan Bart; Jalink, Kees; Velders, Aldrik H; van Leeuwen, Fijs W B
Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents.
Kojima, T; Gando, S; Morimoto, Y; Mashio, H; Goda, Y; Kawahigashi, H; Kemmotsu, O
The aim of this study was to systematically elucidate the effects of tranexamic acid on fibrinolysis and bleeding during and after cardiopulmonary bypass (CPB) surgery. Twenty-two patients undergoing CPB surgery were randomized to receive 100 mg/kg tranexamic acid or an equal volume of saline after anesthesia induction and prior to skin incision. Plasma levels of tissue plasminogen activator (t-PA) antigen and activity, crosslinked fibrin degradation products (D-dimer), alpha2-antiplasmin-plasmin complex, and plasminogen activator inhibitor-1 (PAI-1) antigen were measured. Blood samples were obtained after induction of anesthesia, before, during, and after CPB, at the end of surgery, and the next morning after surgery. Intraoperative and postoperative blood loss during 24 h after surgery was recorded. Patients' demographics were similar between the two groups. No patients suffered from thrombotic complications after surgery. In the tranexamic acid group, fibrinolytic activity and secondary fibrinolysis as measured by t-PA activity and D-dimer were markedly suppressed during CPB surgery (P=.042 and P=.015, respectively). Decreased fibrinolytic activity and fibrinolysis were accompanied by reduction of perioperative bleeding in the tranexamic acid group. We could also find a good positive correlation between the peak levels of t-PA activity and D-dimer (r(2)=.4203, P=.0011). No differences in the t-PA antigen, PAI-1 antigen release, and plasmin inhibition by alpha2-antiplasmin were apparent between the two groups. In a randomized, prospective trial of patients undergoing CPB surgery, we demonstrated that the synthetic antifibrinolytic drug tranexamic acid effectively suppresses fibrinolysis by inhibiting t-PA and plasmin activity with clear reduction of perioperative blood loss. While tranexamic acid had no effects on the other important fibrinolytic inhibitors like PAI-1 and alpha2-antiplasmin.
Undas, A; Celinska-Löwenhoff, M; Löwenhoff, T; Szczeklik, A
Aspirin increases fibrin clot porosity and susceptibility to lysis. It is unknown whether other drugs, in combination with aspirin, used in the treatment of coronary artery disease (CAD) might affect clot structure and resistance to lysis. The aim of the study was to assess the effects of statins, fibrates, or angiotensin-converting enzyme inhibitors (ACEIs) on fibrin clot properties. In a randomized double-blind study, men with advanced CAD taking low-dose aspirin were assigned to receive one of the four drugs: simvastatin 40 mg day(-1) (n = 13), atorvastatin 40 mg day(-1) (n = 12), fenofibrate 160 mg day(-1) (n = 12), and quinapril 10 mg day(-1) (n = 11) for 28 +/- 2 days. Moreover, CAD patients (n = 13) taking aspirin (75 mg day(-1)) for 8 weeks were studied after additional 4 weeks on an open-label basis. Thirty men served as healthy controls. Plasma clot permeability and tissue plasminogen activator-induced fibrinolysis were evaluated at baseline and after drug administration. Permeability increased following the administration of simvastatin (by 20%; P = 0.01), atorvastatin (by 22%; P = 0.001), fenofibrate (by 16%; P = 0.02), and quinapril (by 13%; P = 0.04) like for aspirin (P < 0.001). Turbidity analysis showed that administration of any of the drugs was associated with higher maximum absorbancy, suggesting thicker fibers, and shorter fibrinolysis time (P < 0.001). Post-treatment reduction in lysis time correlated with an increase in clot porosity in all the groups (r from 0.42 to 0.61; P from 0.01 to 0.001). Statins, fibrates, and ACEIs may increase plasma clot permeability and susceptibility to fibrinolysis in CAD patients receiving aspirin. This novel antithrombotic mechanism might contribute to clinical benefits of the drugs tested.
Leng, Weibing; Li, Dezhi; Chen, Liang; Xia, Hongwei; Tang, Qiulin; Chen, Baoqin; Gong, Qiyong; Gao, Fabao; Bi, Feng
Aberrant activation of the Src kinase is implicated in the development of a variety of human malignancies. However, it is almost impossible to monitor Src activity in an in vivo setting with current biochemical techniques. To facilitate the noninvasive investigation of the activity of Src kinase both in vitro and in vivo, we developed a genetically engineered, activatable bioluminescent reporter using split-luciferase complementation. The bioluminescence of this reporter can be used as a surrogate for Src activity in real time. This hybrid luciferase reporter was constructed by sandwiching a Src-dependent conformationally responsive unit (SH2 domain-Srcpep) between the split luciferase fragments. The complementation bioluminescence of this reporter was dependent on the Src activity status. In our study, Src kinase activity in cultured cells and tumor xenografts was monitored quantitatively and dynamically in response to clinical small-molecular kinase inhibitors, dasatinib and saracatinib. This system was also applied for high-throughput screening of Src inhibitors against a kinase inhibitor library in living cells. These results provide unique insights into drug development and pharmacokinetics/phoarmocodynamics of therapeutic drugs targeting Src signaling pathway enabling the optimization of drug administration schedules for maximum benefit. Using both Firefly and Renilla luciferase imaging, we have successfully monitored Src tyrosine kinase activity and Akt serine/threonine kinase activity concurrently in one tumor xenograft. This dual luciferase reporter imaging system will be helpful in exploring the complex signaling networks in vivo. The strategies reported here can also be extended to study and image other important kinases and the cross-talks among them. PMID:26941850
Hargrove, Derek; Lu, Xiuling
Intraperitoneal internal radiation therapy is a cancer treatment option that is employed in situations where surgical resection, systemic chemotherapy, and external beam radiotherapy are not amenable for patients. However, exposure of noncancerous tissues to radiation continues to be a hindrance to safe and effective treatment of patients. In addition, reducing prolonged radiation exposure of personnel during preparation of internal radiation therapy agents makes their manufacture complicated and hazardous. Developments in nanotechnology have provided a platform for targeted treatments that combine dual imaging and treatment capabilities all in one package, while also being robust enough to withstand the intense stresses faced during neutron activation. Here, we describe a method for synthesizing neutron activatable mesoporous silica nanoparticles for use in radiotherapy of metastatic peritoneal cancers while limiting personal exposure to radioactive materials, limiting the leakage of radioactive isotopes caused by nanoparticle degradation during neutron activation, and increasing cancer tissue specificity of radiation.
Tian, Ruisong; Li, Mingjie; Wang, Jin; Yu, Min; Kong, Xiuqi; Feng, Yupeng; Chen, Zeming; Li, Yuxi; Huang, Weiqiang; Wu, Wenjie; Hong, Zhangyong
A newly designed, dual-functional probe based on intracellular activation has been successfully developed for the detection of cancer cells. The probe is nearly non-fluorescent in buffer due to its highly efficient FRET quenching, but it can be specifically activated with dramatic fluorescence enhancement upon intracellular cathepsin B cleavage in target cancer cells after selective internalization via folate receptor-dependent endocytosis. Therefore, this probe enables "turn-on" visualization of cancer cells with desirable specificity and contrast enhancement. This targeted, intracellularly activatable probe exhibits low fluorescence-quenched background when compared with "always-on" probes and avoids non-specific activation by non-specifically expressed enzymes in normal tissue, which normally occurs when using common "turn on" probe design strategies. Therefore, this probe can be potentially applied in intraoperative inspection during clinical cancer surgery with higher contrast and sensitivity.
Wang, Steven T.; Zhegalova, Natalia G.; Gustafson, Tiffany P.; Zhou, Andy; Sher, Joel; Achilefu, Samuel; Berezinand, Oleg Y.
We have developed a new analytical method of evaluating activatable fluorescent probes for ROS detection using integrated fluorescence spectroelectrochemistry. Tafel formalism was applied to describe the process of the probes’ oxidation under electrochemical conditions and identify a novel parameter defined as the threshold oxidation potential. This potential can serve as an approximation to the equilibrium potential and can be utilized for determining the sensitivity of a probe to oxidation. Based upon the measured values of threshold potentials, the order of sensitivity towards oxidation among several mostly used probes was determined to be following (from highest to lowest): 2,7-dichlorodihydrofluorescein > dihydroethidium > dihydrorhodamine 123 > dihydrorhodamine 6G. The presented approach opens up a new direction in synthesizing and screening novel ROS probes with a well-defined sensitivity for in vitro and in vivo applications. PMID:23736882
Nurachman, Zeily; Hermawan, Jatnika; Rachmayanti, Yanti; Baradja, Lubna
Laboratory demonstration, as well as biochemistry lecture, has been used to complement explanation of biochemical processes. The laboratory demonstration is very useful in teaching biochemistry to students who lack background in biology. The experimental model of fibrinolysis described here presents a complex biological reaction in simplified…
Li, Jinbo; Chen, Kai; Liu, Hongguang; Cheng, Kai; Yang, Meng; Zhang, Jiping; Cheng, Jonathan D.; Zhang, Yan; Cheng, Zhen
Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAPα. The ANPFAP consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANPFAP, high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANPFAP. Ex vivo imaging also demonstrated ANPFAP had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANPFAP could serve as a useful NIR optical probe for early detection of FAPα expressing tumors. PMID:22812530
Lane, G; Cuddihy, J; Wright, P; Doherty, D; McShane, A
Patients with an acute myocardial infarction require a rapid response to their symptoms and the earlier fibrinolysis is given (where indicated), the better the outcome. The aim of this study is to compare 'door to needle times' for fibrinolysis in Acute Myocardial Infarction (AMI) in three phases of one year each, at Letterkenny General Hospital. In the PREINTERVENTION year all fibrinolysis was performed in the Coronary Care Unit (CCU). In the INTERVENTION year Emergency Department (ED) fast track fibrinolysis was introduced and in the POST INTERVENTION year most fibrinolysis was performed on fast track in the ED. The time saved by the introduction of ED fibrinolysis was significant, 41 minutes on average per patient. Elderly, female patients were more likely to bypass ED fast track fibrinolysis and to be brought to CCU for fibrinolysis, with attendant delays. This has educational implications in relation to the variation in clinical presentation of AMI with age and sex. The ED fast track fibrinolysis system is recommended as an effective, safe, achievable and worthwhile intervention towards improving 'door to needle times' for fibrinolysis in AMI.
Introduction Nanoparticles for drug delivery to tumors need to satisfy two seemingly conflicting requirements: they should maintain physical and chemical stability during circulation and be able to interact with target cells and release drug at desired locations with no substantial delay. Unique microenvironment of tumors and externally-applied stimuli provide a useful means to maintain a balance between the two requirements. Areas covered We discuss nanoparticulate drug carriers that maintain stable structures in normal conditions but respond to stimuli for spatiotemporal control of drug delivery. We first define the desired effects of extracellular activation of nanoparticles and frequently used stimuli and review examples of extracellularly activated nanoparticles. Expert opinion Several challenges remain in developing extracellularly activatable nanoparticles. First, some of the stimuli-responsive NPs undergo incremental changes in response to stimuli, losing circulation stability. Second, the applicability of stimuli in clinical settings is limited due to the occasional occurrence of the activating conditions in normal tissues. Third, the construction of stimuli-responsive nanoparticles involves increasing complexity in nanoparticle structure and production methods. Future efforts are needed to identify new targeting conditions and increase the contrast between activated and non-activated NPs, while keeping the production methods simple and scalable. PMID:24950343
Kim, Junghyun; Narayan, Roger J; Lu, Xiuling; Jay, Michael
Various approaches have been undertaken to enhance the delivery of therapeutic agents, including tissue-killing radionuclides, into solid tumors. Here we describe the preparation of conical needles composed of Ti and Mo coated by pulsed laser deposition or chemical vapor deposition with elements (Ho and Re) that can readily yield radioactive isotopes following irradiation in a neutron flux. The radioactive needles, whose design were based on solid microneedle arrays used in transdermal drug delivery, can be produced with minimal handling of radioactivity and subsequently inserted into tumors as a means of internal radiation therapy. Ho and Re, were specifically chosen because of their large neutron capture cross-sections as well as the desirable radiotherapeutic properties of the resultant radionuclides. Neutron-absorbing shields were also developed to prevent the production of unwanted radionuclides after neutron irradiation of the needle base materials. Neutron activation calculations showed that therapeutically significant amounts of radionuclides can be produced for treating solid tumors. Stability studies demonstrated that Re did not leach off the Mo needles. These coated neutron-activatable needles offer a new approach to internal radiation therapy of tumors that allows precise tailoring of the absorbed radiation dose delivered to the tumor by controlling the coating thickness and the irradiation time. This article is protected by copyright. All rights reserved. © 2017 Wiley Periodicals, Inc.
Cheng, Shuting; Jiang, Zhou; Zou, Yan; Chen, Chen; Wang, Yuhui; Liu, Yanyou; Xiao, Jing; Guo, Huiling; Wang, Zhengrong
As a main component of circadian genes, clock plays not only an important role in circadian rhythm but also in the regulation of many physiological systems. The dysfunction of clock genes is associated with the development of various disorders. Many studies have investigated the association between clock genes and blood coagulation and the fibrinolytic system. The present study was designed to investigate the effect of downregulation of circulatory Clock on blood coagulation and fibrinolysis at the initial stage of active phase in male mice. Downregulation of the expression of the Clock gene by siRNA and, subsequently, its effect on the thrombotic potential and the expression of relative coagulative and/or fibrinolytic factors were investigated. It was found that the Clock interfered mice were less liable to thrombosis and showed prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) at Zeitgeber time (ZT) 15. Meanwhile, these mice also showed an increase in factor VII (FVII) and a decrease in thrombomodulin (TM) and plasminogen activator inhibitor 1 (PAI-1) at ZT 15 at both transcriptional and translational levels. PT, APTT and mRNA expressions of fvii, tm and pai-1 were analyzed with the least-squares fit of a 24-h cosine function by single cosinor method; no circadian rhythm was determined in PT and APTT, and a higher amplitude of fvii in the Clock RNAi group was found with a circadian phase shift, while lower amplitudes of tm and pai-1 were found in the Clock RNAi group with nearly no phase shift. All these results suggest that downregulation of the Clock gene in circulatory system has an effect on factors involved in both blood coagulation and fibrinolysis resulting in an enhancement in mice. This may be considered as an indication that Clock regulates thrombotic homeostasis through the fibrinolytic system.
Tousoulis, Dimitris; Ntarladimas, Ioannis; Antoniades, Charalambos; Vasiliadou, Carmen; Tentolouris, Costas; Papageorgiou, Nikos; Latsios, George; Stefanadis, Christodoulos
Mild alcohol consumption has been associated with decreased cardiovascular risk, although the underlying mechanisms are still unclear. We compared the acute effects of several alcoholic beverages on endothelial function, inflammatory process and thrombosis/fibrinolysis system in young adults. In this randomized intervention trial, healthy young individuals with no risk factor for atherosclerosis were randomized into 5 equally sized groups and received an equal amount of alcohol (30 g), as red wine (264 ml), white wine (264 ml), beer (633 ml), whisky (79 ml) or water (250 ml). Forearm blood flow was determined by gauge-strain plethysmography, at baseline, 1 and 4 h after alcohol intake. Levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), C-reactive protein (CRP), fibrinogen (Fib), plasminogen activator inhibitor (PAI-1), von Willebrand factor (vWF) and tissue plasminogen activator (tPA) were determined at baseline and 4 h after alcohol consumption. Reactive hyperemia was significantly increased 1 h after beer and red wine consumption (p<0.05 for both), while it returned at baseline at 4 h (p=ns vs baseline) but remained unchanged in all the other groups. vWF was decreased in the beer and red wine groups (p<0.05 for both) only. PAI-1/tPA ratio remained unchanged only in red wine and control group. Inflammatory markers remained unchanged in all the groups. Acute consumption of red wine or beer improves endothelial function and decreases vWF levels, suggesting that the type of beverage may differently affect endothelial function and thrombosis/fibrinolysis system in healthy adults.
Chang, Di; Wang, Yuan-Cheng; Bai, Ying-Ying; Lu, Chun-Qiang; Xu, Ting-Ting; Zhu, Lei; Ju, Shenghong
Matrix metalloproteinases (MMPs) exert a dual effect in ischemic stroke and thus represent an ideal target for detection and therapy. However, to date, all clinical trials of MMP inhibitors have failed, and alternative drug candidates and therapeutic targets are urgently required. Nonetheless, further investigations are limited by the lack of non-invasive imaging techniques. Here, we report a novel, fast and ultrasensitive MMP activatable optical imaging probe for the dynamic visualization of MMP activity in photothrombotic stroke mice. This probe provides a significant signal enhancement in as little as 15 min, with the highest signal intensity occurring at 1 h post-injection, and shows high sensitivity in measuring MMP activity alterations, which makes it specifically suitable for the real-time visualization of MMP activity and drug discovery in preclinical research. Moreover, using this probe, we successfully demonstrate that the regulation of the p38 mitogen-activated protein kinase (MAPK) signal pathway is capable of modulating MMP activity after stroke, revealing a novel regulatory mechanism of postischemic brain damage and overcoming the limitations of traditional therapeutic strategies associated with MMP inhibitors by using a non-invasive molecular imaging method.
Marchenko, V. I.
During periods of high solar activity fibrinolysis and fibrinogenolysis are increased. A direct correlative relationship is established between the indices of fibrinolysis, fibrinogenolysis and solar flares which were recorded two days before the blood was collected for analysis.
Marchenko, V. I.
During periods of high solar activity fibrinolysis and fibrinogenolysis are increased. A direct correlative relationship is established between the indices of fibrinolysis, fibrinogenolysis and solar flares which were recorded two days before the blood was collected for analysis.
Rood, Marcus T M; Oikonomou, Maria; Buckle, Tessa; Raspe, Marcel; Urano, Yasuteru; Jalink, Kees; Velders, Aldrik H; van Leeuwen, Fijs W B
In this proof-of-concept study, a new activatable imaging agent based on two luminophores and two different quenching mechanisms is reported. Both partial and total activation of the luminescence signal can be achieved, either in solution or in vitro. Bond cleavage makes the compound suitable for luminescence lifetime imaging.
Zeng, Leli; Kuang, Shi; Li, Guanying; Jin, Chengzhi; Ji, Liangnian; Chao, Hui
A glutathione (GSH)-activatable ruthenium(ii)-azo photosensitizer was prepared. The complex had low toxicity towards cells under dark conditions. It exhibited excellent phototoxicity under two-photon excitation (810 nm) and thus was developed as a two-photon photodynamic anticancer agent for cancer therapy.
Marchenko, V. I.
It is shown that on days with frontal activity in the atmosphere the levels of fibrinolysis and fibrinogenolysis are increased. The reactions of fibrinolysis and fibrinogenolysis to the passage of warm and cold fronts varies with the season of the year.
Marchenko, V. I.
Analysis of fibrinolysis and fibrinogenolysis indices by month showed an increase in the activity of these processes from winter to summer (1967-1968). At all seasons of the year, fibrinolysis and fibrinogenolysis increase during weather of the cyclonic type with passage of fronts and sharp fluctuations in meteorological factors in the atmosphere.
Marchenko, V. I.
On magnetically-active days, activation of fibrinolysis and fibrinogenolysis is observed. The increase in fibrinolysis and fibrinogenolysis begins on the day of the onset of a magnetic storm, reaching a maximum in 24 hours. Activation is higher on days with magnetic storms with a sudden onset and a C index of 1.5-2.0.
Marchenko, V. I.
Analysis of fibrinolysis and fibrinogenolysis indices by month showed an increase in the activity of these processes from winter to summer (1967-1968). At all seasons of the year, fibrinolysis and fibrinogenolysis increase during weather of the cyclonic type with passage of fronts and sharp fluctuations in meteorological factors in the atmosphere.
Marchenko, V. I.
It is shown that on days with frontal activity in the atmosphere the levels of fibrinolysis and fibrinogenolysis are increased. The reactions of fibrinolysis and fibrinogenolysis to the passage of warm and cold fronts varies with the season of the year.
Marchenko, V. I.
On magnetically-active days, activation of fibrinolysis and fibrinogenolysis is observed. The increase in fibrinolysis and fibrinogenolysis begins on the day of the onset of a magnetic storm, reaching a maximum in 24 hours. Activation is higher on days with magnetic storms with a sudden onset and a C index of 1.5-2.0.
Bardehle, Sophia; Rafalski, Victoria A.; Akassoglou, Katerina
Blood proteins at the neurovascular unit (NVU) are emerging as important molecular determinants of communication between the brain and the immune system. Over the past two decades, roles for the plasminogen activation (PA)/plasmin system in fibrinolysis have been extended from peripheral dissolution of blood clots to the regulation of central nervous system (CNS) functions in physiology and disease. In this review, we discuss how fibrin and its proteolytic degradation affect neuroinflammatory, degenerative and repair processes. In particular, we focus on novel functions of fibrin—the final product of the coagulation cascade and the main substrate of plasmin—in the activation of immune responses and trafficking of immune cells into the brain. We also comment on the suitability of the coagulation and fibrinolytic systems as potential biomarkers and drug targets in diseases, such as multiple sclerosis (MS), Alzheimer’s disease (AD) and stroke. Studying coagulation and fibrinolysis as major molecular pathways that regulate cellular functions at the NVU has the potential to lead to the development of novel strategies for the detection and treatment of neurologic diseases. PMID:26441525
Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao
pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence
Di Pasqua, Anthony J; Huckle, James E; Kim, Jin-Ki; Chung, Younjee; Wang, Andrew Z; Jay, Michael; Lu, Xiuling
Nanoparticles containing stable holmium ((165) Ho) are prepared by nanotemplate engineering and subsequently irradiated in a neutron flux to yield (166) Ho, a beta-emitting radiotherapeutic isotope. After intraperitoneal injection to mice bearing SKOV-3 ovarian tumors, significant tumor accumulation of the (166) Ho-nanoparticles is observed by SPECT imaging indicating the potential of these neutron activatable nanoparticles for internal radiation therapy of ovarian cancer metastases.
Li, Jingjing; Wang, Shan; Wu, Chen; Dai, Yue; Hou, Pingfu; Han, Cuiping; Xu, Kai
Activatable molecular MRI nanoprobe for intracellular GSH sensing was designed. As an alternative to "always on" nanoprobe, activatable imaging nanoprobes which are designed to amplify or boost imaging signals only in response to the targets have attracted more and more attention. In this paper, we designed a novel activatable molecular magnetic resonance imaging (MRI) nanoprobe for tumor cell recognization based on a MRI signal variation induced by the distance change between T1 and T2 contrast agents (CAs) in the presence of glutathione (GSH). To achieve this aim, carboxyl group functionalized iron oxide nanoparticles (Fe3O4 NPs) and polyethylene glycol-coated gadolinium oxide (PEG-Gd2O3) NPs as T2 and T1 MRI CA were connected by cystamine which contains a disulfide linkage. Transmission electron microscopic (TEM), X-ray photoelectron spectroscopy (XPS), energy dispersive spectrometer (EDS), fourier transform infrared spectroscopy (FT-IR), mass spectra and (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) were introduced for their characterizations. The formation of Fe3O4-cystamine-Gd2O3 (Fe3O4-SS-Gd2O3) nanocomplex resulted in a quenched T1 signal due to the near proximity of PEG-Gd2O3 NPs to Fe3O4 NPs and a "light-up" T1 signal with the cleavage of disulfide bond in the presence of GSH. These results provide not only an easy way to realize MRI of tumor cells based on the overexpressed intracellular GSH level, but also a new insight for the design of activatable MRI nanoprobe.
Gong, Ping; Yang, Yueting; Yi, Huqiang; Fang, Shengtao; Zhang, Pengfei; Sheng, Zonghai; Gao, Guanhui; Gao, Duyang; Cai, Lintao
pH is an important control parameter for maintenance of cell viability and tissue functions. pH monitoring provides valuable information on cell metabolic processes and the living environment. In this study, we prepared dual pH-sensitive, fluorescent dye-loaded polypeptide nanoparticles (DPNs) for ratiometric sensing of pH changes in living cells. DPNs contain two types of dyes: N-(rhodamine B) lactam cystamine (RBLC), an acid activatable fluorescent dye with increased fluorescence in an acidic environment, and fluorescein isothiocyanate (FITC), a base activatable fluorescent dye with enhanced fluorescence in an alkaline environment. Hence, DPNs exhibited a dual response signal with strong red fluorescence and weak green fluorescence under acidic conditions; in contrast, they showed strong green fluorescence and almost no red fluorescence under alkaline and neutral conditions. The favorable inverse pH responses of the two fluorescent dyes resulted in ratiometric pH determination for DPNs with an optimized pH-sensitive range of pH 4.5-7.5. Quantitative analysis of the intracellular pH of intact MCF-7 cells has been successfully demonstrated with our nanosensor. Moreover, single acid activatable fluorescent dye doped polypeptide nanoparticles that only contained RBLC can distinguish tumor tissue from normal tissue by monitoring the acidic extracellular environment.
Loof, Torsten G.; Deicke, Christin; Medina, Eva
The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis and is a host defense mechanism that protects the integrity of the vascular system after tissue injury. During bacterial infections, the coagulation system cooperates with the inflammatory system to eliminate the invading pathogens. However, pathogenic bacteria have frequently evolved mechanisms to exploit the hemostatic system components for their own benefit. Streptococcus pyogenes, also known as Group A Streptococcus, provides a remarkable example of the extraordinary capacity of pathogens to exploit the host hemostatic system to support microbial survival and dissemination. The coagulation cascade comprises the contact system (also known as the intrinsic pathway) and the tissue factor pathway (also known as the extrinsic pathway), both leading to fibrin formation. During the early phase of S. pyogenes infection, the activation of the contact system eventually leads to bacterial entrapment within a fibrin clot, where S. pyogenes is immobilized and killed. However, entrapped S. pyogenes can circumvent the antimicrobial effect of the clot by sequestering host plasminogen on the bacterial cell surface that, after conversion into its active proteolytic form, plasmin, degrades the fibrin network and facilitates the liberation of S. pyogenes from the clot. Furthermore, the surface-localized fibrinolytic activity also cleaves a variety of extracellular matrix proteins, thereby enabling S. pyogenes to migrate across barriers and disseminate within the host. This review summarizes the knowledge gained during the last two decades on the role of coagulation/fibrinolysis in host defense against S. pyogenes as well as the strategies developed by this pathogen to evade and exploit these host mechanisms for its own benefit. PMID:25309880
Koshiyama, Y; Ozeki, M; Motoyoshi, A; Fujita, M; Iwaki, M; Aoyama, T
Inhibitory effects of FUT-175 (nafamstat mesilate) on coagulation, platelets and fibrinolysis were examined. FUT-175 prolonged activated partial thromboplastin time, thrombin time and prothrombin time in rabbit plasma. FUT-175 prolonged these coagulation times in human plasma at lower concentration than in rabbit plasma. FUT-175 inhibited platelet aggregation induced by a variety of aggregation agents in rabbit platelet-rich plasma (PRP). In human PRP, FUT-175 inhibited platelet aggregation induced by a variety of aggregation agents at lower concentration than in rabbit PRP. Lipopolysaccharide induced a dose-dependent platelet aggregation in dog PRP. FUT-175 showed an inhibitory effect on this aggregation. FUT-175 inhibited clot retraction in rabbit plasma. The fibrinolysis activity was measured on fibrinolysis of rabbit plasma activated by urokinase. FUT-175 prolonged this fibrinolysis time. Inhibitory effects on coagulation and fibrinolysis were also found ex vivo. FUT-175 prolonged bleeding time in mice. These results indicate that FUT-175 has potent inhibitory effects on coagulation, platelets and fibrinolysis.
Le, Aiping; Zhang, Lunli; Liu, Wei; Li, Xiaopeng; Ren, Jianwei; Ning, An
Abstract A structural equation model was used for verification with chronic schistosomiasis to investigate the coagulation–anticoagulation system imbalance and to deduce the mechanism of D-dimer (D-D) level elevation in patients with advanced schistosome hepatic disease. We detected the plasma levels of tissue-type fiber plasminogen activator (tPA), urokinase type plasminogen activator (uPA), plasmin-antiplasmin complex (PAP), plasminogen (PLG), antithrombin (AT), plasminogen activator inhibitor 1 (PAI1), D-D, factor VIII: C (FVIII:C), antithrombin-III (AT-III), PLG, protein S (PS), and protein C (PC) in the healthy people as control (69), patients with chronic schistosomiasis (150) or advanced chronic schistosomiasis (90). FVIII, PAP, D-D, tPA, and uPA plasma levels were significantly higher in the chronic group than in the control group and were also significantly higher in the advanced group. However, AT-III, PC, PS, AT, PLG, and PAI1 plasma levels in the advanced and chronic groups were significantly lower than those in the control group. With progression of disease in patients with schistosomiasis japonica, a hypercoagulable state is induced by the coagulation–anticoagulation imbalance, eventually leading to patients with high levels of D-D. Furthermore, we established a structural equation model path of a “chronic schistosomiasis disease stage–(coagulation–anticoagulation–fibrinolysis)–D-D.” By using analysis of moment structures (AMOS), it was shown that the chronic schistosomiasis stage was positively related to factor VIII and had negative correlation with AT-III; a good positive correlation with PAP, tPA, and uPA; and a good negative correlation with PLG and PAI1. In addition, our results show that the path coefficient of anticoagulation–fibrinolysis system to the chronic stage of schistosomiasis or D-D levels was significantly higher than that of the coagulation system. In conclusion, the coagulation and fibrinolysis imbalance in
Le, Aiping; Zhang, Lunli; Liu, Wei; Li, Xiaopeng; Ren, Jianwei; Ning, An
A structural equation model was used for verification with chronic schistosomiasis to investigate the coagulation-anticoagulation system imbalance and to deduce the mechanism of D-dimer (D-D) level elevation in patients with advanced schistosome hepatic disease. We detected the plasma levels of tissue-type fiber plasminogen activator (tPA), urokinase type plasminogen activator (uPA), plasmin-antiplasmin complex (PAP), plasminogen (PLG), antithrombin (AT), plasminogen activator inhibitor 1 (PAI1), D-D, factor VIII: C (FVIII:C), antithrombin-III (AT-III), PLG, protein S (PS), and protein C (PC) in the healthy people as control (69), patients with chronic schistosomiasis (150) or advanced chronic schistosomiasis (90). FVIII, PAP, D-D, tPA, and uPA plasma levels were significantly higher in the chronic group than in the control group and were also significantly higher in the advanced group. However, AT-III, PC, PS, AT, PLG, and PAI1 plasma levels in the advanced and chronic groups were significantly lower than those in the control group. With progression of disease in patients with schistosomiasis japonica, a hypercoagulable state is induced by the coagulation-anticoagulation imbalance, eventually leading to patients with high levels of D-D. Furthermore, we established a structural equation model path of a "chronic schistosomiasis disease stage-(coagulation-anticoagulation-fibrinolysis)-D-D." By using analysis of moment structures (AMOS), it was shown that the chronic schistosomiasis stage was positively related to factor VIII and had negative correlation with AT-III; a good positive correlation with PAP, tPA, and uPA; and a good negative correlation with PLG and PAI1. In addition, our results show that the path coefficient of anticoagulation-fibrinolysis system to the chronic stage of schistosomiasis or D-D levels was significantly higher than that of the coagulation system. In conclusion, the coagulation and fibrinolysis imbalance in patients with chronic
Zanardo, Vincenzo; Savio, Valentina; Sabrina, Gavasso; Franzoi, Malida; Zerbinati, Patrizia; Fadin, Mariangela; Tognin, Giulio; Tormene, Daniela; Pagnan, Antonio; Simioni, Paolo
The effect of pre-eclampsia on coagulation and fibrinolysis in newborns is still under investigation. We have evaluated several coagulation and fibrinolysis parameters in umbilical cord blood of 20 newborns from pre-eclamptic women and of 40 newborns from normotensive women with similar gestational age. Additionally, the presence of factor V Leiden and prothrombin G20210A mutation in cord blood has been assessed. Neonates from pre-eclamptic women exhibited significantly lower birth weight (2.48 +/- 0.92 versus 2.88 +/- 0.68 kg, P < 0.05) and were more frequently admitted to the neonatal intensive care unit (45 versus 20%, P < 0.01) as compared with neonates from normotensive women. Cord blood protein C antigen and activated protein C resistance mean levels were slightly higher in the group of neonates from pre-eclamptic mothers. Fibrinogen levels were lower in this group as compared with control newborns (132.17 +/- 46.97 versus 156.08 +/- 49.58 mg%, P < 0.02), and unrelated to birth weight. No significant differences between cases and controls were found in plasminogen activator inhibitor-1 or tissue plasminogen activator cord blood levels. Heterozygous prothrombin 20210A was found in three newborns from normotensive mothers, whereas no factor V Leiden mutation was found in either group. In conclusion, pre-eclampsia seems to have only mild effects on coagulation and fibrinolytic factors in the cord blood of newborns. Since no excess of common polymorphisms predisposing to thrombosis was found in newborns from pre-eclamptic mothers, it is unlikely that the carriership status of these genetic defects of newborns influences the adverse pregnancy/neonatal outcomes.
Condrea, Eugeniu; Timirgaz, Valeriu; Groppa, Stanislav; Codreanu, Ion; Rotaru, Natalia
Objective To evaluate the effectiveness of minimally invasive craniopuncture with local fibrinolysis in the management of supratentorial spontaneous intracerebral hemorrhage (SICH). Methods The study included 218 consecutive patients with supratentorial SICH who were assigned to one of three groups: treated with minimally invasive craniopuncture with local fibrinolysis, treated with craniotomy or other minimally invasive techniques without local fibrinolysis, or receiving conservative management alone. Results Minimally invasive craniopuncture with local fibrinolysis was associated with a lower rate of assisted ventilation, a shorter period of in-hospital stay, a more frequent initiation of early rehabilitation, and a lower mortality rate at all periods of assessment. The overall mortality at 12 months was 19.4% (vs. 50.0 and 33.3% in the two other therapy groups). Lobar (subcortical and cortical) SICHs treated with local fibrinolysis had an overall mortality of 4.8% (vs. 43.5 and 41.7% in the two other therapy groups). On the other hand, SICHs having mixed (basal ganglia and lobar) locations treated with medical therapy alone had an overall mortality of 28.6%, while associated surgery with or without local fibrinolysis increased the overall mortality to over 65%. Conclusions The study demonstrated the applicability of minimally invasive craniopuncture with local fibrinolysis for the management of supratentorial SICHs and the advantages it may have in certain categories of patients. The method proved particularly useful in lobar SICHs, being associated with the lowest mortality. Mixed SICHs do not represent a predilection for surgical interventions; however, the results related to mixed supratentorial locations need confirmation in larger cohorts. PMID:27781045
Naito, Sawa; Yatagai, Chieko; Maruyama, Masugi; Sumi, Hiroyuki
We have previously reported on study results showing that certain types of coffee have the activity to enhance fibrinolysis. This report covers the activity of 10 types of hot water extracts of coffee on human tissue-type plasminogen activator producing cells. Particularly strong activity (29-35 times the control amount) was observed for Blue Mountain, Yunnan and Kilimanjaro beans. It was found that the hot water extracts have anti-thrombin activity, and that coffee components have anti-platelet aggregation activity, although weak. It was revealed that there is no activity affecting tissue-type plasminogen activator producing cells in the coffee components chlorogenic acid, caffeine, quinic acid, trigonelline hydrochloride, 5-(hydroxymethyl)-2-furfuryl and caffeic acid. It was also revealed that there is activity in fractions with a molecular weight of 10,000 or less. This could also be inferred from the fact that oral administration of such fractions of coffee to human subjects resulted in a shortening of their plasma ELT (p<0.05).
Cho, Hong-Jun; Lee, Sung-Jin; Park, Sung-Jun; Paik, Chang H; Lee, Sang-Myung; Kim, Sehoon; Lee, Yoon-Sik
A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an 'always fluorescent' iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.
Gong, Ping; Shi, Bihua; Zhang, Pengfei; Hu, Dehong; Zheng, Mingbin; Zheng, Cuifang; Gao, Duyang; Cai, Lintao
This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the occurrence of a red fluorescence signal due to fluorescence resonance energy transfer (FRET). DNase in biological samples was detected using DFProbes and the fluorescence imaging in living cells was performed using DFprobe-modified Au nanoparticles. The results show that DFProbes have good responses to DNase, and can clearly visualize the degradation of exogenous DNA in cells in real time. The well-designed probes might be useful in tracing the dynamic changes of exogenous DNA and nanocarriers in vitro and in vivo.This work presents a method to visualize the degradation of exogenous DNA in living cells using a novel type of activatable fluorescence imaging probe. Deoxyribonuclease (DNase)-activatable fluorescence probes (DFProbes) are composed of double strands deoxyribonucleic acid (dsDNA) which is labeled with fluorophore (ROX or Cy3) and quencher on the end of one of its strands, and stained with SYBR Green I. In the absence of DNase, DFProbes produce the green fluorescence signal of SYBR Green I. In the presence of DNase, SYBR Green I is removed from the DFProbes and the labeled fluorophore is separated from the quencher owing to the degradation of DFProbes by DNase, resulting in the decrease of the green fluorescence signal and the
Hong, Suk ho; Kim, Hyunjin; Choi, Yongdoo
Here we report indocyanine green (ICG)-loaded hollow mesoporous silica nanoparticles (ICG@HMSNP) as an activatable theranostic platform. Near-infrared fluorescence and singlet oxygen generation of ICG@HMSNP was effectively quenched (i.e. turned off) in its native state because of the fluorescence resonance energy transfer between ICG molecules. Therefore, ICG@HMSNP was nonfluorescent and nonphototoxic in the extracellular region. After the nanoparticles entered the cancer cells via endocytosis, they became highly fluorescent and phototoxic. In addition, intracellular uptake of ICG@HMSNP was 2.75 times higher than that of free ICG, resulting in an enhanced phototherapy of cancer.
Hong, Suk Ho; Kim, Hyunjin; Choi, Yongdoo
Here we report indocyanine green (ICG)-loaded hollow mesoporous silica nanoparticles (ICG@HMSNP) as an activatable theranostic platform. Near-infrared fluorescence and singlet oxygen generation of ICG@HMSNP was effectively quenched (i.e. turned off) in its native state because of the fluorescence resonance energy transfer between ICG molecules. Therefore, ICG@HMSNP was nonfluorescent and nonphototoxic in the extracellular region. After the nanoparticles entered the cancer cells via endocytosis, they became highly fluorescent and phototoxic. In addition, intracellular uptake of ICG@HMSNP was 2.75 times higher than that of free ICG, resulting in an enhanced phototherapy of cancer.
Suchkova, Valentina; Carstensen, Edwin L; Francis, Charles W
Ultrasound (US) accelerates enzymatic fibrinolysis in vitro and in animal models, and may be a useful adjunctive therapy for clinical thrombolysis. Successful clinical application will depend on the selection of appropriate US parameters to optimize fibrinolytic enhancement while limiting adverse effects, including heating. Most studies have been done at megahertz frequencies, but tissue penetration is better and heating less at lower frequencies. We have, therefore, now investigated the effects of continuous-wave and pulsed US on fibrinolysis at midkilohertz frequencies. Fibrinolysis with tissue plasminogen activator (t-PA) was measured by solubilization of radiolabeled fibrin exposed to a calibrated US field in a temperature-controlled water bath. There was significant enhancement of fibrinolysis at frequencies of 27, 40 and 100 kHz, with the greatest effect observed at 27 kHz. The largest effect was observed with continuous-wave US, but significant acceleration was also observed with peak intensities of 1 W/cm(2) duty cycles of 10% and 1%. At a 10% duty cycle, there was approximately 60% of the fibrinolytic enhancement observed with continuous-wave exposure, indicating a clear advantage of pulsing to optimize fibrinolytic effect and limit exposure. We conclude that US in the range of 27 to 100 kHz is effective in accelerating fibrinolysis at intensities and pulsing conditions that minimize the probability of heating and cavitation in clinical applications.
Kodali, Bhavani Shankar; Sa Rego, Monica; Kaynar, A Murat; Urman, Richard D
Amide local anesthetics are known to inhibit coagulation. 2-chloroprocaine is the only ester agent used in obstetric anesthesia. It is used during obstetric emergencies, and also to supplement inadequate epidural block produced by amide local anesthetics. There is no study to date that has evaluated the effect of ester local anesthetics on blood coagulation and fibrinolysis in the parturient. In this study, we obtained blood samples from healthy, term-parturients and mixed them with varying amounts of 2-chloroprocaine for final concentrations ranging from 0.26 to 7.8 mM. Thromboelastograph(®) was used to study the coagulation profile of these samples. Chloroprocaine impaired coagulation in a dose dependent manner, with increased R and K, and decreased MA and α. The difference, when compared to saline controls, reached statistical significance at a dose of 7.8 mM. An additional significant finding was that 2-chloroprocaine also enhanced fibrinolysis. Amide local anesthetics are known to impair coagulation, but 2-chloroprocaine produced significant fibrinolysis in addition to decreasing coagulation. This is the first study to date to demonstrate fibrinolytic properties of an ester local anesthetic. Further study evaluations are required to determine the cause of the variation in fibrinolysis. There is also a need to address the mechanism of increased fibrinolysis observed with 2-chroloprocaine.
Dennis, J; Truong, V; Aïssi, D; Medina-Rivera, A; Blankenberg, S; Germain, M; Lemire, M; Antounians, L; Civelek, M; Schnabel, R; Wells, P; Wilson, M D; Morange, P-E; Trégouët, D-A; Gagnon, F
Essentials Tissue factor pathway inhibitor (TFPI) regulates the blood coagulation cascade. We replicated previously reported linkage of TFPI plasma levels to the chromosome 2q region. The putative causal locus, rs62187992, was associated with TFPI plasma levels and thrombosis. rs62187992 was marginally associated with TFPI expression in human aortic endothelial cells. Click to hear Ann Gil's presentation on new insights into thrombin activatable fibrinolysis inhibitor SUMMARY: Background Tissue factor pathway inhibitor (TFPI) regulates fibrin clot formation, and low TFPI plasma levels increase the risk of arterial thromboembolism and venous thromboembolism (VTE). TFPI plasma levels are also heritable, and a previous linkage scan implicated the chromosome 2q region, but no specific genes. Objectives To replicate the finding of the linkage region in an independent sample, and to identify the causal locus. Methods We first performed a linkage analysis of microsatellite markers and TFPI plasma levels in 251 individuals from the F5L Family Study, and replicated the finding of the linkage peak on chromosome 2q (LOD = 3.06). We next defined a follow-up region that included 112 603 single nucleotide polymorphisms (SNPs) under the linkage peak, and meta-analyzed associations between these SNPs and TFPI plasma levels across the F5L Family Study and the Marseille Thrombosis Association (MARTHA) Study, a study of 1033 unrelated VTE patients. SNPs with false discovery rate q-values of < 0.10 were tested for association with TFPI plasma levels in 892 patients with coronary artery disease in the AtheroGene Study. Results and Conclusions One SNP, rs62187992, was associated with TFPI plasma levels in all three samples (β = + 0.14 and P = 4.23 × 10(-6) combined; β = + 0.16 and P = 0.02 in the F5L Family Study; β = + 0.13 and P = 6.3 × 10(-4) in the MARTHA Study; β = + 0.17 and P = 0.03 in the AtheroGene Study), and contributed to the linkage peak in the F5L Family Study. rs
Lee, Dongwon; Park, Seunggyu; Bae, Soochan; Jeong, Dahee; Park, Minhyung; Kang, Changsun; Yoo, Wooyoung; Samad, Mohammed A.; Ke, Qingen; Khang, Gilson; Kang, Peter M.
Overproduction of hydrogen peroxide (H2O2) causes oxidative stress and is the main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury. Suppression of oxidative stress is therefore critical in the treatment of I/R injury. Here, we report H2O2-activatable antioxidant prodrug (BRAP) that is capable of specifically targeting the site of oxidative stress and exerting anti-inflammatory and anti-apoptotic activities. BRAP with a self-immolative boronic ester protecting group was designed to scavenge H2O2 and release HBA (p-hydroxybenzyl alcohol) with antioxidant and anti-inflammatory activities. BRAP exerted potent antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)- and H2O2-stimulated cells by suppressing the generation of ROS and pro-inflammatory cytokines. In mouse models of hepatic I/R and cardiac I/R, BRAP exerted potent antioxidant, anti-inflammatory and anti-apoptotic activities due to the synergistic effects of H2O2-scavenging boronic esters and therapeutic HBA. In addition, administration of high doses of BRAP daily for 7 days showed no renal or hepatic function abnormalities. Therefore BRAP has tremendous therapeutic potential as H2O2-activatable antioxidant prodrug for the treatment of I/R injuries. PMID:26563741
Yamaguchi, Satoshi; Matsushita, Taku; Izuta, Shin; Katada, Sumika; Ura, Manami; Ikeda, Taro; Hayashi, Gosuke; Suzuki, Yuta; Kobayashi, Koya; Tokunaga, Kyoya; Ozeki, Yasuyuki; Okamoto, Akimitsu
A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm−1 was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the signal activation of the probe was also detected on living cells by stimulated Raman scattering microscopy. The ring-A-opened alkyne steroid analogue, thus, provides a first chemically-activatable Raman probe as a promising tool for potentially unravelling the intracellular formation and trafficking of cholesterol-rich microdomains. PMID:28117375
Spring, Bryan Q.; Abu-Yousif, Adnan O.; Palanisami, Akilan; Rizvi, Imran; Zheng, Xiang; Mai, Zhiming; Anbil, Sriram; Sears, R. Bryan; Mensah, Lawrence B.; Goldschmidt, Ruth; Erdem, S. Sibel; Oliva, Esther; Hasan, Tayyaba
Drug-resistant micrometastases that escape standard therapies often go undetected until the emergence of lethal recurrent disease. Here, we show that it is possible to treat microscopic tumors selectively using an activatable immunoconjugate. The immunoconjugate is composed of self-quenching, near-infrared chromophores loaded onto a cancer cell-targeting antibody. Chromophore phototoxicity and fluorescence are activated by lysosomal proteolysis, and light, after cancer cell internalization, enabling tumor-confined photocytotoxicity and resolution of individual micrometastases. This unique approach not only introduces a therapeutic strategy to help destroy residual drug-resistant cells but also provides a sensitive imaging method to monitor micrometastatic disease in common sites of recurrence. Using fluorescence microendoscopy to monitor immunoconjugate activation and micrometastatic disease, we demonstrate these concepts of “tumor-targeted, activatable photoimmunotherapy” in a mouse model of peritoneal carcinomatosis. By introducing targeted activation to enhance tumor selectively in complex anatomical sites, this study offers prospects for catching early recurrent micrometastases and for treating occult disease. PMID:24572574
Stenberg, B; Risberg, B; Hedman, L; Peterson, H I
Gastric mucosal lesions were induced in rats by pyloric ligature and intragastric instillation of hydrochloric acid. Within 4 h all rats developed focal mucosal lesions. Early regeneration was observed 72 h after release of the pyloric ligature and replacement of the hydrochloric acid by a phosphate buffer. A significantly increased gastric mucosal fibrinolytic activity was found 4 h after pylorus ligation. The submucosal vascular fibrinolysis remained unchanged. Following release of the pyloric ligature the increased mucosal fibrinolysis returned to normal values after 72 h. Intravenous administration of tranexamic acid significantly decreased the mucosal and vascular fibrinolytic activity without influencing the formation of induced gastric lesions. Increased mucosal fibrinolysis is probably not involved in the development of mucosal lesions.
Andrianova, I V; Ionova, V G; Demina, E G; Shabalina, A A; Karabasova, Ia A; Liutova, L I; Povorinskaia, T E; Orekhov, A N
A new form of garlic preparation--long-acting tablets of garlic powder allicor has been studied in patients with cerebral atherosclerosis (CA) complicated by chronic cerebrovascular pathology. A double blind placebo-controlled trial examined allicor effects on hemostasis and fibrinolysis in cross-over groups at two stages. At the first stage patients of group 1 (n = 15) received allicor in a dose 600 mg/day; patients of group 2 (n = 14) were given placebo. At the second stage group 1 received place and group 2 allicor in the same regimen. Before the treatment allicor effects on platelet aggregation and fibrinolysis were studied in vitro (20 patients). Allicor significantly inhibited ADP-induced platelet aggregation in vitro and ex vivo, reduced blood fibrinogen, normalized initially low fibrinolytic activity and fibrinolysis index. Due to the above properties allicor can be used for prevention and treatment of CA complicated by chronic cerebrovascular pathology.
Okafor, Osita N; Gorog, Diana A
Most acute cardiovascular events are attributable to arterial thrombosis. Plaque rupture or erosion stimulates platelet activation, aggregation, and thrombosis, whilst simultaneously activating enzymatic processes that mediate endogenous fibrinolysis to physiologically maintain vessel patency. Interplay between these pathways determines clinical outcome. If proaggregatory factors predominate, the thrombus may propagate, leading to vessel occlusion. However, if balanced by a healthy fibrinolytic system, thrombosis may not occur or cause lasting occlusion. Despite abundant evidence for the fibrinolytic system regulating thrombosis, it has been overlooked compared with platelet reactivity, partly due to a lack of techniques to measure it. We evaluate evidence for endogenous fibrinolysis in arterial thrombosis and review techniques to assess it, including biomarkers and global assays, such as thromboelastography and the Global Thrombosis Test. Global assays, simultaneously assessing proaggregatory and fibrinolytic pathways, could play a role in risk stratification and in identifying impaired fibrinolysis as a potential target for pharmacological modulation.
Chen, Li-Jian; Sun, Shao-Kai; Wang, Yong; Yang, Cheng-Xiong; Wu, Shu-Qi; Yan, Xiu-Ping
Multifunctional nanoprobes that provide diagnosis and treatment features have attracted great interest in precision medicine. Near-infrared (NIR) persistent luminescence nanoparticles (PLNPs) are optimal materials due to no in situ excitation needed, deep tissue penetration, and high signal-to-noise ratio, while activatable optical probes can further enhance signal-to-noise ratio for the signal turn-on nature. Here, we show the design of an activatable multifunctional PLNP/copper sulfide (CuS)-based nanoprobe for luminescence imaging-guided photothermal therapy in vivo. Matrix metalloproteinases (MMPs)-specific peptide substrate (H2N-GPLGVRGC-SH) was used to connect PLNP and CuS to build a MMP activatable system. The nanoprobe not only possesses ultralow-background for in vivo luminescence imaging due to the absence of autofluorescence and optical activatable nature but also offers effective photothermal therapy from CuS nanoparticles. Further bioconjugation of c(RGDyK) enables the nanoprobe for cancer-targeted luminescence imaging-guided photothermal therapy. The good biocompatibility and the multiple functions of highly sensitive tumor-targeting luminescence imaging and effective photothermal therapy make the nanoprobe promising for theranostic application.
St. Peter, Shawn D.; Tsao, Kuojen; Harrison, Christopher; Jackson, Mary Ann; Spilde, Troy L.; Keckler, Scott J.; Sharp, Susan W.; Andrews, Walter S.; Holcomb, George W.; Ostlie, Daniel J.
Purpose Management of empyema has been debated in the literature for decades. Although both primary video-assisted thoracoscopic surgery (VATS) and tube thoracostomy with pleural instillation of fibrinolytics have been shown to result in early resolution when compared to tube thoracostomy alone, there is a lack of comparative data between these modes of management. Therefore, we conducted a prospective, randomized trial comparing VATS to fibrinolytic therapy in children with empyema. Methods After Institutional Review Board approval, children defined as having empyema by either loculation on imaging or more than 10,000 white blood cells/μL were treated with VATS or fibrinolysis. Based on our retrospective data using length of postoperative hospitalization as the primary end point, a sample size of 36 patients was calculated for an α of .5 and a power of 0.8. Fibrinolysis consisted of inserting a 12F chest tube followed by infusion of 4 mg tissue plasminogen activator mixed with 40 mL of normal saline at the time of tube placement followed by 2 subsequent doses 24 hours apart. Results At diagnosis, there were no differences between groups in age, weight, degree of oxygen support, white blood cell count, or days of symptoms. The outcome data showed no difference in days of hospitalization after intervention, days of oxygen requirement, days until afebrile, or analgesic requirements. Video-assisted thoracoscopic surgery was associated with significantly higher charges. Three patients (16.6%) in the fibrinolysis group subsequently required VATS for definitive therapy. Two patients in the VATS group required ventilator support after therapy, one of whom required temporary dialysis. No patient in the fibrinolysis group clinically worsened after initiation of therapy. Conclusions There are no therapeutic or recovery advantages between VATS and fibrinolysis for the treatment of empyema; however, VATS resulted in significantly greater charges. Fibrinolysis may pose less
Arsicot, Matthieu; Della Schiava, Nellie; Boudjelit, Tarek; Rouvière, Olivier; Feugier, Patrick; Lermusiaux, Patrick; Millon, Antoine
The management of acute ischemia due to the thrombosis superficial femoral artery (SFA) stents is complex. In situ arterial fibrinolysis, still not evaluated in this indication, would allow, by lifting the ischemia and uncovering its cause, to avoid thrombectomy, endovascular recanalization, or arterial bypass. The purpose of the study was to evaluate the effectiveness, the complications, and the assisted secondary patency of in situ fibrinolysis for thrombosis of SFA stents. We conducted a retrospective monocentric study with prospective collection of the data. Between October 2011 and December 2014, 86 in situ fibrinolysis procedures were carried out for acute lower limb ischemia. Twelve procedures were carried out for acute ischemia due to the thrombosis of SFA stents. Clinical success was defined by the lifting of acute ischemia. The causes of thromboses, the complications related to the fibrinolysis, and the secondary assisted patency were analyzed. The mean age of the patients was 66.3 (55-90) years. The average length of the stents was 119.3 (18-270) mm. In 10 patients, the thrombosis extended in the full length of the artery. The average time between the implantation of the stent and the initiation of the fibrinolysis was 180 (11-369) days. The average time between the beginning of the symptoms and fibrinolysis was 5 (0-12) days. The average duration of treatment was 46 (24-72) hr. Clinical success was obtained in all the patients. Diagnosed isolated or associated lesions were a progression of the atheromatous disease upstream or downstream of the stent in 6 cases, and an isolated intrastent restenosis in 3 cases. In 2 cases, no obvious cause was found. One or more additional endovascular procedures were carried out in 9 cases at the end of the fibrinolysis, and consisted of a transluminal intrastent angioplasty with an active balloon in 5 cases, an additional stenting in 3 cases, and the stenting of upstream or downstream arteries in 5 cases. Secondary
Hamzah, Azhar Bin Amir; Choo, Yew Maw; Saleem, Fahad; Verma, Ashutosh Kumar
Disseminated Intravascular Coagulation (DIC) develops in patient with prostate cancer, which is manifested by systemic, intracranial, intracavitary or intracutaneous bleeding indicating uncompensated or excessive fibrinolysis (XFL). This case report is a description of a 61-year-old male with metastatic prostate cancer that progressed to manifest DIC. The condition is rare in clinical practice, and even rarer when is coupled with XFL. Treatment was mainly replenishing coagulation factors, platelets and controlling the disease progression with aggressive hormonal therapy. The patient progressed to coagulopathy further with fibrinolysis, hence leading to mortality. This case study discusses the pathophysiology of this complication and various methods to monitor the disease progression are discussed. PMID:28274032
Kudryashov, B. A.; Shapiro, F. B.; Lomovskaya, E. G.; Lyapina, L. A.
The role of the altered hormonal status of an organism in the activation of the anticoagulative system during stress is investigated. The 30 minute immobilization stress was shown to raise significantly the nonenzymatic fibrinolytic activity of blood in rats. Combined with adrenocorticotropin (ACTH) the effect is still greater. Intravenous administration of 0.2 m1 0.01 percent solution of protamine sulphate prevented the nonenzymatic fibrinolysis induced by the stress. Administration of ACTH after protomine sulphate again raised the fibrinolysis. This suggests that ACTH stimulates the release of heparin.
Minnema, M C; ten Cate, H; van Beek, E J; van den Ende, A; Hack, C E; Brandjes, D P
Previous investigations suggested that heparin administration to humans enhances the tissue type plasminogen activator (tPA) levels in blood, but it remains uncertain whether this effect induces fibrinolysis. We studied the effect of therapeutic levels of heparinization on plasma markers for fibrinolysis in patients suspected of pulmonary embolism (PE). Blood samples were taken from 49 consecutive patients; 28 had confirmed PE, 21 had PE excluded. On admission, the plasma levels of plasmin-alpha 2antiplasmin complexes and D-dimer were significantly higher in the patient group with PE compared to those in whom PE was excluded. After heparinization the tPA levels increased in both groups, showing that this effect was not dependent on the initial level of activity of fibrinolysis. In spite of this increment in tPA levels, the concentrations of plasmin-alpha 2antiplasmin complexes and D-dimer decreased. In conclusion, although heparinization in patients with or without pulmonary embolism does lead to elevated tPA:Ag levels, this is not accompanied by enhanced fibrinolysis.
Lai, Zongqiang; Tan, Juntao; Wan, Ruirong; Tan, Jie; Zhang, Zhenghua; Hu, Zixi; Li, Jieping; Yang, Wei; Wang, Yiwei; Jiang, Yafeng; He, Jian; Yang, Nuo; Lu, Xiaoling; Zhao, Yongxiang
It is significant to develop a probe with sensitivity and specificity for the detection of cancer cells. The present study aimed to develop an 'activatable' aptamer-based fluorescence probe (AAFP) to detect cancer cells and frozen cancer tissue. This AAFP consisted of two fragments: aptamer TLS11a that targets HepG2 cells, and two short extending complementary DNA sequences with a 5'- and 3'-terminus that make the aptamer in hairpin structure a capable quencher to fluorophore. The ability of the AAFP to bind specifically to cancer cells was assessed using flow cytometry, fluorescence spectroscopy and fluorescence microscopy. Its ability to bind to frozen cancer tissue was assessed using fluorescence microscopy. As a result, in the absence of cancer cells, AAFP showed minimal fluorescence, reflecting auto-quenching. In the presence of cancer cells, however, AAFP showed a strong fluorescent signal. Therefore, this AAFP may be a promising tool for sensitive and specific detection of cancer.
Liu, Dingbin; Huang, Xinglu; Wang, Zhantong; Jin, Albert; Sun, Xiaolian; Zhu, Lei; Wang, Fu; Ma, Ying; Niu, Gang; HightWalker, Angela R.; Chen, Xiaoyuan
It is still in high demand to develop extremely sensitive and accurate clinical tools for biomarkers of interest for early diagnosis and monitoring of diseases. In this report, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based fluorescence activatable probe for sensing ultra-low levels of prostate-specific antigen (PSA) in patient serum samples. The limit of detection of the newly-developed probe for PSA was pushed down to 0.032 pg/mL, which is more than two orders of magnitude lower than that of the conventional fluorescence probe. The ultrahigh sensitivity of this probe was attributed to the high loading efficiency of the dyes on AuNP surfaces and high fluorescence quenching unquenching abilities of the dye-AuNP pairs. The efficiency and robustness of this probe was investigated in patient serum samples, demonstrating the great potential of this probe in real-world applications. PMID:23683064
We recently developed “photo-unclick chemistry”, a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)2, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)2 had reduced dark toxicity compared with CA4. However, once illuminated, it showed improved toxicity similar to CA4 and displayed bystander effects in vitro. We monitored the time-dependent distribution of Pc-(L-CA4)2 using optical imaging with live mice. We also effectively ablated tumors by the illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and released chemotherapy drug, without any sign of acute systemic toxicity. PMID:24694092
Santra, Santimukul; Jativa, Samuel D.; Kaittanis, Charalambos; Normand, Guillaume; Grimm, Jan; Perez, J. Manuel
Herein we report a novel gadolinium-encapsulating iron oxide nanoparticle-based activatable NMR/MRI nanoprobe. In our design, Gd-DTPA is encapsulated within the polyacrylic acid (PAA) polymer coating of a superparamagnetic iron oxide nanoparticle (IO-PAA) yielding a composite magnetic nanoprobe (IO-PAA-Gd-DTPA) with quenched longitudinal spin-lattice magnetic relaxation (T1). Upon release of the Gd-DTPA complex from the nanoprobe's polymeric coating in acidic media, an increase in the T1 relaxation rate (1/T1) of the composite magnetic nanoprobe was observed, indicating a dequenching of the nanoprobe with a corresponding increase in the T1-weighted MRI signal. When a folate-conjugated nanoprobe was incubated in HeLa cells, a cancer cell line overexpressing folate receptors, an increase in the 1/T1 signal was observed. This result suggests that upon receptor-mediated internalization, the composite magnetic nanoprobe degraded within the cell's lysosome acidic (pH = 5.0) environment, resulting in an intracellular release of Gd-DTPA complex with subsequent T1 activation. No change in T1 was observed when the Gd-DTPA complex was chemically conjugated on the surface of the nanoparticle's polymeric coating or when encapsulated in the polymeric coating of a non-magnetic nanoparticle. These results confirmed that the observed (T1) quenching of the composite magnetic nanoprobe is due to the encapsulation and close proximity of the Gd ion to the nanoparticles superparamagnetic iron oxide (IO) core. In addition, when an anticancer drug (Taxol) was co-encapsulated with the Gd-DTPA within the folate receptor targeting composite magnetic nanoprobe, the T1 activation of the probe coincide with the rate of drug release and corresponding cytotoxic effect in cell culture studies. Taken together, these results suggest that our activatable T1 nanoagent could be of great importance for the detection of acidic tumors and assessment of drug targeting and release by MRI. PMID:22809405
Aboal, Jaime; Núñez, María; Bosch, Daniel; Tirón, Coloma; Brugada, Ramón; Loma-Osorio, Pablo
Long distance from a hospital with a catheterization laboratory is associated with a poorer prognosis in patients who undergo primary angioplasty for ST-elevation myocardial infarction (STEMI). An invasive pharmacologic strategy could offer an alternative treatment for these patients. We aimed to establish whether prognosis was better with primary angioplasty or fibrinolysis for reperfusion in cases of STEMI occurring far from a catheterization laboratory. Prospective registry study of patients with STEMI admitted to our cardiology critical care unit. Patients were included over a 5-year period if they received reperfusion therapy and had required transport of more than 50 km to reach a hospital with a catheterization laboratory. We recorded characteristics of the STEMI event, treatment times, and short- and long-term mortality. The data was used for survival analysis. We registered 584 patients; 194 were treated with primary angioplasty and 390 with fibrinolysis. The mean time between first physician contact and balloon insertion was 160 minutes. The mean time between first physician contact and needle insertion for fibrinolysis was 30 minutes. The 2-year mortality rate was higher in patients treated with angioplasty (12.2%) than with those who underwent fibrinolysis (7.0%) ) (P=.04). Survival analysis showed that risk for death was higher in the primary angioplasty group (hazard ratio, 1.97 (95% CI, 0.64-0.95; P=.001). When STEMI occurs more than 50 km from a catheterization laboratory, reperfusion by means of balloon angioplasty delays care considerably and is associated with a higher mortality rate than reperfusion by fibrinolysis.
Fogari, Roberto; Zoppi, Annalisa; Salvadeo, Sibilla A T; Mugellini, Amedeo; Lazzari, Pierangelo; Santoro, Tara; Derosa, Giuseppe
The aim of this study was to evaluate the effects of imidapril and candesartan on fibrinolysis and insulin sensitivity in normoweight hypertensive patients. After a 2-week wash-out period, 61 patients with mild-to-moderate hypertension were randomized to imidapril or candesartan for 12 weeks. Blood pressure (BP), plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigen activities were evaluated at baseline and during treatment. The patients underwent a euglycemic-hyperinsulinemic clamp (insulin sensitivity was evaluated as glucose infusion rate during the last 30 min) and a desmopressin test (with desmopressin infusion in the brachial artery) to evaluate endothelial ability to release t-PA. Imidapril and candesartan induced similar systolic/diastolic BP reductions (-16/12.6 and -16.1/12.2 mm Hg, respectively, P<0.001 vs. baseline). Imidapril increased glucose infusion rate (+1.1 mg min(-1) per kg, P<0.02), whereas candesartan did not change it. Both drugs decreased PAI-1 antigen activity after 4 weeks of treatment; subsequently, only the decreasing effect of imidapril was sustained throughout the 12 weeks, whereas candesartan increased PAI-1 activity at week 12 (P<0.05 vs. baseline, P<0.01 vs. imidapril). Activity of t-PA decreased with candesartan (from 0.48±0.16 to 0.43±0.14 IU ml(-1), P<0.05) but not with imidapril. Activity of t-PA in response to desmopressin was increased more by imidapril (+4.45 IU ml(-1)) than by candesartan (+2.73 IU ml(-1), P<0.01 vs. imidapril). These results indicate that in normoweight hypertensive patients, despite similar BP reduction, imidapril but not candesartan improved the fibrinolytic balance, suggesting that mechanisms other than Ang II inhibition, possibly including bradykinin-mediated effects on insulin sensitivity and endothelial function, may be responsible for these different effects.
Deira, J; Alberca, I; Lerma, J L; Martín, B; Tabernero, J M
Different immunosuppressive agents, in particular OKT3, have been implicated as causative factors in the risk for renal thrombosis in the period immediately after kidney transplantation. Also, in different types of vascular surgery, a state similar to hypercoagulation has been reported. To assess the extent to which OKT3, cyclosporine A (CsA), and surgery itself affect coagulation and fibrinolysis, a study was conducted of 20 patients divided into two groups: group A, 10 patients received OKT3 (first dose during the induction of anesthesia); and group B, 10 patients received CsA (first dose at least 2 hours before transplantation). Basal determinations and determinations at 2, 4, and 24 hours after the induction of anesthesia were made. No differences were found between the groups with respect to the clinical and usual coagulation parameters. The following were studied in both groups: (1) markers of coagulation activity (prekallikrein [PKK] levels and formation of thrombin-antithrombin complexes [TATc]), (2) inhibitors and suppressors of hemostasis (antithrombin III [AT-III] and protein C [PC] activity), (3) markers of fibrinolysis activation (levels of plasminogen [PLG] and of alpha2-antiplasmin [alpha2-APL]), and (4) markers of endothelial damage (tissue plasminogen activator [TPA] and thrombomodulin [TMD]). In both groups, an important formation of TATc was observed early, together with a decrease in PKK levels and consumption of both AT-III and PC, which reached their lowest levels at 24 hours. This points to an activation of coagulation through the intrinsic route and a secondary consumption of hemostasis inhibitors, both possibly caused by surgery. A consumption of PLG and alpha2-APL was also observed, reflecting stimulation of the fibrinolytic system and a physiological response to the activation of coagulation. A greater release of endothelial TPA was only observed in the patients receiving OKT3 (P < 0.0001), possibly signaling endothelial activation. It is
Wun, T.C.; Capuano, A.
A two-site immunoradiometric assay for tissue plasminogen activator (tPA) antigen has been developed using immunoaffinity purified antibody. Various treatments enhanced the detection of tPA antigen in the plasma samples. Maximum detection was obtained by acidification of plasma to pH 4.8 to 6.5 or addition of 0.5 mol/L of L-lysine or L-arginine. Acidification or addition of lysine to plasma is also required for maximum immunoadsorption of plasma tPA antigen on anti-tPA-Ig-sepharose. These results indicate that plasma tPA antigen is partially cryptic to antibody in untreated plasma. The plasma tPA antigen isolated by immunoadsorption of either untreated plasma or acidified plasma on anti-tPA-Ig-sepharose consists mainly of a 100-kd plasminogen activator species as determined by fibrin-agar zymography. The 100-kd activity is possibly a tPA:inhibitor complex. A standardized sample preparation method was conveniently adopted by mixing 3 vol of plasma and 1 vol of 2 mol/L of L-lysine for the assay. Reconstitution and recovery studies showed that the method is specific and permits full detection of both free tPA and tPA:inhibitor complex. The validity of the assay is further supported by the finding that the spontaneous plasma fibrinolysis previously demonstrated to be dependent on plasma tPA antigen is correlated with tPA antigen content. Using the standardized assay, we found that tPA antigen concentrations in 16 blood bank plasmas are equivalent to 3.7 to 20 ng of 60 kd tPA/mL. In all the plasma tested, more than half of the antigen is undetected unless the plasma is treated as described above.
Olson, John D
D-dimer is the smallest fibrinolysis-specific degradation product found in the circulation. The origins, assays, and clinical use of D-dimer will be addressed. Hemostasis (platelet and vascular function, coagulation, fibrinolysis, hemostasis) is briefly reviewed. D-dimer assays are reviewed. The D-dimer is very sensitive to intravascular thrombus and may be markedly elevated in disseminated intravascular coagulation, acute aortic dissection, and pulmonary embolus. Because of its exquisite sensitivity, negative tests are useful in the exclusion venous thromboembolism. Elevations occur in normal pregnancy, rising two- to fourfold by delivery. D-dimer also rises with age, limiting its use in those >80 years old. There is a variable rise in D-dimer in active malignancy and indicates increased thrombosis risk in active disease. Elevated D-dimer following anticoagulation for a thrombotic event indicates increased risk of recurrent thrombosis. These and other issues are addressed.
Iqbal, A; Morton, C; Kong, K-L
A large and ever-growing number of agents used in anaesthesia can precipitate acute anaphylactic reactions after their administration. Anaphylaxis is a sudden onset (or rapidly progressive), severe systemic allergic reaction, affecting multiple organ systems. The number of people who suffer severe systemic allergic reactions is increasing. The incidence is about 1-3 reactions per 10 000 population per annum, although anaphylaxis is not always recognized; therefore, certain UK studies may underestimate the incidence. In this case report, we present an episode of acute fibrinolysis associated with life-threatening anaphylaxis, demonstrated by thromboelastography (TEG) and resolving spontaneously. This is despite an added fibrinolytic insult in the form of cardiopulmonary bypass. There is a paucity of literature detailing fibrinolysis occurring during anaphylaxis, most likely due to the limited availability of TEG in the acute setting and the primary clinical focus of delivering life-saving interventions.
Aoki, N; Moroi, M; Matsuda, M; Tachiya, K
Human plasma alpha2-plasmin inhibitor in fibrinolytic states was studied using immunochemical methods and radioiodinated plasminogen. The concentration and activity of plasma alpha2-plasmin inhibitor decreased when urokinase was added to plasma in vitro or infused intravenously in man. The decrease was associated with the appearance of plasmin-alpha2-plasmin inhibitor complex which subsequently disappeared from the circulation in a short time. A decrease of other major inhibitors, such as alpha2-macroglobulin and alpha1-antitrypsin, was not observed when the amount of urokinase added or infused was relatively small, and conversion of plasminogen to plasmin was not extensive. The formation of plasmin-alpha2-macroglobulin complex was observed only when plasma plasminogen was activated with a larger amount of urokinase, and after most of the alpha2-plasmin inhibitor was consumed by forming complexes with plasmin. The formation of plasmin-alpha1-antitrypsin complex was not observed even in the highly activated plasma unless exogenous plasmin was added to the plasma. alpha2-Plasmin inhibitor was the only inhibitor of which the concentration in plasma was significantly decreased in patients with disseminated intravascular coagulation and fibrinolysis among the major plasmin inhibitors in plasma. The most reactive inhibitor for regulating plasma fibrinolysis very likely is alpha2-plasmin inhibitor. Images PMID:68962
Winocour, P.D.; Colwell, J.A.
Studies of fibrinolytic activity in diabetes mellitus have produced conflicting results. This may be a result of methodologic insensitivity or of variable contributions of the different blood components to whole blood fibrinolysis. To explore these two possibilities, the authors used a sensitive solid-phase radiometric assay to examine the fibrinolytic activity of whole blood, platelet-rich plasma, leukocytes, and platelet- and leukocyte-poor plasma prepared from control rats and rats with streptozocin-induced diabetes at various times after induction of diabetes. Fibrinolytic activity of whole blood from diabetic rats after 7 days was significantly reduced, and remained reduced after longer durations of diabetes up to 28 days. Platelet-rich plasma from diabetic rats had decreased fibrinolytic activity, which followed the same time course of changes as in whole blood. The platelet contribution to whole blood fibrinolysis was further reduced in vivo after 14 days of diabetes by a reduced whole blood platelet count. In contrast, fibrinolytic activity of leukocytes from diabetic rats became enhanced after 7 days of diabetes. After 49 days of diabetes, the whole blood leukocyte count was reduced, and in vivo would offset the enhanced activity. Plasma fibrinolytic activity was small compared with that of whole blood and was unaltered in diabetic rats. The authors conclude that altered platelet function contributes to decreased fibrinolytic activity of whole blood in diabetic rats, and that this may be partially offset by enhanced leukocyte-mediated fibrinolysis.
Solaz, J; Martínez-Rodrigo, J; Lonjedo, E; Poyatos, C; Vega, M; Palmero, J
Ischemia in the territory of the basilar artery presents with a variable clinical picture of hemiparesia-tetraplegia, progressive deterioration of level of consciousness, irregular respiration and apnea leading to irreversible coma and death in between 75% and 86% of cases. The usual treatment is supportive. We present the case of a 49 year old woman with acute thrombosis of the basilar artery and a progressive course leading to coma. No bulbar lesions were seen on the CT scan done in the Emergency Department. Thrombosis of the basilar artery and permeable bilateral carotid systems were shown on arteriography. There were no contra-indications to fibrinolysis. Following local fibrinolytic treatment with urokinase the patient had full recovery from her neurological disorder and no sequelae. The basilar artery remained permeable six months later. Emergency treatment with cerebral intra-arterial fibrinolysis within the first six hours, in a case of neurological deficit progressing in the basilar artery territory, with persistence of brain-stem functions and no signs of decerebration (provided there are no contra-indications to fibrinolysis and the initial cerebral CT scan shows no bulbar lesions) may save the patient's life, with total or partial recovery of brain-stem function.
Andreenko, G V; Panchenko, V M; Lisina, A N; Liutova, L V
Signs of dysfunction of the coagulation system and fibrinolysis were determined in 45 healthy young individuals who had such risk factors in relation to ischemic heart disease as arterial hypertension, hypercholesterolemia, smoking, aggravated heredity, permanent emotional overstress, etc. These signs were manifested by a tendency to augmentation of blood coagulation and compensatory activation of fibrinolysis. Ischemic-type changes were detected on the ECG after a physical load. It is assumed that dysfunction of the coagulation system and fibrinolysis is an additional risk factor in relation to ischemic heart disease, while derangement of compensatory fibrinolysis tension with the subsequent tension of its components may lead to the development of coronary thrombosis.
Kang, Ji-Yong; Kawaguchi, Daichi; Coin, Irene; Xiang, Zheng; O’Leary, Dennis D. M.; Slesinger, Paul A.; Wang, Lei
SUMMARY Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been employed to manipulate neuronal activity, a method for optical control of neuronal proteins using unnatural amino acids (Uaa) in vivo is lacking. Here, we describe the genetic incorporation of a photoreactive Uaa into the pore of an inwardly-rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel non-conducting, and upon brief light illumination, was released to permit outward K+ current. Expression of this photo-inducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expressed PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. The principles applied here to a potassium channel could be generally expanded to other proteins expressed in the brain to enable optical regulation. PMID:24139041
Zhou, Zijian; Song, Jibin; Tian, Rui; Yang, Zhen; Yu, Guocan; Lin, Lisen; Zhang, Guofeng; Fan, Wenpei; Zhang, Fuwu; Niu, Gang; Nie, Liming; Chen, Xiaoyuan
Reactive oxygen species (ROS)-induced apoptosis is a widely practiced strategy for cancer therapy. Although photodynamic therapy (PDT) takes advantage of the spatial-temporal control of ROS generation, the meticulous participation of light, photosensitizer, and oxygen greatly hinders the broad application of PDT as a first-line cancer treatment option. An activatable system has been developed that enables tumor-specific singlet oxygen ((1) O2 ) generation for cancer therapy, based on a Fenton-like reaction between linoleic acid hydroperoxide (LAHP) tethered on iron oxide nanoparticles (IO NPs) and the released iron(II) ions from IO NPs under acidic-pH condition. The IO-LAHP NPs are able to induce efficient apoptotic cancer cell death both in vitro and in vivo through tumor-specific (1) O2 generation and subsequent ROS mediated mechanism. This study demonstrates the effectiveness of modulating biochemical reactions as a ROS source to exert cancer death. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Umeda, Nobuhiro; Urano, Yasuteru; Nagano, Tetsuo
Caged compound is one of the most powerful tools for spatiotemporal control of biomolecules in cells, which can be activated by irradiation of light. However, ultra violet light, which is required for activation of caged compounds, can damage cells and has poor permeability into tissues. In addition, invisibility of caged compounds makes it difficult to tell distribution of released small molecules. At the conference, we will describe the development of novel caging group and new caged compounds which are fluorescently visible and efficiently activatable with green light. We have found that boron dipyrromethene (BODIPY), known as a widely used fluorophore, is a potential caging group for phenol, carboxyl acid and amine, which can be photolized with irradiation of green light at around 500 nm wavelength. Based on the novel photo-reaction of 4-phenoxy BODIPY derivatives, we have developed caged histamine and applied it to HeLa cells. Photo-irradiation to cells in the presence of caged histamine induced transient increase of calcium ion in cytosol, which was specifically inhibited with pyrilamine, a H1 blocker. Also, we showed that BODIPY-caged compound can be utilized in vivo with tissue-permeable 500 nm green light.
Minibodies show rapider blood clearance than IgGs due to smaller size that improves target-to-background ratio (TBR) in in vivo imaging. Additionally, the ability to activate an optical probe after binding to the target greatly improves the TBR. An optical imaging probe based on a minibody against prostate-specific membrane antigen (PSMA-MB) and conjugated with an activatable fluorophore, indocyanine green (ICG), was designed to fluoresce only after binding to cell-surface PSMA. To further reduce background signal, short polyethylene glycol (PEG) linkers were employed to improve the covalent bonding ratio of ICG. New PSMA-MBs conjugated with bifunctional ICG derivatives specifically visualized PSMA-positive tumor xenografts in mice bearing both PSMA-positive and -negative tumors within 6 h postinjection. The addition of short PEG linkers significantly improved TBRs; however, it did not significantly alter the biodistribution. Thus, minibody-ICG conjugates could be a good alternative to IgG-ICG in the optical cancer imaging for further clinical applications. PMID:24900850
Sheeran, Paul S; Luois, Samantha H; Mullin, Lee B; Matsunaga, Terry O; Dayton, Paul A
Recently, an interest has developed in designing biomaterials for medical ultrasonics that can provide the acoustic activity of microbubbles, but with improved stability in vivo and a smaller size distribution for extravascular interrogation. One proposed alternative is the phase-change contrast agent. Phase-change contrast agents (PCCAs) consist of perfluorocarbons (PFCs) that are initially in liquid form, but can then be vaporized with acoustic energy. Crucial parameters for PCCAs include their sensitivity to acoustic energy, their size distribution, and their stability, and this manuscript provides insight into the custom design of PCCAs for balancing these parameters. Specifically, the relationship between size, thermal stability and sensitivity to ultrasound as a function of PFC boiling point and ambient temperature is illustrated. Emulsion stability and sensitivity can be 'tuned' by mixing PFCs in the gaseous state prior to condensation. Novel observations illustrate that stable droplets can be generated from PFCs with extremely low boiling points, such as octafluoropropane (b.p. -36.7 °C), which can be vaporized with acoustic parameters lower than previously observed. Results demonstrate the potential for low boiling point PFCs as a useful new class of compounds for activatable agents, which can be tailored to the desired application.
Wang, Yongliang; LeVine, Dana N; Gannon, Margaret; Zhao, Yuanchang; Sarkar, Anwesha; Hoch, Bailey; Wang, Xuefeng
Integrin-transmitted cellular forces are critical for platelet adhesion, activation, aggregation and contraction during hemostasis and thrombosis. Measuring and mapping single platelet forces are desired in both research and clinical applications. Conventional force-to-strain based cell traction force microscopies have low resolution which is not ideal for cellular force mapping in small platelets. To enable platelet force mapping with submicron resolution, we developed a force-activatable biosensor named integrative tension sensor (ITS) which directly converts molecular tensions to fluorescent signals, therefore enabling cellular force mapping directly by fluorescence imaging. With ITS, we mapped cellular forces in single platelets at 0.4µm resolution. We found that platelet force distribution has strong polarization which is sensitive to treatment with the anti-platelet drug tirofiban, suggesting that the ITS force map can report anti-platelet drug efficacy. The ITS also calibrated integrin molecular tensions in platelets and revealed two distinct tension levels: 12-54 piconewton (nominal values) tensions generated during platelet adhesion and tensions above 54 piconewton generated during platelet contraction. Overall, the ITS is a powerful biosensor for the study of platelet mechanobiology, and holds great potential in antithrombotic drug development and assessing platelet activity in health and disease. Copyright © 2017 Elsevier B.V. All rights reserved.
Zhao, Ying; Ji, Tianjiao; Wang, Hai; Li, Suping; Zhao, Yuliang; Nie, Guangjun
Design of specific and sensitive imaging probes for targeting tumor microenvironment holds great promise to achieve precise detection and rapid responsiveness to neoplastic tissues. Dysregulated pH, one of the most remarkable hallmarks of tumor microenvironment, can be considered as a good specific trigger for the design of broad-spectrum and local-environment responsive imaging probes. However, the current existing design strategies for pH-responsive systems are insufficient to meet the needs for a rapid and tumor-specific diagnosis. Here we reported a novel biomimetic nanostructure based on oligopeptide self-assembly that can quickly switch into dissociated stage with active fluorescence property from self-assembled stage with quenched fluorescence activity when encountering a subtle pH-change in tumor microenvironment (pH 6.8 vs. 7.4). This oligopeptide-assembly is examined as tumor microenvironment activatable probes for both intratumoral and intravenous in vivo tumor imaging. Through the distinct fluorescent intensities, it is validated that the acidic tumor microenvironment can activate stronger fluorescence signals. The tailor-made self-assembled oligopeptide nanomaterials have the potential for efficient and specific in situ diagnosis of various solid tumors with a weakly acidic microenvironment, which is expected to be of crucial importance for clinical tumor diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.
Hsiao, Po-Yen; Tsai, Chia-Lun; Chen, Ming-Chang; Lin, Yen-Yin; Yang, Shang-Da; Chiang, Ann-Shyn
Scattering and absorption limit light penetration through inhomogeneous tissue. To reduce scattering, biochemists have shifted the wavelengths of excitation light for optogenetic actuators and fluorescent proteins to the orange-red range, while physicists have developed multiphoton technologies for deep tissue stimulation. We have built a rapid multiphoton spectroscopic screening system of genetically encoded red-activatable channelrhodopsin (ReaChR), and considered specific behaviors in transgenic Drosophila melanogaster as readouts to optimize the laser parameters for two-photon optogenetic activation. A wavelength-tunable optical parametric amplifier was adopted as the major light source for widefield two-photon excitation (TPE) of ReaChR. Our assays suggest that the optimized TPE wavelength of ReaChR is 1250 nm. Exploiting its capacity for optogenetic manipulation to induce macroscopic behavioral change, we realized rapid spectroscopic screening of genetically encoded effectors or indicators in vivo, and used modulation of ReaChR in the fly as a successful demonstration of such a system. PMID:26601000
Pouya, Fahima D; Zavar-Reza, Javad; Jalali, Beman A
Methylglyoxal is a reactive α, β dicarbonyl aldehyde compound that originates from various biochemical pathways. Some studies suggest that increased methylglyoxal in blood leads to changes in fibrinolysis; however, the precise mechanism is not clear. The aim of this study was to compare different concentrations of methylglyoxal and aspirin on fibrinolysis in the plasma of healthy individuals in vitro. Different concentrations of methylglyoxal (5, 50, 100, and 500 μmol/l) and aspirin (1, 10, and 100 mg/l) were added to the plasma citrate. They were incubated at 37°C for 24 h. Then, fibrinolysis parameters were analyzed by the turbidimetric procedure at 405 nm. The Independent Samples t-test was utilized to compare them (P < 0.05). Findings revealed that methylglyoxal at 500 μmol/l with aspirin 100 mg/l had significant changes in the maximum lysis velocity (0.163 ± 0.003), half-time lysis (240 ± 10.00), the total lysis time (485 ± 5.00), lag time in lysis (126 ± 5.77), compared with methylglyoxal at 500 μmol/l (0.104 ± 0.005), (276 ± 5.77), (570 ± 10.00), and (186 ± 5.77), respectively (P < 0.05). Methylglyoxal at 500 μmol/l with aspirin 1 mg/l did not significantly change in either parameter (P > 0.05). Methylglyoxal at 100 μmol/l with aspirin 1 mg/l did not significantly change in either fibrinolysis parameter (P > 0.05), compared with methylglyoxal at 100 μmol/l. Methylglyoxal at 5 μmol/l with aspirin (1, 10, 100 mg/l) changed in all fibrinolysis parameters (P < 0.05), compared with methylglyoxal at 5 μmol/l. The other concentrations were compared in the same way. Aspirin (more than 1 mg/l) had more effect on higher concentrations of methylglyoxal. It increased the velocity of lysis of the clot and shortened clot lysis.
Jimenez, Juan J; Iribarren, Jose L; Lorente, Leonardo; Rodriguez, Jose M; Hernandez, Domingo; Nassar, Ibrahim; Perez, Rosalia; Brouard, Maitane; Milena, Antonio; Martinez, Rafael; Mora, Maria L
Introduction Extracorporeal circulation induces hemostatic alterations that lead to inflammatory response (IR) and postoperative bleeding. Tranexamic acid (TA) reduces fibrinolysis and blood loss after cardiopulmonary bypass (CPB). However, its effects on IR and vasoplegic shock (VS) are not well known and elucidating these effects was the main objective of this study. Methods A case control study was carried out to determine factors associated with IR after CPB. Patients undergoing elective CPB surgery were randomly assigned to receive 2 g of TA or placebo (0.9% saline) before and after intervention. We performed an intention-to-treat analysis, comparing the incidence of IR and VS. We also analyzed several biological parameters related to inflammation, coagulation, and fibrinolysis systems. We used SPSS version 12.2 for statistical purposes. Results In the case control study, 165 patients were studied, 20.6% fulfilled IR criteria, and the use of TA proved to be an independent protective variable (odds ratio 0.38, 95% confidence interval 0.18 to 0.81; P < 0.01). The clinical trial was interrupted. Fifty patients were randomly assigned to receive TA (24) or placebo (26). Incidence of IR was 17% in the TA group versus 42% in the placebo group (P = 0.047). In the TA group, we observed a significant reduction in the incidence of VS (P = 0.003), the use of norepinephrine (P = 0.029), and time on mechanical ventilation (P = 0.018). These patients showed significantly lower D-dimer, plasminogen activator inhibitor 1, and creatine-kinase levels and a trend toward lower levels of soluble tumor necrosis factor receptor and interleukin-6 within the first 24 hours after CPB. Conclusion The use of TA attenuates the development of IR and VS after CPB. Trial registration number ISRCTN05718824. PMID:17988379
Nielsen, Vance G; Boyer, Leslie V; Matika, Ryan W; Amos, Quinlan; Redford, Daniel T
In addition to degrading fibrinogen as a source of consumptive coagulopathy, rattlesnake venom has also been demonstrated to enhance fibrinolysis and degrade alpha-2-antiplasmin. The goals of this investigation was to characterize the kinetic fibrinolytic profile of Crotalus atrox venom in the absence and presence of tissue-type plasminogen activator (tPA), and to also ascertain if iron and carbon monoxide (CO, a positive modulator of alpha-2-antiplasmin) could attenuate venom-enhanced fibrinolysis. Utilizing thrombelastographic methods, the coagulation and fibrinolytic kinetic profiles of human plasma exposed to C. atrox venom (0-2 μg/ml) were determined in the absence or presence of tPA (0-100 IU/ml). Then, either separately or in combination, plasma was exposed to iron (ferric chloride, 10 μmol/l) or CO (carbon monoxide-releasing molecule-2, 100 μmol/l) prior to incubation with venom; the plasma sample was subsequently subjected to thrombelastographic analysis with addition of tPA. Venom exposure in the absence of tPA did not result in detectable fibrinolysis. In the presence of tPA, venom markedly enhanced fibrinolysis. Iron and CO, markedly attenuated venom enhancement of fibrinolysis. C. atrox venom enhances tPA-mediated fibrinolysis, and interventions that enhance/protect alpha-2-antiplasmin activity significantly attenuate venom-enhanced fibrinolysis. Future preclinical investigation is required to determine if iron and CO can attenuate venom-mediated degradation of alpha-2-antiplasmin-dependent fibrinolytic resistance.
Lau, C S; McLaren, M; Mackay, I; Belch, J J
OBJECTIVES--(1) To assess if patients with various forms of Raynaud's phenomenon (RP) have abnormal plasma fibrinolysis that may contribute to diminished digital blood flow; (2) to assess whether patients with RP with evidence of endothelial damage have abnormal plasma fibrinolysis; (3) to determine the clinical relevance of abnormalities, if any, in plasma fibrinolysis in patients with RP. METHODS--One hundred and sixty eight patients with significant RP were studied--46 had primary Raynaud's disease (RD), 32 had suspected Raynaud's syndrome secondary to an undifferentiated connective tissue disorder (undifferentiated CTD), 25 had Raynaud's syndrome associated with atherosclerosis (athero RS), and 65 had an underlying connective tissue disease (CTD RS). All attended in the morning after a low fat light breakfast. After a clinical history was obtained, venous blood samples were collected without stasis for assays of plasma fibrinolysis and factor VIII von Willebrand factor antigen (fVIII vWF Ag). Results were compared with those obtained from normal subjects matched for sex and age. As patients with athero RS were significantly older than the other patients with Raynaud's phenomenon, two groups of control subjects were recruited--namely, 'old' and 'young' control subjects. RESULTS--Patients with CTD RS and athero RS had higher concentrations of fVIII vWF Ag (CTD RS median 174.5 range (45-370)% v 100 (38-202)%, p < 0.001; athero RS 182-5 (100-240)% v 100 (50-158)%, p < 0.001). Both had raised fibrinogen (CTD RS 3.25 (1.9-6.8) g/l v 2.4 (1.2-4.2) g/l, p < 0.001; athero RS 3.4 (2.2-6.2) g/l v 2.5 (1.8-3.9) g/l, p < 0.001) and both had diminished fibrinolysis with reduced plasminogen activator activity (CTD RS 79.5 (31-72) mm2 v 92 (37-197) mm2, p < 0.04; athero RS 73 (45-125) mm2 v 98 (41-197) mm2, p < 0.03). Patients with CTD RS also had raised plasminogen activity (3.3 (2.3-5.8) cU/ml v 2.9 (1.5-5.4) cU/ml, p < 0.001). On the contrary, patients with primary RD and
Hämäläinen, H; Rönnemaa, T; Virtanen, A; Lindström, J; Eriksson, J G; Valle, T T; Ilanne-Parikka, P; Keinänen-Kiukaanniemi, S; Rastas, M; Aunola, S; Uusitupa, M; Tuomilehto, J
The aim of this study was to investigate the effects of lifestyle intervention on the levels of plasminogen activator inhibitor (PAI-1) and fibrinogen in subjects participating in the Finnish Diabetes Prevention Study (DPS). In five DPS centres, 321 subjects with impaired glucose tolerance (intervention group, n=163; control group, n=158) had their PAI-1 and fibrinogen levels measured at baseline and at the 1-year follow-up. Additional 3-year follow-up assessments were carried out in a sample of 97 subjects in one of the DPS centres (Turku). The intervention programme included an intensive lifestyle intervention aiming at weight reduction, healthy diet and increased physical activity. During the first intervention year, PAI-1 decreased by 31% in the intervention group but showed no change in the control group (p<0.0001). In the Turku subgroup, the decrease in PAI-1 persisted throughout the 3-year follow-up. Changes in PAI-1 were associated with the number of lifestyle changes made during the first year (p=0.008). Weight reduction was the most important factor explaining the decrease in PAI-1. Changes in fibrinogen levels did not differ between the groups. In addition to the previously reported reduction in the risk of type 2 diabetes in DPS participants with impaired glucose tolerance, the intensive dietary and exercise intervention had beneficial long-term effects on fibrinolysis as indicated by the reduced levels of PAI-1. These results suggest that elevated PAI-1 levels in obese subjects with impaired glucose tolerance are mostly reversible by lifestyle changes, especially those geared to weight reduction.
Cucuianu, M; Fekete, T; Marcusiu, C; Mösler, R; Duţu, A
The variations of dilute blood clot lysis time (DBCLT) in 103 diabetic patients were investigated in terms of insulin dependency, body weight, serum lipids and presence of diabetic vascular diseases. The results showed that DBCLT was significantly longer in the 34 overweight diabetic patients (437 +/- 68 min) than in the 69 diabetics at or below the ideal body weight (240 +/- 28 min) or in the 76 normalipidemic normal weight control subjects (253 +/- 12 min). DBCLT was also longer in the hypertrigliceridemic diabetic patients than in the normolipidemic ones. The mean lysis time was similar in diabetic patients with and without retinopathy. However, a higher level of fibrinolytic inhibitors was found in patients with diabetic small vessel disease. In vitro inhibition of plasma factor XIII by p-chlormercuribenzoate (PCMB) caused an acceleration of DBCLT and the differences between lysis time in the overweight diabetics and in the controls were attenuated. These results suggested that deficient thrombolysis is rather due to overweight and to disturbances of lipid metabolism than to diabets and/or its vascular complications and that enhanced fibrin crosslinking is at least partially responsible for delayed clot lysis.
Palao, Eduardo; Slanina, Tomáš; Muchová, Lucie; Šolomek, Tomáš; Vítek, Libor; Klán, Petr
Carbon monoxide-releasing molecules (CORMs) are chemical agents used to administer CO as an endogenous, biologically active molecule. A precise spatial and temporal control over the CO release is the major requirement for their applications. Here, we report the synthesis and properties of a new generation of transition-metal-free carbon monoxide-releasing molecules based on BODIPY chromophores (COR-BDPs) activatable by visible-to-NIR (up to 730 nm) light. We demonstrate their performance for both in vitro and in vivo experimental settings, and we propose the mechanism of the CO release based on steady-state and transient spectroscopy experiments and quantum chemical calculations.
Lau, Christine L; Zhao, Yunge; Kim, Jiyoun; Kron, Irving L; Sharma, Ashish; Yang, Zequan; Laubach, Victor E; Linden, Joel; Ailawadi, Gorav; Pinsky, David J
Ischemia-reperfusion injury continues to plague the field of lung transplantation, resulting in suboptimal outcomes. In acute lung injury, processes such as ventilator-induced injury, sepsis, or acute respiratory distress syndrome, extravascular fibrin has been shown to promote lung dysfunction and the acute inflammatory response. This study investigates the role of the fibrinolytic cascade in lung ischemia-reperfusion injury and investigates the interplay between the fibrinolytic system and the inflammatory response. Mice lacking the plasminogen activator inhibitor-1 gene (PAI-1 knock out, PAI-1 KO; and thus increased lysis of endogenous fibrin) and wild-type mice underwent in situ left lung ischemia and reperfusion. Fibrin content in the lung was evaluated by immunoblotting. Reperfusion injury was assessed by histologic and physiologic parameters. Proinflammatory mediators were measured in bronchoalveolar lavage fluid and plasma using enzyme-linked immunosorbent assays. Ischemia-reperfusion causes fibrin deposition in murine lungs. Less fibrin was seen in PAI-1 KO mice than in wild-type mice subjected to the same ischemia-reperfusion conditions. By histologic criteria, more evidence of ischemia-reperfusion injury was noted (thickening of the interstium, cellular infiltration in the alveoli) in the wild-type than in PAI-1 KO mice. Physiologic parameters also revealed more ischemia-reperfusion injury in the wild-type than in PAI-1 KO mice. Cytokine and chemokines were elevated more in the wild-type group than the PAI-1 KO group. Lung ischemia-reperfusion injury triggers fibrin deposition in the murine lungs and fibrin creates a proinflammatory environment. Preventing fibrin deposition may reduce ischemia-reperfusion injury and inflammation. This finding may lead to novel treatment strategies for ischemia-reperfusion.
Subra, Caroline; Grand, David; Laulagnier, Karine; Stella, Alexandre; Lambeau, Gérard; Paillasse, Michael; De Medina, Philippe; Monsarrat, Bernard; Perret, Bertrand; Silvente-Poirot, Sandrine; Poirot, Marc; Record, Michel
Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell. PMID:20424270
Zhou, Kejin; Liu, Haoming; Zhang, Shanrong; Huang, Xiaonan; Wang, Yiguang; Huang, Gang; Sumer, Baran D.; Gao, Jinming
Tunable, ultra-pH responsive fluorescent nanoparticles with multichromatic emissions are highly valuable in a variety of biological studies, such as endocytic trafficking, endosome/lysosome maturation, and pH regulation in subcellular organelles. Small differences (e.g., <1 pH unit) and yet finely regulated physiological pH inside different endocytic compartments present a huge challenge to the design of such a system. Herein, we report a general strategy to produce pH-tunable, highly activatable multicolored fluorescent nanoparticles using commonly available pH-insensitive dyes with emission wavelengths from green to near IR range. pH-induced micellization is the primary driving force of fluorescence activation between the ON (unimer) and OFF (micelle) states. Among three possible photochemical mechanisms, homo Förster resonance energy transfer (homo-FRET) was found to be the most facile strategy to render ultra-pH response over the H-dimer and photoinduced electron transfer (PeT) mechanisms. Based on this insight, we selected several fluorophores with small Stoke shifts (<40 nm) and established a panel of multicolored nanoparticles with wide emission range (500-820 nm) and different pH transitions. Each nanoparticle maintained the sharp pH response (ON/OFF <0.25 pH unit) with corresponding pH transition point at pH 5.2, 6.4, 6.9 and 7.2. Incubation of a mixture of multicolored nanoparticles with human H2009 lung cancer cells demonstrated sequential activation of the nanoparticles inside endocytic compartments directly correlating with their pH transitions. This multicolored, pH-tunable nanoplatform offers many exciting opportunities for the study of many important cell physiological processes such as pH regulation and endocytic trafficking of subcellular organelles. PMID:22524413
Gullotti, Emily; Park, Joonyoung; Yeo, Yoon
Purpose To create a poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), where a drug-encapsulating NP core is covered with polyethylene glycol (PEG) in a normal condition but exposes a cell-interactive TAT-modified surface in an environment rich in matrix metalloproteinases (MMPs). Methods PLGA NPs were modified with TAT peptide (PLGA-pDA-TAT NPs) or dual-modified with TAT peptide and a conjugate of PEG and MMP-substrate peptide (Peritumorally activatable NPs, PANPs) via dopamine polymerization. Cellular uptake of fluorescently-labeled NPs was observed with or without a pre-treatment of MMP-2 by confocal microscopy and flow cytometry. NPs loaded with paclitaxel (PTX) were tested against SKOV-3 ovarian cancer cells to evaluate the contribution of surface modification to cellular delivery of PTX. Results While the size and morphology did not significantly change due to the modification, NPs modified with dopamine polymerization were recognized by their dark color. Moreover, TAT-containing NPs (PLGA-pDA-TAT NPs and PANPs) showed changes in surface charge, indicative of effective conjugation of TAT peptide on the surface. PLGA-pDA-TAT NPs and MMP-2-pre-treated PANPs showed relatively good cellular uptake as compared to PLGA NPs, MMP-2-non-treated PANPs, and NPs with non-cleavable PEG. After 3 hour treatment with cells, PTX loaded in cell-interactive NPs showed greater toxicity than that in non-interactive ones as the former could enter cells during the incubation period. However, due to the initial burst drug release, the difference was not as clear as microscopic observation. Conclusions PEGylated polymeric NPs that exposed cell-interactive surface in response to MMP-2 were successfully created by dual modification of PLGA NPs using dopamine polymerization. PMID:23609560
Hirasawa, Takeshi; Iwatate, Ryu J.; Kamiya, Mako; Okawa, Shinpei; Urano, Yasuteru; Ishihara, Miya
Photoacoustic (PA) imaging offers depth-resolved images of optical absorbers with the spatial resolution of ultrasound imaging. To enhance tumour contrast, tumour-specific probes are used as contrast agents. We synthesised a colourless PA probe that is activated in the presence of γ-glutamyltranspeptidase, a cancer-associated enzyme, to show its original colour and fluorescence. We have acquired high specificity fluorescence images of small tumours, using a fluorescent probe based on similar enzymatic reactions. Here, we developed a PA imaging technique to detect the PA probe. In PA imaging, depending on the concentration and excitation wavelength of the probe, the intensities of the probe signals may be lower than those of the background signals produced by intrinsic optical absorbers such as haemoglobin. For probe imaging in the presence of strong background signals, multispectral photoacoustic (MS-PA) imaging was evaluated. In MS-PA imaging, the spectral fitting method, which distinguishes the probe signals from background signals using reference spectra, has been widely used. To compensate for the decrease of fluence due to optical attenuation in biological tissue, we used a simplified compensation method that calculates fluence inside biological tissues by the Monte-Carlo model using published data on optical properties of biological tissues. The validity of the method was confirmed using tissue-mimicking phantoms. Finally, MS-PA imaging of a mouse subcutaneous tumour injected with the activatable probe was demonstrated. In conclusion, our MS-PA imaging technique afforded successful detection of the activated probe in the tumour, and time-increase of PA signals were successfully observed.
Ronald, John A.; Chuang, Hui-Yen; Dragulescu-Andrasi, Anca; Hori, Sharon S.; Gambhir, Sanjiv S.
Earlier detection of cancers can dramatically improve the efficacy of available treatment strategies. However, despite decades of effort on blood-based biomarker cancer detection, many promising endogenous biomarkers have failed clinically because of intractable problems such as highly variable background expression from nonmalignant tissues and tumor heterogeneity. In this work we present a tumor-detection strategy based on systemic administration of tumor-activatable minicircles that use the pan-tumor–specific Survivin promoter to drive expression of a secretable reporter that is detectable in the blood nearly exclusively in tumor-bearing subjects. After systemic administration we demonstrate a robust ability to differentiate mice bearing human melanoma metastases from tumor-free subjects for up to 2 wk simply by measuring blood reporter levels. Cumulative change in reporter levels also identified tumor-bearing subjects, and a receiver operator-characteristic curve analysis highlighted this test’s performance with an area of 0.918 ± 0.084. Lung tumor burden additionally correlated (r2 = 0.714; P < 0.05) with cumulative reporter levels, indicating that determination of disease extent was possible. Continued development of our system could improve tumor detectability dramatically because of the temporally controlled, high reporter expression in tumors and nearly zero background from healthy tissues. Our strategy’s highly modular nature also allows it to be iteratively optimized over time to improve the test’s sensitivity and specificity. We envision this system could be used first in patients at high risk for tumor recurrence, followed by screening high-risk populations before tumor diagnosis, and, if proven safe and effective, eventually may have potential as a powerful cancer-screening tool for the general population. PMID:25713388
Yamanome, T; Yoshida, K; Miura, K; Ogawa, A
A case of successful treatment by local fibrinolysis of a middle cerebral artery embolism caused by a thrombus from a left atrial myxoma is reported. A 62-year-old woman using a pacemaker and suffering from sick sinus syndrome was admitted on December 29th 1996, complaining of transient restlessness. CT and cerebral angiography revealed no abnormal vascular lesions. Eighteen months after the initial episode, she suffered a sudden onset of left hemiparesis and loss of consciousness. CT scan performed during the second episode revealed no lesions and, in particular, no early CT infarction sign, but emergent cerebral angiography revealed a right middle cerebral artery embolic occlusion. Local fibrinolysis using a tissue plasminogen activator was performed within 3 hours after the beginning of the episode, and partial recanalization was obtained within one hour after initiation of the fibrinolytic therapy. On the first hospital day, though CT revealed a small low-density area in the right basal ganglia, motor deficits gradually improved. Considering the possibility of a cardiac source of the embolism, trans-esophageal echocardiography was performed and revealed a left atrial tumor suspected to be a myxoma. It was removed by surgery on the 34th hospital day. Histological examination proved it to be a myxoma. Nine months after local fibrinolytic therapy, the patient returned to work. The diagnosis of cerebral embolism caused by cardiac myxoma is difficult to make at the time when the patient is first examined after admission. It is also hard to discover during emergent cerebral angiography with fibrinolytic therapy. Therefore, in the case of patients with cerebral embolism for which local fibrinolysis is ineffective, it should be presumed that cardiac myxoma is the source of the embolus. Direct PTA alone may be effective for such tumoral embolism.
Chernysh, Irina N.; Everbach, E. Carr; Purohit, Prashant K.; Weisel, John W.
Summary Background Ultrasound accelerates tissue-type plasminogen activator (t-PA)-induced fibrinolysis of clots in vitro and in vivo. Objective To identify mechanisms for the enhancement of t-PA-induced fibrinolysis of clots. Methods Turbidity is an accurate and convenient method, not previously used, to follow the effects of ultrasound. Deconvolution microscopy was used to determine changes in structure, while fluorescence recovery after photobleaching was used to characterize the kinetics of binding/unbinding and transport. Results The ultrasound pulse repetition frequency affected clot lysis times, but there were no thermal effects. Ultrasound in the absence of t-PA produced a slight but consistent decrease in turbidity, suggesting a decrease in fibrin diameter due solely to the action of the ultrasound, likely caused by an increase in protofibril tension because of vibration from ultrasound. Changes in fibrin network structure during lysis with ultrasound were visualized in real time by deconvolution microscopy, revealing that the network becomes unstable when 30–40% of the protein in the network was digested, whereas without ultrasound, the fibrin network was digested gradually and retained structural integrity. Fluorescence recovery after photobleaching during lysis revealed that the off-rate of oligomers from digesting fibers was not much affected but the number of binding/unbinding sites was increased. Conclusions Ultrasound causes a decrease in the diameter of the fibers due to tension as a result of vibration, leading to increased binding sites for plasmin(ogen)/t-PA. The positive feedback of this structural change together with increased mixing/transport of t-PA/plasmin(ogen) is likely to account for the observed enhancement of fibrinolysis by ultrasound. PMID:25619618
Levashov, M Yu; Aisina, R B; Gershkovich, K B; Varfolomeyev, S D
Stimulation of Lys-plasminogen (Lys-Pg) and Glu-plasminogen (Glu-Pg) activation under the action of staphylokinase and Glu-Pg activation under the action of preformed plasmin-staphylokinase activator complex (Pm-STA) by low concentrations and inhibition by high concentrations of omega-amino acids (>90-140 mM) were found. Maximal stimulation of the activation was observed at concentrations of L-lysine, 6-aminohexanoic acid (6-AHA), and trans-(4-aminomethyl)cyclohexanecarboxylic acid 8.0, 2.0, and 0.8 mM, respectively. In contrast, the Lys-Pg activation rate by Pm-STA complex sharply decreased when concentrations of omega-amino acids exceeded the above-mentioned values. It was found that formation of Pm-STA complex from a mixture of equimolar concentrations of staphylokinase and Glu-Pg or Lys-Pg is stimulated by low concentrations (maximal at 10 mM) of 6-AHA. Negligible increase in the specific activities of plasmin and Pm-STA complex was detected at higher concentrations of 6-AHA (to maximal at 70 and 50 mM, respectively). Inhibitory effects of omega-amino acids on the rate of fibrinolysis induced by staphylokinase, Pm-STA complex, and plasmin were compared. It was found that inhibition of staphylokinase-induced fibrinolysis by omega-amino acids includes blocking of the reactions of Pm-STA complex formation, plasminogen activation by this complex, and lysis of fibrin by forming plasmin as a result of displacement of plasminogen and plasmin from the fibrin surface. Thus, the slow stage of Pm-STA complex formation plays an important role in the mechanism of action of omega-amino acids on Glu-Pg activation and fibrinolysis induced by staphylokinase. In addition to alpha-->beta change of Glu-Pg conformation, stimulation of Pm-STA complex formation leads to increase in Glu-Pg activation rate in the presence of low concentrations of omega-amino acids. Inhibition of Pm-STA complex formation on fibrin surface by omega-amino acids is responsible for appearance of long lag
MacKay, N.; Ferguson, J. C.; McNicol, G. P.
The effects of the venoms of Naja melanoleuca, Naja nigricollis, and Ophiophagus hannah on blood coagulation, platelet aggregation, and fibrinolysis were studied in vitro. All three venoms were shown to be anticoagulant. This action appeared to be due to an effect on both the extrinsic and blood thromboplastin mechanisms. Platelet aggregation in Chandler's tubes and adenosine diphosphate reactivity were inhibited by the three venoms, although in the case of Ophiophagus hannah venom they were inhibited only with intermediate concentrations. The three venoms possessed proteolytic properties, but when incorporated into purified caseinolytic systems and euglobulin clot lysis systems inhibition of plasmin activity was observed. PMID:5814735
Optical fluorescence-guided imaging is increasingly used to guide surgery and endoscopic procedures. Sprayable enzyme-activatable probes are particularly useful because of high target-to-background ratios that increase sensitivity for tiny cancer foci. However, green fluorescent activatable probes suffers from interference from autofluorescence found in biological tissue. Dynamic imaging followed by the kinetic analysis could be detected local enzyme activity and used to differentiate specific fluorescence arising from an activated probe in a tumor from autofluorescence in background tissues especially when low concentrations of the dye are applied to detect tiny cancer foci. Serial fluorescence imaging was performed using various concentrations of γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) which was sprayed on the peritoneal surface with tiny implants of SHIN3-dsRed ovarian cancer tumors. Temporal differences in signal between specific green fluorescence in cancer foci and non-specific autofluorescence in background tissue was measured and processed into three kinetic maps reflecting maximum fluorescence signal (MF), wash-in rate (WIR), and area under the curve (AUC), respectively. Especially at lower concentrations, kinetic maps derived from dynamic fluorescence imaging were clearly superior to unprocessed images for detection small cancer foci.
Hsu, Benedict You Wei; Ng, Michael; Tan, Aaron; Connell, John; Roberts, Thomas; Lythgoe, Mark; Zhang, Yu; Wong, Siew Yee; Bhakoo, Kishore; Seifalian, Alexander M; Li, Xu; Wang, John
Stimuli-responsive nanoprobes that combine both fluorescence and magnetic resonance imaging (MRI) are anticipated to be highly beneficial for tumor visualization with high imaging sensitivity. By employing an interfacial templating scheme, a pH-activatable fluorescence/MRI dual-modality imaging nanoprobe is successfully developed based on the coencapsulation of MnO nanoparticles and coumarin-545T inside a hybrid silica nanoshell. To promote cancer cell targeting with high-specificity, the nanoprobes are also conjugated with folic acid to establish a greater affinity for cancer cells that over-express folate receptors on their cell membrane. In the new nanosystem, MnO nanoparticles are shown to function as an efficient fluorescence quencher of coumarin-545T prior to cellular uptake. However, fluorescence recovery is achieved upon acidic dissolution of the MnO nanoparticles following receptor-mediated endocytosis into the low pH compartments of the cancer cells. Meanwhile, the Mn(2+) ions thus released are also shown to exert a strong T1 contrast enhancement in the cancer cells. Therefore, by demonstrating the dual-activatable MRI and fluorescence imaging in response to the low pH conditions, it is envisioned that these nanoprobes would have tremendous potential for emerging cancer-imaging modalities such as image-guided cancer therapy.
Schutt, Carolyn; Ibsen, Stuart; Zahavy, Eran; Aryal, Santosh; Kuo, Stacey; Esener, Selin; Berns, Michael; Esener, Sadik
A major challenge facing nanoparticle-based delivery of chemotherapy agents is the natural and unavoidable accumulation of these particles in healthy tissue resulting in local toxicity and dose-limiting side effects. To address this issue, we have designed and characterized a new prodrug nanoparticle with controllable toxicity allowing a locally-delivered light trigger to convert the payload of the particle from a low to a high toxicity state. The nanoparticles are created entirely from light-activatable prodrug molecules using a nanoprecipitation process. The prodrug is a conjugate of doxorubicin and photocleavable biotin (DOX-PCB). These DOX-PCB nanoparticles are 30 times less toxic to cells than doxorubicin, but can be activated to release pure therapeutic doxorubicin when exposed to 365 nm light. These nanoparticles have an average diameter of around 100 nm and achieve the maximum possible prodrug loading capacity since no support structure or coating is required to prevent loss of prodrug from the nanoparticle. These light activatable nanoparticles demonstrate tunable toxicity and can be used to facilitate future therapy development whereby light delivered specifically to the tumor tissue would locally convert the nanoparticles to doxorubicin while leaving nanoparticles accumulated in healthy tissue in the less toxic prodrug form.
Montrose, Kristopher; Yang, Yi; Krissansen, Geoffrey W
Here we describe a structure-function analysis of the cell-penetrating peptide Xentry derived from the X-protein of the hepatitis B virus. Remarkably, the tetrapeptide core LCLR retains the cell-penetrating ability of the parental peptide LCLRPVG, as either an L- or D-enantiomer. Substitution of the cysteine with leucine revealed that the cysteine is essential for activity. In contrast, the C-terminal arginine could be substituted in the L-isomer with lysine, histidine, glutamic acid, glutamine, and asparagine, though the resulting peptides displayed distinct cell-type-specific uptake. Substitution of the leucines in the D-isomer with other hydrophobic residues revealed that leucines are optimal for activity. Surprisingly, linear di- and tetra-peptide forms of Xentry are not cell-permeable. Protease-activatable forms of Xentry were created by fusing Xentry to itself via a protease-cleavable peptide, or by attaching a heparin mimic peptide to the N-terminus. These novel activatable forms of Xentry were only taken up by MCF-7 cells after cleavage by matrix metalloproteinase 9, and could be used to deliver drugs specifically to tumours.
Montrose, Kristopher; Yang, Yi; Krissansen, Geoffrey W.
Here we describe a structure-function analysis of the cell-penetrating peptide Xentry derived from the X-protein of the hepatitis B virus. Remarkably, the tetrapeptide core LCLR retains the cell-penetrating ability of the parental peptide LCLRPVG, as either an L- or D-enantiomer. Substitution of the cysteine with leucine revealed that the cysteine is essential for activity. In contrast, the C-terminal arginine could be substituted in the L-isomer with lysine, histidine, glutamic acid, glutamine, and asparagine, though the resulting peptides displayed distinct cell-type-specific uptake. Substitution of the leucines in the D-isomer with other hydrophobic residues revealed that leucines are optimal for activity. Surprisingly, linear di- and tetra-peptide forms of Xentry are not cell-permeable. Protease-activatable forms of Xentry were created by fusing Xentry to itself via a protease-cleavable peptide, or by attaching a heparin mimic peptide to the N-terminus. These novel activatable forms of Xentry were only taken up by MCF-7 cells after cleavage by matrix metalloproteinase 9, and could be used to deliver drugs specifically to tumours. PMID:24811205
Hu, Dehong; Sheng, Zonghai; Gao, Guanhui; Siu, Fungming; Liu, Chengbo; Wan, Qian; Gong, Ping; Zheng, Hairong; Ma, Yifan; Cai, Lintao
Photodynamic therapy (PDT) is a noninvasive and effective approach for cancer treatment. The main bottlenecks of clinical PDT are poor selectivity of photosensitizer and inadequate oxygen supply resulting in serious side effects and low therapeutic efficiency. Herein, a thermal-modulated reactive oxygen species (ROS) strategy using activatable human serum albumin-chlorin e6 nanoassemblies (HSA-Ce6 NAs) for promoting PDT against cancer is developed. Through intermolecular disulfide bond crosslinking and hydrophobic interaction, Ce6 photosensitizer is effectively loaded into the HSA NAs, and the obtained HSA-Ce6 NAs exhibit excellent reduction response, as well as enhanced tumor accumulation and retention. By the precision control of the overall body temperature instead of local tumor temperature increasing from 37 °C to 43 °C, the photosensitization reaction rate of HSA-Ce6 NAs increases 20%, and the oxygen saturation of tumor tissue raise 52%, significantly enhancing the generation of ROS for promoting PDT. Meanwhile, the intrinsic fluorescence and photoacoustic properties, and the chelating characteristic of porphyrin ring can endow the HSA-Ce6 NAs with fluorescence, photoacoustic and magnetic resonance triple-modal imaging functions. Upon irradiation of low-energy near-infrared laser, the tumors are completely suppressed without tumor recurrence and therapy-induced side effects. The robust thermal-modulated ROS strategy combined with albumin-based activatable nanophotosensitizer is highly potential for multi-modal imaging-guided PDT and clinical translation.
Haeckel, Akvile; Appler, Franziska; Ariza de Schellenberger, Angela; Schellenberger, Eyk
Increased effectiveness and reduced side effects are general goals in drug research, especially important in cancer therapy. The aim of this study was to design a long-circulating, activatable cytostatic drug that is completely producible in E. coli. Crucial for this goal was the novel unstructured polypeptide XTEN, which acts like polyethylene glycol (PEG) but has many important advantages. Most importantly, it can be produced in E. coli, is less immunogenic, and is biodegradable. We tested constructs containing a fragment of Killin as cytostatic/cytotoxic element, a cell-penetrating peptide, an MMP-2 cleavage site for specific activation, and XTEN for long blood circulation and deactivation of Killin. One of three sequence variants was efficiently expressed in E. coli. As typical for XTEN, it allowed efficient purification of the E. coli lysate by a heat step (10 min 75°C) and subsequent anion exchange chromatography using XTEN as purification tag. After 24 h XTEN-Killin reduced the number of viable cells of HT-1080 tumor cell line to 3.8 ±2.0% (p<0.001) compared to untreated controls. In contrast, liver derived non-tumor cells (BRL3A) did not show significant changes in viability. Our results demonstrate the feasibility of completely producing a complex protease-activatable, potentially long-circulating cytostatic/cytotoxic prodrug in E. coli—a concept that could lead to efficient production of highly multifunctional drugs in the future. PMID:27295081
Morisaki, Akimasa; Nakahira, Atsushi; Sasaki, Yasuyuki; Hirai, Hidekazu; Okada, Yuko; Suehiro, Shigefumi; Shibata, Toshihiko
OBJECTIVES Guidelines recommend the avoidance of direct return of pericardial blood based on evidence from coronary surgery. A continuous auto-transfusion system (CATS) can be a good alternative to cardiotomy suction by reinfusing aspirated pericardial blood without the necessity of intermittent collection. To clarify the effects of direct return of pericardial blood in aortic valve replacement (AVR), we compared the effects of cardiotomy suction and an alternative CATS on perioperative coagulofibrinolysis and inflammation systems, and clinical outcomes. METHODS In 40 AVR operations between April 2009 and April 2011, the retransfusion method of pericardial blood during cardiopulmonary bypass (CPB) was allocated to the use of cardiotomy suction (non-Cell-Saver group, n = 20) or CATS (Cell-Saver group, n = 20) under identical protocols of anticoagulation and transfusion. The blood from the left ventricular vent was returned to the venous reservoir. We obtained blood samples at nine points up to the morning after surgery. RESULTS Perioperative values for coagulofibrinolysis markers, such as thrombin–antithrombin III complex, fibrinogen degeneration products, D-dimer and plasmin-α2 plasmin inhibitor complex, were significantly lower in the Cell-Saver group than those in the non-Cell-Saver group from 1 h after the initiation of cardiopulmonary bypass to 3 or 6 h after termination of cardiopulmonary bypass (P < 0.05 for all markers). A fibrinolysis inhibition marker of plasminogen activator inhibitor-1 and the inflammation markers of interleukin-6, 8 and 10 as well as tumour necrosis factor-α were not significantly different. The amount of packed red blood cells required after the termination of CPB was significantly less in the Cell-Saver group compared with that in the non-Cell-Saver group (P = 0.004). There were no significant differences in the other clinical outcomes between the two groups. CONCLUSIONS In AVR, the avoidance of direct return of pericardial blood
Ramipril and Losartan Exert a Similar Long-Term Effect upon Markers of Heart Failure, Endogenous Fibrinolysis, and Platelet Aggregation in Survivors of ST-Elevation Myocardial Infarction: A Single Centre Randomized Trial.
Marinšek, Martin; Sinkovič, Andreja
Blocking the renin-angiotensin-aldosterone system in ST-elevation myocardial infarction (STEMI) patients prevents heart failure and recurrent thrombosis. Our aim was to compare the effects of ramipril and losartan upon the markers of heart failure, endogenous fibrinolysis, and platelet aggregation in STEMI patients over the long term. After primary percutaneous coronary intervention (PPCI), 28 STEMI patients were randomly assigned ramipril and 27 losartan, receiving therapy for six months with dual antiplatelet therapy (DAPT). We measured N-terminal proBNP (NT-proBNP), ejection fraction (EF), plasminogen-activator-inhibitor type 1 (PAI-1), and platelet aggregation by closure times (CT) at the baseline and after six months. Baseline NT-proBNP ≥ 200 pmol/mL was observed in 48.1% of the patients, EF < 55% in 49.1%, and PAI-1 ≥ 3.5 U/mL in 32.7%. Six-month treatment with ramipril or losartan resulted in a similar effect upon PAI-1, NT-proBNP, EF, and CT levels in survivors of STEMI, but in comparison to control group, receiving DAPT alone, ramipril or losartan treatment with DAPT significantly increased mean CT (226.7 ± 80.3 sec versus 158.1 ± 80.3 sec, p < 0.05). Ramipril and losartan exert a similar effect upon markers of heart failure and endogenous fibrinolysis, and, with DAPT, a more efficient antiplatelet effect in long term than DAPT alone.
Husson, Steven J.; Liewald, Jana F.; Schultheis, Christian; Stirman, Jeffrey N.; Lu, Hang; Gottschalk, Alexander
Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH. PMID:22815873
Payelle, G; Maiza, D; Coffin, O; Alachkar, F; Alweis, S; Courtheoux, P; Khayat, M C; Gérard, J L; Théron, J
Between March 1987 and March 1993 we used pulsed transthrombotic fibrinolysis to treat 58 symptomatic thrombotic occlusions of lower limb bypass grafts in 45 patients. There were 17 suprainguinal grafts and 28 infrainguinal grafts. Treatment consisted of pulsed infusion of fibrinolytic agents into the thrombus followed by continuous infusion using an electric pump. Minor percutaneous or surgical procedures were often associated. The mean delay to treatment was 7 days. The mean duration of treatment was 150 +/- 66 minutes. Immediate patency was achieved in 88% of cases with no significant difference between suprainguinal and infrainguinal grafts. The clinical success rate was 55%. Actuarial patency at 1 year was 54% +/- 11% for suprainguinal grafts and 26% +/- 7% for infrainguinal grafts. The probability of patency was much lower in patients whose grafts had been implanted within 3 months before occlusion and in patients in whom an adjuvant procedure had not been performed. This study demonstrates that, in cases not requiring immediate surgery, pulsed transthrombotic fibrinolysis can achieve durable patency by treating both the bypass and distal arterial network. This technique allows identification of lesions causing thrombosis and adaptation of treatment specifically to these lesions.
Chaikeeratisak, Vorrapon; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee
Proteomic analysis of the hemocytic proteins of Penaeus monodon (Pm) has previously shown that alpha-2-macroglobulin (A2M) was among the proteins that showed substantially altered expression levels upon Vibrio harveyi infection. Therefore, in this study its potentially important role in the response of shrimp to bacterial infection was further characterized. The yeast two-hybrid system revealed that the receptor binding domain of PmA2M interacted with the carboxyl-terminus of one or both of the transglutaminase type II isoforms, which are key enzymes involved in the shrimp clotting system. In accord with this, PmA2M was found to be localized on the extracellular blood clots and to colocalize with clottable proteins. RNA interference (RNAi)-mediated knockdown of A2M transcript levels reduced the PmA2M transcript levels (∼94%) and significantly reduced the bacterial seizing ability of the clotting system, resulting in an up to 3.3-fold higher number of V. harveyi that systemically disseminated into the circulatory system at 5 min post-infection before subsequent clearance by the immune system. Furthermore, an appearance of PmA2M depleted clots in the presence of V. harveyi strikingly demonstrated fibrinolysis zones surrounding the bacteria. This study provides the first evidence of the vital role of PmA2M in enhancing bacterial sequestration by protecting blood clots against fibrinolysis. PMID:23082160
Jacovina, Andrew T.; Deora, Arunkumar B.; Ling, Qi; Broekman, M. Johan; Almeida, Dena; Greenberg, Caroline B.; Marcus, Aaron J.; Smith, Jonathan D.; Hajjar, Katherine A.
When plasma levels of homocysteine (HC), a thiol amino acid formed upon methionine demethylation, exceed 12 μM, individuals are at increased risk of developing large vessel atherothrombosis and small vessel dysfunction. The annexin A2 complex (termed “A2”) is the cell surface coreceptor for plasminogen and TPA and accelerates the catalytic activation of plasmin, the major fibrinolytic agent in mammals. We previously showed that HC prevents A2-mediated, TPA-dependent activation of plasminogen in vitro by disulfide derivatization of the “tail” domain of A2. We also demonstrated that fibrinolysis and angiogenesis are severely impaired in A2-deficient mice. We now report here that, although hyperhomocysteinemic mice had a normal coagulation profile and normal platelet function, fibrin accumulated in their tissues due to reduced perivascular fibrinolytic activity and angiogenesis was impaired. A2 isolated from hyperhomocysteinemic mice failed to fully support TPA-dependent plasmin activation. However, infusion of hyperhomocysteinemic mice with fresh recombinant A2, which localized to neoangiogenic endothelial cells, resulted in normalization of angiogenesis and disappearance of peri- and intravascular fibrin. We therefore conclude that hyperhomocysteinemia impairs postnatal angiogenesis by derivatizing A2, preventing perivascular fibrinolysis, and inhibiting directed endothelial cell migration. These findings provide a mechanistic explanation for microvascular dysfunction and macrovascular occlusion in individuals with hyperhomocysteinemia. PMID:19841537
Verma, S K; Jain, Vartika; Katewa, S S
Elettaria cardamomum (L.) Maton. (Small cardamom) fruit powder was evaluated for its antihypertensive potential and its effect on some of the cardiovascular risk factors in individuals with stage 1 hypertension. Twenty, newly diagnosed individuals with primary hypertension of stage 1 were administered 3 g of cardamom powder in two divided doses for 12 weeks. Blood pressure was recorded initially and at 4 weeks interval for 3 months. Blood samples were also collected initially and at 4 weeks interval for estimation of lipid profile, fibrinogen and fibrinolysis. Total antioxidant status, however, was assessed initially and at the end of the study. Administration of 3 g cardamom powder significantly (p<0.001) decreased systolic, diastolic and mean blood pressure and significantly (p<0.05) increased fibrinolytic activity at the end of 12th week. Total antioxidant status was also significantly (p<0.05) increased by 90% at the end of 3 months. However, fibrinogen and lipid levels were not significantly altered. All study subjects experienced a feeling of well being without any side-effects. Thus, the present study demonstrates that small cardamom effectively reduces blood pressure, enhances fibrinolysis and improves antioxidant status, without significantly altering blood lipids and fibrinogen levels in stage 1 hypertensive individuals.
Kaye, Sarrah; Johnson, Shawn; Rios, Carlos; Fletcher, Daniel J
Prepatent Otostrongylus arteritis results in hemorrhagic diathesis in free-ranging Northern elephant seals (Mirounga angustirostris) attributed to aberrant larval migration of the lungworm, Otostrongylus circumlitus. Clinical signs are often nonspecific, including lethargy, anorexia, and blepharospasm, but can progress to spontaneous frank hemorrhage and death within 72 hours of onset. Previously published case reports describe coagulopathy with prolonged PT and APTT, normal to elevated platelet counts, normal antithrombin concentrations, and low concentrations of fibrinogen degradation products. Disseminated intravascular coagulation was proposed as the cause of hemorrhage, but is inconsistent with some of the reported clinicopathologic changes. The purpose of this study was to compare plasmatic coagulation and fibrinolysis in healthy and Otostrongylus-affected elephant seals, in order to identify potential therapy. We hypothesized that hyperfibrinolysis contributed to hemorrhage in these cases. Citrated plasma samples were collected from 3- to 4-month-old Northern elephant seals in a wildlife rehabilitation hospital. The sampled population included 25 healthy, prerelease seals and 32 clinically ill seals diagnosed with presumptive Otostrongylus arteritis. Twenty-one of the included seals had Otostrongylus infestation confirmed at necropsy. Standard coagulation tests and plasma thromboelastography were performed for a complete assessment of coagulation and fibrinolysis. Northern elephant seals with definitive Otostrongylus infestation were hypocoagulable and hypofibrinolytic compared to healthy controls. Results were most consistent with disseminated intravascular coagulation. Treatment with antifibrinolytic drugs to control hemorrhage may be unrewarding; alternative therapies such as plasma transfusions or coagulation factor concentrates should be investigated. © 2017 American Society for Veterinary Clinical Pathology.
Lopes, João Madeira; Victorino, Rui M. M.; Meneses Santos, João
Disseminated intravascular coagulation (DIC) is the most frequent coagulation disorder associated with metastatic prostate adenocarcinoma. However, DIC with enhanced fibrinolysis as an initial presentation of prostate cancer is extremely rare. The appropriate treatment to control bleeding in these situations is challenging, controversial, and based on isolated case reports in the literature. A 66-year-old male presented at the emergency department with acute severe spontaneous ecchymoses localized to the limbs, laterocervical hematoma, and hemothorax. Prostate specific antigen level was 385 μg/L, bone scintigraphy revealed multiple bone metastases, and prostate biopsy confirmed adenocarcinoma (Gleason 9; 4 + 5). Laboratory investigation showed a pattern of enhanced fibrinolysis rather than the more common intravascular coagulation mechanism. Epsilon aminocaproic acid in monotherapy was initiated with a clear and rapid control of bleeding manifestations. This rare case of massive bleeding due to DIC with enhanced fibrinolysis as the first manifestation of prostate cancer suggests that in selected cases where the acute bleeding dyscrasia is clearly associated with a dominant fibrinolysis mechanism it is possible to use an approach of monotherapy with antifibrinolytics. PMID:27803823
Leeper, Christine M; Neal, Matthew D; McKenna, Christine J; Gaines, Barbara A
To trend fibrinolysis after injury and determine the influence of traumatic brain injury (TBI) and massive transfusion on fibrinolysis status. Admission fibrinolytic derangement is common in injured children and adults, and is associated with poor outcome. No studies examine fibrinolysis days after injury. Prospective study of severely injured children at a level 1 pediatric trauma center. Rapid thromboelastography was obtained on admission and daily for up to 7 days. Standard definitions of hyperfibrinolysis (HF; LY30 ≥3), fibrinolysis shutdown (SD; LY30 ≤0.8), and normal (LY30 = 0.9-2.9) were applied. Antifibrinolytic use was documented. Outcomes were death, disability, and thromboembolic complications. Wilcoxon rank-sum and Fisher exact tests were performed. Exploratory subgroups included massively transfused and severe TBI patients. In all, 83 patients were analyzed with median (interquartile ranges) age 8 (4-12) and Injury Severity Score 22 (13-34), 73.5% blunt mechanism, 47% severe TBI, 20.5% massively transfused. Outcomes were 14.5% mortality, 43.7% disability, and 9.8% deep vein thrombosis. Remaining in or trending to SD was associated with death (P = 0.007), disability (P = 0.012), and deep vein thrombosis (P = 0.048). Median LY30 was lower on post-trauma day (PTD)1 to PTD4 in patients with poor compared with good outcome; median LY30 was lower on PTD1 to PTD3 in TBI patients compared with non-TBI patients. HF without associated shutdown was not related to poor outcome, but extreme HF (LY30 >30%, n = 3) was lethal. Also, 50% of massively transfused patients in hemorrhagic shock demonstrated SD physiology on admission. All with HF (fc31.2%) corrected after hemostatic resuscitation without tranexamic acid. Fibrinolysis shutdown is common postinjury and predicts poor outcomes. Severe TBI is associated with sustained shutdown. Empiric antifibrinolytics for children should be questioned; thromboelastography-directed selective use should be considered for
Chapman, Michael P; Moore, Ernest E; Ramos, Christopher R; Ghasabyan, Arsen; Harr, Jeffrey N; Chin, Theresa L; Stringham, John R; Sauaia, Angela; Silliman, Christopher C; Banerjee, Anirban
The acute coagulopathy of trauma is present in up to one third of patients by the time of admission, and the recent CRASH-2 and MATTERs trials have focused worldwide attention on hyperfibrinolysis as a component of acute coagulopathy of trauma. Thromboelastography (TEG) is a powerful tool for analyzing fibrinolyis, but a clinically relevant threshold for defining hyperfibrinolysis has yet to be determined. Recent data suggest that the accepted normal upper bound of 7.5% for 30-minute fibrinolysis (LY30) by TEG is inappropriate in severe trauma, as the risk of death rises at much lower levels of clot lysis. We wished to determine the validity of this hypothesis and establish a threshold value to treat fibrinolysis, based on prediction of massive transfusion requirement and risk of mortality. Patients with uncontrolled hemorrhage, meeting the massive transfusion protocol (MTP) criteria at admission (n = 73), represent the most severely injured trauma population at our center (median Injury Severity Score [ISS], 30; interquartile range, 20-38). Citrated kaolin TEG was performed at admission blood samples from this population, stratified by LY30, and evaluated for transfusion requirement and 28-day mortality. The same analysis was conducted on available field blood samples from all non-MTP trauma patients (n = 216) in the same period. These represent the general trauma population. Within the MTP-activating population, the cohort of patients with LY30 of 3% or greater was shown to be at much higher risk for requiring a massive transfusion (90.9% vs. 30.5%, p = 0.0008) and dying of hemorrhage (45.5% vs. 4.8%, p = 0.0014) than those with LY30 less than 3%. Similar trends were seen in the general trauma population. LY30 of 3% or greater defines clinically relevant hyperfibrinolysis and strongly predicts the requirement for massive transfusion and an increased risk of mortality in trauma patients presenting with uncontrolled hemorrhage. This threshold value for LY30
Chen, Zhixing; Wei, Lu; Zhu, Xinxin; Min, Wei
It is highly desirable to be able to optically probe biological activities deep inside live organisms. By employing a spatially confined excitation via a nonlinear transition, multiphoton fluorescence microscopy has become indispensable for imaging scattering samples. However, as the incident laser power drops exponentially with imaging depth due to scattering loss, the out-of-focus fluorescence eventually overwhelms the in-focal signal. The resulting loss of imaging contrast defines a fundamental imaging-depth limit, which cannot be overcome by increasing excitation intensity. Herein we propose to significantly extend this depth limit by multiphoton activation and imaging (MPAI) of photo-activatable fluorophores. The imaging contrast is drastically improved due to the created disparity of bright-dark quantum states in space. We demonstrate this new principle by both analytical theory and experiments on tissue phantoms labeled with synthetic caged fluorescein dye or genetically encodable photoactivatable GFP.
Park, Dongjin; Ahn, Kyung-Ohk; Jeong, Kyung-Chae; Choi, Yongdoo
Here, we fabricated polypyrrole nanoparticles (PPys) (termed HA10-PPy, HA20-PPy, and HA40-PPy) doped with different average molecular weight hyaluronic acids (HAs) (10, 20, and 40 kDa, respectively), and evaluated the effect of molecular weight of doped HA on photothermal induction, fluorescence quenching, and drug loading efficiencies. Doxorubicin-loaded HA-doped PPys (DOX@HA-PPys) could be used for imaging and therapy of triple-negative breast cancer (TNBC). Fluorescence turn-on, stimuli-responsive drug release, and photo-induced heating of DOX@HA-PPys enabled not only activatable fluorescence imaging but also subsequent chemo/photothermal dual therapy for TNBC. In particular, we illustrated the potential usefulness of the photothermal effect of the nanoparticles for overcoming chemoresistance in TNBC.
Conway de Macario, E; Ellis, J; Guzman, R; Rotman, B
Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 22.214.171.124), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,
de Macario, E C; Ellis, J; Guzman, R; Rotman, B
Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 126.96.36.199), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody, PMID:416439
Hirasawa, Takeshi; Okawa, Shinpei; Iwatate, Ryu J.; Kamiya, Mako; Urano, Yasuteru; Ishihara, Miya
Multispectral photoacoustic (MS-PA) imaging has been researched to image molecular probes in the presence of strong background signals produced from intrinsic optical absorbers. Spectral fitting method (SFM) discriminates probe signals from background signals by fitting the PA spectra that are calculated from MS-PA images to reference spectra of the probe and background, respectively. Because hemoglobin is a dominant optical absorber in visible to near-infrared wavelength range, absorption spectra of hemoglobin have been widely used as reference background spectra. However, the spectra of background signals produced from heterogeneous biological tissue differ from the reference background spectra due to presence of other intrinsic optical absorbers and effect of optical scattering. Due to the difference, the background signals partly remain in the probe images. To image the probe injected in subcutaneous tumors of mice clearly, we added the melanosome absorption spectrum to the reference background spectra because skin contains nonnegligible concentration of melanosome and the spectrum is very similar to the scattering spectrum of biological tissue. The probe injected in the subcutaneous tumor of mice was an enzyme-activatable probe which show their original colors only in the presence of γ-glutamyltranspeptidase, an enzyme associated with cancer. The probes have been successfully used for rapid fluorescence imaging of cancer. As a result of MS-PA imaging, by considering the melanosome absorption spectrum, the background signals were successfully suppressed and then clearer probe image was obtained. Our MS-PA imaging method afforded successful imaging of tumors in mice injected with activatable PA probes.
Chen, Mei-Chin; Lin, Zhi-Wei; Ling, Ming-Hung
Because of the aggressive and recurrent nature of cancers, repeated and multimodal treatments are often necessary. Traditional cancer therapies have a risk of serious toxicity and side effects. Hence, it is crucial to develop an alternative treatment modality that is minimally invasive, effectively treats cancers with low toxicity, and can be repeated as required. We developed a light-activatable microneedle (MN) system that can repeatedly and simultaneously provide photothermal therapy and chemotherapy to superficial tumors and exert synergistic anticancer effects. This system consists of embeddable polycaprolactone MNs containing a photosensitive nanomaterial (lanthanum hexaboride) and an anticancer drug (doxorubicin; DOX), and a dissolvable poly(vinyl alcohol)/polyvinylpyrrolidone supporting array patch. Because of this supporting array, the MNs can be completely inserted into the skin and embedded within the target tissue for locoregional cancer treatment. When exposed to near-infrared light, the embedded MN array uniformly heats the target tissue to induce a large thermal ablation area and then melts at 50 °C to release DOX in a broad area, thus destroying tumors. This light-activated heating and releasing behavior can be precisely controlled and switched on and off on demand for several cycles. We demonstrated that the MN-mediated synergistic therapy completely eradicated 4T1 tumors within 1 week after a single application of the MN and three cycles of laser treatment. No tumor recurrence and no significant body weight loss of mice were observed. Thus, the developed light-activatable MN with a unique embeddable feature offers an effective, user-friendly, and low-toxicity option for patients requiring long-term and multiple cancer treatments.
Chen, L B; Buchanan, J M
The fibrinolytic activity of proteases secreted by chick embryo fibroblasts infected with Rous sarcoma virus was studied by use of a procedure in which a fibrin clot was formed with highly purified fibrinogen and thrombin above the cell layer. This procedure results in the formation of fibrin that is apparently a more suitable substrate for studies on fibrinolysis than is fibrin prepared by other methods. Since neither plasminogen nor serum were included in the assay system in the present studies, the fibrinolytic activity observed cannot be ascribed to the conversion of the plasminogen in serum to plasmin by a plasminogen activator produced by transformed cells. Our procedure, therefore, measures proteolytic activities other than those reported by previous investigators. Maintenance of some of the transformed phenotypes of Rous sarcoma virus transformed chick embryo fibroblasts such as morpholigical change and increased rate of glucose uptake apparently does not depend on the presence of plasminogen in the culture medium. Images PMID:165484
Wang, Hong; Hong, Chan E; Lewis, Joshua P; Zhu, Yanbei; Wang, Xing; Chu, Xin; Backman, Joshua; Hu, Ziying; Yang, Peixing; Still, Christopher D; Gerhard, Glenn S; Fu, Mao
Two genetic variants (rs3798220 and rs10455872) in the apolipoprotein (a) gene (LPA) have been implicated in cardiovascular disease (CVD), presumably through their association with Lipoprotein (a) (Lp(a)) levels. While Lp(a) is recognized as a lipoprotein with atherogenic and thrombogenic characteristics, it is unclear whether or not the two Lp(a)-associated genetic variants are also associated with markers of thrombosis (i.e. plasminogen levels and fibrinolysis). In the present study, we genotyped the two genetic variants in 2919 subjects of the Old Order Amish (OOA) and recruited 146 subjects according the carrier and non-carrier status for rs3798220 and rs10455872, and also matched for gender and age. We measured plasma Lp(a) and plasminogen levels in these subjects, and found that the concentrations of plasma Lp(a) were 2.62 and 1.73 fold higher in minor allele carriers of rs3798220 and rs10455872, respectively, compared with non-carriers (P = 2.04 × 10(-17) and P = 1.64 × 10(-6), respectively). By contrast, there was no difference in plasminogen concentrations between carriers and non-carriers of rs3798220 and rs10455872. Furthermore, we observed no association between carrier status of rs3798220 or rs10455872 with clot lysis time. Finally, plasminogen mRNA expression in liver samples derived from 76 Caucasian subjects was not significantly different between carriers and non-carriers of these two genetic variants. Our results provide further insight into the mechanism of action behind two genetic variants previously implicated in CVD risk and show that these polymorphisms are not major modulating factors for plasma plasminogen levels and fibrinolysis.
Wang, Hong; Hong, Chan E.; Lewis, Joshua P.; Zhu, Yanbei; Wang, Xing; Chu, Xin; Backman, Joshua; Hu, Ziying; Yang, Peixin; Still, Christopher D.; Gerhard, Glenn S.; Fu, Mao
Two genetic variants (rs3798220 and rs10455872) in the apolipoprotein (a) gene (LPA) have been implicated in cardiovascular disease (CVD), presumably through their association with lipoprotein (a) [Lp(a)] levels. While Lp(a) is recognized as a lipoprotein with atherogenic and thrombogenic characteristics, it is unclear whether or not the two Lp(a)-associated genetic variants are also associated with markers of thrombosis (i.e., plasminogen levels and fibrinolysis). In the present study, we genotyped the two genetic variants in 2919 subjects of the Old Order Amish (OOA) and recruited 146 subjects according to the carrier and noncarrier status for rs3798220 and rs10455872, and also matched for gender and age. We measured plasma Lp(a) and plasminogen levels in these subjects, and found that the concentrations of plasma Lp(a) were 2.62- and 1.73-fold higher in minor allele carriers of rs3798220 and rs10455872, respectively, compared with noncarriers (P = 2.04 × 10−17 and P = 1.64 × 10−6, respectively). By contrast, there was no difference in plasminogen concentrations between carriers and noncarriers of rs3798220 and rs10455872. Furthermore, we observed no association between carrier status of rs3798220 or rs10455872 with clot lysis time. Finally, plasminogen mRNA expression in liver samples derived from 76 Caucasian subjects was not significantly different between carriers and noncarriers of these two genetic variants. Our results provide further insight into the mechanism of action behind two genetic variants previously implicated in CVD risk and show that these polymorphisms are not major modulating factors for plasma plasminogen levels and fibrinolysis. PMID:27605514
Chin, Theresa L; Moore, Ernest E; Moore, Hunter B; Gonzalez, Eduardo; Chapman, Michael P; Stringham, John R; Ramos, Christopher R; Banerjee, Anirban; Sauaia, Angela
The mechanisms driving trauma-induced coagulopathy (TIC) remain to be defined, and its therapy demands an orchestrated replacement of specific blood products. Thrombelastography (TEG) is a tool to guide the TIC multicomponent therapy. Principal component analysis (PCA) is a statistical approach that identifies variable clusters; thus, we hypothesize that PCA can identify specific combinations of TEG-generated values that reflect TIC mechanisms. Adult trauma patients admitted from September 2010 to October 2013 for whom a massive transfusion protocol was activated were included. Rapid TEG values obtained within the first 6 hours after injury were included in the PCA. PCA components with an eigenvalue >1 were retained, and, within components, variable loadings (equivalent to correlation coefficients) >|60| were considered significant. Component scorings for each patient were calculated and clinical characteristics of patients with high and low scores were compared. Of 98 enrolled patients, 67% were male and 70% suffered blunt trauma. Median age was 41 years (interquartile range 28-55) and median Injury Severity Score was 31.5 (interquartile range 24-43). PCA identified three principal components (PCs) that together explained 93% of the overall variance. PC1 reflected global coagulopathy with depletion of platelets and fibrinogen whereas PC3 indicated hyperfibrinolysis. PC2 may represent endogenous anticoagulants such as the activation of protein C. PCA suggests depletion coagulopathy is independent from fibrinolytic coagulopathy. Furthermore, the distribution of mortality suggests that low levels of fibrinolysis may be beneficial in a select group of injured patients. These data underscore the potential of risk for concurrent presumptive treatment for preserved depletion coagulopathy and possible fibrinolysis. Copyright © 2014 Mosby, Inc. All rights reserved.
Lorimier, S; Bouthors, S; Droulle, C; Maquin, D L; Maquart, F X; Gillery, P; Emonard, H; Hornebeck, W
We previously demonstrated that human gingival fibroblasts (HGF), but not their dermal counterparts, when seeded in retracting fibrin lattices induced intense fibrinolysis that was observed at the earliest stages of contraction and led to complete matrix degradation by day 7 of culture. Our aim was to examine the influence of mechanical forces in such fibrinolytic processes. HGF were seeded in retracting (R) e.g. free floating or non retracting (NR) e.g. anchored fibrin lattices (FL). Cultures were analysed from day 1-12 by phase contrast microscopy and scanning electron microscopy (s.e.m.). Levels of fibrin degradation products (FDP) and tissue plasminogen activator (tPA) accumulating in culture media were quantified by ELISA. Urokinase (uPA) and gelatinase A (MMP2) were identified by zymographic techniques. At the s.e.m. level, vacuolization around some HGF was noticed at the earliest stages of culture for RFL and complete degradation of lattices occurred at day 7. Formation of lysed matrix cavity was far less intense in NRFL even after 12 days of culture. FDP amounts at day 4 of culture were equal to 79 +/- 14 and 8.5 +/- 0.6 micrograms/10(5) cells for RFL and NRFL, respectively; tPA levels were equal to 5.8 +/- 0.6 (RFL) and 2.1 +/- 0.3 ng/10(5) cells (NRFL) and differences were still evident at day 7. The kinetics of tPA production were identical in either retracting fibrin or collagen lattices. On the contrary, uPA and proMMP2 productions were similar in RFL and NRFL. Isometric forces, but not the matrix support, were responsible for accelerated tPA production and fibrinolysis in HGF populated lattices.
Shenkman, Boris; Budnik, Ivan; Einav, Yulia; Hauschner, Hagit; Andrejchin, Mykhaylo; Martinowitz, Uriel
Trauma-induced coagulopathy (TIC) is commonly seen among patients with severe injury. The dynamic process of TIC is characterized by variability of the features of the disease. A model of TIC was created. Hemodilution was produced by mixing the blood with 40% Tris/saline solution, fibrinolysis by treating the blood with 160 ng/mL tPA, acidosis by adding 1.2 mg/mL lactic acid achieving pH 7.0 to 7.1, and hypothermia by running the assay at 31°C. Intact blood tested at 37°C served as control. Clot formation was evaluated using rotation thromboelastometry. Platelet adhesion and aggregation were assayed at a shear rate of 1800 s using Impact-R device. Clotting time was not affected by any of the TIC constituents used. Clotting initiation was reduced by hemodilution and further reduced by additive hypothermia. The propagation phase of blood clotting was reduced by hemodilution, further reduced by additive hypothermia, and maximally reduced if additionally combined with fibrinolysis. No effect of fibrinolysis on clot propagation was observed at 37°C. Maximum clot firmness was reduced by hemodilution, further reduced by additive fibrinolysis, and maximally reduced if additionally combined with hypothermia. No effect of hypothermia on clot strength was observed in the absence of fibrinolysis. Platelet adhesion (percentage of surface coverage) and aggregation (aggregate size) under flow condition were reduced by hemodilution and further reduced by additive acidosis. Introduction of tPA to diluted blood had no effect on platelet function. The study revealed a differential effect of TIC constituents-hemodilution, hypothermia, fibrinolysis, and acidosis-on clot formation and platelet function. The effect of one factor may influence that of another factor. These data may be helpful to better understand the pathogenesis of TIC and to elaborate an individually tailored treatment strategy. A new model of TIC is created. Contribution of various constituents to pathogenesis of
An, Peng; Yu, Zhipeng; Lin, Qing
The design and synthesis of a new class of laser light activatable tetrazoles with extended π-systems is reported. Upon 405 nm laser light irradiation, these bithiophene-substituted tetrazoles underwent extremely fast 1,3-dipolar cycloaddition reactions with dimethyl fumarate with second-order rate constants approaching 4000 M(-1) s(-1). The resulting pyrazoline cycloadducts exhibited solvent-dependent red fluorescence, making these tetrazoles potentially useful as fluorogenic probes for detecting alkenes in vivo.
Miao, Qingqing; Lyu, Yan; Ding, Dan; Pu, Kanyi
Despite the great potential of photoacoustic imaging in the life sciences, the development of smart activatable photoacoustic probes remains elusive. On page 3662, K. Pu and co-workers report a facile nanoengineering approach based on semiconducting oligomer nano-particles to develop ratiometric photoacoustic probes with amplified brightness and enhanced sensing capability for accurate photoacoustic mapping of pH in the tumors of living mice.
González-Miguel, Javier; Morchón, Rodrigo; Siles-Lucas, Mar; Simón, Fernando
The interaction between blood-borne pathogens and fibrinolysis is one of the most important mechanisms that mediate invasion and the establishment of infectious agents in their hosts. However, overproduction of plasmin (final product of the route) has been related in other contexts to proliferation and migration of the arterial wall cells and degradation of the extracellular matrix. We have recently identified fibrinolysis-activating antigens from Dirofilaria immitis, a blood-borne parasite whose key pathological event (proliferative endarteritis) is produced by similar mechanisms to those indicated above. The objective of this work is to study how two of this antigens [actin (ACT) and fructose-bisphosphate aldolase (FBAL)] highly conserved in pathogens, activate fibrinolysis and to establish a relationship between this activation and the development of proliferative endarteritis during cardiopulmonary dirofilariasis. We demonstrate that both proteins bind plasminogen, enhance plasmin generation, stimulate the expression of the fibrinolytic activators tPA and uPA in endothelial cell cultures and are located on the surface of the worm in contact with the host’s blood. ELISA, western blot and immunofluorescence techniques were employed for this purpose. Additionally, the implication of lysine residues in this interaction was analyzed by bioinformatics. The involvement of plasmin generated by the ACT/FBAL and plasminogen binding in cell proliferation and migration, and degradation of the extracellular matrix were shown in an “in vitro” model of endothelial and smooth muscle cells in culture. The obtained results indicate that ACT and FBAL from D. immitis activate fibrinolysis, which could be used by the parasite like a survival mechanism to avoid the clot formation. However, long-term overproduction of plasmin can trigger pathological events similar to those described in the emergence of proliferative endarteritis. Due to the high degree of evolutionary
Menzel, Kathleen; Hilberg, Thomas
Increased age is associated with a higher risk of thrombotic events. The aim of this study was to investigate the age-related changes in hemostasis before and after moderate exercise controlled by individual anaerobic threshold as recommended for rehabilitation training. In this study, 24 young (25 +/- 1 years) and 24 middle-aged healthy nonsmokers (48 +/- 1 years) underwent an individualized exercise test with 80% of individual anaerobic threshold (young individuals: 127 +/- 6 W; middle-aged individuals: 128 +/- 5 W; values are expressed as mean +/- standard error of mean) for 60 minutes. The blood samples were collected before and after the exercise. The age-related higher (P < or = .05) levels could be detected in factors II, VII, VIII, IX, XI, XII, prothrombin fragment 1+2, in tissue plasminogen activator antigen and activity, as well as in plasminogen. The relative exercise-induced increases in these parameters were similar in both groups, although beginning at a higher level for those in the middle-aged group.A statistically enhanced increase after exercise in the middle-aged group could be shown in prothrombin fragment 1+2 (young individuals: 98 +/- 6 to 102 +/- 6 pmol/L; middle-aged individuals: 138 +/- 7 to 156 +/- 8 pmol/L) and in thrombin-antithrombin complex (young individuals: 2.2 +/- 0.1 to 3.1 +/- 0.2 microg/L; middle-aged individuals: 2.4 +/- 0.3 to 3.9 +/- 0.6 microg/L); the latter only showing a tendency. The data show the age-related changes with a rise in blood coagulation and fibrinolysis in a healthy middle-aged group compared with younger participants. Moderate exercise leads to comparably relative increases in hemostatic parameters but starting at higher levels. However, the exercise-induced thrombin generation (prothrombin fragment 1+2) is enhanced in the middle-aged participants in comparison with younger participants, but may be compensated by a sufficient fibrinolysis, and therefore the hemostatic system remains in balance.
Laine, Outi K; Koskela, Sirpa M; Outinen, Tuula K; Joutsi-Korhonen, Lotta; Huhtala, Heini; Vaheri, Antti; Hurme, Mikko A; Jylhävä, Juulia; Mäkelä, Satu M; Mustonen, Jukka T
Thrombocytopenia and altered coagulation characterize all hantavirus infections. To further assess the newly discovered predictive biomarkers of disease severity during acute Puumala virus (PUUV) infection, we studied the associations between them and the variables reflecting coagulation, fibrinolysis and endothelial activation. Nineteen hospital-treated patients with serologically confirmed acute PUUV infection were included. Acutely, plasma levels of pentraxin-3 (PTX3), cell-free DNA (cf-DNA), complement components SC5b-9 and C3 and interleukin-6 (IL-6) were recorded as well as platelet ligands and markers of coagulation and fibrinolysis. High values of plasma PTX3 associated with thrombin formation (prothrombin fragments F1+2; r = 0.46, P = 0.05), consumption of platelet ligand fibrinogen (r = -0.70, P < 0.001) and natural anticoagulants antithrombin (AT) (r = -0.74, P < 0.001), protein C (r = -0.77, P < 0.001) and protein S free antigen (r = -0.81, P < 0.001) and a decreased endothelial marker ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 domain 13) (r = -0.48, P = 0.04). Plasma level of AT associated with C3 (r = 0.76, P < 0.001), IL-6 (r = -0.56, P = 0.01) and cf-DNA (r = -0.47, P = 0.04). High cf-DNA coincided with increased prothrombin fragments F1+2 (r = 0.47, P = 0.04). Low C3 levels reflecting the activation of complement system through the alternative route predicted loss of all natural anticoagulants (for protein C r = 0.53, P = 0.03 and for protein S free antigen r = 0.64, P = 0.004). Variables depicting altered coagulation follow the new predictive biomarkers of disease severity, especially PTX3, in acute PUUV infection. The findings are consistent with the previous observations of these biomarkers also being predictive for low platelet count and underline the cross-talk of inflammation and coagulation systems in acute PUUV infection.
Ramipril and Losartan Exert a Similar Long-Term Effect upon Markers of Heart Failure, Endogenous Fibrinolysis, and Platelet Aggregation in Survivors of ST-Elevation Myocardial Infarction: A Single Centre Randomized Trial
Marinšek, Martin; Sinkovič, Andreja
Introduction. Blocking the renin-angiotensin-aldosterone system in ST-elevation myocardial infarction (STEMI) patients prevents heart failure and recurrent thrombosis. Our aim was to compare the effects of ramipril and losartan upon the markers of heart failure, endogenous fibrinolysis, and platelet aggregation in STEMI patients over the long term. Methods. After primary percutaneous coronary intervention (PPCI), 28 STEMI patients were randomly assigned ramipril and 27 losartan, receiving therapy for six months with dual antiplatelet therapy (DAPT). We measured N-terminal proBNP (NT-proBNP), ejection fraction (EF), plasminogen-activator-inhibitor type 1 (PAI-1), and platelet aggregation by closure times (CT) at the baseline and after six months. Results. Baseline NT-proBNP ≥ 200 pmol/mL was observed in 48.1% of the patients, EF < 55% in 49.1%, and PAI-1 ≥ 3.5 U/mL in 32.7%. Six-month treatment with ramipril or losartan resulted in a similar effect upon PAI-1, NT-proBNP, EF, and CT levels in survivors of STEMI, but in comparison to control group, receiving DAPT alone, ramipril or losartan treatment with DAPT significantly increased mean CT (226.7 ± 80.3 sec versus 158.1 ± 80.3 sec, p < 0.05). Conclusions. Ramipril and losartan exert a similar effect upon markers of heart failure and endogenous fibrinolysis, and, with DAPT, a more efficient antiplatelet effect in long term than DAPT alone. PMID:27064499
Reikerås, Olav; Borgen, Pål
Background Traumatic injury induces changes in mediators of inflammation and coagulation, but the pivotal roles of inflammation and coagulation has not been precisely clarified. Therefore we have studied markers of inflammation and coagulation after a standardized musculoskeletal trauma like total hip replacement surgery. Methods We allocated 21 patients aged 50 to 84 years who underwent total hip replacement surgery. Releases of TNF-α, IL-1β, IL-6, IL-8 and IL-10 and protrombin fragment F1.2 and plasmin-antiplasmin complex (PAP) were examined during surgery and up 6 days postoperatively, and systemic releases were compared to pre-operative values. Surgery induced significant increments in serum levels of IL-6 at 6 hours and at 1 day after surgery and in levels of IL-8 at 6 hours after surgery. There were no significant changes in serum levels of TNF-α, IL-1β or IL-10. There were significant increments in blood levels of F1.2 and PAP up to 6 days postoperatively with highest levels at 6 hours after surgery. There were only week correlations between IL-6 and IL-8 and F1.2 and PAP. Conclusion Major musculoskeletal surgery causes changes of the inflammatory, coagulatory and fibrinolytic cascades in stable patients, but with no correlations between inflammation and coagulation and fibrinolysis. PMID:25364904
Boluijt, Jacoline; Meijers, Joost CM; Rinkel, Gabriel JE; Vergouwen, Mervyn DI
Delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been associated with microthrombosis, which can result from activated hemostasis, inhibited fibrinolysis, or both. We systematically searched the PUBMED and EMBASE databases to identify hemostatic or fibrinolytic parameters that can be used for the prediction or diagnosis of DCI, or that inform on the pathogenesis of DCI and may serve as treatment targets. We included 24 studies that fulfilled predefined criteria and described 39 biomarkers. Only one study fulfilled predefined criteria for high quality. Since no parameter on admission was associated with DCI and in none of the included studies blood was drawn at the time of clinical deterioration, none of the studied parameters can presently be used for the prediction or diagnosis of DCI. Regarding the pathogenesis of DCI, it was shown that compared with patients without DCI those with DCI had higher levels of von Willebrand factor and platelet activating factor in plasma 5 to 9 days after aSAH, membrane tissue factor in cerebrospinal fluid 5 to 9 days after aSAH, and D-dimer in plasma 11 to 14 days after aSAH. Confirmation in high-quality studies is needed to investigate whether these parameters can serve as targets for new intervention studies. PMID:25690473
Boluijt, Jacoline; Meijers, Joost C M; Rinkel, Gabriel J E; Vergouwen, Mervyn D I
Delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) has been associated with microthrombosis, which can result from activated hemostasis, inhibited fibrinolysis, or both. We systematically searched the PUBMED and EMBASE databases to identify hemostatic or fibrinolytic parameters that can be used for the prediction or diagnosis of DCI, or that inform on the pathogenesis of DCI and may serve as treatment targets. We included 24 studies that fulfilled predefined criteria and described 39 biomarkers. Only one study fulfilled predefined criteria for high quality. Since no parameter on admission was associated with DCI and in none of the included studies blood was drawn at the time of clinical deterioration, none of the studied parameters can presently be used for the prediction or diagnosis of DCI. Regarding the pathogenesis of DCI, it was shown that compared with patients without DCI those with DCI had higher levels of von Willebrand factor and platelet activating factor in plasma 5 to 9 days after aSAH, membrane tissue factor in cerebrospinal fluid 5 to 9 days after aSAH, and D-dimer in plasma 11 to 14 days after aSAH. Confirmation in high-quality studies is needed to investigate whether these parameters can serve as targets for new intervention studies.
Yuasa, Masato; Mignemi, Nicholas A.; Nyman, Jeffry S.; Duvall, Craig L.; Schwartz, Herbert S.; Okawa, Atsushi; Yoshii, Toshitaka; Bhattacharjee, Gourab; Zhao, Chenguang; Bible, Jesse E.; Obremskey, William T.; Flick, Matthew J.; Degen, Jay L.; Barnett, Joey V.; Cates, Justin M.M.; Schoenecker, Jonathan G.
Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair. PMID:26214526
Sutton, J Mark; Wayne, Jonathan; Scott-Tucker, Anthony; O'Brien, Susan M; Marks, Philip M H; Alexander, Frances C G; Shone, Clifford C; Chaddock, John A
Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.
Zhao, Xu; Yang, Cheng-Xiong; Chen, Li-Gong; Yan, Xiu-Ping
The integrated functions of diagnostics and therapeutics make theranostics great potential for personalized medicine. Stimulus-responsive therapy allows spatial control of therapeutic effect only in the site of interest, and offers promising opportunities for imaging-guided precision therapy. However, the imaging strategies in previous stimulus-responsive therapies are `always on' or irreversible `turn on' modality, resulting in poor signal-to-noise ratios or even `false positive' results. Here we show the design of dual-stimuli-responsive and reversibly activatable nanoprobe for precision tumour-targeting and fluorescence-guided photothermal therapy. We fabricate the nanoprobe from asymmetric cyanine and glycosyl-functionalized gold nanorods (AuNRs) with matrix metalloproteinases (MMPs)-specific peptide as a linker to achieve MMPs/pH synergistic and pH reversible activation. The unique activation and glycosyl targetibility makes the nanoprobe bright only in tumour sites with negligible background, while AuNRs and asymmetric cyanine give synergistic photothermal effect. This work paves the way to designing efficient nanoprobes for precision theranostics.
OROSCO, RYAN K.; SAVARIAR, ELAMPRAKASH N.; WEISSBROD, PHILIP A.; DIAZ-PEREZ, JULIO A.; BOUVET, MICHAEL; TSIEN, ROGER Y.; NGUYEN, QUYEN T.
Background and Objectives Molecularly targeted fluorescent molecules may help detect tumors that are unseen by traditional white-light surgical techniques. We sought to evaluate a fluorescent ratiometric activatable cell penetrating peptide (RACPP) for tumor detection in a transgenic model of PTC. Methods Thirteen BRAFV600E mice with PTC were studied—seven injected intravenously with RACPP, four controls with saline. Total thyroidectomy was performed with microscopic white-light visualization. Fluorescent imaging of post-thyroidectomy fields was performed, and tissue with increased signal was removed and evaluated for PTC. Final samples were analyzed by a pathologist blinded to conditions. Vocal cord function was evaluated postoperatively with video laryngoscopy. Results The average in situ ratiometric (Cy5/Cy7) thyroid tumor-to-background contrast ratio was 2.27 +/−0.91. Fluorescence-guided clean-up following thyroidectomy identified additional tumor in 2 of 7 RACPP animals (smallest dimension 1.2 mm), and decreased the number of animals with residual tumor from 4 to 3. All retained tumor foci on final pathology were smaller than 0.76 mm. Intact vocal abduction was present in all of the RACPP animals. Conclusions RACPPs successfully targeted PTC in a transgenic thyroidectomy model, and allowed for residual tumor detection that reduced positive margins beyond what was possible with white-light surgery alone. PMID:26799257
Hussain, Timon; Savariar, Elamprakash N.; Diaz-Perez, Julio A.; Messer, Karen; Pu, Minya; Tsien, Roger Y.; Nguyen, Quyen T.
Background We evaluated the use of intraoperative fluorescence guidance by enzymatically cleavable ratiometric activatable cell-penetrating peptide (RACPPPLGC(Me)AG) containing Cy5 as a fluorescent donor and Cy7 as a fluorescent acceptor for salivary gland cancer surgery in a mouse model. Methods Surgical resection of small parotid gland cancers in mice was performed with fluorescence guidance or white light (WL) imaging alone. Tumor identification accuracy, operating time and tumor free survival were compared. Results RACPP guidance aided tumor detection (positive histology in 90% (27/30) vs. 48% (15/31) for WL, p<0.001). A ~25% ratiometric signal increase as the threshold to distinguish between tumor and adjacent tissue, yielded >90% detection sensitivity and specificity. Operating time was reduced by 54% (p<0.001), tumor free survival was increased with RACPP guidance (p=0.025). Conclusions RACPP provides real-time intraoperative guidance leading to improved survival. Ratiometric signal thresholds can be set according to desired detection accuracy levels for future RACPP applications. PMID:25521629
Wang, Zhaohui; Luo, Min; Mao, Chengqiong; Wei, Qi; Zhao, Tian; Li, Yang; Huang, Gang; Gao, Jinming
Efficient delivery of biomacromolecules (e.g., proteins, nucleic acids) into cell cytosol remains a critical challenge for the development of macromolecular therapeutics or diagnostics. To date, most common approaches to assess cytosolic delivery rely on fluorescent labeling of macromolecules with an "always on" reporter and subcellular imaging of endolysosomal escape by confocal microscopy. This strategy is limited by poor signal-to-noise ratio and only offers low throughput, qualitative information. Herein we describe a quantitative redox-activatable sensor (qRAS) for the real-time monitoring of cytosolic delivery of macromolecules. qRAS-labeled macromolecules are silent (off) inside the intact endocytic organelles, but can be turned on by redox activation after endolysosomal disruption and delivery into the cytosol, thereby greatly improving the detection accuracy. In addition to confocal microscopy, this quantitative sensing technology allowed for a high-throughput screening of a panel of polymer carriers toward efficient cytosolic delivery of model proteins on a plate reader. The simple and versatile qRAS design offers a useful tool for the investigation of new strategies for endolysosomal escape of biomacromolecules to facilitate the development of macromolecular therapeutics for a variety of disease indications. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Thompson, Stephen Dessi, John; Self, Colin H.
The construction and in vitro testing of a photo-activatable anti-tumour immuno-regulatory antibody is described. In this 'cloaked' folated anti-CD3 antibody conjugate, the folate portion of the conjugate is free to bind to folate receptor expressing cancer cells, whilst the anti-CD3 activity is effectively rendered inert by a coating of photo-labile 2-nitrobenzyl groups. On irradiation with UV-A light the activity of the anti-CD3 antibody is restored, not only when it is required, but more importantly, only where it is required. The conjugate can then attract killer T-cells to the surface of the tumour cells and kill them. Unirradiated normal tissues, to which the conjugate has been targeted by specific and non-specific binding, remain unharmed. We believe that these 'photo-switchable' conjugates could be used to markedly improve the targeting of the immune response to folate receptor (FR) expressing ovarian and breast cancers whilst minimising the side effects in the rest of the body.
Mittermayr, M; Streif, W; Haas, T; Fries, D; Velik-Salchner, C; Klingler, A; Innerhofer, P
The study was conducted to explore the effects of colloid and crystalloid solutions on activation of fibrinolysis during orthopaedic surgery and to determine whether fluids facilitate clot dissolution at a particular fibrinolytic activity. Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured in plasma samples of 66 orthopaedic patients randomly receiving gelatin solution, hydroxyethyl starch (HES) (130/0.4), or exclusively Ringer's lactate solution. Plasma obtained before induction of anaesthesia (undiluted) and at the end of surgery (diluted) was exposed to recombinant tissue plasminogen activator (r-tPA) in vitro and analysed by modified thrombelastography (ROTEM). There were similar changes in t-PA and PAI-1 concentrations in the gelatin, HES, and Ringer's lactate groups. When compared with the effect of r-tPA on undiluted plasma samples, the presence of colloids prompted faster clot dissolution than did Ringer's lactate solution. Lysis index at 30 min decreased significantly [median (min/max); P vs Ringer's lactate solution] to 43 (1/82)% (P=0.007), 14 (3/70)% (P<0.001), and 91 (34/97)%, lysis onset time decreased to 1269 (1054/1743) s (P=0.007), 972 (490/1565) s (P<0.001), and 1970 (1260/2165) s, and lysis time to 2469 (1586/3303) s (P=0.019), 2002 (1569/3600) s (P=0.006), and 3012 (2017/3600) s in the gelatin, HES, and Ringer's lactate groups, respectively. The type of i.v. fluid used does not influence endogenously occurring fibrinolytic activity in patients undergoing major orthopaedic surgery. However, during hyperfibrinolysis, the presence of HES or gelatin solution facilitates clot disintegration to a greater extent than Ringer's lactate solution, because the weaker clots formed with colloids dissolve faster.
Westbury, Sarah K; Duval, Cédric; Philippou, Helen; Brown, Rebecca; Lee, Kurtis R; Murden, Sherina L; Phillips, Emma; Reilly-Stitt, Christopher; Whalley, Daniel; Ariëns, Robert A; Mumford, Andrew D
Genetic fibrinogen (FGN) variants that are associated with bleeding or thrombosis may be informative about fibrin polymerisation, structure and fibrinolysis. We report a four generation family with thrombosis and heritable dysfibrinogenaemia segregating with a c.[1541delC];[=] variation in FGA (FGN-Perth). This deletion predicts a truncated FGN αC-domain with an unpaired terminal Cys at residue 517 of FGN-Aα. In keeping with this, SDS-PAGE of purified FGN-Perth identified a truncated FGN-Aα chain with increased co-purification of albumin, consistent with disulphide bonding to the terminal Cys of the variant FGN-Aα. Clot visco-elastic strength in whole blood containing FGN-Perth was greater than controls and tPA-mediated fibrinolysis was delayed. In FGN-Perth plasma and in purified FGN-Perth, there was markedly reduced final turbidity after thrombin-mediated clot generation. Consistent with this, FGN-Perth formed tighter, thinner fibrin fibres than controls indicating defective lateral aggregation of protofibrils. Clots generated with thrombin in FGN-Perth plasma were resistant to tPA-mediated fibrinolysis. FGN-Perth clot also displayed impaired tPA-mediated plasmin generation but incorporated α2-antiplasmin at a similar rate to control. Impaired fibrinolysis because of defective plasmin generation potentially explains the FGN-Perth clinical phenotype. These findings highlight the importance of the FGN αC-domain in the regulation of clot formation and fibrinolysis.
Lucking, Andrew J; Gibson, Kyle R; Paterson, Elspeth E; Faratian, Dana; Ludlam, Christopher A; Boon, Nicholas A; Fox, Keith A A; Newby, David E
Using a clinical model of deep arterial injury, we assessed the ability of exogenous and endogenous tissue plasminogen activator (t-PA) to limit acute in situ thrombus formation. Ex vivo thrombus formation was assessed in the Badimon chamber at low and high shear rates in 2 double-blind randomized cross-over studies of 20 healthy volunteers during extracorporeal administration of recombinant t-PA (0, 40, 200, and 1000 ng/mL) or during endogenous t-PA release stimulated by intra-arterial bradykinin infusion in the presence or absence of oral enalapril. Recombinant t-PA caused a dose-dependent reduction in thrombus area under low and high shear conditions (P<0.001 for all). Intra-arterial bradykinin increased plasma t-PA concentrations in the chamber effluent (P<0.01 for all versus saline) that was quadrupled in the presence of enalapril (P<0.0001 versus placebo). These increases were accompanied by an increase in plasma D-dimer concentration (P<0.005 for all versus saline) and, in the presence of enalapril, a reduction in thrombus area in the low shear (16±5; P=0.03) and a trend toward a reduction in the high shear chamber (13±7%; P=0.07). Using a well-characterized clinical model of coronary arterial injury, we demonstrate that endogenous t-PA released from the vascular endothelium enhances fibrinolysis and limits in situ thrombus propagation. These data support a crucial role for the endogenous fibrinolytic system in vivo and suggest that continued exploration and manipulation of its therapeutic potential are warranted.
Yildirim, Dogan; Hut, Adnan; Avaroglu, Huseyin Imam; Erdem, Duygu Ayfer; Cekic, Erdinc; Erozgen, Fazilet
Background Laparoscopic cholecystectomies (LC) are generally performed in a 12 mmHg-pressured pneumoperitoneum in a slight sitting position. Considerable thromboembolism risk arises in this operation due to pneumoperitoneum, operation position and risk factors of patients. We aim to investigate the effect of pneumoperitoneum pressure on coagulation and fibrinolysis under general anesthesia. Material and Methods Fifty American Society of Anesthesiologist (ASA) I–III patients who underwent elective LC without thromboprophlaxis were enrolled in this prospective study. The patients were randomly divided into two groups according to the pneumoperitoneum pressure during LC: the 10 mmHg group (n = 25) and the 14 mmHg group. Prothrombin time (PT), thrombin time (TT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT) and blood levels of d-dimer and fibrinogen were measured preoperatively (pre), one hour (post1) and 24 h (post24) after the surgery. Moreover, alanine amino transferase, aspartate amino transferase and lactate dehydrogenase were measured before and after the surgery. These parameters were compared between and within the groups. Results PT, TT, aPTT, INR, and D-dimer and fibrinogen levels significantly increased after the surgery in both of the groups. D-dimer level was significantly higher in 14-mmHg group at post24. Conclusion Both the 10-mmHg and 14-mmHg pressure of pneumoperitoneum may lead to affect coagulation tests and fibrinogen and D-dimer levels without any occurrence of deep vein thrombosis, but 14-mmHg pressure of pneumoperitoneum has a greater effect on D-dimer. However, lower pneumoperitoneum pressure may be useful for the prevention of deep vein thrombosis. PMID:27651988
Donmez, Turgut; Uzman, Sinan; Yildirim, Dogan; Hut, Adnan; Avaroglu, Huseyin Imam; Erdem, Duygu Ayfer; Cekic, Erdinc; Erozgen, Fazilet
Laparoscopic cholecystectomies (LC) are generally performed in a 12 mmHg-pressured pneumoperitoneum in a slight sitting position. Considerable thromboembolism risk arises in this operation due to pneumoperitoneum, operation position and risk factors of patients. We aim to investigate the effect of pneumoperitoneum pressure on coagulation and fibrinolysis under general anesthesia. Fifty American Society of Anesthesiologist (ASA) I-III patients who underwent elective LC without thromboprophlaxis were enrolled in this prospective study. The patients were randomly divided into two groups according to the pneumoperitoneum pressure during LC: the 10 mmHg group (n = 25) and the 14 mmHg group. Prothrombin time (PT), thrombin time (TT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT) and blood levels of d-dimer and fibrinogen were measured preoperatively (pre), one hour (post1) and 24 h (post24) after the surgery. Moreover, alanine amino transferase, aspartate amino transferase and lactate dehydrogenase were measured before and after the surgery. These parameters were compared between and within the groups. PT, TT, aPTT, INR, and D-dimer and fibrinogen levels significantly increased after the surgery in both of the groups. D-dimer level was significantly higher in 14-mmHg group at post24. Both the 10-mmHg and 14-mmHg pressure of pneumoperitoneum may lead to affect coagulation tests and fibrinogen and D-dimer levels without any occurrence of deep vein thrombosis, but 14-mmHg pressure of pneumoperitoneum has a greater effect on D-dimer. However, lower pneumoperitoneum pressure may be useful for the prevention of deep vein thrombosis.
Ajjan, R A; Standeven, K F; Khanbhai, M; Phoenix, F; Gersh, K C; Weisel, J W; Kearney, M T; Ariëns, R A S; Grant, P J
The purpose of this study was to investigate the direct effects of aspirin on fibrin structure/function. Chinese Hamster Ovary cell lines stably transfected with fibrinogen were grown in the absence (0) and presence of increasing concentrations of aspirin. Fibrinogen was purified from the media using affinity chromatography, and clots were made from recombinant protein. Mean final turbidity [OD(+/-SEM)] was 0.083(+/-0.03), 0.093(+/-0.002), 0.101(+/-0.005), and 0.125(+/-0.003) in clots made from 0, 1, 10, and 100 mg/L aspirin-treated fibrinogen, respectively (P<0.05). Permeability coefficient (Ks cm2 x 10(-8)) was 1.68(+/-0.29) and 4.13(+/-0.33) comparing fibrinogen produced from cells grown with 0 mg/L and 100 mg/L aspirin respectively (P<0.05). Scanning electron microscopy confirmed a looser clot structure and increased fiber thickness of clots made from aspirin-treated fibrinogen, whereas rheometer studies showed a significant 30% reduction in clot rigidity. Fibrinolysis was quicker in clots made from aspirin-treated fibrinogen. Ex vivo studies in 3 normal volunteers given 150 mg aspirin daily for 1 week demonstrated similar changes in clot structure/function. Aspirin directly altered clot structure resulting in the formation of clots with thicker fibers and bigger pores, which are easier to lyse. This study clearly demonstrates an alternative mode of action for aspirin, which should be considered in studies evaluating the biochemical efficacy of this agent.
Teblum, Andrew; Basumallick, Srijita; Shah, Rikhav; Mitra, Rajendra N.; Banerjee, Subhash; Santra, Swadeshmukul
We report design and synthesis of a series of activatable "OFF/ON" CdS:Mn/ZnS quantum dot (Qdot) based sensing probes. The Qdot "OFF" state represent the "quenched state" where the Qdot fluorescence is quenched by ligands attached to Qdot surface. Fluorescence quenching is likely due to ligand assisted electron transfer process. Qdot fluorescence is restored when the electron transfer process is stopped. Using this activatable Qdots, we have successfully demonstrated usefulness of these Qdot probes for reliable detection of toxic cadmium ions in solution, selective detection of glutathione and sensitive detection of intracellular cancer drug release event. In this paper, we will discuss a simple but robust method of making water-soluble CdS:Mn/ZnS Qdots at the room-temperature. Two different water-soluble biomolecules, the N-acetyl cysteine (NAC) and the glutathione (GSH) were used as surface coating ligands. This is a singlestep, one-pot synthesis where the Qdot nanocrystals were grown in the presence of the biomolecules. These Qdots were characterized by fluorescence spectroscopy. Stability of the GSH coated Qdots and the NAC coated Qdots were studied by treating with ethylenediaminetetraacetic acid (EDTA, a strong chelating agent for Zn and Cd ions). Our results show that fluorescence properties of Qdots are affected by the type of surface coated ligands. In comparison to the GSH coated Qdots, the NAC coated Qdots show broad but strong emission towards near infra-red region. When treated with EDTA, fluorescence property of the GSH coated Qdot was affected less than the NAC coated Qdots. This preliminary study shows that NAC coated Qdots could potentially be used to develop activatable ("OFF/ON") probes for potential deep-tissue imaging applications. Similarly, the GSH coated Qdots could be applied for probing desired analytes or for bioimaging purposes in environmentally harsh conditions.
Zhang, Zongren; Fan, Jinda; Cheney, Philip P; Berezin, Mikhail Y; Edwards, W Barry; Akers, Walter J; Shen, Duanwen; Liang, Kexian; Culver, Joseph P; Achilefu, Samuel
We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near-infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding k(cat) and K(M), values of 1.02 +/- 0.06 s(-1) and 15 +/- 3 microM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in > 2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.
Jana, Pradipta; Maiti, Smarajit; Kahn, Nighat N; Sinha, Asru K
Estriol, an oestrogen, at 0.6 nmol/l was reported to inhibit ADP-induced platelet aggregation through nitric oxide synthesis. As nitric oxide has been reported to cause fibrinolysis due to the activation of plasminogen to plasmin, the role of estriol as a fibrinolytic agent was investigated. Also, the mechanism of estriol-induced nitric oxide synthesis in anucleated platelets was investigated. The estriol-induced lysis of platelet-rich plasma (PRP) clot was determined by photography of the clot lysis and by the assay of fibrin degradation products in the lysate and was obtained by SDS-PAGE. Nitric oxide was determined by methemoglobin method. The platelet membrane protein was isolated from the platelets by using Triton X-100 (0.05% v/v). The binding of estriol to the protein was determined by Scatchard plot by using an ELISA for estriol. Estriol at 0.6 nmol/l was found to lyse the clotted PRP due to fibrinolysis that produced fibrin degradation products in the lysate. The amino acid analysis of the platelet membrane protein, which resembles with nitric oxide synthase (NOS) activity, was activated nearly 10-fold over the control in the presence of estriol and was identified to be a human serum albumin precursor (Mr. 69 kDa) that binds to estriol with Kd1 of 6.0 × 10 mol/l and 39 ± 2 molecules of estriol bound the NOS molecule. The estriol-induced nitric oxide is capable of inducing fibrinolysis of the clotted PRP. The binding of estriol to platelet membrane NOS activated the enzyme in the absence of DNA in the platelet.
Levi, M; Roem, D; Kamp, A M; de Boer, J P; Hack, C E; ten Cate, J W
It has been shown that the most important inhibitor of plasmin is alpha 2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed. To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin. It was confirmed that alpha 2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than alpha 2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-alpha 1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with alpha 2-macroglobulin and with antithrombin III were significantly elevated. In conclusion, we confirmed the important role of alpha 2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Shi, Hui; Ye, Xiaosheng; He, Xiaoxiao; Wang, Kemin; Cui, Wensi; He, Dinggeng; Li, Duo; Jia, Xuekun
Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo ``nano-doctors'' that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as the model, in vitro and in vivo studies of A549 lung cancer verified that the ATNP greatly improved imaging contrast and specific destruction, suggesting a robust and versatile theranostic strategy for personalized medicine in future.Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo ``nano-doctors'' that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as
Mohanty, Tirthankar; Karlsson, Christofer; Mörgelin, Matthias; Frick, Inga-Maria; Malmström, Johan; Björck, Lars
Streptococcal pharyngitis is among the most common bacterial infections, but the molecular mechanisms involved remain poorly understood. Here we investigate the interactions among three major players in streptococcal pharyngitis: streptococci, plasma, and saliva. We find that saliva activates the plasma coagulation system through both the extrinsic and the intrinsic pathways, entrapping the bacteria in fibrin clots. The bacteria escape the clots by activating host plasminogen. Our results identify a potential function for the intrinsic pathway of coagulation in host defense and a corresponding role for fibrinolysis in streptococcal immune evasion. PMID:27456827
Kostousov, Vadim; Wang, Yao-Wei W; Cotton, Bryan A; Wade, Charles E; Holcomb, John B; Matijevic, Nena
Hyperfibrinolysis has been identified as a mechanism of trauma coagulopathy associated with poor outcome. The aim of the study was to create a trauma coagulopathy model (TCM) with a hyperfibrinolysis thrombelastography (TEG) pattern similar to injured patients and test the effects of different resuscitation fluids and antifibrinolytics on fibrinolysis. TCM was established from whole blood by either 15% dilution with isotonic saline, lactated Ringer's, Plasma-Lyte, 5% albumin, Voluven, Hextend, 6% dextran in isotonic saline or 30% dilution with lactated Ringer's plus Voluven and supplementation with tissue factor and tissue plasminogen activator (tPA). These combinations resulted in a TCM that could then be 'treated' with tranexamic acid (TXA) or 6-aminocaproic acid (ACA). Clot formation was evaluated by TEG. Whole-blood dilution by 15% with crystalloids and albumin in the presence of tissue factor plus tPA resulted in an abnormal TEG pattern and increased fibrinolysis, as did dilution with synthetic colloids. TXA 1 μg/ml or ACA 10 μg/ml were sufficient to suppress fibrinolysis when TCM was diluted 15% with lactated Ringer's, but 3 μg/ml of TXA or 30 μg/ml of ACA were needed for fibrinolysis inhibition induced by simultaneous euvolemic dilution with lactated Ringer's plus Voluven by 30%. A total of 15% dilution of whole blood in the presence of tissue factor plus tPA results in a hyperfibrinolysis TEG pattern similar to that observed in severely injured patients. Synthetic colloids worsen TEG variables with a further increase of fibrinolysis. Low concentrations of TXA or ACA reversed hyperfibrinolysis, but the efficient concentrations were dependent on the degree of fibrinolysis and whole-blood dilution.
Mitsunaga, Makoto; Kosaka, Nobuyuki; Choyke, Peter L; Young, Matthew R; Dextras, Christopher R; Saud, Shakir M; Colburn, Nancy H; Sakabe, Masayo; Nagano, Tetsuo; Asanuma, Daisuke; Urano, Yasuteru; Kobayashi, Hisataka
Screening colonoscopy to monitor for early colitis-associated colon cancer (CAC) is difficult due to the aberrant mucosal patterns associated with long-standing colitis. The aim of this study was to develop a rapid fluorescent detection method for use during colonoscopy for improving the detection of CAC utilising a topically applied enzymatically activatable probe (gGlu-HMRG) which fluoresces in the presence of γ-glutamyltranspeptidase (GGT), an enzyme associated with cancer. Expression of GGT in colon cell lines was examined with fluorescence microscopy and flow cytometry. A mouse model (azoxymethane/dextran sulphate sodium) of CAC was used and mice were examined with white light and fluorescence colonoscopy before and after topical gGlu-HMRG administration. Expression of GGT, although variable, was higher in human colon cancer cells than normal human colon cells. Using fluorescence colonoscopy in mice, gGlu-HMRG fluorescent lesions were detected 5 min after topical administration and fluorescence persisted for at least 30 min. Fluorescence guided biopsy revealed all fluorescent lesions that contained cancer or dysplasia (n=16), whereas three out of 12 non-fluorescent lesions contained low grade dysplasia and others did not contain neoplastic histology. Microscopic inflammatory infiltration also had variable fluorescence but in general was much lower (∼10-fold) in signal than cancer. Repeat fluorescence endoscopy allowed individual tumours to be monitored. These results suggest that gGlu-HMRG can improve endoscopic detection of CAC with a higher target to background ratio than conventional white light colonoscopy. This could be of benefit to patients with long-standing colitis who must undergo repeated screening colonoscopies.
Thompson, Stephen; Dessi, John; Self, Colin H
The administration of anti-CD3 antibodies, either unmodified or in bispecific formats, has been shown to kill tumors. However, their activity needs to be carefully controlled. We have approached this problem by inhibiting their anti-CD3 activity until it is required. Folated anti-human CD3 antibody bispecific conjugates were therefore synthesised in which the folate portion of the conjugates remained free to bind to folate receptor (FR) expressing cancer cells, whilst their anti-CD3 activity was reversibly inhibited. On irradiation with UV-A light, the T-cell binding activity of the anti-CD3 antibody can be restored only when and where it is required, i.e., adjacent to a tumor. Conjugate bound to FR expressed on normal tissues in other parts of the body remains inactive. This report describes the preclinical in vivo testing of these conjugates in transgenic mice whose T-cells express human CD3 molecules. When the 'cloaked' conjugates were reactivated in the region of the primary tumor, both primary tumor growth and liver metastasis were markedly reduced. That the deliberate targeting of T-cell activity locally to the primary tumor also resulted in reduced distant metastatic growth was a key finding. Light-activatable bispecific antibody conjugates similar to those described here offer a means to control T-cell targeting with a much higher degree of specificity to tumors because they minimize potentially dangerous and unwanted side effects in non-illuminated areas. The addition of light-specific targeting to the inherent tumor specific targeting of therapeutic antibody conjugates could result in the development of safer treatments for patients.
Carrillo, Xavier; Fernandez-Nofrerias, Eduard; Rodriguez-Leor, Oriol; Oliveras, Teresa; Serra, Jordi; Mauri, Josepa; Curos, Antoni; Rueda, Ferran; García-García, Cosme; Tresserras, Ricard; Rosas, Alba; Faixedas, Maria Teresa; Bayes-Genis, Antoni
The preferred reperfusion strategy for early ST elevation myocardial infarction (STEMI, defined as time from symptoms onset ≤120 min) in non-capable percutaneous coronary intervention (PCI) centres remains controversial. We sought to compare mortality of in situ fibrinolysis vs. PCI transfer in a real-life consecutive cohort of early STEMI. Prospective multicentre STEMI registry (Catalonia 'Codi IAM' network) of all-comers in a non-capable PCI centre with symptom onset to first medical contact (FMC) <120 min. Two groups were identified: in situ fibrinolysis and transfer to a PCI-capable centre. Primary endpoint was 30-day mortality. We included 2470 patients, of whom 2227 (90.2%) and 243 (9.8%) comprised the transfer and fibrinolysis groups, respectively. In the fibrinolysis group, diagnostic and system delays were shorter (24 vs. 31 min, P < 0.001; 45 vs. 119 min, P < 0.001, respectively). Thirty-day mortality was 7.7 and 5.1% in fibrinolysis and transfer groups, respectively (P = 0.09). However, patients in the transfer group whose time FMC-device was achieved within 140 min were associated with significantly lower mortality (2.0% for FMC-device <99 min, and 4.6% for FMC-device 99-140 min; P < 0.01 and P = 0.03, respectively vs. fibrinolysis). In multivariable logistic regression analysis, reperfusion with fibrinolysis was an independent 30-day mortality predictive factor (odds ratio: 1.91, 95% confidence interval: 1.01-3.50; P = 0.04), together with age and Killip-Kimball class (both P < 0.001). In early STEMI patients assisted in non-capable PCI centres, in situ fibrinolysis had worse prognosis than patient transfer. Transfer to a PCI-capable centre seems recommended in patients with FMC-device delay <140 min. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: firstname.lastname@example.org.
Watkins, Gregory A.; Jones, Ella Fung; Shell, M. Scott; VanBrocklin, Henry F.; Pan, Mei-Hsiu; Hanrahan, Stephen M.; Feng, Jin Jin; He, Jiang; Sounni, Nor Eddine; Dill, Ken A.; Contag, Christopher H.; Coussens, Lisa M.; Franc, Benjamin L.
Matrix Metalloproteinase-14 (MT1-MMP or MMP-14) is a membrane-associated protease implicated in a variety of tissue remodeling processes and a molecular hallmark of select metastatic cancers. The ability to detect MMP-14 in vivo would be useful in studying its role in pathologic processes and may potentially serve as a guide for the development of targeted molecular therapies. Four MMP-14 specific probes containing a positively charged cell penetrating peptide (CPP) d-arginine octamer (r8) linked with a MMP-14 peptide substrate and attenuating sequences with glutamate (8e, 4e) or glutamate-glycine (4eg and 4egg) repeating units were modeled using an AMBER force field method. The probe with 4egg attenuating sequence exhibited the highest CPP/attenuator interaction, predicting minimized cellular uptake until cleaved. The in vitro MMP-14-mediated cleavage studies using the human recombinant MMP-14 catalytic domain revealed an enhanced cleavage rate that directly correlated with the linearity of the embedded peptide substrate sequence. Successful cleavage and uptake of a technetium-99m labeled version of the optimal probe was demonstrated in MMP-14 transfected human breast cancer cells. Two- fold reduction of cellular uptake was found in the presence of a broad spectrum MMP inhibitor. The combination of computational chemistry, parallel synthesis and biochemical screening, therefore, shows promise as a set of tools for developing new radiolabeled probes that are sensitive to protease activity. PMID:19109023
Stewart, Lauren K.; Peitz, Geoffrey W.; Nordenholz, Kristen E.; Courtney, D. Mark; Kabrhel, Christopher; Jones, Alan E.; Rondina, Matthew T.; Diercks, Deborah B.; Klinger, James R.
Acute pulmonary embolism (PE) can diminish patient quality of life (QoL). The objective was to test whether treatment with tenecteplase has an independent effect on a measurement that reflects QoL in patients with submassive PE. This was a secondary analysis of an 8-center, prospective randomized controlled trial, utilizing multivariate regression to control for predefined predictors of worsened QoL including: age, active malignancy, history of PE or deep venous thrombosis (DVT), recurrent PE or DVT, chronic obstructive pulmonary disease and heart failure. QoL was measured with the physical component summary (PCS) of the SF-36. Analysis included 76 patients (37 randomized to tenecteplase, 39 to placebo). Multivariate regression yielded an equation f(8, 67), P<0.001, with R2 = 0.303. Obesity had the largest effect on PCS (β = −8.6, P<0.001), with tenecteplase second (β = 4.73, P = 0.056). After controlling for all interactions, tenecteplase increased the PCS by +5.37 points (P = 0.027). In patients without any of the defined comorbidities, the coefficient on the tenecteplase variable was not significant (−0.835, P = 0.777). In patients with submassive PE, obesity had the greatest influence on QoL, followed by use of fibrinolysis. Fibrinolysis had a marginal independent effect on patient QoL after controlling for comorbidities, but was not significant in patients without comorbid conditions. PMID:25433511
Stock, Klaus Wilhelm; Jacob, Augustinus Ludwig; Schnabel, Karl Jakob; Bongartz, Georg; Steinbrich, Wolfgang
Purpose: To report the results of thrombus fragmentation in combination with local fibrinolysis using recombinant human-tissue plasminogen activator (rtPA) in patients with massive pulmonary embolism. Methods: Five patients with massive pulmonary embolism were treated with thrombus fragmentation followed by intrapulmonary injection of rtPA. Clot fragmentation was performed with a guidewire, angiographic catheter, and balloon catheter. Three patients had undergone recent surgery; one of them received a reduced dosage of rtPA. Results: All patients survived and showed clinical improvement with a resultant significant (p < 0.05) decrease in the pulmonary blood pressure (mean systolic pulmonary blood pressure before treatment, 49 mmHg; 4 hr after treatment, 28 mmHg). Angiographic follow-up in three patients revealed a decrease in thrombus material and an increase in pulmonary perfusion. Two patients developed retroperitoneal hematomas requiring transfusion. Conclusion: Clot fragmentation and local fibrinolysis with rtPA was an effective therapy for massive pulmonary embolism. Bleeding at the puncture site was a frequent complication.
Mutoh, S; Kobayashi, M; Hirata, J; Itoh, N; Maki, M; Komatsu, Y; Yoshida, A; Sasa, H; Kuroda, K; Kikuchi, Y
Urinary kallikrein and kallikrein activity significantly decreased in cases of preeclampsia (u-kall./CRE.index 42.39 +/- 9.66 ng/mg, u-kall. act./CRE.index 0.26 +/- 0.06 ng/min/mg), and urinary kininase II and kininase activity significantly increased (u-kininase/CRE.index 10.91 +/- 1.26 x 10(-3) IU/min/mg, u-kininase act./CRE.index 506.37 +/- 178.45 pg/min/mg) when compared with those of normal gravidas from 28 weeks to 42 weeks of gestation (u-kall./CRE.index 189.31 +/- 14.17 ng/mg, u-kall. act./CRE index 1.08 +/- 0.10 ng/min/mg, u-kininase/CRE.index 6.24 +/- 0.31 x 10(-3) IU/min/mg, u-kininase act./CRE.index 15.64 +/- 0.10 pg/min/mg). Urinary FPA, B beta 5-42, alpha 2-PI, and alpha 2PI-plasmin-complex (PIC) significantly increased in preeclampsia (u-FPA/CRE.index 23.59 +/- 8.47 ng/mg, u-B beta/CRE.index 105.26 +/- 29.30 ng/mg, u-alpha 2PI/CRE.index 121.53 +/- 43.57 ng/mg, u-PIC/CRE index 278.39 +/- 60.50 ng/mg) when compared with those of normal control group (u-FPA/CRE.index 0.92 +/- 0.04 ng/mg, u-B beta/CRE.index 12.15 +/- 0.44 ng/mg, u-alpha 2PI/CRE.index 4.18 +/- 0.33 ng/mg, u-PIC/CRE.index 5.98 +/- 1.15 ng/mg). Urinary urokinase markedly increased and urinary D-dimer was detected in severe cases of preeclampsia (u-UK/CRE.index 58.20 +/- 43.69 ng/mg, u-D-dimer 54.76 +/- 9.89 ng/ml) when compared with those of normal control group. These findings suggest that deficiency in urinary kinin excretion may induce hypertension in addition to the changes of urinary coagulation-fibrinolysis system that represents the occurrence of either the endothelial cell injury in the glomerulus or the renal tulbular damage in mild cases of preeclampsia, eventually resulting in the intra-renal vascular coagulation.
Piotrowski, Christine; Ihling, Christian H; Sinz, Andrea
Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner.
Shao, Qi; Hackel, Benjamin J.; Thomas, David D.; Ashkenazi, Shai
Abstract. Activatable photoacoustic probes efficiently combine the high spatial resolution and penetration depth of ultrasound with the high optical contrast and versatility of molecular imaging agents. Our approach is based on photoacoustic probing of the excited-state lifetime of methylene blue (MB), a fluorophore widely used in clinical therapeutic and diagnostic applications. Upon aggregation, static quenching between the bound molecules dramatically shortens their lifetime by three orders of magnitude. We present preliminary results demonstrating the ability of photoacoustic imaging to probe the lifetime contrast between monomers and dimers with high sensitivity in cylindrical phantoms. Gradual dimerization enhancement, driven by the addition of increasing concentrations of sodium sulfate to a MB solution, showed that lifetime-based photoacoustic probing decreases linearly with monomer concentration. Similarly, the addition of 4 mM sodium dodecyl sulfate, a concentration that amplifies MB aggregation and reduces the monomer concentration by more than 20-fold, led to a signal decrease of more than 20 dB compared to a solution free of surfactant. These results suggest that photoacoustic imaging can be used to selectively detect the presence of monomers. We conclude by discussing the implementation of the monomer–dimer contrast mechanism for the development of an enzyme-specific activatable probe. PMID:23640075
Tansi, Felista L; Rüger, Ronny; Böhm, Claudia; Kontermann, Roland E; Teichgraeber, Ulf K; Fahr, Alfred; Hilger, Ingrid
The underlying data demonstrates that fibroblast activation protein (FAP) paves the way for fibrosarcoma cells, which require the proteolysis of the extracellular matrix (ECM) and basement membranes to intravasate from implanted subcutaneous primary tumors into blood vessels, be transported to distant organs where they extravasate from the blood vessels, reattach and proliferate to metastases. The data additionally shows that FAP, when overexpressed on fibrosarcoma cells induces their invasion and formation of spontaneous metastases in multiple organs, particularly after subcutaneous co-implantation of the FAP-expressing and wildtype fibrosarcoma. The raw and processed data presented herein is related to a research article entitled "Potential of activatable FAP-targeting immunoliposomes in intraoperative imaging of spontaneous metastases" (F.L. Tansi, R. Rüger, C. Böhm, R.E. Kontermann, U.K. Teichgraeber, A. Fahr, I. Hilger, 2016) . Furthermore, evidence for the detection of FAP-expressing tumor cells and cells of the tumor stroma by activatable FAP-targeting liposomes is presented in this dataset.
Shi, Hui; Ye, Xiaosheng; He, Xiaoxiao; Wang, Kemin; Cui, Wensi; He, Dinggeng; Li, Duo; Jia, Xuekun
Although nanomaterial-based theranostics have increased positive expectations from cancer treatment, it remains challenging to develop in vivo "nano-doctors" that provide high-contrast image-guided site-specific therapy. Here we designed an activatable theranostic nanoprobe (ATNP) via self-assembly of activatable aptamer probes (AAPs) on Au@Ag/Au nanoparticles (NPs). As both quenchers and heaters, novel Au@Ag/Au NPs were prepared, showing excellent fluorescence quenching and more effective near-infrared photothermal therapy than Au nanorods. The AAP comprised a thiolated aptamer and a fluorophore-labeled complementary DNA; thus, the ATNP with quenched fluorescence in the free state could realize signal activation through target binding-induced conformational change of the AAP, and then achieve on-demand treatment under image-guided irradiation. By using S6 aptamer as the model, in vitro and in vivo studies of A549 lung cancer verified that the ATNP greatly improved imaging contrast and specific destruction, suggesting a robust and versatile theranostic strategy for personalized medicine in future.
Zhu, Xiandi; Sun, Yn; Chen, Di; Li, Jingfeng; Dong, Xia; Wang, Jie; Chen, Huaiwen; Wang, Ying; Zhang, Fulei; Dai, Jinaxin; Pirraco, Rogério P; Guo, Shangjing; Marques, Alexandra P; Reis, Rui L; Li, Wei
This manuscript describes a synergistic therapy for mastocarcinoma by pH and temperature dual-sensitive nanogel, and effects of microstructure, composition and properties of nanogel on the cellular response mechanism. The extracellular internalization of nanogels was obviously enhanced, due to the passive targeting function at T>VPTT. Interestingly, the increased cytotoxicity was further synergistically enhanced by an unexpected apoptosis as evoked by the 5-fluorouracil loaded nanogel (FLNG). The systemically evaluation of the effectors generated from different sub-cellular organelles including endosome, lysosome, autophagosome confirmed that it was a lysomal dependent apoptosis. Such specific apoptosis was mainly attributed to its activatable protonated PEI at low pH, which caused lysosomal membrane destruction and lysosomal enzyme cathepsin B (Cat B) leakage. This Cat B was then translocated to the mitochondria resulting in mitochondrial membrane permeability increase and mitochondrial membrane potential (MMP) decrease, followed by cytochrome c (Cyt C) release. Cyt C was the main molecule that evoked apoptosis as reflected by overexpression of caspase 9. Additionally, such lysosome dependent, apoptosis was further enhanced by the passive cellular targeting at T>VPTT. Thus, the tumor growth inhibition was synergistically enhanced by the extracellular temperature dependent passive targeting and intracellular pH activatable lysosomal dependent apoptosis.
Guisado-Alonso, D; Fayos-Vidal, F; Martí-Fàbregas, J; Prats-Sánchez, L; Marín-Bueno, R; Martínez-Domeño, A; Delgado-Mederos, R; Camps-Renom, P
Speed of administration conditions the effectiveness of intravenous fibrinolysis in treating acute ischaemic stroke. To reduce the risk of haemorrhagic complications, the intervention is contraindicated in certain cases, such as where the International Normalised Ratio (INR) is ≥ 1.7. This study aimed to determine the reliability of point-of-care INR readings (POC-INR) taken using the CoaguChek(®) XS portable coagulometer compared to laboratory results (L-INR). We conducted a retrospective observational study of consecutive patients admitted to our centre with acute ischaemic stroke and who were treated with intravenous fibrinolysis, over a period of 4 years. Patients' INR was measured with a portable coagulometer and in the laboratory. Results were compared using the paired-sample t test; using L-INR results as a reference value, ROC analysis was performed to determine POC-INR with greater predictive value. The study included 210 patients with a mean age of 74.3±11.5 years old; 18 (8.6%) were taking vitamin K antagonist oral anticoagulants (OAC). There were no significant differences between the 2 INR measurements in the population as a whole (POC-INR-L-INR difference: 0.001±0.085; P=.82). In subgroup analysis, the results coincided for patients taking OACs (0.001±0.081; P=.42) and those with L-INR ≤ 1.2 (0.008±0.081; P=.16). For L-INR>1.2, however, the portable coagulometer underestimated INR (0.058±0.095; P=.01). Through ROC analysis, POC-INR < 1.6 was found to be the cut-off point with greatest sensitivity (100%) and specificity (98.97%) for identifying patients eligible for intravenous fibrinolysis (L-INR < 1.7). POC-INR shows a good correlation with L-INR. Our results suggest that the best threshold to predict an L-INR < 1.7 is POC-INR < 1.6. Internal validation studies for POC-INR should be considered in all treatment centres. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.
Godier, Anne; Parmar, Kiran; Manandhar, Karuna; Hunt, Beverley J
Acute traumatic coagulopathy is characterised by fibrinolysis and low fibrinogen. It is unclear how much fibrinogenolysis contributes to reduce fibrinogen levels. The study aim was to: investigate in vitro the effects of tissue-plasminogen activator (t-PA) and tranexamic acid (TXA) on coagulation and fibrinolysis. Whole blood was spiked with varying t-PA concentrations. Clauss fibrinogen levels and thrombelastography (TEG, Haemonetics) were performed, including functional fibrinogen level (FLEV). TXA effects were assessed using four TXA concentrations. Recorded parameters from kaolin activated TEG included maximal amplitude (MA), clot strength (G), percentage lysis (LY). Plasmin-antiplasmin complex (PAP), endogenous thrombin potential (ETP), prothrombin fragment 1+2 (PF1+2), factor V and factor VIII levels were all measured. t-PA induced fibrinolysis: it increased PAP and LY, but decreased MA and G. t-PA induced fibrinogenolysis, with a concentration-dependant decrease in fibrinogen from 2.7 (2.6-3.1) to 0.8 (0.8-0.9) g/L with 60 nM t-PA. FLEV and fibrinogen levels were well correlated. High t-PA doses increased PF1+2, decreased ETP of 19% and FVIII of 63% but not FV. TXA had no effect on plasmin generation as evidenced by no change in PAP. It corrected LY, MA and G and partly protected fibrinogen against fibrinogenolysis: 0.03 mg/mL TXA reduced the fibrinogen fall induced by t-PA 20 nM from 43% to 14%. TXA halved the FVIII fall and increased ETP. t-PA induced plasminogen activation and fibrinogenolysis in a concentration-dependant manner. TXA did not affect plasmin activation but reduced fibrinogenolysis. These results suggest that TXA given early in bleeding patients may prevent fibrinogenolysis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.
Activatable bispecific liposomes bearing fibroblast activation protein directed single chain fragment/Trastuzumab deliver encapsulated cargo into the nuclei of tumor cells and the tumor microenvironment simultaneously.
Tansi, Felista L; Rüger, Ronny; Böhm, Claudia; Steiniger, Frank; Kontermann, Roland E; Teichgraeber, Ulf K; Fahr, Alfred; Hilger, Ingrid
Molecular targeting plays a significant role in cancer diagnosis and therapy. However, the heterogeneity of tumors is a limiting obstacle for molecular targeting. Consequently, clinically approved drug delivery systems such as liposomes still rely on passive targeting to tumors, which does not address tumor heterogeneity. In this work, we therefore designed and elucidated the potentials of activatable bispecific targeted liposomes for simultaneous detection of fibroblast activation protein (FAP) and the human epidermal growth factor receptor 2 (HER2). The bispecific liposomes were encapsulated with fluorescence-quenched concentrations of the near-infrared fluorescent dye, DY-676-COOH, making them detectable solely post processing within target cells. The liposomes were endowed with a combination of single chain antibody fragments specific for FAP and HER2 respectively, or with the FAP single chain antibody fragment in combination with Trastuzumab, which is specific for HER2. The Trastuzumab based bispecific formulation, termed Bi-FAP/Tras-IL revealed delivery of the encapsulated dye into the nuclei of HER2 expressing cancer cells and caused cell death at significantly higher rates than the free Trastuzumab. Furthermore, fluorescence imaging and live microscopy of tumor models in mice substantiated the delivery of the encapsulated cargo into the nuclei of target tumor cells and tumor stromal fibroblasts. Hence, they convey potentials to address tumor plasticity, to improve targeted cancer therapy and reduce Trastuzumab resistance in the future. This work demonstrates the design of activatable bispecific liposomes aimed to target HER2, a poor prognosis tumor marker in many tumor types, and fibroblast activation protein (FAP), a universal tumor marker overexpressed on tumor fibroblasts and pericytes of almost all solid tumors. Encapsulating liposomes with a quenched concentration of a NIRF dye which only fluoresced after cellular degradation and activation enabled
Kini, R. Manjunatha; Koh, Cho Yeow
Snake venom metalloproteases, in addition to their contribution to the digestion of the prey, affect various physiological functions by cleaving specific proteins. They exhibit their activities through activation of zymogens of coagulation factors, and precursors of integrins or receptors. Based on their structure–function relationships and mechanism of action, we have defined classification and nomenclature of functional sites of proteases. These metalloproteases are useful as research tools and in diagnosis and treatment of various thrombotic and hemostatic conditions. They also contribute to our understanding of molecular details in the activation of specific factors involved in coagulation, platelet aggregation and matrix biology. This review provides a ready reference for metalloproteases that interfere in blood coagulation, fibrinolysis and platelet aggregation. PMID:27690102
Thelwell, Craig; Williams, Stella C.; Silva, Marta M. C. G.; Szabó, László; Kolev, Krasimir
Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time. PMID:20966169
Longstaff, Colin; Thelwell, Craig; Williams, Stella C; Silva, Marta M C G; Szabó, László; Kolev, Krasimir
Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.
Da’dara, Akram A.; de Laforcade, Armelle M.
Schistosomes are parasitic platyhelminths that currently infect over 200 million people and cause the chronic debilitating disease schistosomiasis. While these large intravascular parasites can disturb blood flow, surprisingly they do not appear to provoke thrombus formation around them in vivo. In order to determine if the worms can alter their local environment to impede coagulation, we incubated adult worms (50 pairs) in murine blood (500 μl) for 1 h at 37 °C and, using thromboelastography (TEG), we compared the coagulation profile of the blood with control blood that never contained worms. Substantial differences were apparent between the two profiles. Blood that had been exposed to schistosomes clotted more slowly and yielded relatively poor, though stable, thrombi; all TEG measures of blood coagulation (R, K, α-angle, MA, G and TMA) differed significantly between conditions. No fibrinolysis (as determined by LY30 and LY60 values) was detected in either case. The observed TEG profile suggests that the worms are acting as local anti-coagulants. Blood recovered from schistosome-infected mice, however, does not behave in this way. At an early time point post infection (4-weeks), the TEG profile of infected murine blood is essentially the same as that of control blood. However at a later time point (7-weeks) infected murine blood clots significantly faster than control blood but these clots also break down faster. The R, K, α-angle, and TMA measures of coagulation are all significantly different between the control versus infected mice as are the LY30 and LY60 values. This profile is indicative of a hypercoagulable state with fibrinolysis and is akin to that seen in human patients with advanced schistosomiasis. PMID:26573180
Deng, Lingquan; Norberg, Oscar; Uppalapati, Suji; Yan, Mingdi; Ramström, Olof
A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length. PMID:21423935
Spring, Bryan Q.; Palanisami, Akilan; Hasan, Tayyaba
Molecular-targeted probes are emerging with applications for optical biopsy of cancer. An underexplored potential clinical use of these probes is to monitor residual cancer micrometastases that escape cytoreductive surgery and chemotherapy. Here, we show that leukocytes, or white blood cells, residing in nontumor tissues-as well as those infiltrating micrometastatic lesions-uptake cancer cell-targeted, activatable immunoconjugates nonspecifically, which limits the accuracy and resolution of micrometastasis recognition using these probes. Receiver operating characteristic analysis of freshly excised tissues from a mouse model of peritoneal carcinomatosis suggests that dual-color imaging, adding an immunostain for leukocytes, offers promise for enabling accurate recognition of single cancer cells. Our results indicate that leukocyte identification improves micrometastasis recognition sensitivity and specificity from 92 to 93%-for multicellular metastases >20 to 30 μm in size-to 98 to 99.9% for resolving metastases as small as a single cell.
Ding, Jianxun; Li, Di; Zhuang, Xiuli; Chen, Xuesi
Two pH-activatable star-shaped prodrugs are synthesized through the condensation reaction between Y- or dumbbell-shaped poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) (PEG-PLGA) copolymer and acid-sensitive cis-aconityl-doxorubicin. The prodrugs self-assemble into micelles with favorable hydrodynamic radii and relatively low critical micelle concentrations. In vitro DOX release from prodrug micelles is accelerated by the decrease of the PLGA content or at the late endosomal pH. The efficient cellular uptake and intracellular DOX release of the prodrug micelles are confirmed and the improved long-term anti-proliferative activities of prodrug micelles are revealed. These features suggest that the prodrugs provide a favorable approach to construct effective polymeric drug delivery systems for malignancy therapy.
Deng, Lingquan; Norberg, Oscar; Uppalapati, Suji; Yan, Mingdi; Ramström, Olof
A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S- and O-linked structures with intermediate linker length.
Thapa, Pritam; Li, Mengjie; Bio, Moses; Rajaputra, Pallavi; Nkepang, Gregory; Sun, Yajing; Woo, Sukyung; You, Youngjae
Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several cancers, including ovarian, breast, pancreatic, and nonsmall cell lung cancer. However, it causes systemic side effects when administered parenterally. Photodynamic therapy (PDT) is a new strategy for treating local cancers using light and photosensitizer. Unfortunately, PDT is often followed by recurrence due to incomplete ablation of tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker. Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically reduced. However, once illuminated with far-red light, the prodrug effectively killed SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue penetrable and clinically useful far-red light, which kills the cancer cells through the combined effects of PDT and site-specific PTX chemotherapy.
Al-Mawsawi, Laith Q.; Fikkert, Valery; Dayam, Raveendra; Witvrouw, Myriam; Burke, Terrence R.; Borchers, Christoph H.; Neamati, Nouri
Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128AACWWAGIK136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors. PMID:16785440
Xie, Guoqiang; Zhang, Han; Wu, Yaxi; Yang, Lixia
Background Percutaneous coronary intervention (PCI), fibrinolysis and the combination of both methods are current therapeutic options for patients with ST-segment elevation myocardial infarction (STEMI). Methods We searched PubMed, EMBASE, Google scholar and Cochrane Controlled Trials Register for randomized controlled trials (RCTs) evaluating the efficacy and safety of PCI after fibrinolysis within 24 hours, which was compared with primary PCI alone and ischemia-guided or delayed PCI. Meta-analysis was conducted using Review Manager 5.30 following the methods described by the Cochrane library. Results A total of 16 studies including 10,034 patients were enrolled. As compared with primary PCI alone group, the short-term mortality (5.8% vs 4.5%, RR 1.29, 95% confidence interval [CI] 1.00–1.65) and re-infarction rate (4.1% vs 2.7%, RR 1.46, 95%CI 1.05–2.03) were higher in the immediate PCI group (median/mean time ≤ 2 h after fibrinolysis). However, the short-term mortality and re-infarction rate showed no statistically significant differences in the early PCI group (2–24 hours after fibrinolysis). The rate of major bleeding events was higher both in the immediate PCI (6.3% vs 4.4%, RR 1.43, 95%CI 1.11–1.85) and the early PCI group (6.4% vs 4.4%, RR 1.46, 95%CI 1.03–2.06) as compared with primary PCI alone group. As compared with ischemia-guided or delayed PCI, early PCI was associated with significantly reduced re-infarction (2.4% vs 4.0%, RR 0.61, 95%CI 0.41–0.92) and recurrent ischemia (1.5% vs 5.3%, RR 0.29, 95%CI 0.12–0.70) at short-term. And the reduced re-infarction rate was also observed at long-term. Conclusions Early PCI after fibrinolysis, with a relatively broader time for PCI preparation, can bring the similar effects with primary PCI alone and is better than ischemia-guided or delayed PCI in STEMI patients with symptom onset < 12 h who cannot receive timely PCI. However, immediate PCI after fibrinolysis is detrimental. PMID:26523834
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Bagai, Akshay; Tan, Mary; Di Mario, Carlo; Halvorsen, Sigrun; Cantor, Warren J; Le May, Michel R; Fernandez-Aviles, Francisco; Scheller, Bruno; Armstrong, Paul W; Borgia, Francesco; Piscione, Federico; Sánchez, Pedro L; Westerhout, Cynthia M; Goodman, Shaun G; Yan, Andrew T
The efficacy of a routine invasive strategy early after fibrinolysis in relation to baseline risk status is unclear. We sought to characterize the interaction between patient risk and treatment with routine invasive strategy early after fibrinolysis for ST-segment elevation myocardial infarction. We pooled 2,974 patients from 7 randomized trials of fibrinolysis-treated patients with ST-segment elevation myocardial infarction comparing a routine early invasive strategy with a standard approach of percutaneous coronary intervention (PCI) guided by recurrent ischemia or need for rescue. Cox proportional hazards regression was used to examine the interaction between baseline patient risk classified by Thrombolysis in Myocardial Infarction risk score (low/intermediate: ≤ 5 [n = 2,697] vs high: > 5 [n = 277]) and treatment with routine early invasive strategy. Time to PCI after fibrinolysis was longer among patients randomized to standard treatment compared with routine early invasive strategy in the low/intermediate-risk strata (median 11.4 vs 3.5 hours), but was only marginally different between the 2 groups in the high-risk strata (median 4.1 vs 3.5 hours). There was a significant interaction between treatment assignment and risk status for the composite of 30-day death or reinfarction (P = .01). Compared with standard treatment, routine early invasive strategy was associated with lower 30-day death/reinfarction in the low/intermediate-risk stratum (7.5% vs 4.0%, P < .001), but not in the high-risk stratum (14.9% vs 19.6%, P = .45). Although clearly beneficial among the larger subgroup of patients at low/intermediate risk, the benefit of a routine early invasive strategy was not evident in the smaller subgroup of higher-risk patients in the context of an increased requirement for urgent PCI in the comparative standard treatment arm. Copyright © 2014 Elsevier Inc. All rights reserved.
Torres, Márcia Regina Simas Gonçalves; Sanjuliani, Antonio Felipe
Obesity is characterized by chronic subclinical inflammation, which is critical to endothelial dysfunction. Weight loss, induced by lifestyle interventions, is associated with a decline in biomarkers of inflammation and endothelial dysfunction. There is little evidence that high dietary calcium intake may reduce inflammation and improve endothelial function. The purpose of this study was to evaluate the effects of weight loss from a high-calcium energy-reduced diet on biomarkers of inflammation, fibrinolysis, and endothelial function in obese individuals. In this randomized clinical trial, we analyzed the data from 35 obese adults who lost at least 3% of initial body weight, during a period of 16 wk of energy restriction (-800 Kcal/d). Individuals were randomized into the following dietary regimens: (1) a high calcium diet (HCD; 1200-1300 mg/d) or (2) a low-calcium diet (LCD; <500 mg/d). After 16 wk of intervention subjects on HCD compared with those on LCD exhibited greater reduction in waist circumference and waist-to-hip ratio. Participants on HCD presented a significant reduction in all biomarkers of endothelial dysfunction evaluated in the study (intracellular adhesion molecule-1, vascular cell adhesion molecule 1, and E-Selectin), whereas subjects on LCD showed a significant decrease in intracellular adhesion molecule-1 and E-Selectin. Biomarkers of inflammation and fibrinolysis were reduced in both diets, although without reaching statistical significance. The reduction in all markers of inflammation, fibrinolysis, and endothelial dysfunction was similar in both diets. The findings of this study suggest that increased calcium intake during weight loss has no benefits with respect to biomarkers of inflammation, fibrinolysis, and endothelial function. Copyright © 2013 Elsevier Inc. All rights reserved.
[Evaluation of selected parameters of blood coagulation and fibrinolysis system in patients undergoing total hip replacement surgery with normovolemic hemodilution procedure and standard enoxaparine prophylaxis].
Piecuch, Wiesław; Sokołowska, Bozena; Dmoszyńska, Anna; Furmanik, Franciszek
The aim of the study was to evaluate selected blood coagulation and fibrinolysis parameters in patients undergoing total hip replacement surgery with normovolemic hemodilution and standard enoksaparine profilaxis. The study included 66 patients undergoing hip replacement surgery. The group consisted of 51 women and 15 men, within the age range of 47-78, the mean age was 64. In 32 (subgroup II) patients the surgery was performed with the use of normovolemic hemodilution, in 34 (subgroup I) the hemodilution procedure was not applied. The enoksaparine as prophylaxis started 12 hours prior to surgery and continued during hospitalisation. The examination of the coagulation system was performed: on the day of the operation in the morning, on the day of the operation in the evening and on the first day after operation. We determined the concentrations of TAT and PAP complexes, prothrombin fragments 1 + 2 (F1 + 2) and d-dimers (DD). 1) during total hip replacement surgery and particularly in the period of the first 12 hours after the procedure marked activation of coagulation and fibrinolysis occurRed; 2) the application of the hemodilution procedure does not influence significantly the degree of coagulation and fibrinolysis disorders in the perioperative period, but could reduced incidence of thromboembolic complications in the postoperative period.
Schultz, M; Millo, J; Levi, M; Hack, C; Weverling, G; Garrard, C; van der Poll, T
Methods: Non-directed bronchial lavage (NBL) was performed on alternate days in patients expected to require mechanical ventilation for more than 5 days. A total of 28 patients were studied, nine of whom developed VAP. Results: In patients who developed VAP a significant increase in thrombin generation was observed in the airways, as reflected by a rise in the levels of thrombin-antithrombin complexes in NBL fluid accompanied by increases in soluble tissue factor and factor VIIa concentrations. The diagnosis of VAP was preceded by a decrease in fibrinolytic activity in NBL fluid. Indeed, before VAP was diagnosed clinically, plasminogen activator activity levels in NBL fluid gradually declined, which appeared to be caused by a sharp increase in NBL fluid levels of plasminogen activator inhibitor 1. Conclusion: VAP is characterised by a shift in the local haemostatic balance to the procoagulant side, which precedes the clinical diagnosis of VAP. PMID:14760153
Shapiro, F. B.; Baskova, I. P.; Cherkesova, L. U.; Lyapina, L. A.; Goldovskaya, M. D.
The specific inhibitor of thrombin, hirudin, was used for studying the mechanism of the activating effect of ACTH and adrenalin on nonenzymatic fibrinolytic activity (NEFA), the latter characterizing the function of the anticoagulation system (ACS). Simultaneous administration of ACTH and hirudin to animals subjected to immobilization stress did not reduce the effect of ACTH on NEFA, while simultaneous administration of adrenalin and hirudin revealed a diminished effect of the former. This suggests different mechanisms of ACTH and adrenalin effects upon NEFA: the stimulating effect of norepinephrine is realized through throminogenesis followed by activation of the ACS function and by increased NEFA and therefore inhibitable by hirudin which forms an inactive complex with thrombin. In fact the stimulating effect of ACTH upon NEFA is brought about specifically by another route than thrombinogenesis and thus occurs in the presence of hirudin. Hirudin itself has no effect upon NEFA.
Sikora, Joanna; Broncel, Marlena; Markowicz, Magdalena; Chałubiński, Maciej; Wojdan, Katarzyna; Mikiciuk-Olasik, Elżbieta
A diet rich in berries is believed to play a distinct role in the prevention of metabolic diseases associated with obesity. So far, there have been no published clinical observations evaluating the influence of Aronia melanocarpa on hemostasis. The aim of our study was to investigate the effects of A. melanocarpa extract (AM) supplementation on platelet aggregation, clot formation, and lysis in patients with metabolic syndrome (MS). Middle-aged non-medicated subjects with MS (n = 38) and 14 healthy volunteers were included in this study. Patients with MS were treated with 100 mg of AM three times daily for 2 months. We observed a significant reduction in the concentration of TC, LDL-C, and TG after AM supplementation. Beneficial changes in coagulation parameters were also observed. After 1 month of AM administration, we noticed significant inhibition of platelet aggregation. However, this effect became less pronounced after 2 months of supplementation. In the case of coagulation induced by endogenic thrombin, a significant decrease in the overall potential for coagulation was induced after 1 or 2 months of supplementation. Moreover, after 1 month of AM extract supplementation, we observed a beneficial reduction in the overall potential for clot formation and fibrinolysis. We observed the normalization of hemostasis parameters in MS patients after both 1 and 2 months of AM administration. After 1 month of AM supplementation, we found favorable changes in regards to the overall potential for plasma clotting, clot formation, and lysis, as well as in the lipid profiles of subjects.
Leitão, D P; Polizello, A C; Rothschild, Z
Fibrinolytic and coagulation properties of capybara (Hydrochaeris hydrochaeris, LINNAEUS, 1766) plasma were analysed and the results compared to the guinea-pig (Cavia porcellus), a close relative. Capybara fibrinogen was isolated and fibrinolysis of its plasma was carried out in a homologous system and with bovine fibrin. Undiluted plasma did not have fibrinolytic activity on fibrin plates; euglobulins gave a dose-related response. Zymography of capybara and guinea-pig plasma gave the same patterns of activity as human or bovine plasma. Human urokinase (UK) and tissue plasminogen activator (t-PA) produced lysis in capybara fibrin plates. Streptokinase (SK) (500 IU/ml) did not activate capybara or guinea-pig plasma. In this system, human plasma was extensively activated. Coagulation tests for both species of rodent were prolonged. The capybara showed values for prothrombin time (PT) shorter than activated thromboplastin time (APTT). The guinea-pig, as already shown, had longer PT values. Factors X and VII were very low for capybara and guinea-pig when tested using reference curves and diagnostic kits for human plasma. It is suggested that the capybara could be a valuable laboratory animal considering its size and closeness to the guinea-pig, and this could allow for the provision of materials from one single animal when convenient or necessary.
Sánchez, C M; Suárez, M A; Nebra, A; Gutiérrez, I; Guallart, A; Millaste, A
Traumatic brain injury (TBI) and spontaneous intracerebral hemorrhage (SIH) are associated with acute brain injury. Both increase intracranial pressure because of hemorrhages which activate the coagulation system. The relationship between acute brain injury and coagulation activation can be explained by the tissue factor release from the damaged brain to the circulation. This problem has been addressed in several studies, though detailed investigations are lacking mainly in SIH. To study the activation of hemostasis in a group of patients with TBI and with SIH. Patients and methods. Prospective observational study. Seventy-five intensive care patients were divided in two groups. The first group (n = 45) included TBI and the second (n = 30) SIH. There were 40 healthy persons as a control group. Thrombin-antithrombin III complex, D-dimer and 1 + 2 thrombin fragment levels were measured. All the studied markers were significantly higher in the two patient groups. Thrombin-antithrombin III complex and D-dimer were significantly higher in TBI. We have confirmed a relationship between higher levels of markers and the injury severity, measured with the Glasgow coma scale. Acute brain injury secondary to TBI and SIH activates coagulation system and fibrinolysis early. This activation attempts to achieve a hemostasia status. The activation is more intense in patients with low Glasgow coma scores on admission.
Váradi, Balázs; Kolev, Krasimir; Tenekedjiev, Kiril; Mészáros, Gyöngyi; Kovalszky, Ilona; Longstaff, Colin; Machovich, Raymund
The massive presence of phospholipids is demonstrated in frozen sections of human arterial thrombi. Purified platelet phospholipids and synthetic phospholipids retard in vitro tissue-type plasminogen activator (tPA)-induced fibrinolysis through effects on plasminogen activation and plasmin function. The inhibition of plasminogen activation on the surface of fibrin correlates with the fraction of anionic phospholipid. The phospholipids decrease the amount of tPA penetrating into the clot by 75% and the depth of the reactive surface layer occupied by the activator by up to 30%, whereas for plasmin both of these parameters decrease by approximately 50%. The phospholipids are not only a diffusion barrier, they also bind the components of the fibrinolytic system. Isothermal titration calorimetry shows binding characterized with dissociation constants in the range 0.35-7.64 microm for plasmin and tPA (lower values with more negative phospholipids). The interactions are endothermic and thermodynamically driven by an increase in entropy, probably caused by the rearrangements in the ordered gel structure of the phospholipids (in line with the stronger inhibition at gel phase temperatures compared with liquid crystalline phase temperatures). These findings show a phospholipid barrier, which should be overcome during lysis of arterial thrombi.
Barnard, Sunelle A.; Loots, Du Toit; Rijken, Dingeman C.
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
Pieters, Marlien; Barnard, Sunelle A; Loots, Du Toit; Rijken, Dingeman C
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
Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise
Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025
Güngör, Ömer; Altınbaş Özpınar, Gül; Durmuş, Mahmut; Ahsen, Vefa
Silicon(iv) phthalocyanines ( and ) bearing two calixarene groups as axial ligands were synthesized. Surprisingly, both phthalocyanines were obtained as two different isomers ( and ) depending on the distance between calixarene benzene groups and the phthalocyanine ring. DFT and TD-DFT computations were performed to model plausible structures of these isomers and to simulate electronic absorption spectra. These isomers converted into each other depending on the polarity of the used solvent, temperature and light irradiation. The photophysical and photochemical properties of each isomer were investigated in dimethylsulfoxide (DMSO) for the determination of photodynamic therapy (PDT) activities of these compounds. The more blue-shifted isomers ( and ) showed higher fluorescence quantum yields and singlet oxygen generation compared to more red-shifted counterparts ( and ). This behavior is extremely important for developing activatable photosensitizers for cancer treatment by PDT. Although these photosensitizers produce lower singlet oxygen in normal cells, they produce higher singlet oxygen (six times higher for ) in cancer cells since these photosensitizers converted to more blue-shifted isomers by using light irradiation.
Jeng, J R; Jeng, C Y; Sheu, W H; Lee, M M; Huang, S H; Shieh, S M
The fibrinolytic and metabolic changes associated with gemfibrozil treatment of hypertriglyceridemia were evaluated in 16 patients with type IV hyperlipidemia by criteria of triglyceride levels > 250 mg/dl and total cholesterol levels < 220 mg/dl. The plasma triglyceride level was significantly lower (323 +/- 71 vs 189 +/- 57 mg/dl; p = 0.000) and high-density lipoprotein cholesterol level significantly higher (33.5 +/- 4.6 vs 38.0 +/- 6.7 mg/dl; p = 0.005) after 3 to 4 months of gemfibrozil treatment. However, the glucose and insulin metabolism measured by oral glucose challenge and insulin suppression tests showed no significant changes after gemfibrozil therapy. In contrast, plasma plasminogen activator inhibitor-1 antigen (36.9 +/- 12.4 vs 27.3 +/- 11.4 ng/ml; p = 0.008) and activity (15.5 +/- 5.5 vs 11.8 +/- 3.0 IU/ml; p = 0.009) and tissue plasminogen activator antigen (13.2 +/- 4.0 vs 10.4 +/- 3.7 ng/ml; p = 0.007) were significantly depressed, and tissue plasimogen activator activity (0.57 +/- 0.31 vs 0.69 +/- 0.38 IU/ml; p = 0.015) was significantly elevated by gemfibrozil. The data indicate that lowering plasma triglyceride and raising high-density lipoprotein cholesterol levels by gemfibrozil treatment also improved the fibrinolytic system without changes of insulin resistance and glucose intolerance in patients with isolated hypertriglyceridemia.
Taylor, John R; Fox, Erin E; Holcomb, John B; Rizoli, Sandro; Inaba, Kenji; Schreiber, Martin A; Brasel, Karen; Scalea, Thomas M; Wade, Charles E; Bulger, Eileen; Cotton, Bryan A
Among bleeding patients, we hypothesized that the hyperfibrinolytic (HF) phenotype would be associated with the highest mortality, while shutdown (SD) patients would have the greatest complication burden. Severely injured patients predicted to receive a massive transfusion at 12 level-1 trauma centers were randomized to one of two transfusion ratios as described in the PROPPR trial. Fibrinolysis phenotypes were determined based on admission clot lysis at 30 minutes (LY30): SD ≤0.8%, physiologic (PHYS) 0.9-2.9% and HF ≥3%. Univariate and multivariate analysis was performed. Logistic regression was used to adjust for age, gender, arrival physiology, shock, injury severity, center-effect and treatment arm. Among the 680 patients randomized, 547(80%) had admission TEG values available to determine fibrinolytic phenotypes. Compared to SD and PHYS, HF patients had higher ISS (25 vs. 25. vs. 34), greater base deficit (-8 vs. -6 vs. -12) and were more uniformly hypocoagulable on admission by PT, PTT and TEG values; all p<0.001. HF patients also received more RBC, plasma and platelets (at 3, 6 and 24 hours), had fewer ICU, ventilator and hospital-free days, and had higher 24-hr and 30-d mortality. There were no differences in complications between the three phenotypes. Multivariate logistic regression demonstrated that HF on admission was associated with a 3-fold higher mortality (OR 3.06, 95% C.I. 1.57-5.95, p=0.001) CONCLUSIONS: Previous data have shown that both the SD and HF phenotypes are associated with increased mortality and complications in the general trauma population. However, in a large cohort of bleeding patients, HF as confirmed to be a much more lethal and resource intense phenotype. These data suggest that further research into the understanding of SD and HF is warranted to improve outcomes in this patient population. Prognostic, II.
Jung, Seunguk; Jung, Cheolkyu; Bae, Yun Jung; Choi, Byung Se; Kim, Jae Hyoung; Lee, Sang-Hwa; Chang, Jun Young; Kim, Beom Joon; Han, Moon-Ku; Bae, Hee-Joon; Kwon, Bae Ju; Cha, Sang-Hoon
Background and Purpose Recent advances in intra-arterial techniques and thrombectomy devices lead to high rate of recanalization. However, little is known regarding the effect of the evolvement of endovascular revascularization therapy (ERT) in acute basilar artery occlusion (BAO). We compared the outcome of endovascular mechanical thrombectomy (EMT) versus intra-arterial fibrinolysis (IAF)-based ERT in patients with acute BAO. Methods After retrospectively reviewed a registry of consecutive patients with acute ischemic stroke who underwent ERT from September 2003 to February 2015, 57 patients with acute BAO within 12 hours from stroke onset were enrolled. They were categorized as an IAF group (n=24) and EMT group (n=33) according to the primary technical option. We compared the procedural and clinical outcomes between the groups. Results The time from groin puncture to recanalization was significantly shorter in the EMT group than in the IAF group (48.5 [25.3 to 87.8] vs. 92 [44 to 179] minutes; P=0.02) The rate of complete recanalization was significantly higher in the EMT group than in the IAF group (87.9% vs 41.7%; P<0.01). The good outcome of the modified Rankin Scale score≤2 at 3 months was more frequent in the EMT group than in the IAF group, but it was not statistically significant (39.4% vs 16.7%; P=0.06). Conclusions EMT-based ERT in patients with acute BAO is superior to IAF-based ERT in terms of the reduction of time from groin puncture to recanalization and the improvement of the rate of complete recanalization. PMID:27283281
Shakur, Haleema; Fawole, Bukola; Kuti, Modupe; Olayemi, Oladapo; Bello, Adenike; Ogunbode, Olayinka; Kotila, Taiwo; Aimakhu, Chris O; Huque, Sumaya; Gregg, Meghann; Roberts, Ian
Background: Postpartum haemorrhage (PPH) is a leading cause of maternal death. Tranexamic acid has the potential to reduce bleeding and a large randomized controlled trial of its effect on maternal health outcomes in women with PPH (The WOMAN trial) is ongoing. We will examine the effect of tranexamic acid on fibrinolysis and coagulation in a subset of WOMAN trial participants. Methods. Adult women with clinically diagnosed primary PPH after vaginal or caesarean delivery are eligible for inclusion in the WOMAN trial. In a sub-group of trial participants, blood samples will be collected at baseline and 30 minutes after the first dose of tranexamic acid or matching placebo. Our primary objective is to evaluate the effect of tranexamic acid on fibrinolysis. Fibrinolysis will be assessed by measuring D-dimers and by rotational thromboelastometry (ROTEM). Secondary outcomes are international normalized ratio (INR), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, haemoglobin and platelets. We aim to include about 180 women from the University College Hospital, Ibadan in Nigeria. Discussion: This sub-study of WOMAN trial participants should provide information on the mechanism of action of tranexamic acid in women with postpartum haemorrhage. We present the trial protocol and statistical analysis plan. The trial protocol was registered prior to the start of patient recruitment. The statistical analysis plan was completed before un-blinding. Trial registration: The trial was registered: ClinicalTrials.gov, Identifier NCT00872469 https://clinicaltrials.gov/ct2/show/NCT00872469; ISRCTN registry, Identifier ISRCTN76912190 http://www.isrctn.com/ISRCTN76912190 (Registration date: 22/03/2012).
Background: Postpartum haemorrhage (PPH) is a leading cause of maternal death. Tranexamic acid has the potential to reduce bleeding and a large randomized controlled trial of its effect on maternal health outcomes in women with PPH (The WOMAN trial) is ongoing. We will examine the effect of tranexamic acid on fibrinolysis and coagulation in a subset of WOMAN trial participants. Methods. Adult women with clinically diagnosed primary PPH after vaginal or caesarean delivery are eligible for inclusion in the WOMAN trial. In a sub-group of trial participants, blood samples will be collected at baseline and 30 minutes after the first dose of tranexamic acid or matching placebo. Our primary objective is to evaluate the effect of tranexamic acid on fibrinolysis. Fibrinolysis will be assessed by measuring D-dimers and by rotational thromboelastometry (ROTEM). Secondary outcomes are international normalized ratio (INR), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, haemoglobin and platelets. We aim to include about 180 women from the University College Hospital, Ibadan in Nigeria. Discussion: This sub-study of WOMAN trial participants should provide information on the mechanism of action of tranexamic acid in women with postpartum haemorrhage. We present the trial protocol and statistical analysis plan. The trial protocol was registered prior to the start of patient recruitment. The statistical analysis plan was completed before un-blinding. Trial registration: The trial was registered: ClinicalTrials.gov, Identifier NCT00872469 https://clinicaltrials.gov/ct2/show/NCT00872469; ISRCTN registry, Identifier ISRCTN76912190 http://www.isrctn.com/ISRCTN76912190 (Registration date: 22/03/2012). PMID:28317031
Deng, Zu-Yue; Shan, Wei-Guang; Wang, Shen-Feng; Hu, Meng-Mei; Chen, Yan
Astaxanthin (ASTX) is a xanthophyll carotenoid that reduces hemostasis in hyperlipidemic organisms. Its antihemostatic mechanisms remain unclear. The effects of ASTX on coagulation, the fibrinolytic system and platelet aggregation were investigated in hyperlipidemic rats. Different doses of ASTX (5, 10 and 30 mg/kg/day, p.o.) were administered for four weeks to high-fat diet-induced hyperlipidemic rats. Serum lipid and lipoprotein levels were measured with an automatic biochemical analyzer. The prothrombin time (PT), activated partial thromboplastin time (APTT) and maximum platelet aggregation rate (MAR) were determined by a coagulation analyzer. The activities of the tissue-type plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1) and endothelial nitric oxide synthase (eNOS), as well as the levels of thromboxane B(2) [TXB(2)], 6-keto prostaglandin F(1α) [6-keto-PGF(1α)] and platelet granule membrane protein (GMP-140), were measured with enzyme-linked immunosorbent assay kits. Gene and protein expression levels were analyzed by reverse transcriptase polymerase chain reaction and Western blot, respectively. ASTX (30 mg/kg) treatment in hyperlipidemic rats reduced serum TG (0.58 ± 0.14 versus 1.12 ± 0.24 mmol/L), serum TC (1.77 ± 0.22 versus 2.24 ± 0.21 mmol/L), serum LDL-C (1.13 ± 0.32 versus 2.04 ± 0.48 mmol/L), serum MDA (69%), plasma MAR (55%), serum TXB2/6-keto-PGF1α (34%) and serum GMP-140 levels (25%), plasma PAI-1 activity (48%) and downregulated the mRNA (33%) and protein (23%) expression of aorta eNOS, the mRNA (79%) and protein (72%) expression levels of aorta PAI-1. However, ASTX (30 mg/kg/d) treatment increased serum SOD activity (2.1 fold), serum GPx activity (1.8 fold), plasma PT (1.3 fold), plasma APTT (1.7 fold), serum NO (1.4-fold), serum 6-keto-PGF1α (1.3 fold). ASTX reduced blood coagulation and platelet aggregation and promoted fibrinolytic activity in hyperlipidemic rats
Negreva, Mariya; Georgiev, Svetoslav; Vitlianova, Katerina
Abstract There are no studies to date on the early changes in the hemostasis profile of patients with paroxysmal atrial fibrillation (PAF). Given the key role of the fibrinolytic system in maintaining blood fluidity, our aim was to examine its activity in patients with clinical manifestation of the disease <24 hours. We studied 51 nonanticoagulated patients with a first episode of the disease (26 men, 25 women; mean age 59.84 ± 1.60 years) and 52 controls (26 men, 26 women; mean age 59.50 ± 1.46 years) who matched the patients in terms of gender, age, comorbidities, and conducted treatment. Using enzyme-linked immunoassays and colorimetric assays we assessed the plasminogen activity, tissue plasminogen activator level (t-PA), plasminogen activator inhibitor 1 activity (PAI-1), α2-antiplasmin activity (α2-AP), D-dimer level, and vitronectin level. Blood samples were collected immediately after hospitalization. Patients were hospitalized between the second and twenty fourth hours (mean 8.14 ± 0.76 hours) after the onset of PAF. Compared to controls, plasminogen (159.40 ± 4.81 vs 100.2 ± 2.88%, P < 0.001) and t-PA levels (11.25 ± 0.35 vs 6.05 ± 0.31 ng/mL, P < 0.001) were significantly higher in the patient group. PAI-1 activity (7.33 ± 0.37 vs 15.15 ± 0.52 AU/mL, P < 0.001) and α2-AP (112.9 ± 2.80 vs 125.60 ± 3.74%, P < 0.05) as well as vitronectin plasma levels (134.7 ± 5.83 vs 287.3 ± 10.44 mcg/mL, P < 0.001) were lower in the PAF group. Conversely, the levels of D-dimer in patients were significantly higher (0.53 ± 0.07 vs 0.33 ± 0.02 ng/mL, P < 0.05). Early changes in the fibrinolytic system occur in PAF, suggesting their close relationship with the manifestation of the disease. There is high plasma fibrinolytic activity, during the very first 24 hours of the disease, which is most likely a pathophysiological response to the intensified procoagulation
Gu, Kaizhi; Liu, Yajing; Guo, Zhiqian; Lian, Cheng; Yan, Chenxu; Shi, Ping; Tian, He; Zhu, Wei-Hong
Leucine aminopeptidase (LAP), one of the important proteolytic enzymes, is intertwined with the progress of many pathological disorders as a well-defined biomarker. To explore fluorescent aminopeptidase probe for quantitative detection of LAP distribution and dynamic changes, herein we report a LAP-targeting near-infrared (NIR) fluorescent probe (DCM-Leu) for ratiometric quantitative trapping of LAP activity in different kinds of living cells. DCM-Leu is composed of a NIR-emitting fluorophore (DCM) as a reporter and l-leucine as a triggered moiety, which are linked together by an amide bond specific for LAP cleavage. High contrast on the ratiometric NIR fluorescence signal can be achieved in response to LAP activity, thus enabling quantification of endogenous LAP with "build-in calibration" as well as minimal background interference. Its ratiometric NIR signal can be blocked in a dose-dependent manner by bestatin, an LAP inhibitor, indicating that the alteration of endogenous LAP activity results in these obviously fluorescent signal responses. It is worth noting that DCM-Leu features striking characteristics such as a large Stokes shift (∼205 nm), superior selectivity, and strong photostability responding to LAP. Impressively, not only did we successfully exemplify DCM-Leu in situ ratiometric trapping and quantification of endogenous LAP activity in various types of living cells, but also, with the aid of three-dimensional confocal imaging, the intracellular LAP distribution is clearly observed from different perspectives for the first time, owing to the high signal-to-noise of ratiometric NIR fluorescent response. Collectively, these results demonstrate preclinical potential value of DCM-Leu serving as a useful NIR fluorescent probe for early detection of LAP-associated disease and screening inhibitor.
Qiu, Xudong; Johnson, James R.; Wilson, Bradley S.; Gammon, Seth T.; Piwnica-Worms, David; Barnett, Edward M.
Peptide probes for imaging retinal ganglion cell (RGC) apoptosis consist of a cell-penetrating peptide targeting moiety and a fluorophore-quencher pair flanking an effector caspase consensus sequence. Using ex vivo fluorescence imaging, we previously validated the capacity of these probes to identify apoptotic RGCs in cell culture and in an in vivo rat model of N-methyl- D-aspartate (NMDA)-induced neurotoxicity. Herein, using TcapQ488, a new probe designed and synthesized for compatibility with clinically-relevant imaging instruments, and real time imaging of a live rat RGC degeneration model, we fully characterized time- and dose-dependent probe activation, signal-to-noise ratios, and probe safety profiles in vivo. Adult rats received intravitreal injections of four NMDA concentrations followed by varying TcapQ488 doses. Fluorescence fundus imaging was performed sequentially in vivo using a confocal scanning laser ophthalmoscope and individual RGCs displaying activated probe were counted and analyzed. Rats also underwent electroretinography following intravitreal injection of probe. In vivo fluorescence fundus imaging revealed distinct single-cell probe activation as an indicator of RGC apoptosis induced by intravitreal NMDA injection that corresponded to the identical cells observed in retinal flat mounts of the same eye. Peak activation of probe in vivo was detected 12 hours post probe injection. Detectable fluorescent RGCs increased with increasing NMDA concentration; sensitivity of detection generally increased with increasing TcapQ488 dose until saturating at 0.387 nmol. Electroretinography following intravitreal injections of TcapQ488 showed no significant difference compared with control injections. We optimized the signal-to-noise ratio of a caspase-activatable cell penetrating peptide probe for quantitative non-invasive detection of RGC apoptosis in vivo. Full characterization of probe performance in this setting creates an important in vivo imaging
Lu, Yongliang; Feng, Wenming; Sun, Xinrong; Tang, Chengwu; Wang, Xiang; Shen, Mo
Hepatic metastasis is one of the critical progressions of colon cancer. Blocking this process is key to prolonging survival time in cancer patients. Studies on activatable cell-penetrating peptides (dtACPPs) have demonstrated their potential as gene carriers. It showed high tumor cell-targeting specificity and transfection efficiency and low cytotoxicity in the in vitro settings of drug delivery. However, using this system to silence target genes to inhibit metastasis in colorectal cancer cells has not been widely reported and requires further investigation. In this study, we observed that expression of Rac1, a key molecule for cytoskeletal reorganization, was higher in hepatic metastatic tumor tissue compared with prime colon cancer tissue and that patients with high Rac1-expressing colon cancer showed shorter survival time. Base on these findings, we created dtACPP-PEG-DGL (dtACPPD)/shRac1 nanoparticles and demonstrated that they downregulated Rac1 expression in colon cancer cells. Moreover, we observed inhibitory effects on migration, invasion and adhesion in HCT116 colorectal cancer cells in vitro, and our results showed that Rac1 regulated colon cancer cell matrix adhesion through the regulation of cytofilament dynamics. Moreover, mechanically, repression of Rac1 inhibiting cells migration and invasion by enhancing cell to cell adhesion and reducing cell to extracellular matrix adhesion. Furthermore, when atCDPPD/shRac1 nanoparticles were administered intravenously to a HCT116 xenograft model, significant tumor metastasis to the liver was inhibited. Our results suggest that atCDPP/shRac1 nanoparticles may enable the blockade of hepatic metastasis in colon cancer. PMID:27791203
Xiang, Bai; Jia, Xue-Li; Qi, Jin-Long; Yang, Li-Ping; Sun, Wei-Hong; Yan, Xiao; Yang, Shao-Kun; Cao, De-Ying; Du, Qing; Qi, Xian-Rong
As a potent therapeutic agent, small interfering RNA (siRNA) has been exploited to silence critical genes involved in tumor initiation and progression. However, development of a desirable delivery system is required to overcome the unfavorable properties of siRNA such as its high degradability, molecular size, and negative charge to help increase its accumulation in tumor tissues and promote efficient cellular uptake and endosomal/lysosomal escape of the nucleic acids. In this study, we developed a new activatable cell-penetrating peptide (ACPP) that is responsive to an acidic tumor microenvironment, which was then used to modify the surfaces of siRNA-loaded liposomes. The ACPP is composed of a cell-penetrating peptide (CPP), an acid-labile linker (hydrazone), and a polyanionic domain, including glutamic acid and histidine. In the systemic circulation (pH 7.4), the surface polycationic moieties of the CPP (polyarginine) are “shielded” by the intramolecular electrostatic interaction of the inhibitory domain. When exposed to a lower pH, a common property of solid tumors, the ACPP undergoes acid-catalyzed breakage at the hydrazone site, and the consequent protonation of histidine residues promotes detachment of the inhibitory peptide. Subsequently, the unshielded CPP would facilitate the cellular membrane penetration and efficient endosomal/lysosomal evasion of liposomal siRNA. A series of investigations demonstrated that once exposed to an acidic pH, the ACPP-modified liposomes showed elevated cellular uptake, downregulated expression of polo-like kinase 1, and augmented cell apoptosis. In addition, favorable siRNA avoidance of the endosome/lysosome was observed in both MCF-7 and A549 cells, followed by effective cytoplasmic release. In view of its acid sensitivity and therapeutic potency, this newly developed pH-responsive and ACPP-mediated liposome system represents a potential platform for siRNA-based cancer treatment. PMID:28405163
Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid
Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP
Satake, K; Ha, S; Hiura, A; Nishiwaki, H; Haku, A; Umeyama, K
The coagulation disturbance observed during severe acute pancreatitis before and after the infusion of a new synthetic low molecular weight protease inhibitor (Fut-175) was compared. The coagulo-fibrinolytic changes after acute pancreatitis was induced by the intraductal injection of an autologous bile and trypsin mixture showed decreased platelet counts, decreased plasma fibrinogen levels, prolonged partial prothrombin time and increased fibrinogen degradation products. In addition, markers of hypercoagulation showed increased fibrin-peptide A and decreased antithrombin III. The two markers of fibrinolysis showed increased B beta 15-42 immunoreactive peptide and decreased alpha 2 antiplasmin. After the infusion of Fut-175, the coagulo-fibrinolytic abnormalities, which were observed during severe acute pancreatitis without infusion of Fut-175, were improved. Furthermore, Fut-175 could suppress the rise in fibrino-peptide A and B beta 15-42 immunoreactive peptide and decrease in antithrombin III and alpha 2 antiplasmin. Thus, Fut-175 seems to be an effective inhibitor of protease-mediated hypercoagulation and fibrinolysis in severe acute pancreatitis.
Cox, A C
Deinagkistrodon acutus venom contains a collection of anticoagulant proteins that has been reported to prevent prothrombinase assembly (Teng and Seegers, 1981, Thromb. Res. 23, 255). A partial sequence indicates that these proteins are related to the functionally equivalent protein in Trimeresurus flavoviridis (Atoda et al., 1991, J. Biochem. 106, 808). Inhibition of prothrombinase, the complex of Factors Xa and Va combined with phospholipids, is expressed in bovine, human, and rat plasmas as indicated by an assay dependent on only prothrombinase activity. The concentration dependence of inhibition of prothrombin conversion by different combinations of the components of bovine prothrombinase under the same conditions yielded estimates of apparent dissociation constants of 104 nM and 2 nM for complexes of the inhibitor with Factor Xa and with Factors Xa and Va, respectively. Because this inhibitor does not prevent Factor Xa alone from converting prothrombin, but blocks the other combinations, we conclude the inhibitor prevents the complex of Factors Xa and Va from binding to phospholipid surfaces and to prothrombin. The inhibitor also blocks the activation of Factor X by Factor VIIa and thromboplastin as well. However, the inhibitor has no effect on thrombin-induced clotting or fibrinolysis induced by either plasminogen activator or streptokinase. Therefore, this inhibitor has several properties required of an anticoagulant, therapeutic agent.
Hatfield, M. Jason; Potter, Philip M.
Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191
Tharkur, Jeremy; Teblum, Andrew; Basumallick, Srijita; Shah, Rikhav; Cantarero, Karishma; Maity, Niharika; Rifai, Sara; Doshi, Mona; Gesquiere, Andre J.; Santra, Swadeshmukul
In recent years, activatable Quantum Dots (AQdots) are gaining popularity in a number of chemical and biological sensing applications. A basic design of AQdot probes involves a suitable quencher which is capable of altering optical properties of the Qdots. In our previous studies we have shown that CdS:Mn/ZnS fluorescence can be effectively quenched using small molecule quenchers (such as dopamine, chemotherapeutic drug) as well as iron oxide nanoparticle via electron/energy transfer process. We have also shown that the quenched Qdot fluorescence can be restored when the Qdots are separated from the quencher. Using Qdot based activatable probes, we detected intracellular drug release event. Qdot fluorescence was restored upon interaction with the intracellular glutathione (GSH). In this paper, we report a GSH induced quenching of water-soluble N-Acetyl Cysteine (NAC) surface-conjugated Cds:Mn/ZnS Qdots. Quenching of NAC-Qdots was due to aggregation of Qdots in solution. This aggregation induced fluorescence quenching phenomenon resembles with the self-quenching phenomenon of traditional organic fluorescence dyes at high concentrations. UV-VIS and fluorescence emission spectroscopy data support the interaction and binding of GSH with the NAC-Qdots. Increase in particle size due to GSH induced aggregation of NAC-Qdots was confirmed by the Dynamic Light Scattering (DLS) data.
The Comparison of the Outcomes between Primary PCI, Fibrinolysis, and No Reperfusion in Patients ≥ 75 Years Old with ST-Segment Elevation Myocardial Infarction: Results from the Chinese Acute Myocardial Infarction (CAMI) Registry
Peiyuan, He; Jingang, Yang; Haiyan, Xu; Xiaojin, Gao; Ying, Xian; Yuan, Wu; Wei, Li; Yang, Wang; Xinran, Tang; Ruohua, Yan; Chen, Jin; Lei, Song; Xuan, Zhang; Rui, Fu; Yunqing, Ye; Qiuting, Dong; Hui, Sun; Xinxin, Yan; Runlin, Gao; Yuejin, Yang
Background Only a few randomized trials have analyzed the clinical outcomes of elderly ST-segment elevation myocardial infarction (STEMI) patients (≥ 75 years old). Therefore, the best reperfusion strategy has not been well established. An observational study focused on clinical outcomes was performed in this population. Methods Based on the national registry on STEMI patients, the in-hospital outcomes of elderly patients with different reperfusion strategies were compared. The primary endpoint was defined as death. Secondary endpoints included recurrent myocardial infarction, ischemia driven revascularization, myocardial infarction related complications, and major bleeding. Multivariable regression analysis was performed to adjust for the baseline disparities between the groups. Results Patients who had primary percutaneous coronary intervention (PCI) or fibrinolysis were relatively younger. They came to hospital earlier, and had lower risk of death compared with patients who had no reperfusion. The guideline recommended medications were more frequently used in patients with primary PCI during the hospitalization and at discharge. The rates of death were 7.7%, 15.0%, and 19.9% respectively, with primary PCI, fibrinolysis, and no reperfusion (P < 0.001). Patients having primary PCI also had lower rates of heart failure, mechanical complications, and cardiac arrest compared with fibrinolysis and no reperfusion (P < 0.05). The rates of hemorrhage stroke (0.3%, 0.6%, and 0.1%) and other major bleeding (3.0%, 5.0%, and 3.1%) were similar in the primary PCI, fibrinolysis, and no reperfusion group (P > 0.05). In the multivariable regression analysis, primary PCI outweighs no reperfusion in predicting the in-hospital death in patients ≥ 75 years old. However, fibrinolysis does not. Conclusions Early reperfusion, especially primary PCI was safe and effective with absolute reduction of mortality compared with no reperfusion. However, certain randomized trials were
Primary angioplasty vs. fibrinolysis in very old patients with acute myocardial infarction: TRIANA (TRatamiento del Infarto Agudo de miocardio eN Ancianos) randomized trial and pooled analysis with previous studies.
Bueno, Héctor; Betriu, Amadeo; Heras, Magda; Alonso, Joaquín J; Cequier, Angel; García, Eulogio J; López-Sendón, José L; Macaya, Carlos; Hernández-Antolín, Rosana
To compare primary percutaneous coronary intervention (pPCI) and fibrinolysis in very old patients with ST-segment elevation myocardial infarction (STEMI), in whom head-to-head comparisons between both strategies are scarce. Patients ≥75 years old with STEMI <6 h were randomized to pPCI or fibrinolysis. The primary endpoint was a composite of all-cause mortality, re-infarction, or disabling stroke at 30 days. The trial was prematurely stopped due to slow recruitment after enrolling 266 patients (134 allocated to pPCI and 132 to fibrinolysis). Both groups were well balanced in baseline characteristics. Mean age was 81 years. The primary endpoint was reached in 25 patients in the pPCI group (18.9%) and 34 (25.4%) in the fibrinolysis arm [odds ratio (OR), 0.69; 95% confidence interval (CI) 0.38-1.23; P = 0.21]. Similarly, non-significant reductions were found in death (13.6 vs. 17.2%, P = 0.43), re-infarction (5.3 vs. 8.2%, P = 0.35), or disabling stroke (0.8 vs. 3.0%, P = 0.18). Recurrent ischaemia was less common in pPCI-treated patients (0.8 vs. 9.7%, P< 0.001). No differences were found in major bleeds. A pooled analysis with the two previous reperfusion trials performed in older patients showed an advantage of pPCI over fibrinolysis in reducing death, re-infarction, or stroke at 30 days (OR, 0.64; 95% CI 0.45-0.91). Primary PCI seems to be the best reperfusion therapy for STEMI even for the oldest patients. Early contemporary fibrinolytic therapy may be a safe alternative to pPCI in the elderly when this is not available.
Primary angioplasty vs. fibrinolysis in very old patients with acute myocardial infarction: TRIANA (TRatamiento del Infarto Agudo de miocardio eN Ancianos) randomized trial and pooled analysis with previous studies
Bueno, Héctor; Betriu, Amadeo; Heras, Magda; Alonso, Joaquín J.; Cequier, Angel; García, Eulogio J.; López-Sendón, José L.; Macaya, Carlos; Hernández-Antolín, Rosana; Bueno, Héctor; Hernández-Antolín, Rosana; Alonso, Joaquín J.; Betriu, Amadeo; Cequier, Angel; García, Eulogio J.; Heras, Magda; López-Sendón, José L.; Macaya, Carlos; Azpitarte, José; Sanz, Ginés; Chamorro, Angel; López-Palop, Ramón; Sionis, Alex; Arós, Fernando; García-Fernández, Eulogio; Rubio, Rafael; Hernández, Felipe; Tascón, Juan Carlos; Moreu, José; Betriu, Amadeu; Heras, Magda; Hernández-Antolín, Rosana; Fernández-Ortiz, Antonio; Morís, César; de Posada, Ignacio Sánchez; Cequier, Ángel; Esplugas, Enrique; Melgares, Rafael; Bosa, Francisco; García-González, Martín Jesús; Lezáun, Román; Carmona, José Ramón; Vázquez, José Manuel; Castro-Beiras, Alfonso; Picart, Joan García; de Rozas, José Domínguez; Fernández, José Díaz; Vázquez, Felipe Fernández; Alonso, Norberto; Zueco, José Javier; San José, José María; San Román, Alberto; Hernández, Carolina; García, José María Hernández; Alcántara, Ángel García; Bethencourt, Armando; Fiol, Miquel; Mancisidor, Xabier; Mancisidor, Xabier; Ruiz, Rafael; Hidalgo, Rafael; Sobrino, Nicolás; Maqueda, Isidoro González; Torres, Alfonso; Arós, Fernando; Amaro, Antonio; Jaquet, Michel
Aims To compare primary percutaneous coronary intervention (pPCI) and fibrinolysis in very old patients with ST-segment elevation myocardial infarction (STEMI), in whom head-to-head comparisons between both strategies are scarce. Methods and results Patients ≥75 years old with STEMI <6 h were randomized to pPCI or fibrinolysis. The primary endpoint was a composite of all-cause mortality, re-infarction, or disabling stroke at 30 days. The trial was prematurely stopped due to slow recruitment after enroling 266 patients (134 allocated to pPCI and 132 to fibrinolysis). Both groups were well balanced in baseline characteristics. Mean age was 81 years. The primary endpoint was reached in 25 patients in the pPCI group (18.9%) and 34 (25.4%) in the fibrinolysis arm [odds ratio (OR), 0.69; 95% confidence interval (CI) 0.38–1.23; P = 0.21]. Similarly, non-significant reductions were found in death (13.6 vs. 17.2%, P = 0.43), re-infarction (5.3 vs. 8.2%, P = 0.35), or disabling stroke (0.8 vs. 3.0%, P = 0.18). Recurrent ischaemia was less common in pPCI-treated patients (0.8 vs. 9.7%, P< 0.001). No differences were found in major bleeds. A pooled analysis with the two previous reperfusion trials performed in older patients showed an advantage of pPCI over fibrinolysis in reducing death, re-infarction, or stroke at 30 days (OR, 0.64; 95% CI 0.45–0.91). Conclusion Primary PCI seems to be the best reperfusion therapy for STEMI even for the oldest patients. Early contemporary fibrinolytic therapy may be a safe alternative to pPCI in the elderly when this is not available. Clinicaltrials.gov # NCT00257309. PMID:20971744
... LE, Heslop HE, Weitz JI, Anastasi J, eds. Hematology: Basic Principles and Practice . 6th ed. Philadelphia, PA: ... 2/1/2016 Updated by: Todd Gersten, MD, Hematology/Oncology, Florida Cancer Specialists & Research Institute, Wellington, FL. ...
Kaufmann, Joel C D; Krause, Benjamin S; Grimm, Christiane; Ritter, Eglof; Hegemann, Peter; Bartl, Franz J
Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. ChR variants with a red-shifted absorption maximum, such as the modified Volvox carteri ChR1 red-activatable channelrhodopsin ("ReaChR," λmax = 527 nm), are of particular interest because longer wavelengths allow optical excitation of cells in deeper layers of organic tissue. In all ChRs investigated so far, proton transfer reactions and hydrogen bond changes are crucial for the formation of the ion-conducting pore and the selectivity for protons versus cations, such as Na(+), K(+), and Ca(2+) (1). By using a combination of electrophysiological measurements and UV-visible and FTIR spectroscopy, we characterized the proton transfer events in the photocycle of ReaChR and describe their relevance for its function. 1) The central gate residue Glu(130) (Glu(90) in Chlamydomonas reinhardtii (Cr) ChR2) (i) undergoes a hydrogen bond change in D → K transition and (ii) deprotonates in K → M transition. Its negative charge in the open state is decisive for proton selectivity. 2) The counter-ion Asp(293) (Asp(253) in CrChR2) receives the retinal Schiff base proton during M-state formation. Starting from M, a photocycle branching occurs involving (i) a direct M → D transition and (ii) formation of late photointermediates N and O. 3) The DC pair residue Asp(196) (Asp(156) in CrChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15-syn-retinal at the expense of 15-anti, which is the predominant isomer in the wild type, and abolishes the peak current in electrophysiological measurements. This suggests that the peak current is formed by 15-anti species, whereas 15-syn species contribute only to the stationary current. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Majumdar, Poulomi; Cui, Xiaoneng; Xu, Kejing; Zhao, Jianzhang
stimuli-activatable transition metal complexes.
Knapik, Jadwiga; Korabiowska, Monika; Hönig, Johannes F; Grohmann, Ulrike; Undas, Anetta; Bartkowski, Stanislaw B; Brinck, Ulrich
Selected parameters of blood coagulation and fibrinolysis were evaluated in 20 patients with squamous cell carcinoma of the oral cavity (stages T2/4 N0/3 M0). The control group consisted of 10 healthy age-matched men. The clotting time, calcium plasma clotting time, prothrombin time index and fibrinogen levels in the patients were similar to those observed in the controls. Increased fibrin degradation products (FDP) to 480 and 80 microg/min were found in patients before and after surgical treatment, respectively. We conclude that, in patients treated surgically because of carcinoma of the oral cavity, there might be increased activity of the fibrinolytic system both prior to and after operation.
Schmidt, E B; Nielsen, L K; Pedersen, J O; Kornerup, H J; Dyerberg, J
We have studied the effect of dietary supplementation with 4 g of n-3 polyunsaturated fatty acids (n-3 PUFA) daily for 6 wk on plasma lipids, haemostasis and monocyte chemotaxis in 10 patients with untreated hypertension. Total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides did not change, but the ratio of total to HDL-cholesterol was significantly reduced after the fish oil supplement. Platelet function was unaltered by intake of n-3. Plasma fibrinogen and fibronectin decreased after supplementation with n-3 PUFA, while the effects on fibrinolysis were equivocal. Monocyte chemotaxis was reduced by the supplement. These data lend support to a role for an increased intake of n-3 PUFA in the management of patients with hypertension.
Roy, Shreosee; Saha, Kaushik; Mukherjee, Krishnendu; Dutta, Santanu; Mukhopadhyay, Debasis; Das, Indranil; Raychaudhuri, Gargi
Coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) is associated with intense activation of hemostatic mechanisms. But the precise knowledge of the effects of eliminating CPB in patients undergoing off-pump coronary artery bypass grafting (CABG) are not well established. The present study was carried out to compare and document the changes in selected coagulation and fibrinolysis variables in patients undergoing on-pump and off-pump CABG (OPCAB). A total of 42 patients of on-pump and 31 patients of off-pump CABG were selected for the study. Platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), Fibrinogen and D-dimer levels were measured immediately, 24 h and 7 days after operation and compared with the baseline preoperative values. Statistical analysis was done by mixed ANOVA for repeated measures and Post-hoc tests using the Bonferroni correction, Chi square and unpaired t test. All the parameters were significantly changed (P < 0.05) with the time. Platelet counts, fibrinogen and D-dimer levels were significantly different between on-pump and off-pump CABG patients on immediate and 24 h postoperative period and attained almost same level after 7 days of operation. Fibrinogen level and platelet counts were increased after a sharp fall in the immediate post-operative period whereas D-dimer levels were persistently increased with a sharp peak of rise in the immediate post-operative period in on-pump group. On-pump surgery was associated with excessive fibrinolytic activity immediately after operation. The off-pump group demonstrated less activation of coagulation and fibrinolysis and delayed postoperative response that became almost equal to the on-pump group in the later postoperative period.
Falsoleiman, H.; Fatehi, G. H.; Dehghani, M; Shakeri, M. T.; Bayani, Baktash; Ahmadi, Mostafa; Rohani, Atoosheh
Objective: The aim of the present study was to compare the short-term and 6-month clinical outcome, and survival in patients older than 60 years with ST-elevation myocardial infarction randomized to either primary percutaneous coronary intervention (PPCI) or thrombolysis. Materials and Methods: 82 patients with STEMI older than 60 years were randomized to either primary PCI or thrombolysis from September 2006 to August 2008. Angiograms were reviewed by two interventionalists not involved in the study. Patients randomized to primary PCI received Aspirin and 600 mg Clopidogrel. Heparin was administered in conjunction with PCI. Patients randomized to thrombolysis received Aspirin followed by streptokinase infusion for one hour. Rescue PCI was considered if there was ongoing pain and ST-segment resolution was <50% at 90 min. after initiation of thrombolysis or chest pain recurred with ST-segment elevation within 24 hours. All patients were followed up for 6 months. End points were reinfarction and cardiac death using competing-risks regression estimation. Results: The mean time from hospital admission to start of streptokinase infusion was 31 ± 15 min and door to balloon time was 70 ± 25 min. There was no significant difference between the groups in the number of deaths and reinfarctions at 6 months. As expected, the fibrinolysis group had a higher rate of revascularization and heart failure. Conclusion: The higher rates of heart failure and need for revascularization in the fibrinolysis group reinforces benefits of PPCI in patients older than 60 years. PPCI in those who are 60 years and above with AMI is safe and cost effective. PMID:23439588
Huq, Muhammad Aminul; Takeyama, Naoshi; Harada, Makoto; Miki, Yasuo; Takeuchi, Akinori; Inoue, Sousuke; Nakagawa, Takashi; Kanou, Hideki; Hirakawa, Akihiko; Noguchi, Hiroshi
Impaired fibrinolysis is associated with a higher incidence of both multiple organ dysfunction and mortality in the intensive care unit (ICU). Plasminogen activator inhibitor (PAI)-1 is the chief inhibitor of fibrinolysis. We investigated the influence of the 4G/5G polymorphism (rs1799768) of the PAI-1 gene on the plasma PAI-1 level and the outcome of critically ill patients. In 41 consecutive patients admitted to the ICU, PAI-1 gene polymorphism was assessed, plasma PAI-1 and arterial lactate concentrations were measured and clinical severity scores were recorded. Homozygotes for the 4G allele had higher plasma levels of PAI-1 antigen. The mean ± SD PAI-1 antigen level was 193.31 ± 167.93 ng/ml for the 4G/4G genotype, 100.67 ± 114.16 ng/ml for the 4G/5G genotype and 0.43 ± 0.53 ng/ml for the 5G/5G genotype. There was a significant correlation between plasma PAI-1 and arterial lactate concentrations, as well as between PAI-1 and severity scores. The mortality rate was 63, 33 and 0% for patients with the 4G/4G, 4G/5G and 5G/5G genotypes, respectively. These results demonstrate that the 4G/5G polymorphism of the PAI-1 gene affects the plasma PAI-1 concentration, which could impair fibrinolysis and cause organ failure, and thus the presence of the 4G allele increases the risk of death. Copyright © 2011 S. Karger AG, Basel.
Impaired activation of the fibrinolytic system in children with Henoch-Schönlein purpura: beneficial effect of hydrocortisone plus Sigma-aminocaproic acid therapy on disappearance rate of cutaneous vasculitis and fibrinolysis.
Prandota, J; Pankow-Prandota, L; Kotecki, L
/mL, respectively, t = 2.33, P <.045). Both treatment regimens significantly improved fibrinolysis, which manifested as a shortening of ELT, but the mean values of this parameter remained within normal range. After treatment with H plus EACA, the skin lesions started to disappear significantly faster compared with the H alone regimen (2.28 +/- 0.45 days vs. 4.12 +/- 1.05, t = 4.41, P <.0023). In four of six patients receiving H plus EACA therapy, an approximately 20% decrease of systolic and diastolic arterial blood pressure lasting for 5 to 7 hours after administration of EACA was observed. These results may suggest that children with HSP have impaired plasma fibrinolytic activity and that an increased release of plasminogen activator inhibitor-1 (PAI-1) might be, at least in part, responsible for this phenomenon. Concomitant use of H (approximately 10 mg/kg/d) plus EACA (approximately 100 mg/kg/d) for a few days opens new therapeutic possibilities in some children with HSP.
Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.
Malone, Christopher D.; Olson, Emilia S.; Mattrey, Robert F.; Jiang, Tao; Tsien, Roger Y.; Nguyen, Quyen T.
Purpose The ability to detect small malignant lesions with magnetic resonance imaging (MRI) is limited by inadequate accumulations of Gd with standard chelate agents. To date, no T1-targeted agents have proven superiority to Gd chelates in their ability to detect small tumors at clinically relevant field strengths. Activatable cell-penetrating peptides and their Gd-loaded dendrimeric form (ACPPD-Gd) have been shown to selectively accumulate in tumors. In this study we compared the performance of ACPPD-Gd vs. untargeted Gd chelates to detect small tumors in rodent models using a clinical 3T-MR system. Materials and Methods This study was approved by the Institutional-Animal Care-and-Use Committee. 2 of 4 inguinal breast fat pads of 16 albino-C57BL/6 mice were inoculated with tumor Py8119 cells and the other 2 with saline at random. MRI at 3T was performed at 4, 9, and 14 days after inoculation on 8 mice 24-hours after injection of 0.036mmol Gd/kg (ACPPD-Gd), and before and 2–3 minutes after 0.1 mmol/kg gadobutrol on the other 8 mice. T1-weighted (T1w) tumor signal normalized to muscle, was compared among the non-contrast, gadobutrol, and ACPPD-Gd groups using ANOVA. Experienced and trainee readers blinded to experimental conditions assessed for the presence of tumor in each of the 4 breast regions. Receiver operator characteristic (ROC) curves and area-under-curve (AUC) values were constructed and analyzed. Results Tumors ≥1mm3 were iso-intense to muscle without contrast on T1w sequences. They enhanced diffusely and homogeneously by 57±20% (p<0.001) 24 hours after ACPPD-Gd and by 25±13% (p<0.001) immediately after gadobutrol. The nearly 2-fold difference was similar for small tumors (1-5mm3) (45±19% vs. 19±18%, p = 0.03). ACPPD-Gd tended to improve tumor detection by an experienced reader (AUC 0.98 vs 0.91) and significantly more for a trainee (0.93 vs. 0.82, p = 0.02) compared to gadobutrol. This improvement was more pronounced when obvious tumors (>5mm3
Kunadian, Babu; Vijayalakshmi, Kunadian; Dunning, Joel; Sutton, Andrew; de Belder, Mark A
The large randomized trials on rescue angioplasty (rPCI) have been interpreted by some as showing differing results. We compared the protocols, demographics and 6-month clinical outcomes of the MERLIN trial with the RESCUE I and REACT trials to assess their differences. The RESCUE I trial did not involve the use of stenting or glycoprotein IIb/IIIa inhibitors, and patients with previous MI were excluded. The recruitment rate in MERLIN was 30.7 patients per center per year vs. 3.3 in REACT and 2.4 in the RESCUE I trial. The patients in the MERLIN trial were older. Streptokinase was used more commonly in the MERLIN trial: 96% vs. 60% in REACT. In the rPCI arm, pain onset to lysis time was longer, 180 vs. 140 minutes in MERLIN, but lysis to angiography (146 vs. 274 minutes) and pain onset to angiography (327 vs. 414 minute) times were shorter compared with the REACT trial. In the rPCI arms, mortality at 6 months was lower in the REACT trial than in the MERLIN trial although the difference was not statistically significant. This numeric difference in mortality is unlikely to be explained by the differences in the use of glycoprotein IIb/IIIa inhibitors or stenting. In the rPCI arms, reinfarction rates were higher in the MERLIN trial (7.8% vs. 2.1%), possibly due to lower rates of stenting (50.3% vs. 68.5%) and less use of glycoprotein IIb/IIIa inhibitors (3.3% vs. 43.4%). The three trials used different definitions of heart failure, with a trend towards a reduction in heart failure in all three studies. These trials differ in various aspects. The RESCUE I trial enrolled lower-risk patients than the MERLIN trial and was done without the use of stents and glycoprotein IIb/IIIa inhibitors. The MERLIN trial is widely applicable to patients receiving streptokinase and had no age limit. REACT patients were younger and were enrolled more selectively. Although some outcomes were similar, differences in patient recruitment and protocols may have accounted for the numerical
Barazzone, C; Belin, D; Piguet, P F; Vassalli, J D; Sappino, A P
Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury. PMID:8981909
Hasumuma, Ryoichi; Kawaguchi, Kiichiro; Kikuchi, Sei-ichi; Sugiyama, Tsuyoshi; Kumazawa, Yoshio
The effects of isoflavones and of a derivative of soybeans fermented with Bacillus subtilis, designated Nattoesse, on the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha) and fibrinolysis were investigated in vivo. The dietary supplement Nattoesse contains several isoflavones. Therefore, we examined the effects of individual isoflavones (daidzein, daidzin, genistein, and genistin) on the LPS-induced production of TNF-alpha. Intraperitoneal injections of daidzein, daidzin, and genistin (but not of genistein before a challenge with LPS) resulted in significant depression of serum levels of TNF-alpha in mice. Daidzein had the strongest activity in this assay. Oral administration of daidzein to mice also had a significant suppressive effect, as compared with that of the Citrus flavanone naringin. In galactosamine-sensitized mice, by contrast, the suppression of LPS-induced lethal shock by daidzein was very weak. Nattoesse did not inhibit the production of TNF-alpha nor did it prevent lethal shock. However, oral administration of Nattoesse to mice significantly suppressed LPS-induced increases in scores of the fibrin degradation product, and the effect was both dose- and time-dependent. Thus, it appears that Nattoesse has fibrinolytic activity during LPS-induced circulatory failure.
Jevtic, Dragana; Zecevic, Zeljko; Veljkovic, Dobrila; Dopsaj, Violeta; Radojicic, Zoran; Elezovic, Ivo
Prediction of veno-occlusive disease (VOD), its precise diagnosis, and treatment have been the subject of various studies, but still remain unclear. Our goal was to investigate the levels of activated coagulation and fibrinolysis markers and natural anticoagulants in pediatric patients with VOD after hematopoietic stem cell transplantation (HSCT). We investigated 47 pediatric patients: 20 with neuroblastoma, 17 with leukemias, and 10 with lymphomas and measured the values of antithrombin (AT), protein C (PC), fibrinogen (FI), thrombin AT complex, prothrombin fragments 1+2 (F1+2), and D-dimer from day -7 to day +30 post-HSCT. Patients were monitored for the occurrence of VOD, and it occurred in 10 patients at a median post-HSCT day of 17.5 (range: 2 to 28 d). In the VOD group, at baseline the levels of FI were significantly lower, and on days +7 and +14 a relevant difference existed in F1+2 levels. The levels of PC were significantly lower on day +14. Logistic multivariate regression analysis between the groups showed significantly different D-dimer levels on day +14. On day +30, the levels of PC, AT, and F1+2 were different between these 2 groups of patients. The levels of D-dimer and F1+2 were increased, and PC and FI decreased before the clinical onset of VOD. The parameter differences may have a predictive value in VOD onset, which makes them candidates to be routinely monitored in patients after HSCT.
Alwakeel, J; Gader, A M; Hurieb, S; al-Momen, A K; Mitwalli, A; Abu Aisha, H
Coagulation inhibitors and fibrinolytic parameters were studied in twelve patients on continuous ambulatory peritoneal dialysis (CAPD) and ten patients on haemodialysis (HD). Patients on CAPD exhibited higher levels of ATIII and proteins C and S than those on HD. No significant differences were noted in tPA and PAI levels. Both groups of patients showed higher levels of tPA than controls. Besides, patients on HD had significantly lower levels of ATIII and protein C than controls. PAI levels in both patient groups were similar to those of the controls, but tPA levels were higher in patients than in controls. These results indicate that HD is associated with marked diminution in the circulating levels of coagulation inhibitors. This is in contrast to CAPD patients who showed elevated levels of these inhibitors, despite their significant loss in the dialysate. The finding of enhanced fibrinolysis in both patient groups may be a natural protective mechanism against the development of a thrombotic tendency.
Messa, G; Blardi, P; La Placa, G; Puccetti, L; Ghezzi, A
In this study the profibrinolytic activity of two single oral doses of mesoglycan was evaluated. Furthermore, a mathematical model describing the patterns of the resulting phenomena was applied. Ten patients with impaired fibrinolytic system (euglobulin lysis time > 180 min) were enrolled in the study. In the morning following a 24 hour fast period, the patients were given orally a single dose (100 and 50 mg) of mesoglycan and placebo, with an interval of 48 hours between each treatment. The following parameters were evaluated at the time 0 and after 2, 4, 6, 8, 10 and 12 hours from each administration: tissue plasminogen activator (t-PA) and its inhibitor PAI-1, euglobulin lysis time, plasminogen and alfa-2-antiplasmin as indexes of the fibrinolytic system; aPTT, TT, fibrinogen as indexes of the hemostatic-coagulative system. Mesoglycan showed a dose-dependent profibrinolytic activity, that was also present after placebo but in a less entity. The mathematical study confirms the experimental observations and thus may allow to describe, with a high degree of approximation, the in vivo pharmacology of mesoglycan through the use of the mathematical function.
Bork, Konrad; Kleist, Rouven; Hardt, Jochen; Witzke, Günther
In a subgroup of hereditary angioedema (HAE) patients with normal C1-esterase inhibitor levels, HAE is caused by a Thr309Lys mutation in the coagulation factor XII (F12) gene. The aim of this study was to examine elements of the kallikrein-kinin system ('contact system') and the downstream-linked coagulation, complement and fibrinolytic systems in the plasma of six patients with HAE caused by the Thr309Lys mutation and healthy probands. Blood samples were taken from participants during the symptom-free interval between attacks. Samples were analyzed for activity and concentrations of components of the kallikrein-kinin system and linked enzyme systems. The mean FXII clotting activity was 90% in patients with the FXII mutation, and the concentration of FXIIa was 4.1 ng/ml; this did not differ from healthy probands. Mean prekallikrein amidolytic activity and high-molecular-weight kininogen clotting activity were 130 and 144%, respectively, both higher than in healthy probands. The mean kallikrein-like activity of the HAE patients was 11.4 U/l and did not differ from healthy probands. There was no difference in FXII surface activation by silicon dioxide or in kallikrein-like activity with and without activation by dextran sulfate. Contrary to the results of a recently published study, no indication that the Thr309Lys mutation causes a 'gain-of-function' of FXIIa was observed in this investigation.
Mosad, Eman; Elsayh, Khalid I; Eltayeb, Azza A
Inflammation and coagulation occur concomitantly in sepsis. Thrombin activates platelet that leads to P-selectin translocation, which upregulate tissue factor (TF) generation. Tissue factor pathway inhibitor (TFPI) is an anticoagulant that modulates coagulation induced by TF. The term non-overt disseminated intravascular coagulation (DIC) refers to a state of affairs prevalent before the occurrence of overt DIC. It was suggested that an initiation of treatment in non-overt DIC has better outcome than overt DIC. This study investigated the role of TFPI level, P-selectin, and thrombin activation markers in non-overt and overt DIC induced by sepsis and its relationship to outcome and organ dysfunction as measured by the Sequential Organ Failure Assessment (SOFA) score. It included 176 patients with sepsis. They were admitted to the pediatric intensive care unit (ICU).They included 144 cases of non-overt DIC and 32 cases of overt DIC. There was a significant difference in hemostatic markers, platelet count, partial thromboplastin time (PTT), P-selectin, thrombin activation markers, TFPI, and DIC score between overt and non-overt DIC in both groups. It was noticed that P-selectin was positively correlated with DIC score, fibrinogen consumption, fibrinolysis (D-dimer), thrombin activation markers, and TFPI. Tissue factor pathway inhibitor was significantly correlated with fibrinolysis, DIC score, and prothrombin fragment 1+2. Sequential Organ Failure Assessment score was correlated with DIC score and other hemostatic markers in patients with overt DIC. To improve the outcome of patients with DIC, there is a need to establish more diagnostic criteria for non-overt-DIC. Plasma levels of TFPI and P-selectin may be helpful in this respect.
van den Boogaard, Florry E; Hofstra, Jorrit J; Brands, Xanthe; Levi, Marcel M; Roelofs, Joris J T H; Zaat, Sebastiaan A J; Van't Veer, Cornelis; van der Poll, Tom; Schultz, Marcus J
Critically ill patients are at a constant risk of direct (e.g., by pneumonia) or indirect lung injury (e.g., by sepsis). Excessive alveolar fibrin deposition is a prominent feature of lung injury, undermining pulmonary integrity and function. We examined the effect of local administration of recombinant human tissue factor pathway inhibitor (rh-TFPI), a natural anticoagulant, in two well-established models of lung injury in rats. Rats received intratracheal instillation of Pseudomonas aeruginosa, causing direct lung injury, or they received an intravenous injection of Escherichia coli lipopolysaccharide (LPS), causing indirect lung injury. Rats were randomized to local treatment with rh-TFPI or placebo through repeated nebulization. Challenge with P. aeruginosa or LPS was associated with increased coagulation and decreased fibrinolysis in bronchoalveolar lavage fluid (BALF) and plasma. Rh-TFPI levels in BALF increased after nebulization, whereas plasma rh-TFPI levels remained low and systemic TFPI activity was not affected. Nebulization of rh-TFPI attenuated pulmonary and systemic coagulation in both models, without affecting fibrinolysis. Nebulization of rh-TFPI modestly reduced the inflammatory response and bacterial growth of P. aeruginosa in the alveolar compartment. Local treatment with rh-TFPI does not alter systemic TFPI activity; however, it attenuates both pulmonary and systemic coagulopathy. Furthermore, nebulized rh-TFPI modestly reduces the pulmonary inflammatory response and allows increased bacterial clearance in rats with direct lung injury caused by P. aeruginosa.
Wong, Cheuk-Kit; Leon de la Barra, Sophia; Herbison, Peter
The current European and American Guidelines differ with regard to the recommended level for the use of routine early angiography after fibrinolysis for STEMI. Previous meta-analyses on randomized controlled trials have supported the routine early approach, but its advantage may be because of an excessively low angiography rate among patients in the non-routine strategy arm of the trials. We update the meta-analysis and apply meta-regression to evaluate whether the difference in outcome between the 2 randomized arms could be explained by the angiography rates in the non-routine strategy arm. Because reinfarction and recurrent ischemia are often the reported indication for angiography, we only use mortality endpoint in our meta-regression analysis. Among the eight trials included with 3195 patients, the angiography rate in the non-routine strategy arms ranges from 15% to 100%. The overall odds ratio for 30-day mortality comparing the routine early angiography arm vs the non-routine arm is 0.86 (95% confidence interval 0.60-1.24). On the plot listing the eight trials according to angiography rates, there is no visual trend in the odds ratio estimates for mortality when comparing the 2 treatment strategies as angiography rate decreases. In meta-regression analysis, angiography rate does not predict 30-day mortality (p=0.461). For STEMI, mortality endpoint trumps the softer endpoints of recurrent infarction and ischemia. The current study shows that the equipoise between the routine early invasive versus the non-routine strategy on 30-day mortality cannot be explained by the variable performance of angiography in the non-routine strategy arm. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
Beyer, U; Krönung, S K; Leha, A; Walter, L; Dobbelstein, M
The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression. PMID:26024393
Beyer, U; Krönung, S K; Leha, A; Walter, L; Dobbelstein, M
The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression.
Kiciński, Paweł; Przybylska-Kuć, Sylwia; Dybała, Andrzej; Myśliński, Wojciech; Pastryk, Jolanta; Tomaszewski, Tomasz; Mosiewicz, Jerzy
Obstructive sleep apnoea (OSA) induces thrombophilia and reduces fibrinolysis. Alpha-2-antiplasmin (a-2-AP) and plasminogen activator inhibitor 1 (PAI-1) are major inhibitors of the fibrinolytic system. Increased concentrations of these factors are associated with a higher risk of cardiovascular diseases. The aim of this study was to assess plasma a-2-AP and PAI-1 in patients with OSA and evaluate correlations with the polysomnographic record and selected risk factors of cardiovascular diseases. The study group comprised 45 patients with OSA, and the control group consisted of 19 patients who did not meet the diagnostic criteria of OSA. Plasma a-2-AP and PAI-1 concentrations were assessed by enzyme-linked immunosorbent assay (ELISA). In the study group, the median value of plasma a-2-AP was higher than that of the control group (157.34 vs. 11.89 pg/ml, respectively, P<0.0001). A-2-AP concentration increased proportionally to the severity of OSA. The concentration of a-2-AP was positively correlated with the apnoea-hypopnoea index (AHI), apnoea index (AI), respiratory disturbances time (RDT), and desaturaion index (DI), and negatively correlated with mean and minimal oxygen saturation (SpO2 mean, SpO2 min, respectively). The median value of PAI-1 was higher in the study group than the control group (12.55 vs. 5.40 ng/ml, respectively, P = 0.006) and increased along with OSA severity. PAI-1 concentration was positively correlated with AHI, AI, RDT, DI, and body mass index (BMI) and negatively correlated with SpO2 mean and SpO2 min. Higher plasma concentrations of a-2-AP and PAI-1 in patients with OSA indicated that these patients had increased prothrombotic activity. OSA increases the risk of cardiovascular complications as it enhances prothrombotic activity. PMID:27861608
Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...
Knot, E A; Drijfhout, H R; ten Cate, J W; de Jong, E; Iburg, A H; Kahlé, L H; Grijm, R
We describe the metabolism of purified human alpha 2-plasmin inhibitor in patients with liver cirrhosis to determine whether low plasma concentrations of alpha 2-plasmin inhibitor are the result of impaired synthesis or increased catabolism or both. A kinetic study was performed with 131I-alpha 2-plasmin inhibitor as a sensitive parameter of fibrinolysis in 14 patients with histologically proved liver cirrhosis compared with six healthy control subjects. Eight patients had macronodular cirrhosis (with positive hepatitis B surface antigen), and six had micronodular cirrhosis as a result of alcohol abuse. None of the patients had clinical signs of ascites, and in all the disease was stabilized. alpha 2-Plasmin inhibitor levels biologically and immunologically measured were decreased in all patients. Ten microCi 131I-alpha 2PI was injected intravenously, the disappearance of plasma radioactivity was measured, and turnover data were calculated according to the function x(t) = A1e-alpha 1t + A2e-alpha 2t + Be-beta t. Mean (+/- SD) turnover data in the control subjects were plasma radioactivity half-life 60.1 +/- 5.3 hours, fractional catabolic rate constant of the plasma pool 0.0318 +/- 0.0106 hr-1, and absolute catabolic (synthetic) rate constant 2.10 +/- 0.60 mg/kg/day. The alpha 1-phase was 1.26 +/- 0.23, and the transcapillary influx constant (k2,1) was 0.974 +/- 0.109 hr-1. In the patients, plasma radioactivity half-life was 58.7 +/- 12.09 hr, and fractional catabolic rate constant of the plasma pool 0.0283 +/- 0.0043 hr-1. The alpha 1-phase 4.74 +/- 6.48 and the transcapillary influx (k2,1) 3.08 +/- 3.9 hr-1 were both significantly increased compared with control values (p less than 0.05 and p less than 0.05, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Leroux, Philippe; Omouendze, Priscilla L; Roy, Vincent; Dourmap, Nathalie; Gonzalez, Bruno J; Brasse-Lagnel, Carole; Carmeliet, Peter; Leroux-Nicollet, Isabelle; Marret, Stéphane
Intracerebral-intraventricular hemorrhages (ICH/IVH) in very preterm neonates are responsible for high mortality and subsequent disabilities. In humans, tissue plasminogen activator (t-PA) initiates fibrinolysis and activates endoluminal-endothelial receptors; dysfunction of the t-PA inhibitor (PAI-1) results in recurrent hemorrhages. We used PAI-1 knockout (PAI-1) mice to examine the role of t-PA in age-dependent intracranial hemorrhages as a possible model of preterm ICH/IVH. Intracortical injection of 2 μL of phosphate-buffered saline produced a small traumatic injury and a high rate of hemorrhage in PAI-1 pups at postnatal day 3 (P3) or P5, whereas it had no effect in wild-type neonates. This resulted in white matter and cortical lesions, ventricle enlargement, hyperlocomotion, and altered cortical levels of serotonin and dopamine in the adult PAI mice. N-methyl-D-aspartate receptor blockers, plasmin- and matrix metalloproteinases inhibitors reduced hemorrhage and tissue lesions. In contrast to P3 to P5, no significant hemorrhages were induced in P10 PAI-1 pups and there were no behavioral or neurochemical alterations in adulthood. These data suggest that microvascular immaturity up to P5 in mice is a determinant factor required for t-PA-dependent vascular rupture. Neonatal PAI-1 mice could be a useful ICH/IVH model for studying the ontogenic window of vascular immaturity and vascular protection against later neurodisabilities.
Joseph, Kusumam; Tholanikunnel, Baby G.; Wolf, Bethany; Bork, Konrad; Kaplan, Allen P.
Background Hereditary angioedema with normal C1 inhibitor levels (HAE-N) is associated with a Factor XII mutation in 30% of subjects; however, the role of this mutation in the pathogenesis of angioedema is unclear. Objective We sought evidence of abnormalities in the pathways of bradykinin formation and bradykinin degradation in the plasma of patients with HAE-N both with and without the mutation. Methods Bradykinin was added to plasma, and its rate of degradation was measured by using ELISA. Plasma autoactivation was assessed by using a chromogenic assay of kallikrein formation. Plasminogen activator inhibitors (PAIs) 1 and 2 were also measured by means of ELISA. Results PAI-1 levels varied from 0.1 to 4.5 ng/mL (mean, 2.4 ng/mL) in 23 control subjects, from 0.0 to 2 ng/mL (mean, 0.54 ng/mL) in patients with HAE-N with a Factor XII mutation (12 samples), and from 0.0 to 3.7 ng/mL (mean, 1.03 ng/mL) in patients with HAE-N without a Factor XII mutation (11 samples). PAI-2 levels varied from 25 to 87 ng/mL (mean, 53.8 ng/mL) in control subjects and were 0 to 25 ng/mL (mean, 4.3 ng/mL) in patients with HAE-N with or without the Factor XII mutation. Autoactivation at a 1:2 dilution was abnormally high in 8 of 17 patients with HAE-N (4 in each subcategory) and could be corrected by supplemental C1 inhibitor in 4 of them. Bradykinin degradation was markedly abnormal in 1 of 23 patients with HAE-N and normal in the remaining 22 patients. Conclusions Bradykinin degradation was normal in all but 1 of 23 patients with HAE-N studied. By contrast, there was a marked abnormality in PAI-2 levels in patients with HAE-N that is not seen in patients with C1 inhibitor deficiency. PAI-1 levels varied considerably, but a statistically significant difference was not seen. A link between excessive fibrinolysis and bradykinin generation that is estrogen dependent is suggested. PMID:26395818
Yu, Chao; Luan, Yun; Wang, Zejun; Zhao, Jingjie; Xu, Chengwei
Human thrombin activatable fibrinolysis inhibitor (TAFI), also known as carboxypeptidase B2 (CPB2), is a procarboxypeptidase enzyme. The purpose of the present study was to observe the expression of TAFI in breast cancer (BC) and breast cancer cell (BCC) lines and to investigate the effect of TAFI suppression by small interfering (si)RNA gene silencing on invasion and migration of BCC lines. A significant increase in TAFI level was identified by immunohistochemical analysis in BC tissues compared with normal breast tissues. TAFI suppression also inhibited cell viability, invasion and migration ability as demonstrated by MTT, Transwell chamber, and wound scratch assays, respectively (P<0.05). The data suggested that suppression of TAFI by siRNA inhibits invasion and migration of breast cancer cells and that TAFI may be a new target for breast cancer therapy.
Matsui, H; Takahashi, Y; Matsunaga, T; Tanaka-Horie, T; Minowa, H; Sugimoto, M; Tsukino, R; Mii, Y; Giddings, J; Yoshioka, A
We report the arthroscopic treatment of pigmented villonodular synovitis (PVNS) in a 13-year-old Japanese boy with congenital partial deficiency of plasminogen activator inhibitor-1 (PAI-1). He was admitted to our hospital with recurrent haemarthrosis of his right knee. Characteristic abnormalities of fibrinolysis included shortened euglobulin lysis time, low PAI-1 activity and low PAI-1 antigen levels. In addition, levels of "active PAI" in the plasma, which is a measure of total PAI bound to exogenous plasminogen activator, were very low. These parameters remained low after venous occlusion. The diagnosis of PVNS was established by synovial membrane biopsy, and arthroscopic synovectomy was performed with adjuvant administration of intravenous tranexamic acid. Subsequent bleeding episodes have been well controlled by oral administration of tranexamic acid on demand.
Magdoud, Kalthoum; Herbepin, Viviana G; Touraine, Renaud; Almawi, Wassim Y; Mahjoub, Touhami
Plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis, and the common promoter region variants -675G/A (4G/5G) and -844G/A are associated with increased thrombotic risk. Despite evidence linking altered fibrinolysis with adverse pregnancy events, including idiopathic recurrent pregnancy loss (RPL), the contribution of PAI-1 variants to RPL risk remains controversial. We investigated the association between the PAI-1 -844G/A and 4G/5G (-675G/A) variants with altered risk of RPL. This was a case-control study involving 304 women with confirmed RPL and 371 age- and ethnically matched control women. PAI-1 genotyping was performed by PCR single-specific primer -675 (G/A) and real-time PCR (-844G/A) analysis. Minor allele frequency (MAF) of 4G/5G (P < 0.001), but not -844G/A (P = 0.507), was higher in RPL cases. PAI-1 4G/5G single-nucleotide polymorphism (SNP) was significantly associated with RPL under additive, dominant, and recessive genetic models; no association of -844G/A with RPL was seen irrespective of the genetic model tested. Taking common -844G/5G haplotype as reference (OR = 1.00), multivariate analysis confirmed the association of 4G-containing -844A/4G (P < 0.001) and -844G/4G (P = 0.011) haplotypes with increased RPL risk. 4G/5G, but not -844G/A, PAI-1 variant is associated with an increased risk of RPL. © 2013 John Wiley & Sons Ltd.
Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi
Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446
Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.
This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764
Landsem, A; Fure, H; Mollnes, T E; Nielsen, E W; Brekke, O L
C1-inhibitor (C1-INH), a serine protease inhibitor in plasma plays a central role in the cross-talk among the complement, coagulation, fibrinolytic and kallikrein-kinin systems. However, previous reports indicate thrombotic risks in children following supraphysiological dosing with C1-INH. To investigate the role of supraphysiological C1-INH concentrations in clot development with and without addition of Escherichia coli (E. coli) in fresh human whole blood using thromboelastometry. Blood was collected in citrate tubes, and C1-INH (3.0 to 47.6μM) or human serum albumin (HSA) was added as a control. Activated partial thromboplastin time (aPTT) was analysed in the plasma. The analyses non-activated thromboelastometry (NATEM), extrinsic (EXTEM) or intrinsic thromboelastometry (INTEM) were performed using rotational thromboelastometry. C1-INH increased aPTT 1.8-fold (p< 0.05), whereas HSA had no effect. C1-INH increased NATEM clotting time (CT) from 789s to 2025 s (p< 0.05) in a dose-dependent manner. C1-INH reduced the NATEM alpha angle from 47 to 28° (p<0.05) and increased the NATEM clot formation time from 261s to 595s (p< 0.05). E. coli significantly reduced the NATEM CT after 120min of incubation. C1-INH prevented E. coli-induced activation (p< 0.05). C1-INH significantly increased the INTEM CT (p< 0.05), but had no effect on EXTEM CT. C1-INH (47.6μM) significantly reduced fibrinolysis measured as NATEM and EXTEM lysis indices LI60. Supraphysiological C1-INH concentrations have dose-dependent anticoagulant effects in human whole blood in vitro. At very high levels C1-INH also inhibits fibrinolysis. Copyright © 2016 Elsevier Ltd. All rights reserved.
Leach, R. D.
The effect of long and short-term venous hypertension upon lymph fibrinogen concentrations was studied in an attempt to explain the peri-capillary deposition of fibrin reported in patients with post-phlebitic syndromes. The clearance of radioactive fibrinogen/thrombin clots from the subcutaneous tissues of rats and human volunteers was also studied. Both long- and short-term venous hypertension were found to increase fibrinogen transport across the interstitial space by more than 600%. Not only was there evidence of fibrinolytic activity in the lymph but after long-term venous hypertension alpha 2 antiplasmin activity was also detectable. Skin biopsies from the venous hypertensive ankles showed deposition of interstitial fibrin. The clearance of radioactive fibrinogen/thrombin clots from the subcutaneous tissues of the rat was found to be delayed if the rats were given epsilon amino caproic acid but it could not be increased with stanozolol. In human subjects it was found that patients with lipodermatosclerosis had delayed clot clearance and retarded blood fibrinolytic activity when compared with normal volunteers and patients with uncomplicated varicose veins. The principle cause why tall men are more subject to ulcers than short men, Dr Young conceived to be then length of the column of blood in their veins; which by its pressure, renders the legs less able to recover when hurt by any violence. Images Fig. 1 Fig. 2 Fig. 5 PMID:6742738
acLDL uptake? In order to quantitate the number of sprouts in each chamber with Dil-labeled acLDL, we would want to know that BPE , FGF, or VEGF treatment...mm 2 tissue culture flasks, treated with BPE , VEGF, and/or FGF similarly to the explants, and exposed to AlexaFluor 488 - labeled acLDL (Molecular...10 BPE Mammary vessels were placed in fibrin matrices in 3 & 11 VEGF, 50 ng/ml assays 1-8 and aortic rings were similarly treated in 4& 12 FGF ng/ml
Piazza, Gregory; Hohlfelder, Benjamin; Jaff, Michael R; Ouriel, Kenneth; Engelhardt, Tod C; Sterling, Keith M; Jones, Noah J; Gurley, John C; Bhatheja, Rohit; Kennedy, Robert J; Goswami, Nilesh; Natarajan, Kannan; Rundback, John; Sadiq, Immad R; Liu, Stephen K; Bhalla, Narinder; Raja, M Laiq; Weinstock, Barry S; Cynamon, Jacob; Elmasri, Fakhir F; Garcia, Mark J; Kumar, Mark; Ayerdi, Juan; Soukas, Peter; Kuo, William; Liu, Ping-Yu; Goldhaber, Samuel Z
This study conducted a prospective, single-arm, multicenter trial to evaluate the safety and efficacy of ultrasound-facilitated, catheter-directed, low-dose fibrinolysis, using the EkoSonic Endovascular System (EKOS, Bothell, Washington). Systemic fibrinolysis for acute pulmonary embolism (PE) reduces cardiovascular collapse but causes hemorrhagic stroke at a rate exceeding 2%. Eligible patients had a proximal PE and a right ventricular (RV)-to-left ventricular (LV) diameter ratio ≥0.9 on chest computed tomography (CT). We included 150 patients with acute massive (n = 31) or submassive (n = 119) PE. We used 24 mg of tissue-plasminogen activator (t-PA) administered either as 1 mg/h for 24 h with a unilateral catheter or 1 mg/h/catheter for 12 h with bilateral catheters. The primary safety outcome was major bleeding within 72 h of procedure initiation. The primary efficacy outcome was the change in the chest CT-measured RV/LV diameter ratio within 48 h of procedure initiation. Mean RV/LV diameter ratio decreased from baseline to 48 h post-procedure (1.55 vs. 1.13; mean difference, -0.42; p < 0.0001). Mean pulmonary artery systolic pressure (51.4 mm Hg vs. 36.9 mm Hg; p < 0.0001) and modified Miller Index score (22.5 vs. 15.8; p < 0.0001) also decreased post-procedure. One GUSTO (Global Utilization of Streptokinase and Tissue Plasminogen Activator for Occluded Coronary Arteries)-defined severe bleed (groin hematoma with transient hypotension) and 16 GUSTO-defined moderate bleeding events occurred in 15 patients (10%). No patient experienced intracranial hemorrhage. Ultrasound-facilitated, catheter-directed, low-dose fibrinolysis decreased RV dilation, reduced pulmonary hypertension, decreased anatomic thrombus burden, and minimized intracranial hemorrhage in patients with acute massive and submassive PE. (A Prospective, Single-arm, Multi-center Trial of EkoSonic® Endovascular System and Activase for Treatment of Acute Pulmonary Embolism (PE) [SEATTLE II]; NCT
Van de Ven, P.; Fritz, P.; Pellet, R.
A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.
A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ. PMID:19873496
Kenny, Susan; Steele, Islay; Lyons, Suzanne; Moore, Andrew R.; Murugesan, Senthil V.; Tiszlavicz, Laszlo; Dimaline, Rod; Pritchard, D. Mark; Varro, Andrea
Gastric mucosal health is maintained in response to potentially damaging luminal factors. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt protective mechanisms leading to bleeding and ulceration. The plasminogen activator system has been implicated in fibrinolysis following gastric ulceration, and an inhibitor of this system, plasminogen activator inhibitor (PAI)-1, is expressed in gastric epithelial cells. In Helicobacter pylori-negative patients with normal gastric histology taking aspirin or NSAIDs, we found elevated gastric PAI-1 mRNA abundance compared with controls; the increase in patients on aspirin was independent of whether they were also taking proton pump inhibitors. In the same patients, aspirin tended to lower urokinase plasminogen activator mRNA. Immunohistochemistry indicated PAI-1 localization to epithelial cells. In a model system using MKN45 or AGS-GR cells transfected with a PAI-1 promoter-luciferase reporter construct, we found no evidence for upregulation of PAI-1 expression by indomethacin, and, in fact, cyclooxygenase products such as PGE2 and PGI2 weakly stimulated expression. Increased gastric PAI-1 mRNA was also found in mice following gavage with ethanol or indomethacin, but plasma PAI-1 was unaffected. In PAI-1−/− mice, gastric hemorrhagic lesions in response to ethanol or indomethacin were increased compared with C57BL/6 mice. In contrast, in PAI-1-H/Kβ mice in which PAI-1 is overexpressed in parietal cells, there were decreased lesions in response to ethanol and indomethacin. Thus, PAI-1 expression is increased in gastric epithelial cells in response to mucosal irritants such as aspirin and NSAIDs probably via an indirect mechanism, and PAI-1 acts as a local autoregulator to minimize mucosal damage. PMID:23494120
Huang, Wen-Tan; Akhter, Hasina; Jiang, Chunsun; MacEwen, Mark; Ding, Qiang; Antony, Veena; Thannickal, Victor John; Liu, Rui-Ming
Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder with unknown cause and no effective treatment. The incidence of and mortality from IPF increase with age, suggesting that advanced age is a major risk factor for IPF. The mechanism underlying the increased susceptibility of the elderly to IPF, however, is unknown. In this study, we show for the first time that the protein level of plasminogen activator inhibitor 1 (PAI-1), a protease inhibitor which plays an essential role in the control of fibrinolysis, was significantly increased with age in mouse lung homogenate and lung fibroblasts. Upon bleomycin challenge, old mice experienced augmented PAI-1 induction and lung fibrosis as compared to young mice. Most interestingly, we show that fewer (myo)fibroblasts underwent apoptosis and more (myo)fibroblasts with increased level of PAI-1 accumulated in the lung of old than in young mice after bleomycin challenge. In vitro studies further demonstrate that fibroblasts isolated from lungs of old mice were resistant to H2O2 and tumor necrosis factor alpha-induced apoptosis and had augmented fibrotic responses to TGF-β1, compared to fibroblasts isolated from young mice. Inhibition of PAI-1 activity with a PAI-1 inhibitor, on the other hand, eliminated the aging-related apoptosis resistance and TGF-β1 sensitivity in isolated fibroblasts. Moreover, we show that knocking down PAI-1 in human lung fibroblasts with PAI-1 siRNA significantly increased their sensitivity to apoptosis and inhibited their responses to TGF-β1. Together, the results suggest that increased PAI-1 expression may underlie the aging-related sensitivity to lung fibrosis in part by protecting fibroblasts from apoptosis. Published by Elsevier Inc.
Małecki, Rafał; Gacka, Małgorzata; Kuliszkiewicz-Janus, Małgorzata; Jakobsche-Policht, Urszula; Kwiatkowski, Jacek; Adamiec, Rajmund; Undas, Anetta
Patients with increased thromboembolic risk tend to form denser fibrin clots which are relatively resistant to lysis. We sought to investigate whether essential thrombocythemia (ET) is associated with altered fibrin clot properties in plasma. Ex vivo plasma fibrin clot permeability coefficient (Ks), turbidimetry and clot lysis time (CLT) were measured in 43 consecutive patients with ET (platelet count from 245 to 991 × 10(3)/µL) and 50 control subjects matched for age, sex and comorbidities. Fibrinolysis proteins and inhibitors together with platelet activation markers were determined. Reduced Ks (-38%, p < 0.0001) and prolonged CLT (+34%, p < 0.0001) were observed in ET. The differences remained significant after adjustment for fibrinogen and platelet count. ET was associated with a slightly shorter lag phase (-5%, p = 0.01) and higher maximum absorbency of the turbidimetric curve (+6%, p < 0.001). The ET patients had higher plasma P-selectin by 193% (p < 0.00001) and platelet factor 4 (PF4) by 173% (p < 0.00001), with higher P-selectin observed in 19 (44%) patients with JAK-2 gene V617F mutation. Higher t-PA (+20%, p < 0.001), 23% higher plasminogen activator inhibitor-1, PAI-1 (+23%, p < 0.01) and unaltered thrombin-activatable fibrinolysis inhibitor, plasminogen and α2-antiplasmin activity were found in the ET group. Ks inversely correlated with fibrinogen, PF4 and C-reactive protein. CLT positively correlated only with PAI-1. Patients with ET display prothrombotic plasma fibrin clot phenotype including impaired fibrinolysis, which represents a new prothrombotic mechanism in this disease.
Haiko, Johanna; Laakkonen, Liisa; Juuti, Katri; Kalkkinen, Nisse; Korhonen, Timo K
Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis.
Shammaa, Dina M R; Sabbagh, Amira S; Taher, Ali T; Zaatari, Ghazi S; Mahfouz, Rami A R
Plasminogen activator inhibitor-1 (PAI-1) is an inhibitor of fibrinolysis. Increased plasma PAI-1 levels play an essential role in the pathogenesis of cardiovascular risk and other diseases associated with thrombosis. The 4G/5G polymorphism of the PAI-1 promoter region has been extensively studied in different populations. We studied 160 healthy unrelated Lebanese individuals using a reverse hybridization PCR assay to detect the 5G/5G, 4G/5G and, 4G/4G genotypes of the PAI-1 gene and the frequencies of the 4G and 5G alleles. We found that 4G/5G genotype was the most prevalent (45.6%) followed by 5G/5G (36.9%) and 4G/4G (17.5%). The frequencies of the 4G and 5G alleles were calculated to be 0.403 and 0.597, respectively. Compared to other ethnic communities, the Lebanese population was found to harbour a relatively high prevalence of the rare 4G allele. This, in turn, may predispose this population to develop cardiovascular diseases and other thrombotic clinical conditions. This study aids to enhance our understanding of the genetic features of the Lebanese population.
Huntington, James A
The serine protease thrombin is the effector enzyme of blood coagulation. It has many activities critical for the formation of stable clots, including cleavage of fibrinogen to fibrin, activation of platelets and conversion of procofactors to active cofactors. Thrombin carries-out its multiple functions by utilising three special features: a deep active site cleft and two anion binding exosites (exosite I and II). Similarly, thrombin inhibitors have evolved to exploit the unique features of thrombin to achieve rapid and specific inactivation of thrombin. Exogenous thrombin inhibitors come from several different protein families and are generally found in the saliva of haematophagous animals (blood suckers) as part of an anticoagulant cocktail that allows them to feed. Crystal structures of several of these inhibitors reveal how peptides and proteins can be targeted to thrombin in different and interesting ways. Thrombin activity must also be regulated by endogenous inhibitors so that thrombi do not occlude blood flow and cause thrombosis. A single protein family, the serpins, provides all four of the endogenous thrombin inhibitors found in man. The crystal structures of these serpins bound to thrombin have been solved, revealing a similar exosite-dependence on complex formation. In addition to forming the recognition complex, serpins destroy the structure of thrombin, allowing them to be released from cofactors and substrates for clearance. This review examines how the special features of thrombin have been exploited by evolution to achieve inhibition of the ultimate coagulation protease.
Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.
Dardi, I; Kouvatsos, T; Jabbour, S A
Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM.
Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen
Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920
Napp, Joanna; Dullin, Christian; Müller, Friedemann; Uhland, Kerstin; Petri, Jean Bernhard; van de Locht, Andreas; Steinmetzer, Torsten; Alves, Frauke
Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin-like transmembrane serine protease, as potential target for novel inhibitor-based tumor therapies. We applied time-domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC-1 pancreatic tumor model in nude mice. We show strong and tumor-specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT-Ab*Cy5.5) only to primary AsPC-1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT-Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat-panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY-681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor-bearing mice, and that application of synthetic active-site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor-based cancer therapies.
He, Lianying; Asai, Suzuka; Kawamura, Takeshi; Kimbara, Noriaki; Tada, Toyohiro; Okada, Hidechika; Okada, Noriko
Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.
Minnema, M C; Ten Cate, H; Hack, C E
In 1991 it was demonstrated that, besides factor XII, thrombin is capable of activating factor XI in vitro. Thrombin-dependent activation of factor XI is an integral part of the revised theoretical model of coagulation in which coagulation is initiated by the extrinsic pathway and maintained by thrombin-induced activation of clotting factors V, VIII, and XI. In this review, special interest is given to the new role of factor XI in coagulation, with emphasise on data supporting the concept of thrombin-mediated factor XI activation in vivo. Furthermore, activation of factor XI in human disease, especially atherosclerotic disease, measured by newly developed immunologic assays, is discussed. The relation of factor XI to fibrinolysis through activation of the carboxypeptidase, thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin provides an explanation for the bleeding tendency observed in factor XI-deficient patients. The probable link with factor XI-mediated TAFI activation may have clinical and therapeutic consequences and deserves further study.
Minnema, M C; Friederich, P W; Levi, M; von dem Borne, P A; Mosnier, L O; Meijers, J C; Biemond, B J; Hack, C E; Bouma, B N; ten Cate, H
Recent in vitro studies have shown that fibrinolytic activity may be attenuated by a thrombin-activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin, generated via the intrinsic pathway of coagulation in a factor XI-dependent way. Thus factor XI may play a role in the regulation of endogenous fibrinolysis. The aim of this study was to investigate the effect of in vivo inhibition of factor XI and TAFI in an experimental thrombosis model in rabbits. Incorporation of anti-factor XI antibodies in jugular vein thrombi resulted in an almost twofold increase in endogenous thrombolysis compared with a control antibody. A similar effect was observed when the anti-factor XI antibody was administered systemically. Inhibition of TAFI activity also resulted in a twofold increase in clot lysis whereas inhibition of both factor XI and TAFI activity had no additional effect. Thus, we provide the first in vivo evidence for enhanced thrombolysis through inhibition of clotting factor XI, demonstrating a novel role for the intrinsic pathway of coagulation. Furthermore we demonstrate that inhibition of TAFI had a similar effect on thrombolysis. We postulate that inhibition of factor XI activity enhances thrombolysis because of diminished indirect activation of TAFI.
Lwaleed, Bashir A; Greenfield, Robert S; Cooper, Alan J
The presence of fibrin degradation products, thrombin-like enzyme, prothrombin fragments, thrombin-activatable fibrinolysis inhibitor, plasmin and other active components of blood coagulation and fibrinolysis in seminal plasma has been reported. In the present study we investigate the presence of thrombomodulin in human semen. Using an Imubind thrombomodulin enzyme-linked immunosorbent assay (American Diagnostica Inc., Stamford, Connecticut, USA), seminal thrombomodulin levels were measured in 47 semen specimens obtained from subfertile individuals, normally fertile individuals, semen donors as well as vasectomized individuals, and in a further group defined by normality in several parameters derived from the World Health Organization fertility criteria. Conventional semen parameters were analysed in all semen samples. Thrombomodulin is quantifiable in human semen at a concentration lower than that normally found in citrated blood plasma samples. Slightly higher levels were seen for fertile stratifications compared with infertile individuals but without significant difference, given the numbers accrued. A vasectomized group showed the lowest value. In conclusion, our results establish the presence of thrombomodulin in human semen and suggest its production both upstream and downstream from the level of a vasectomy lesion.
Petersen, Karl-Uwe; Rea, Catherine J.; Harpell, Lori; Powell, Sandra; Lillicrap, David; Nesheim, Michael E.; Sørensen, Benny
Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)–deficient plasma by an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)–dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor–initiated fibrin formation and tissue-plasminogen activator–induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombin-Solulin complex were determined to be 1.53 and 0.02/μM/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses. PMID:22234684
Waschki, Benjamin; Watz, Henrik; Holz, Olaf; Magnussen, Helgo; Olejnicka, Beata; Welte, Tobias; Rabe, Klaus F; Janciauskiene, Sabina
Introduction Plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of fibrinolysis, is associated with thrombosis, obesity, insulin resistance, dyslipidemia, and premature aging, which all are coexisting conditions of chronic obstructive pulmonary disease (COPD). The role of PAI-1 in COPD with respect to metabolic and cardiovascular functions is unclear. Methods In this study, which was nested within a prospective cohort study, the serum levels of PAI-1 were cross-sectionally measured in 74 stable COPD patients (Global Initiative for Chronic Obstructive Lung Disease [GOLD] Stages I–IV) and 18 controls without lung disease. In addition, triglycerides, high-density lipoprotein cholesterol, fasting plasma glucose, waist circumference, blood pressure, smoking status, high-sensitive C-reactive protein (hs-CRP), adiponectin, ankle–brachial index, N-terminal pro-B-type natriuretic peptide, and history of comorbidities were also determined. Results The serum levels of PAI-1 were significantly higher in COPD patients than in controls, independent of a broad spectrum of possible confounders including metabolic and cardiovascular dysfunction. A multivariate regression analysis revealed triglyceride and hs-CRP levels to be the best predictors of PAI-1 within COPD. GOLD Stages II and III remained independently associated with higher PAI-1 levels in a final regression analysis. Conclusion The data from the present study showed that the serum levels of PAI-1 are higher in patients with COPD and that moderate-to-severe airflow limitation, hypertriglyceridemia, and systemic inflammation are independent predictors of an elevated PAI-1 level. PAI-1 may be a potential biomarker candidate for COPD-specific and extra-pulmonary manifestations. PMID:28356730
Schleef, R R; Higgins, D L; Pillemer, E; Levitt, L J
We evaluated an elderly patient with a lifelong history of severe bleeding after surgery or trauma and with evidence of persistent hyperfibrinolysis. Routine coagulation studies were normal. Serum plasminogen (40%, normal 72-128%) and alpha 2-antiplasmin (55%, normal 70-145%) activities were decreased. Euglobulin clot lysis was abnormally shortened (50 min) and normalized in vitro with epsilon-aminocaproic acid (EACA). The patient was treated with EACA with prompt cessation of bleeding. Patient tissue-plasminogen activator (t-PA) levels in serum were normal (4.7 ng/ml, control 3.5-7.2) as detected by a two-site immunoradiometric assay (IRMA). Patient fibrinolytic inhibitor activities were assessed by incubating 125I-labeled t-PA with either whole blood or serum followed by SDS-PAGE and autoradiography to identify the resultant protease/protease inhibitor complexes. In comparison to blood samples obtained from normal donors, patient plasma and serum demonstrated reduced binding of a fast-acting plasminogen activator inhibitor to 125I-labeled t-PA. Immunoprecipitation experiments indicated diminished complex formation between type 1 plasminogen activator inhibitor (PAI-1) in patient serum and 125I-labeled t-PA. Low patient PAI-1 activity was confirmed in serum (0.36 U/ml, control 0.87-1.81; n = 3) and in platelet lysates using a functional IRMA to quantitate PAI-1 binding to immobilized t-PA. However, patient serum PAI-1 antigen was within the normal range when analyzed by IRMA (31.8 ng/ml, control 19.6-42.2); this result was confirmed in both serum and platelets by Western blot (n = 3). Mixing experiments using purified PAI-1 as well as patient and control sera did not show evidence for an inhibitor against PAI-1. We conclude that this patient's bleeding diathesis was due to hyperfibrinolysis and defective PAI-1. This patient provides the first demonstration of a link between decreased in vivo PAI-1 activity and disordered hemostasis, and supports a role for PAI-1
Sanderson, P E; Naylor-Olsen, A M
Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.
Batabyal, Subrata; Cervenka, Gregory; Ha, Ji Hee; Kim, Young-tae; Mohanty, Samarendra
Currently, the use of optogenetic sensitization of retinal cells combined with activation/inhibition has the potential to be an alternative to retinal implants that would require electrodes inside every single neuron for high visual resolution. However, clinical translation of optogenetic activation for restoration of vision suffers from the drawback that the narrow spectral sensitivity of an opsin requires active stimulation by a blue laser or a light emitting diode with much higher intensities than ambient light. In order to allow an ambient light-based stimulation paradigm, we report the development of a ‘white-opsin’ that has broad spectral excitability in the visible spectrum. The cells sensitized with white-opsin showed excitability at an order of magnitude higher with white light compared to using only narrow-band light components. Further, cells sensitized with white-opsin produced a photocurrent that was five times higher than Channelrhodopsin-2 under similar photo-excitation conditions. The use of fast white-opsin may allow opsin-sensitized neurons in a degenerated retina to exhibit a higher sensitivity to ambient white light. This property, therefore, significantly lowers the activation threshold in contrast to conventional approaches that use intense narrow-band opsins and light to activate cellular stimulation. PMID:26360377
Batabyal, Subrata; Cervenka, Gregory; Ha, Ji Hee; Kim, Young-Tae; Mohanty, Samarendra
Currently, the use of optogenetic sensitization of retinal cells combined with activation/inhibition has the potential to be an alternative to retinal implants that would require electrodes inside every single neuron for high visual resolution. However, clinical translation of optogenetic activation for restoration of vision suffers from the drawback that the narrow spectral sensitivity of an opsin requires active stimulation by a blue laser or a light emitting diode with much higher intensities than ambient light. In order to allow an ambient light-based stimulation paradigm, we report the development of a 'white-opsin' that has broad spectral excitability in the visible spectrum. The cells sensitized with white-opsin showed excitability at an order of magnitude higher with white light compared to using only narrow-band light components. Further, cells sensitized with white-opsin produced a photocurrent that was five times higher than Channelrhodopsin-2 under similar photo-excitation conditions. The use of fast white-opsin may allow opsin-sensitized neurons in a degenerated retina to exhibit a higher sensitivity to ambient white light. This property, therefore, significantly lowers the activation threshold in contrast to conventional approaches that use intense narrow-band opsins and light to activate cellular stimulation.
Simon, V.; Ceccaldi, A.; Lévy, L.
With almost 150,000 deaths every year in France (26 times more than on the roads), cancers represent the second cause of mortality after cardiovascular disease. In 2002, 10 million new cases of cancer were registered in the world and there were 6 million deaths (of which 40% in developed countries). Moreover, the World Health Organisation (WHO) predicts a significant increase in the number of new cases between now and 2020 (+40% in developed countries and +100% in developing countries), due mainly to longer life expectations, change in behaviour, and degradation of the environment. The word ‘cancer’ is a generic term covering a group of more than a hundred diseases, all characterised by the organism losing control over the proliferation of certain cells. These cells then develop in an anarchic way, eventually constituting a tumour which invades surrounding tissue and in many cases ends up disseminating to distant tissues (metastases).
Blardi, P; Messa, G; Puccetti, L; La Placa, G; Ghezzi, A
We evaluated the mesoglycan effects on the coagulative-fibrinolytic system in 10 patients with euglobulin lysis time (ELT) over 180 minutes. A mathematical model was used to analyze such phenomena. 100 mg of mesoglycan was administered to 10 patients for 14 days. The following parameters were evaluated: tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), euglobulin lysis time (ELT), plasminogen, alpha 2 antiplasmin, prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin clotting time (TCT), and fibrinogen. Those parameters were evaluated on the first and on the last day of the mesoglycan treatment at the following times: 0 (basal), 2, 4, 6, 8, 10 and 12 hours. Our results suggest that the mesoglycan is able to reduce a profibrinolytic activity without any influence on the coagulative-fibrinolytic system, at the baseline conditions and after chronic administration. The pharmacodynamic study and the statistical analysis using our mathematic model resulted to be statistically significant.
In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.
Kubota, Naoto; Kadowaki, Takashi
SGLT2 is a glucose transporter which plays an important role for reabsorption of urinary glucose depending on the sodium concentration gradient. SGLT2 is mainly present in apical site of S1 segment of renal proximal tubule and accounts for approximately 90% of total urinary glucose reabsorption. SLC5a2, which codes SGLT2, is also known as the causative gene of familial renal glucosuria. SGLT2 inhibitors are attracting attention as newly developed oral anti-diabetic agents which improve glucose intolerance and also have an anti-obese effect by promoting urinary glucose excretion (UGE), which is a different pharmacological effect from other conventional anti-diabetic agents. In this review, we will discuss the effect of SGLT2 inhibitor on the regulation of glucose and lipid metabolism in type 2 diabetes.
Aversa, Antonio; Bruzziches, Roberto; Francomano, Davide; Natali, Marco; Lenzi, Andrea
Normal vascular endothelium is essential for the synthesis and release of substances affecting vascular tone (e.g. nitric oxide; NO), cell adhesion (e.g. endothelins, interleukins), and the homeostasis of clotting and fibrinolysis (e.g. plasminogen inhibitors, von Willebrand factor). The degeneration of endothelial integrity promotes adverse events (AEs) leading to increased atherogenesis and to the development of vascular systemic and penile end-organ disease. Testosterone (T) is an important player in the regulation of vascular tone through non-genomic actions exerted via blockade of extracellular-calcium entry or activation of potassium channels; also, adequate T concentrations are paramount for the regulation of phosphodiesterase type-5 (PDE5) expression and finally, for the actions exerted by hydrogen sulphide, a gas involved in the alternative pathway controlling vasodilator responses in penile tissue. It is known that an age-related decline of serum T is reported in approximately 20 to 30% of men whereas T deficiency is reported in up to 50% of men with metabolic syndrome or diabetes. A number of laboratory and human studies have shown the combination of T and other treatments for erectile dysfunction (ED), such as PDE5 inhibitors, to be more beneficial in patients with ED and hypogonadism, who fail monotherapy for sexual disturbances. The aim of this review is to show evidence on the role of T and PDE5 inhibitors, alone or in combination, as potential boosters of endothelial function in internal medicine diseases associated with reduced T or NO bioavailability, i.e. metabolic syndrome, obesity, diabetes, coronary artery disease, hyperhomocysteinemia, that share common risk factors with ED. Furthermore, the possibility of such a strategy to prevent endothelial dysfunction in men at increased cardiovascular risk is discussed. PMID:21789066
The protease enzyme is essential for HIV to make copies of itself. So far, research has failed to find a protease inhibitor that works against HIV. It is believed that, regardless of what type of protease inhibitor someone takes, it will need to be supplemented with other anti-HIV drugs. Three protease inhibitors have thus far been found to be safe, although long-term effects are unknown. These drugs are saquinavir, ABT-538, and L-735,524 produced by Hoffman-LaRoche, Abbott, and Merck respectively. Clinical trials of saquinavir are promising but it has not been shown to be the knock-out drug needed. ABT-538 has high bioavailability, but studies are showing it can cause liver and eye damage. L-735,524 studies are showing that resistance develops quite quickly. Future studies at higher doses are expected. To obtain information on protease studies currently looking for participants, contact The Network. Information on other approved, alternative, and experimental drugs is also available.
During the last fifty years, scale inhibition has gone from an art to a science. Scale inhibition has changed from simple pH adjustment to the use of optimized dose of designer polymers from multiple monomers. The water-treatment industry faces many challenges due to the need to conserve water, availability of only low quality water, increasing environmental regulations of the water discharge, and concern for human safety when using acid. Natural materials such as starch, lignin, tannin, etc., have been replaced with hydrolytically stable organic phosphates and synthetic polymers. Most progress in scale inhibition has come from the use of synergistic mixtures and copolymerizing different functionalities to achieve specific goals. Development of scale inhibitors requires an understanding of the mechanism of crystal growth and its inhibition. This paper discusses the historic perspective of scale inhibition and the development of new inhibitors based on the understanding of the mechanism of crystal growth and the use of powerful tools like molecular modeling to visualize crystal-inhibitor interactions.
Increasing pharmacy costs are among the fastest growing segments of the health care budget. Health plans are focusing on appropriately managing pharmaceutical costs, both from a long-term global perspective and a short-term approach emphasizing newly marketed products. Over the next six months, cox-2 inhibitors are expected to be approved by the FDA. This new class of drugs, investigated as a safer alternative to non-steroidal anti-inflammatory drugs (NSAIDs), is among the most highly anticipated medications to hit the marketplace. How health plans react to the launch of cox-2 inhibitors may serve as an example for future pharmacy management efforts. A proactive policy regarding the use of cox-2 inhibitors may be challenging, but should include: Reviewing clinical information; evaluating the cost of the new drug; and identifying appropriate patient selection criteria. The available management strategies include precertification, a tiered co-payment system, restricting prescriptions to a provider specialty, retrospective physician profiling, and physician education.
Wojewodzka-Zelezniakowicz, Marzena; Gromotowicz-Poplawska, Anna; Kisiel, Wioleta; Konarzewska, Emilia; Szemraj, Janusz; Ladny, Jerzy Robert; Chabielska, Ewa
The aim of this study was to investigate the effects of plasma and tissue angiotensin-converting enzyme inhibitors (ACE-Is) against propofol-induced endothelial dysfunction and to elucidate the involved mechanisms in vitro. We examined the effects of propofol (50 μM), quinaprilat and enalaprilat (10(-5) M) on fibrinolysis (t-PA, PAI-1, TAFI antigen levels), oxidative stress parameters (H2O2 and MDA antigen levels and SOD and NADPH oxidase mRNA levels) and nitric oxide bioavailability (NO2/NO3 concentration and NOS expression at the level of mRNA) in human umbilical vein endothelial cells (HUVECs). We found that both ACE-Is promoted similar endothelial fibrinolytic properties and decreased oxidative stress in vitro. Propofol alone increased the release of antifibrinolytic and pro-oxidative factors from the endothelium and increased mRNA iNOS expression. We also found that the incubation of HUVECs in the presence of propofol following ACE-Is pre-incubation caused weakness of the antifibrinolytic and pro-oxidative potential of propofol and this effect was similar after both ACE-Is. This observation suggests that the studied ACE-Is exerted protective effects against endothelial cell dysfunction caused by propofol, independently of hemodynamics.
Orbe, Josune; Sánchez-Arias, Juan A; Rabal, Obdulia; Rodríguez, José A; Salicio, Agustina; Ugarte, Ana; Belzunce, Miriam; Xu, Musheng; Wu, Wei; Tan, Haizhong; Ma, Hongyu; Páramo, José A; Oyarzabal, Julen
Growing evidence suggests that matrix metalloproteinases (MMP) are involved in thrombus dissolution; then, considering that new therapeutic strategies are required for controlling hemorrhage, we hypothesized that MMP inhibition may reduce bleeding by delaying fibrinolysis. Thus, we designed and synthesized a novel series of MMP inhibitors to identify potential candidates for acute treatment of bleeding. Structure-based and knowledge-based strategies were utilized to design this novel chemical series, α-spiropiperidine hydroxamates, of potent and soluble (>75 μg/mL) pan-MMP inhibitors. The initial hit, 12, was progressed to an optimal lead 19d. Racemic 19d showed a remarkable in vitro phenotypic response and outstanding in vivo efficacy; in fact, the mouse bleeding time at 1 mg/kg was 0.85 min compared to 29.28 min using saline. In addition, 19d displayed an optimal ADME and safety profile (e.g., no thrombus formation). Its corresponding enantiomers were separated, leading to the preclinical candidate 5 (described in Drug Annotations series, J. Med. Chem. 2015, ).
Gruber, Florian; Hufnagl, Peter; Hofer-Warbinek, Renate; Schmid, Johannes A; Breuss, Johannes M; Huber-Beckmann, Renate; Lucerna, Markus; Papac, Nikolina; Harant, Hanna; Lindley, Ivan; de Martin, Rainer; Binder, Bernd R
Plasminogen activator inhibitor 1 (PAI-1) is the main fibrinolysis inhibitor, and high plasma levels are associated with an increased risk for vascular diseases. Inflammatory cytokines regulate PAI-1 through a hitherto unclear mechanism. Using reporter gene analysis, we could identify a region in the PAI-1 promoter that contributes to basal expression as well as to tumor necrosis factor alpha (TNFalpha) induction of PAI-1 in endothelial cells. Using this region as bait in a genetic screen, we could identify Nur77 (NAK-1, TR3, NR4A1) as an inducible DNA-binding protein that binds specifically to the PAI-1 promoter. Nur77 drives transcription of PAI-1 through direct binding to an NGFI-B responsive element (NBRE), indicating monomeric binding and a ligand-independent mechanism. Nur77, itself, is transcriptionally up-regulated by TNFalpha. High expression levels of Nur77 and its colocalization with PAI-1 in atherosclerotic tissues indicate that the described mechanism for PAI-1 regulation may also be operative in vivo.
Ohkura, Naoki; Oishi, Katsutaka; Kihara-Negishi, Fumiko; Atsumi, Gen-ichi; Tatefuji, Tomoki
Background: Brazilian propolis has many biological activities including the ability to help prevent thrombotic diseases, but this particular effect has not been proven. Plasma levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, increase under inflammatory conditions such as infection, obesity and atherosclerosis and such elevated levels predispose individuals to a risk of developing thrombotic diseases. Aim: This study aimed to determine the effects of a diet containing Brazilian propolis on lipopolysaccharide (LPS)-induced increases in plasma PAI-1 levels. Materials and Methods: Mice were fed with a diet containing 0.5% (w/w) Brazilian propolis for 8 weeks. Thereafter, the mice were subcutaneously injected with saline containing 0.015 mg/kg of LPS and sacrificed 4 h later. Results: Orally administered Brazilian propolis significantly suppressed the LPS-induced increase in PAI-1 antigen and its activity in mouse plasma. Conclusion: This study indicated that Brazilian propolis contains natural products that can decrease thrombotic tendencies in mice. PMID:27757277
Iribarren, Jose L; Jimenez, Juan J; Hernández, Domingo; Brouard, Maitane; Riverol, Debora; Lorente, Leonardo; de La Llana, Ramiro; Nassar, Ibrahim; Perez, Rosalia; Martinez, Rafael; Mora, Maria L
Plasminogen activator inhibitor 1 (PAI-1) attenuates the conversion of plasminogen to plasmin. Polymorphisms of the PAI-1 gene are associated with varying PAI-1 levels and risk of prothrombotic events in nonsurgical patients. The purpose of this study, a secondary analysis of a clinical trial, was to investigate whether PAI-1 genotype affects the efficacy of tranexamic acid (TA) in reducing postoperative chest tube blood loss of patients undergoing cardiopulmonary bypass. Fifty patients were classified according to PAI-1 genotype (4G/4G, 4G/5G, or 5G/5G). Twenty-four received 2 g TA before and after cardiopulmonary bypass, whereas 26 received placebo. The authors recorded data related to coagulation, fibrinolysis, and bleeding before surgery, at admission to the intensive care unit (0 h), and 4 and 24 h later. In patients not receiving TA, those with the 5G/5G genotype had significantly higher chest tube blood loss and transfusion requirements compared with patients with the other genotypes at all time points. Patients with the 5G/5G genotype receiving TA showed significantly lower blood loss compared with the placebo group. There were no significant differences in blood loss or transfusion requirements between patients with the 4G/4G genotype when TA was used. Plasminogen activator inhibitor-1 5G/5G homozygotes who did not receive TA showed significantly greater postoperative bleeding than patients with other PAI-1 genotypes. 5G/5G homozygotes who received TA showed the greatest blood-sparing benefit.
Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.
Development of a plasminogen activator inhibitor (PAI-1) assay and comparison of plasma PAI-1 activity in hyperlipidemic/dyslipidemic dogs with either hyperadrenocorticism or diabetes mellitus, and healthy dogs.
Wong, Cheryl J; Koch, Michael; Behling-Kelly, Erica L
Thrombosis is a serious complication of many canine diseases and may be related to decreased fibrinolytic potential. Plasminogen activator inhibitor-1 (PAI-1) is the key regulator of fibrinolysis with increased levels demonstrated in states of pro-thrombosis and abnormal lipid metabolism. Our objective was to develop and validate a canine PAI-1 activity assay and test whether dogs with hyperadrenocorticism or diabetes mellitus that are hyperlipidemic/dyslipidemic have increased plasma PAI-1 activity. Functionally active PAI-1 in the plasma sample was incubated with recombinant tissue plasminogen activator (tPA), allowing the formation of a 1:1 stoichiometric inactive complex. Residual unbound tPA was then reacted with excess plasminogen in the presence of a colorimetric plasmin substrate. Plasmin production is quantified by computing the area under the curve of time (x) vs optical density (y) plot and converted to tPA IU/mL by comparison to a calibration curve of tPA standards. PAI-1 activity was determined by calculating the proportion of exogeneous tPA suppressed by PAI-1 in plasma. Assay verification included assessment of linearity, specificity, precision, sensitivity, and stability. PAI-1 activity was increased in hyperlipidemic compared to healthy dogs, but there was no significant difference between dogs with hyperadrenocorticism and diabetes mellitus. A near significant decrease in activity was detected in thawed plasma stored for 20h at 4°C. Our successfully validated assay offers a new tool for investigating fibrinolysis in dogs. Investigation of PAI-1 activity in dogs with other diseases associated with an increased risk of thrombosis would be valuable. Future studies of PAI-1 activity should consider its lability.
Ye, Tao; Hui, Chunngai
Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.
Pérez-Martínez, P; Adarraga-Cansino, M D; Fernández de la Puebla, R A; Blanco-Molina, A; Delgado-Lista, J; Marín, C; Ordovás, J M; López-Miranda, J; Pérez-Jiménez, F
The objective of the study was to determine whether Plasminogen Activator Inhibitor Type 1 (PAI-1) -675 4G/5G polymorphism is associated with the response of functional plasma PAI-1 concentrations to changes in the amount and quality of dietary fat in healthy subjects. PAI-1 is the major inhibitor of fibrinolysis, and a lower level of fibrinolytic activity could be implicated in an increased risk of IHD. Fifty-nine healthy Spanish volunteers (ten 4G/4G homozygotes, twenty-eight heterozygotes 4G/5G and twenty-one 5G/5G homozygotes) consumed three diets for periods of 4 weeks each: a SFA-rich diet (38 % fat, 20 % SFA), followed by a carbohydrate-rich diet (30 % fat, 55 % carbohydrate) and a MUFA-rich diet (38 % fat, 22 % MUFA) according to a randomized crossover design. At the end of each dietary period plasma lipid and functional plasma PAI-1 concentrations were determined. Subjects carrying the 4G allele (4G/4G and 4G/5G) showed a significant decrease in PAI-1 concentrations after the MUFA diet, compared with the SFA-rich and carbohydrate-rich diets (genotype x diet interaction: P = 0.028). 5G/5G homozygotes had the lowest plasma PAI-1 concentrations compared with 4G/4G and 4G/5G subjects (genotype: P = 0.002), without any changes as a result of the amount and the quality of the dietary fat. In summary, no differences in plasma PAI-1 concentration response were found after changes in dietary fat intake in 5G/5G homozygotes, although these subjects displayed the lowest concentrations of PAI-1. On the other hand, carriers of the 4G allele are more likely to hyper-respond to the presence of MUFA in the diet because of a greater decrease in PAI-1 concentrations.
Wojtowicz-Praga, S M; Dickson, R B; Hawkins, M J
The matrix metalloproteinases (MMPs) are a family of at least fifteen secreted and membrane-bound zinc-endopeptidases. Collectively, these enzymes can degrade all of the components of the extracellular matrix, including fibrallar and non-fibrallar collagens, fibronectin, laminin and basement membrane glycoproteins. MMPs are thought to be essential for the diverse invasive processes of angiogenesis and tumor metastasis. Numerous studies have shown that there is a close association between expression of various members of the MMP family by tumors and their proliferative and invasive behavior and metastatic potential. In some of human cancers a positive correlation has also been demonstrated between the intensity of new blood vessel growth (angiogenesis) and the likelihood of developing metastases. Thus, control of MMP activity in these two different contexts has generated considerable interest as a possible therapeutic target. The tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring proteins that specifically inhibit matrix metalloproteinases, thus maintaining balance between matrix destruction and formation. An imbalance between MMPs and the associated TIMPs may play a significant role in the invasive phenotype of malignant tumors. TIMP-1 has been shown to inhibit tumor-induced angiogenesis in experimental systems. These findings raised the possibility of using an agent that affects expression or activity of MMPs as an anti-cancer therapy. TIMPs are probably not suitable for pharmacologic applications due to their short half-life in vivo. Batimastat (BB-94) and marimastat (BB-2516) are synthetic, low-molecular weight MMP inhibitors. They have a collagen-mimicking hydroxamate structure, which facilitates chelation of the zinc ion in the active site of the MMPs. These compounds inhibit MMPs potently and specifically. Batimastat was the first synthetic MMP inhibitor studied in humans with advanced malignancies, but its usefulness has been limited by
Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain prolonged clinical benefit. Many questions remain, however, as to the best sequence of the two types of AIs and of the other available agents, including tamoxifen and fulvestrant, in different patient groups. PMID:16100523
Villalba, José M.; Alcaín, Francisco J.
Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD+ for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activity. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin-based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging-related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex®, SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases. PMID:22730114
Muetze, Sabine; Eggermann, Thomas; Leeners, Brigitte; Birke, Cornelia; Kuse, Sabine; Ortlepp, Jan Rudolf; Rudnik-Schoeneborn, Sabine; Zerres, Klaus; Rath, Werner
Plasminogen activator inhibitor-1 (PAI-1) is a major inhibitor of fibrinolysis, and a single nucleotide insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene has been identified. Subjects homozygous for the 4G allele have the highest PAI-levels due to increased PAI-1 gene transcription. Pre-eclampsia, and one of its most severe forms, the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome, are characterized by increased placental thrombosis based on a procoagulatory state in the mother. Several studies have investigated the role of the PAI-1 4G/5G polymorphism in pre-eclampsia, but no study has focused especially on HELLP syndrome. Therefore we aimed to assess the association between HELLP syndrome and the 4G/5G polymorphism in the PAI-1 gene. Genotyping of the PAI-1 4G/5G promoter polymorphism was performed in 102 Caucasian women with HELLP syndrome and 102 Caucasian women with uncomplicated pregnancies. The 4G/4G genotype was more frequent in women with HELLP syndrome than in controls (35.3% vs. 22.5%, respectively) but this difference was not significantly different (P = 0.129). The frequency of the 4G allele was 0.588 in patients and 0.515 in controls. These data suggest that women carrying a 4G/4G genotype of the PAI-1 gene are not at increased risk for developing HELLP syndrome and are thus in line with the majority of previous studies on the association between the PAI-1 4G/5G polymorphism and pre-eclampsia.
Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...
Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C
HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159
Semeraro, Nicola; Ammollo, Concetta T.; Semeraro, Fabrizio; Colucci, Mario
Sepsis is almost invariably associated with haemostatic abnormalities ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by massive thrombin formation and widespread microvascular thrombosis, partly responsible of the multiple organ dysfunction syndrome (MODS), and subsequent consumption of platelets and coagulation proteins causing, in most severe cases, bleeding manifestations. There is general agreement that the key event underlying this life-threatening sepsis complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. Mechanistically, the latter, together with the micro-organism and its derivatives, causes DIC by 1) up-regulation of procoagulant molecules, primarily tissue factor (TF), which is produced mainly by stimulated monocytes-macrophages and by specific cells in target tissues; 2) impairment of physiological anticoagulant pathways (antithrombin, protein C pathway, tissue factor pathway inhibitor), which is orchestrated mainly by dysfunctional endothelial cells (ECs); and 3) suppression of fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Notably, clotting enzymes non only lead to microvascular thrombosis but can also elicit cellular responses that amplify the inflammatory reactions. Inflammatory mediators can also cause, directly or indirectly, cell apoptosis or necrosis and recent evidence indicates that products released from dead cells, such as nuclear proteins (particularly extracellular histones), are able to propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenetic mechanisms of DIC and MODS may have important implications for the
Ni, Wei-Wei; Cao, Meng-Da; Huang, Wen; Meng, Ling; Wei, Ji-Fu
Tryptase is one of the main serine-proteinases located in the secretory granules of mast cells, and is released through degranulation, which is involved in the pathogenesis of allergic inflammatory disease, cardiovascular diseases, lung fibrosis and tumor. Therefore, inhibitors targeting tryptase may represent a new direction for the treatment of allergic inflammatory disease and other diseases. Areas covered: In this article, we discussed the history and development of tryptase inhibitors and described a variety of tryptase inhibitors via their structures and biological importance in clinical studies and drug development for tryptase-related diseases. Expert opinion: Initial tryptase inhibitors based on indole structure as the hydrophobic substituent on a benzylamine-piperidine template have low specificity and poor bioavailability. Therefore, designing new and specific inhibitors targeting tryptase should be involved in future clinical studies. Modifications toward indoles with varying N-substitution, introducing an amide bond, and growing the chain length contribute to an increase in the specific selectivity and potency of tryptase inhibitors. Tryptase has become the research hotspot to explore many related diseases. Therefore, there has been growing appreciation for the potential importance of the tryptase inhibitors as a target for treating these diseases.
White, Carl W.; Rancourt, Raymond C.; Veress, Livia A.
Acute lung injury due to sulfur mustard (SM) inhalation causes formation of airway fibrin casts that obstruct airways at multiple levels, leading to acute respiratory failure and death. These pathophysiological effects are seen in rodent models of acute SM vapor inhalation, as well as in human victims of acute SM inhalation. In rat models, the initial steps in activation of the coagulation system at extravascular sites depend on tissue factor (TF) expression by airway cells, especially in the microparticle fraction, and these effects can be inhibited by TF pathway inhibitor (TFPI) protein. Not only does the procoagulant environment of the acutely injured lung contribute to airway cast formation, but these lesions persist in airways because of the activation of multiple antifibrinolytic pathways, including plasminogen activator inhibitor-1 (PAI-1), thrombin-activatable fibrinolysis inhibitor (TAFI), and α2-antiplasmin (α2AP). Airway administration of tissue plasminogen activator (tPA) can overwhelm these effects and be save lives by preventing fibrin-dependent airway obstruction, gas-exchange abnormalities, and respiratory failure. In human survivors of SM inhalation, fibrotic processes, including bronchiolitis obliterans and interstitial fibrosis of the lung, are among the most disabling chronic lesions. Antifibrotic therapies may prove useful in preventing either or both of these forms of chronic lung damage. PMID:27384912
Gorar, Suheyla; Alioglu, Bulent; Ademoglu, Esranur; Uyar, Seyit; Bekdemir, Handan; Candan, Zehra; Saglam, Beylan; Koc, Gonul; Culha, Cavit; Aral, Yalcin
Impact of gestational diabetes mellitus (GDM) on the coagulation system, dynamics involved at a pathophysiological level and the exact mechanism remain unclear. To evaluate the association between diabetes-related parameters and hemostatic factors to search for a tendency of thrombosis in GDM. Nineteen pregnant women who had GDM, 16 healthy pregnant and 13 healthy nonpregnant controls admitted to the Endocrinology outpatient clinics were enrolled in the study. Fasting and postprandial glucose, hemoglobin A1c and insulin levels, and insulin resistance; fructosamine, thrombin activatable fibrinolysis inhibitor (TAFI), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor Type-1 (PAI-1), tissue-type plasminogen activator (t-PA), fibrinogen, plasminogen and hemoglobin levels, platelet counts, prothrombin time (PT), and activated partial thromboplastin time (aPTT) were studied. One-way analysis of variance, Kruskal-Wallis, and post hoc Tukey honestly significant difference or Conover's nonparametric multiple comparison tests for comparison of the study groups. PT and aPTT were significantly lower in GDM patients compared to controls (P < 0.05), whereas fibrinogen and plasminogen levels were significantly higher in this group compared to both nonpregnant and healthy pregnant controls (P < 0.05 for each). TAFI, TFPI, PAI-1, and tissue t-PA levels were not significantly different among groups. Our findings indicate tendency to develop thrombosis in GDM similar to diabetes mellitus; but more comprehensive studies with larger sample size are needed to determine the relationship between GDM and hemostasis.
Klíma, Petr; Laňková, Martina; Zažímalová, Eva
Here we present an overview of what is known about endogenous plant compounds that act as inhibitors of hormonal transport processes in plants, about their identity and mechanism of action. We have also summarized commonly and less commonly used compounds of non-plant origin and synthetic drugs that show at least partial 'specificity' to transport or transporters of particular phytohormones. Our main attention is focused on the inhibitors of auxin transport. The urgent need to understand precisely the molecular mechanism of action of these inhibitors is highlighted.
Pepeu, Giancarlo; Giovannini, Maria Grazia
Cholinesterase inhibitors (ChEIs) were introduced in the therapy of Alzheimer Disease (AD) in the nineteen nineties with great expectations. The hopes and large interest raised by these drugs are well demonstrated by 12,000 references listed by PubMed under 'ChEI' for 1995-2007. The list is reduced to 2500 if we confine ourselves to 'ChEIs and dementia'. Of them, about 500 were published in the last two years. Whereas an increase in brain acetylcholine and an improvement of cognitive deficits have been consistently demonstrated in animal models of AD, from aging rats to transgenic mice, the clinical effectiveness of ChEIs has been and is still a matter of contrasting opinions. These range from the negative conclusions of the AD2000 trial on donepezil, claiming that it is not cost effective, with benefits below a minimally relevant threshold, to the NICE appraisal of 2007 declaring that donepezil, rivastigmine, galantamine are efficacious for mild to moderate AD, irrespective of their different selectivity for acetyl- (AChE) and butyrylcholinesterase (BuChE). The possibility that ChEIs may exert their effects through mechanisms beyond cholinesterase inhibition has been envisaged. However, according to the information presented in this review, the "classical" ChEIs, donepezil, rivastigmine and galantamine, show no pharmacological actions beyond cholinesterase inhibition which may play an important role in their therapeutic efficacy. The diverging opinions on clinical efficacy do not discourage from developing new ChEIs, and particularly the so called multifunctional ChEIs. They represent the future of the cholinergic therapy for AD but other indications for these drugs may be considered, including vascular dementia, mild cognitive impairment, and the ethically sensitive improvement of memory and learning in healthy subjects.
Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853
Hui, Chunngai; Ye, Tao
Lysine methyltransferase which catalyze methylation of histone and non-histone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery. PMID:26258118
Hörl, W H
Treatment with ACE inhibitors results in kidney protection due to reduction of systemic blood pressure, intraglomerular pressure, an antiproliferative effect, reduction of proteinuria and a lipid-lowering effect in proteinuric patients (secondary due to reduction of protein excretion). Elderly patients with diabetes melitus, coronary heart disease or peripheral vascular occlusion are at risk for deterioration of kidney function due to a high frequency of renal artery stenosis in these patients. In patients with renal insufficiency dose reduction of ACE inhibitors is necessary (exception: fosinopril) but more important is the risk for development of hyperkalemia. Patients at risk for renal artery stenosis and patients pretreated with diuretics should receive a low ACE inhibitor dosage initially ("start low - go slow"). For compliance reasons once daily ACE inhibitor dosage is recommended.
Tanaka-Azevedo, A. M.; Morais-Zani, K.; Torquato, R. J. S.; Tanaka, A. S.
Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor Xa. Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals. PMID:20976270
Abdel-Magid, A F; Maryanoff, C A; Mehrman, S J
Influenza neuraminidase inhibitors provide a means to combat flu, a potentially very serious disease. For the first time, there is a way to treat influenza by blocking the influenza enzyme neuraminidase to stop or slow the progression of infection. The diverse structures and synthesis of several influenza neuraminidase inhibitors are discussed. Contributions from chemical process development groups are highlighted for those compounds that have reached the market, such as zanamivir and oseltamivir phosphate.
Kyosseva, S V; Kyossev, Z N; Elbein, A D
Trehazolin, a new trehalase inhibitor isolated from the culture broth of Micromonospora, was reported to be a highly specific inhibitor for porcine and silk worm trehalases with IC50 values of 5.5 x 10(-9) and 3.7 x 10(-9) M, respectively (O. Ando, H. Satake, K. Itoi, A. Sato, M. Nakajima, S. Takashi, H. Haruyama, Y. Ohkuma, T. Kinoshita, and R. Enokita (1991) J. Antibiot. 44, 1165-1168). We also found that trehazolin is a very powerful and quite specific inhibitor against purified pig kidney trehalase, giving an IC50 value of 1.9 x 10(-8) M. Lineweaver-Burk plots showed that this compound was a competitive inhibitor of the trehalase. However, even at concentrations of 200 micrograms/ml, trehazolin did not inhibit the rat intestinal maltase or sucrase, yeast alpha-glucosidase or almond beta-glucosidase. Validoxylamine A and validamycin A, two other trehalase inhibitors, showed potent competitive inhibition against purified pig kidney trehalase, with IC50 values of 2.4 x 10(-9) and 2.5 x 10(-4) M, respectively. On the other hand, validoxylamine A was almost inactive against rat intestinal sucrase and maltase, with some inhibition being observed at millimolar concentration. A number of other glucosidase inhibitors, such as MDL 25637, castanospermine, and deoxynojirimycin were also tested against the purified trehalase and showed reasonable inhibitory activity.
Swarbreck, Scott B; Secor, Dan; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel
The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis.
Scheer, Frank A J L; Shea, Steven A
Serious adverse cardiovascular events peak in the morning, possibly related to increased thrombosis in critical vessels. Plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis, is a key circulating prothrombotic factor that rises in the morning in humans. We tested whether this morning peak in PAI-1 is caused by the internal circadian system or by behaviors that typically occur in the morning, such as altered posture and physical activity. Twelve healthy adults underwent a 2-week protocol that enabled the distinction of endogenous circadian effects from behavioral and environmental effects. The results demonstrated a robust circadian rhythm in circulating PAI-1 with a peak corresponding to ∼6:30 am. This rhythm in PAI-1 was 8-times larger than changes in PAI-1 induced by standardized behavioral stressors, including head-up tilt and 15-minute cycle exercise. If this large endogenous morning peak in PAI-1 persists in vulnerable individuals, it could help explain the morning peak in adverse cardiovascular events.
Sashindranath, Maithili; Sales, Eunice; Daglas, Maria; Freeman, Roxann; Samson, Andre L; Cops, Elisa J; Beckham, Simone; Galle, Adam; McLean, Catriona; Morganti-Kossmann, Cristina; Rosenfeld, Jeffrey V; Madani, Rime; Vassalli, Jean-Dominique; Su, Enming J; Lawrence, Daniel A; Medcalf, Robert L
The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix
Ammollo, Concetta Tiziana; Semeraro, Nicola; Carratù, Maria Rosaria; Colucci, Mario; Semeraro, Fabrizio
The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins.
Byrnes, James R.; Duval, Cédric; Wang, Yiming; Hansen, Caroline E.; Ahn, Byungwook; Mooberry, Micah J.; Clark, Martha A.; Johnsen, Jill M.; Lord, Susan T.; Lam, Wilbur A.; Meijers, Joost C. M.; Ni, Heyu; Ariëns, Robert A. S.
Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size. PMID:26324704
Byrnes, James R; Duval, Cédric; Wang, Yiming; Hansen, Caroline E; Ahn, Byungwook; Mooberry, Micah J; Clark, Martha A; Johnsen, Jill M; Lord, Susan T; Lam, Wilbur A; Meijers, Joost C M; Ni, Heyu; Ariëns, Robert A S; Wolberg, Alisa S
Factor XIII(a) [FXIII(a)] stabilizes clots and increases resistance to fibrinolysis and mechanical disruption. FXIIIa also mediates red blood cell (RBC) retention in contracting clots and determines venous thrombus size, suggesting FXIII(a) is a potential target for reducing thrombosis. However, the mechanism by which FXIIIa retains RBCs in clots is unknown. We determined the effect of FXIII(a) on human and murine clot weight and composition. Real-time microscopy revealed extensive RBC loss from clots formed in the absence of FXIIIa activity, and RBCs exhibited transient deformation as they exited the clots. Fibrin band-shift assays and flow cytometry did not reveal crosslinking of fibrin or FXIIIa substrates to RBCs, suggesting FXIIIa does not crosslink RBCs directly to the clot. RBCs were retained in clots from mice deficient in α2-antiplasmin, thrombin-activatable fibrinolysis inhibitor, or fibronectin, indicating RBC retention does not depend on these FXIIIa substrates. RBC retention in clots was positively correlated with fibrin network density; however, FXIIIa inhibition reduced RBC retention at all network densities. FXIIIa inhibition reduced RBC retention in clots formed with fibrinogen that lacks γ-chain crosslinking sites, but not in clots that lack α-chain crosslinking sites. Moreover, FXIIIa inhibitor concentrations that primarily block α-, but not γ-, chain crosslinking decreased RBC retention in clots. These data indicate FXIIIa-dependent retention of RBCs in clots is mediated by fibrin α-chain crosslinking. These findings expose a newly recognized, essential role for fibrin crosslinking during whole blood clot formation and consolidation and establish FXIIIa activity as a key determinant of thrombus composition and size.
Davies, Joanna; Kadir, Rezan A
Under normal physiological circumstances menstruation is a highly regulated, complex process that is under strict hormonal control. During normal menstruation, progesterone withdrawal initiates menstruation. The cessation of menstrual bleeding is achieved by endometrial haemostasis via platelet aggregation, fibrin deposition and thrombus formation. Local endocrine, immunological and haemostatic factors interact at a molecular level to control endometrial haemostasis. Tissue factor and thrombin play a key role locally in the cessation of menstrual bleeding through instigation of the coagulation factors. On the other hand, fibrinolysis prevents clot organisation within the uterine cavity while plasminogen activator inhibitors (PAI) and thrombin-activatable fibrinolysis inhibitors control plasminogen activators and plasmin activity. Abnormalities of uterine bleeding can result from imbalance of the haemostatic factors. The most common abnormality of uterine bleeding is heavy menstrual bleeding (HMB). Modern research has shown that an undiagnosed bleeding disorder, in particular von Willebrand disease (VWD) and platelet function disorders, can be an underlying cause of HMB. This has led to a change in the approach to the management of HMB. While full haemostatic assessment is not required for all women presenting with HMB, menstrual score and bleeding score can help to discriminate women who are more likely to have a bleeding disorder and benefit from laboratory haemostatic evaluation. Haemostatic agents (tranexamic acid and DDAVP) enhance systemic and endometrial haemostasis and are effective in reducing menstrual blood loss in women with or without bleeding disorders. Further research is required to enhance our understanding of the complex interactions of haemostatic factors in general, and specifically within the endometrium. This will lead to the development of more targeted interventions for the management of abnormal uterine bleeding in the future.
Batt, Anna R; St Germain, Commodore P; Gokey, Trevor; Guliaev, Anton B; Baird, Teaster
The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.
Mosolov, V V; Kolosova, G V; Valueva, T A; Dronova, L A
The trypsin inhibitor from Gleditsia triacanthos (L.) seeds was purified by affinity chromatography on a column with trypsin-Sepharose 4B. The isolated inhibitor is a single-chain protein with molecular weight of about 20 000. The inhibitor suppresses bovine trypsin at a molar rate of 1 : 1, but weakly inhibits chymotrypsin in a non-stoichiometric manner. Some properties of the isolated inhibitor closely resembled those of soybean trypsin inhibitor (Kunitz).
Brand, B; Graf, L
Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.
Sumi, H; Nakajima, N; Mihara, H
1. Extracts with physiological saline solution were obtained from about 20 species of invertebrates and seaweed. Tosyl-L-Arg-MeOH hydrolysing and fibrin plate lytic activity were detected in the invertebrates Stichopus japonicus, Crassost gigas, Tapes japonica, and Kintai-gai as well as the seaweed Codiales codium. 2. These activities were all labile against heat (at 65 degrees C for 1 hr). Except for the extract from Stichopus japonicus, lytic activities against fibrin plates with and without plasminogen were similar. 3. The extract from S. japonicus showed plasminogen activating potency as well as the existence of urokinase (UK) activity enhancing factor. 4. On the other hand, the extract of the seaweed Hizikia fusiformis showed a strong UK inhibiting activity. 5. A fraction of fibrinolytic enzyme was obtained from the extract of S. japonicus by absorption to the celite affinity chromatography. It was orally administered to rabbits at a dosage of 40 mg/kg/day. 6. Fibrinolytic activity was determined periodically on the eugloblin fraction of plasma samples collected from these animals. 7. As compared with the pretreatment value, the activity increased about 2 times (P less than 0.01) and 3 times (P less than 0.005) after 4 and 8 weeks, respectively, of the treatment. 8. After 8 weeks of treatment, the kidney of treated rabbits was extracted with 2 M KCl. The activity of tissue plasminogen activator (free-type TPA) was revealed to be enhanced significantly (P less than 0.001) in the extracts. 9. The fibrinolytic enzyme increased in the blood was recognized by zymography to be mainly the UK type plasminogen activator with mol. wt of 53,000.
Piazza, Gregory; Fanikos, John; Zayaruzny, Maksim; Goldhaber, Samuel Z.
SUMMARY The number of acutely ill hospitalized medical patients at risk for acute venous thromboembolism (VTE) has not been well defined. Therefore, we used the 2003 United States Healthcare Cost and Utilization Project (HCUP) Nationwide Inpatient Sample database to estimate VTE events among hospitalized medical patients. We then modeled the potential reduction in VTE with universal utilization of appropriate pharmacological thromboprophylaxis. We calculated that 8,077,919 acutely ill hospitalized medical patients were at risk for VTE. Heart failure, respiratory failure, pneumonia, and cancer were the most common medical diagnoses. We estimated that 196,134 VTE-related events occurred in 2003, afflicting two out of every 100 acutely ill hospitalized medical patients. These VTE-related events were comprised of 122,235 symptomatic deep vein thromboses, 32,654 symptomatic episodes of pulmonary embolism, and 41,245 deaths due to VTE. In our model, rates of pharmacological thromboprophylaxis prescription were low for various acute medical illnesses, ranging from 15.3% to 49.2%. However, with universal thromboprophylaxis, 114,174 VTE-related events would have been prevented. In conclusion, acutely ill medical patients represent a large population vulnerable to the development of VTE during hospitalization. The number of VTE-related events would be halved with universal thromboprophylaxis. Further efforts focused on improving VTE prevention strategies in hospitalized medical patients are warranted. PMID:19718471
Mishchenko, S V
In experiments in vitro on a man, cat and rat thrombocyte-free plasma it was determined that the polarized light over 5 cm distance from the object (6 min exposure) causes the inhibition of fibrinolytic activity of euglobulin fraction. It was shown that the fibrinolytic inhibition under the influence of the polarized light is connected with its antiplasmin effect. The importance of the fibrinolytic reaction for the course of inflammation process and tissue regeneration after injury and the role of therapy with polarized light in these reactions is discussed.
Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.
This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.
Danford, M. D.
The effect of single salts, as well as multicomponent mixtures, on corrosion inhibition was studied for type 1010 steel; for 5052, 1100, and 2219-T87 aluminum alloys; and for copper. Molybdate-containing inhibitors exhibit an immediate, positive effect for steel corrosion, but an incubation period may be required for aluminum before the effect of a given inhibitor can be determined. The absence of oxygen was found to provide a positive effect (smaller corrosion rate) for steel and copper, but a negative effect for aluminum. This is attributed to the two possible mechanisms by which aluminum can oxidize. Corrosion inhibition is generally similar for oxygen-rich and oxygen-free environments. The results show that the electrochemical method is an effective means of screening inhibitors for the corrosion of single metals, with caution to be exercised in the case of aluminum.
Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt
This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.
Dobbs, J.B.; Brown, J.M.
This paper describes a method of inhibiting the formation of scales such as barium and strontium sulfate in low pH aqueous systems, and calcium carbonate in systems containing high concentrations of dissolved iron. The solution, chemically, involves treating the aqueous system with an inhibitor designed to replace organic-phosphonates. Typical low pH aqueous systems where the inhibitor is particularly useful are oilfield produced-water, resin bed water softeners that form scale during low pH, acid regeneration operations. Downhole applications are recommended where high concentrations of dissolved iron are present in the produced water. This new approach to inhibition replaces typical organic phosphonates and polymers with a non-toxic, biodegradable scale inhibitor that performs in harsh environments.
Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.
This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.
Antonov, A.V.; Petrenko, V.G.; Frolova, R.P.; Kurinnaya, S.N.
Activated sludge and froth from the biological treatment of coke plant waste waters has been determined to be a corrosion inhibitor in both neutral and acidic media, due to the presence of unreacted coking derived inhibitors, bacteriological formation of inhibitors, bacterial organisms, humic-type organics and traces of germanium, zinc, mercury and manganese. The corrosive liquids tested were, river water, technical system water, gas cooler aqueous condensate, gas collector condensate and coking waste water before and after treatment, the substrate being St 3 steel plates (45 X 45 X 5 M) (time 24-30 hr (acid media) and 934 hr (neutral media)). The activated sludge (25 g/l) reduced acid media corrosion rate by 10/sup 3/, the protective effect being 99% for the test liquids: Sludge is more effective than the froth.
Vaszilcsin, Nicolae; Ordodi, Valentin; Borza, Alexandra
This paper presents a method of expired or unused drugs valorization as corrosion inhibitors for metals in various media. Cyclic voltammograms were drawn on platinum in order to assess the stability of pharmaceutically active substances from drugs at the metal-corrosive environment interface. Tafel slope method was used to determine corrosion rates of steel in the absence and presence of inhibitors. Expired Carbamazepine and Paracetamol tablets were used to obtain corrosion inhibitors. For the former, the corrosion inhibition of carbon steel in 0.1 mol L(-1) sulfuric acid solution was about 90%, whereas for the latter, the corrosion inhibition efficiency of the same material in the 0.25 mol L(-1) acetic acid-0.25 mol L(-1) sodium acetate buffer solution was about 85%.
Bonyadi, M; Shaghaghi, Z; Haghi, M; Dastgiri, S
Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by intermittent episodes of fever with serositis, arthritis, or eriseplemya. Plasminogen activator inhibitor 1 (PAI-1) is a key element in the inhibition of fibrinolysis by inactivating tissue-type and urokinase-type plasminogen activators. We evaluated the association of PAI-1 -675 4G/5G polymorphism with the severity of FMF disease. For this purpose, 89 FMF patients with M694V homozygous mutation and 95 healthy controls from Iranian Azeri Turks were selected. Detection of this polymorphism was performed by polymerase chain reaction using allele-specific primers. No significant association was found between patients and control group. However, these data showed that FMF patients with M694V homozygous mutation carrying 4G/4G genotype have a reduced risk for development of pleuritis (odds ratios (OR) 0.36; 95 % confidence intervals (CI) 0.5-0.85; P value = 0.007) compared with 5G/5G homozygotes who have increased risk for development of amyloidosis (OR = 2.46; 95 %CI = 1.29-4.72; P value = 0.001), pleuritis (OR = 2.55; 95 %CI = 1.31-4.99; P value = 0.001), and fever (OR = 4.68; 95 %CI = 2.04-10.96; P value = 0.000). Furthermore, the allelic frequency of the 4G among the patients with pleuritis was significantly low (OR = 0.5, 95 % CI = 0.27-0.92, P value = 0.008). Our data suggest a protective role for the 4G allele against pleuritis in FMF patients with M694V homozygous mutation in this cohort. More evaluation of this polymorphism may be important and require further studies.
Urano, T; Ihara, H; Umemura, K; Suzuki, Y; Oike, M; Akita, S; Tsukamoto, Y; Suzuki, I; Takada, A
In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.
Kim, Hong-Soo; Hwang, Kyu-Yoon; Chung, Il-Kwon; Park, Sang-Heum; Lee, Moon-Ho; Kim, Sun-Joo; Hong, Sae-Yong
Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) may be involved in the pathogenesis of peptic ulcers through suppression of fibrinolysis. This study was designed to investigate associations of t-PA and PAI-1 genes with clinical features of the patients with bleeding gastric ulcers. Eighty-four patients with peptic ulcers and 100 controls were studied between January 1998 and April 2000. We used polymerase chain reaction and endonuclease digestion to genotype for 4G/5G polymorphism in the promoter region of the PAI-1 gene and the Alurepeat insertion/deletion (I/D) polymorphism in intron h of the t-PA gene. Various clinical features, including lesion site, bleeding event, recurrence of ulcer, and rebleeding, were assessed using a multiple logistic regression model. The genotype distributions of both the t-PA and PAI-1 genes did not differ between the patient and control groups. The occurrence of the I/D or D/D genotype of t-PA was significantly higher in cases of duodenal ulcer (adjusted OR=4.39, 95% CI=1.12-17.21). When a dominant effect (i.e., 4G/4G or 4G/5G versus 5G/5G) of the 4G allele was assumed, the PAI-1 4G/4G genotype was independently associated with rebleeding after hemostasis (adjusted OR=5.07, 95% CI=1.03-24.87). Our data suggest that t-PA gene polymorphism is associated with duodenal ulcers, and that the PAI-1 gene may be a risk factor leading to recurrent bleeding after initial hemostasis. PMID:12589088
Di Nardo, Maria Antonietta; Annunziata, Maria Laura; Ammirabile, Massimiliano; Di Minno, Matteo Nicola Dario; Ruocco, Anna Lilia; De Falco, Marianna; Di Lieto, Andrea
The study investigated the impact of gonadotropin-releasing hormone analogue (GnRH-a) on coagulation and fibrinolytic activities and its effectiveness in the prevention of pelvic adhesion after myomectomy. Thirty-two infertile women underwent myomectomy followed by adhesion evaluation surgery with a second-look laparoscopy. Before myomectomy, 15 women were treated with triptorelin acetate for 3 months and 17 received no treatment. Plasminogen activator inhibitor (PAI), thrombin activatable fibrinolysis inhibitor (TAFI), protein C (PC), plasminogen, α2-antiplasmin were determined by enzyme-linked immunosorbent assays and the activity of coagulation factors V and VIII by coagulometric methods. Patients treated with GnRH-a showed significant decrease in PAI, TAFI, factors V, and VIII (P < .05) and increased PC (P < .05), but no significant change in plasminogen and α2-antiplasmin levels compared with control group. The incidence, extent, and severity of adhesions were significantly lower in GnRH-a-treated patients compared with control group (P < .05), suggesting a possible critical role of the GnRH-a therapy in preventing postoperative adhesion development.
Meltzer, Mirjam E; Lisman, Ton; de Groot, Philip G; Meijers, Joost C M; le Cessie, Saskia; Doggen, Carine J M; Rosendaal, Frits R
Elevated plasma clot lysis time (CLT) increases risk of venous and arterial thrombosis. It is unclear which fibrinolytic factors contribute to thrombosis risk. In 743 healthy control subjects we investigated determinants of CLT. By comparison with 770 thrombosis patients, we assessed plasma levels of fibrinolytic proteins as risk factors for a first thrombosis. Plasminogen activator inhibitor-1 (PAI-1) levels were the main determinants of CLT, followed by plasminogen, thrombin-activatable fibrinolysis inhibitor (TAFI), prothrombin, and alpha2-antiplasmin. Fibrinogen, factor VII, X, and XI contributed minimally. These proteins explained 77% of variation in CLT. Levels of the fibrinolytic factors were associated with thrombosis risk (odds ratios, highest quartile vs lowest, adjusted for age, sex, and body mass index: 1.6 for plasminogen, 1.2 for alpha2-antiplasmin, 1.6 for TAFI, 1.6 for PAI-1, and 1.8 for tissue plasminogen activator [t-PA]). Adjusting for acute-phase proteins attenuated the risk associated with elevated plasminogen levels. The risk associated with increased t-PA nearly disappeared after adjusting for acute-phase proteins and endothelial activation. TAFI and PAI-1 remained associated with thrombosis after extensive adjustment. In conclusion, CLT reflects levels of all fibrinolytic factors except t-PA. Plasminogen, TAFI, PAI-1, and t-PA are associated with venous thrombosis. However, plasminogen and t-PA levels may reflect underlying risk factors.
Gromotowicz, Anna; Osmólska, Urszula; Mantur, Maria; Szoka, Piotr; Zakrzeska, Agnieszka; Szemraj, Janusz; Chabielska, Ewa
Recent studies have focused on a new wave of interest in aldosterone due mainly to its growing profile as a local messenger in pathology of the cardiovascular system, rather than its hormonal action. In the last few years strong evidence for a correlation between raised aldosterone level and haemostasis disturbances leading to increased risk of cardiovascular events has been provided. It has been demonstrated that aldosterone contributes to endothelial dysfunction, fibrinolytic disorders and oxidative stress augmentation. It was also shown that chronic aldosterone treatment results in enhanced experimental arterial thrombosis. Our study in a venous model of thrombosis in normotensive rats confirmed that even a short-lasting increase in aldosterone level intensified thrombus formation. One-hour aldosterone infusion shortened bleeding time; increased platelet adhesion to collagen; reduced tissue factor, thrombin activatable fibrinolysis inhibitor, and plasminogen activator inhibitor; and increased plasminogen activator plasma level. A fall in plasma nitric oxide metabolite concentration with a decrease in aortic nitric oxide synthase mRNA level was also observed. Moreover, aldosterone increased hydrogen peroxide and malonyl dialdehyde plasma concentration and augmented NADPH oxidase and superoxide dismutase aortic expression. Therefore, the mechanism of aldosterone prothrombotic action is multiple and involves primary haemostasis activation, procoagulative and antifibrinolytic action, NO bioavailability impairment and oxidative stress augmentation. The effects of aldosterone were not fully abolished by mineralocorticoid receptor blockade, suggesting the involvement of alternative mechanisms in the prothrombotic aldosterone action.
Martí-Fàbregas, Joan; Borrell, Montserrat; Silva, Yolanda; Delgado-Mederos, Raquel; Martínez-Ramírez, Sergi; de Juan-Delago, Manuel; Tirado, Isabel; Alejaldre, Aída; Marín, Rebeca; Martí-Vilalta, Josep-Lluis; Fontcuberta, Jordi
We tested the hypothesis that proteins of hemostasia could be associated with hematoma growth (HG) in patients with acute intracerebral hemorrhage. We prospectively studied patients with spontaneous supratentorial intracerebral hemorrhage within the first 6 hours after the onset of symptoms. HG was defined as an increase > 33% in the volume of hematoma on CT obtained 24 to 72 hours after the onset of symptoms in comparison with the CT obtained at admission. We collected admission and follow-up blood samples. We measured fibrinogen, factor XIII, thrombin activatable fibrinolysis inhibitor, plasminogen activator inhibitor, plasminogen, α₂-antiplasmin, tissue plasminogen activator, d-dimer, thrombomodulin, thrombin-antithrombin complex, and plasmin-antiplasmin complex. We included 90 patients with a mean age of 71 ± 10.8 years; 61% were men. HG was observed in 35 (39%) of the patients. Mean baseline and follow-up protein measurements showed no difference between the groups with and without HG. The analysis of variance showed that factor XIII activity decreased in the non-HG group in the 24 to 72 hours sample, whereas it increased in the HG group (P = 0.001). Factor XIII was the only measured protein related to HG. The levels at the follow-up sample decreased in the non-HG group and increased in the HG group. Further studies are needed to confirm this association.
Undas, Anetta; Zubkiewicz-Usnarska, Lidia; Helbig, Grzegorz; Woszczyk, Dariusz; Kozińska, Justyna; Dmoszyńska, Anna; Dębski, Jakub; Podolak-Dawidziak, Maria; Kuliczkowski, Kazimierz
Induction therapy in patients with multiple myeloma increases the risk of thromboembolism. We have recently shown that multiple myeloma patients tend to form denser fibrin clots displaying poor lysability. We investigated the effect of induction therapy on fibrin clot properties in multiple myeloma patients. Ex-vivo plasma fibrin clot permeability, turbidity, susceptibility to lysis, thrombin generation, factor VIII and fibrinolytic proteins were compared in 48 multiple myeloma patients prior to and following 3 months of induction therapy, mainly with cyclophosphamide-thalidomide-dexamethasone regimen. Patients on thromboprophylaxis with aspirin or heparins were eligible. A 3-month induction therapy resulted in improved clot properties, that is higher clot permeability, compaction, shorter lag phase and higher final turbidity, along with shorter clot lysis time and higher rate of D-dimer release from fibrin clots than the baseline values. The therapy also resulted in lower thrombin generation, antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), but elevated factor VIII. Progressive disease was associated with lower posttreatment clot permeability and lysability. Despite thromboprophylaxis, two patients developed ischemic stroke and 10 had venous thromboembolism. They were characterized by pretreatment lower clot permeability, prolonged clot lysis time, longer lag phase, higher peak thrombin generation, TAFI and plasminogen activator inhibitor -1. Formation of denser plasma fibrin clots with reduced lysability and increased thrombin generation at baseline could predispose to thrombotic complications during induction treatment in multiple myeloma patients. We observed improved fibrin clot properties and thrombin generation in multiple myeloma patients except those with progressive disease.
Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds. PMID:24308725
Dysregulation of protein phosphorylation, especially on tyrosine residues, plays a crucial role in development and progression of hematological malignancies. Since remarkable success in imatinib therapy of CML and Ph+ALL, extensive efforts have made to explore candidate molecular targets and next breakthrough drugs. Now that next generation ABL kinase inhibitors are available for CML, the therapeutic algorithm has been revolutionized. As for AML and lymphoid malignancies, many kinase inhibitors targeting FLT3, BTK and aurora-A are on early and late clinical trials, and a number of promising drugs including ibrutinib are picked up for further evaluation.
Cui, Jie; Hu, Yun-Feng; Feng, Xie-Min; Tian, Tao; Guo, Ya-Huan; Ma, Jun-Wei; Nan, Ke-Jun; Zhang, Hong-Yi
Epidermal growth factor receptor (EGFR) inhibitor treatment is a strategy for cancer therapy. However, innate and acquired resistance is a major obstacle of the efficacy. Autophagy is a self-digesting process in cells, which is considered to be associated with anti-cancer drug resistance. The activation of EGFR can regulate autophagy through multiple signal pathways. EGFR inhibitors can induce autophagy, but the specific function of the induction of autophagy by EGFR inhibitors remains biphasic. On the one hand, autophagy induced by EGFR inhibitors acts as a cytoprotective response in cancer cells, and autophagy inhibitors can enhance the cytotoxic effects of EGFR inhibitors. On the other hand, a high level of autophagy after treatment of EGFR inhibitors can also result in autophagic cell death lacking features of apoptosis, and the combination of EGFR inhibitors with an autophagy inducer might be beneficial. Thus, autophagy regulation represents a promising approach for improving the efficacy of EGFR inhibitors in the treatment of cancer patients.
Incalcaterra, Egle; Meli, Francesco; Muratori, Ida; Corrado, Egle; Amato, Corrado; Canino, Baldassare; Ferrara, Filippo
Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of plasminogen activator. The functional 4G/5G polymorphism of the gene coding for PAI-1 may affect PAI-1 plasmatic activity, influencing the imbalance between coagulation and fibrinolysis cascades. In this prospective cohort analytic study, we investigated the role of this single nucleotide polymorphism in the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome. In a group of 168 patients with post-surgical deep vein thrombosis of the legs, we analyzed the 4G/5G polymorphism in the promoter of PAI-1 gene and plasmatic PAI-1 activity. Enrolled patients were divided in two groups: patients with 4G/5G polymorphism and increased PAI-1 activity (n=85) and patients without 4G/5G polymorphism and normal PAI-1 activity (n=83). All patients were treated according to current protocols and re-examined after 3, 12 and 36 months in order to evaluate the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome. We found a significantly increased PAI activity in carrier of the 4G allele, who experienced much more frequently a persistence of thrombosis after 3, 12 and 36 months and/or the development of post-thrombosis syndrome, in spite of the anticoagulant treatment. These data not only confirm the role played by PAI-1 activity and by the 4G/5G SNP of the PAI-1 gene, but also suggest that current therapeutic protocols, recommending the administration of low weight molecular heparin and oral anticoagulant for the treatment of deep vein thrombosis, could be non sufficient for patients genetically predisposed to a less efficient clot lysis. Copyright © 2013. Published by Elsevier Ltd.
Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M
Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466
development for ration and commercial items that deteriorate from Maillard browning effects while in storage. Figure 15: Avocado 96 h after being...MAILLARD BROWNING by Nicole Favreau Farhadi Lauren Pecukonis and Jacqueline LeBlanc December 2013 Final Report...2009- September 2011 4. TITLE AND SUBTITLE NATURAL INHIBITORS OF MAILLARD BROWNING 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT
Humphries, T. S.
Combinations of borates, nitrates, phosphates, silicates, and sodium MBT protect aluminum from corrosion in fresh water. Most effective combinations contained sodium phosphate and were alkaline. These inhibitors replace toxic chromates which are subject to governmental restrictions, but must be used in larger quantities. Experimental exposure times varied from 1 to 14 months depending upon nature of submersion solution.
Képiró, Miklós; Várkuti, Boglárka H.; Bodor, Andrea; Hegyi, György; Drahos, László; Kovács, Mihály; Málnási-Csizmadia, András
Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 μM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions. PMID:22647605
Garuti, Laura; Roberti, Marinella; Bottegoni, Giovanni
Benzimidazole is a common kinase inhibitor scaffold and benzimidazole-based compounds interact with enzymes by multiple binding modes. In some cases, the benzimidazole acts as part of the hinge-binding motif, in others it has a scaffolding role without evidence for direct hinge binding. Several of these compounds are ATP-competitive inhibitors and show high selectivity by exploiting unique structural properties that distinguish one kinase from the majority of other kinases. However, the high specificity for a single target is not always sufficient. Thus another approach, called multi-target therapy, has been developed over the last few years. The simultaneous inhibition of various kinases may be useful because the disease is attacked at several relevant targets. Moreover, if a kinase becomes drug-resistant, a multitargeted drug can act on the other kinases. Some benzimidazole derivatives are multi-target inhibitors. In this article benzimidazole inhibitors are reported with their mechanisms of action, structure-activity relationship (SAR) and biological properties.
Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min
Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.
Cao, Guangli; Ximenes, Eduardo; Nichols, Nancy N; Zhang, Leyu; Ladisch, Michael
Removal of enzyme inhibitors released during lignocellulose pretreatment is essential for economically feasible biofuel production. We tested bio-abatement to mitigate enzyme inhibitor effects observed in corn stover liquors after pretreatment with either dilute acid or liquid hot water at 10% (w/v) solids. Bio-abatement of liquors was followed by enzymatic hydrolysis of cellulose. To distinguish between inhibitor effects on enzymes and recalcitrance of the substrate, pretreated corn stover solids were removed and replaced with 1% (w/v) Solka Floc. Cellulose conversion in the presence of bio-abated liquors from dilute acid pretreatment was 8.6% (0.1x enzyme) and 16% (1x enzyme) higher than control (non-abated) samples. In the presence of bio-abated liquor from liquid hot water pretreated corn stover, 10% (0.1x enzyme) and 13% (1x enzyme) higher cellulose conversion was obtained compared to control. Bio-abatement yielded improved enzyme hydrolysis in the same range as that obtained using a chemical (overliming) method for mitigating inhibitors. Copyright © 2013 Elsevier Ltd. All rights reserved.
Chandrasekar, Balakumaran; Hong, Tram Ngoc; van der Hoorn, Renier A L
Activity-based protein profiling (ABPP) has emerged as a powerful proteomic approach to study the active proteins in their native environment by using chemical probes that label active site residues in proteins. Traditionally, ABPP is classified as either comparative or competitive ABPP. In this protocol, we describe a simple method called convolution ABPP, which takes benefit from both the competitive and comparative ABPP. Convolution ABPP allows one to detect if a reduced signal observed during comparative ABPP could be due to the presence of inhibitors. In convolution ABPP, the proteomes are analyzed by comparing labeling intensities in two mixed proteomes that were labeled either before or after mixing. A reduction of labeling in the mix-and-label sample when compared to the label-and-mix sample indicates the presence of an inhibitor excess in one of the proteomes. This method is broadly applicable to detect inhibitors in proteomes against any proteome containing protein activities of interest. As a proof of concept, we applied convolution ABPP to analyze secreted proteomes from Pseudomonas syringae-infected Nicotiana benthamiana leaves to display the presence of a beta-galactosidase inhibitor.
Colović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M
Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer's disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases.
Schudt, Christian; Hatzelmann, Armin; Beume, Rolf; Tenor, Hermann
The first pharmacological investigations of phosphodiesterase (PDE) inhibitors were developed with the clinical efficacies of drugs isolated from coffee, cacao and tea but only later their relevant ingredients were identified as xanthines that act as PDE. With its diuretic, inotropic and bronchodilating clinical efficacy, use of theophylline anticipated the clinical goals, which were later approached with the first-generation of weakly selective PDE inhibitors in the period from 1980 to 1990. Pharmacological and clinical research with these early compounds provided a vast pool of information regarding desired and adverse actions - although most of these new drugs had to be discontinued due to severe adverse effects. The pharmacological models for cardiac, vascular and respiratory indications were analysed for their PDE isoenzyme profiles, and when biochemical and molecular biological approaches expanded our knowledge of the PDE superfamily, the purified isoenzymes that were now available opened the door for more systematic studies of inhibitors and for generation of highly selective isoenzyme-specific drugs. The development of simple screening models and clinically relevant indication models reflecting the growing knowledge about pathomechanisms of disease are summarised here for today's successful application of highly selective PDE3, PDE4 and PDE5 inhibitors. The interplay of serendipitous discoveries, the establishment of intelligent pharmacological models and the knowledge gain by research results with new substances is reviewed. The broad efficacies of new substances in vitro, the enormous biodiversity of the PDE isoenzyme family and the sophisticated biochemical pharmacology enabled Viagra to be the first success story in the field of PDE inhibitor drug development, but probably more success stories will follow.
Baskova, I P; Zavalova, L L
The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.
Proton pump inhibitors are the one of the most widely used drugs in the world. Hypomagnesemic hypoparathyroidism has been reported with different proton pump inhibitors with prolonged oral use. We report the first reported case of possible such effect with intravenous preparation of proton pump inhibitor. This case report raises awareness among physicians worldwide of this often unknown association, as life-threatening cardiac and neuromuscular complications can arise with unrecognized hypocalcemia and hypomagnesemia with proton pump inhibitors.
Proton pump inhibitors are the one of the most widely used drugs in the world. Hypomagnesemic hypoparathyroidism has been reported with different proton pump inhibitors with prolonged oral use. We report the first reported case of possible such effect with intravenous preparation of proton pump inhibitor. This case report raises awareness among physicians worldwide of this often unknown association, as life-threatening cardiac and neuromuscular complications can arise with unrecognized hypocalcemia and hypomagnesemia with proton pump inhibitors. PMID:26069375
Orbe, J; Alexandru, N; Roncal, C; Belzunce, M; Bibiot, P; Rodriguez, J A; Meijers, J C M; Georgescu, A; Paramo, J A
Thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI-/-) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI-/-. to examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI-/- mice. Thrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry. Compared with saline, tPA significantly increased the infarct size in TAFI-/- mice (p<0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI-/- mice (p<0.05). Interestingly, higher numbers of MPs were found in TAFI-/- plasma as compared to wild type, after stroke (p<0.05). TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production. Copyright © 2015 Elsevier Ltd. All rights reserved.
van Mens, Thijs E.; Liang, Hai-Po H.; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; May, Jennifer; Zhan, Min; Yang, Qiuhui; Foeckler, Jamie; Kalloway, Shawn; Sood, Rashmi; Karlson, Caren Sue; Weiler, Hartmut
Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd−/−) mice to term. Half of the Thbd−/− offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd−/− animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd−/− mice. However, Thbd−/− females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy. PMID:28920104
Fomovska, Alina; Wood, Richard D.; Mui, Ernest; Dubey, Jitenter P.; Ferriera, Leandra R.; Hickman, Mark R.; Lee, Patricia J.; Leed, Susan E.; Auschwitz, Jennifer M.; Welsh, William J.; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima
Toxoplasma gondii(T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose anti-apicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles. PMID:22970937
Fomovska, Alina; Wood, Richard D; Mui, Ernest; Dubey, Jitenter P; Ferreira, Leandra R; Hickman, Mark R; Lee, Patricia J; Leed, Susan E; Auschwitz, Jennifer M; Welsh, William J; Sommerville, Caroline; Woods, Stuart; Roberts, Craig; McLeod, Rima
Toxoplasma gondii (T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose antiapicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles.
Ivanenkov, Yan A; Chufarova, Nina V
Arginase is an enzyme that metabolizes L-arginine to L-ornithine and urea. In addition to its fundamental role in the hepatic ornithine cycle, it also influences the immune systems in humans and mice. Arginase participates in many inflammatory disorders by decreasing the synthesis of nitric oxide and inducing fibrosis and tissue regeneration. L-arginine deficiency, which is modulated by myeloid cell arginase, suppresses T-cell immune response. This mechanism plays a fundamental role in inflammation-associated immunosuppression. Pathogens can synthesize their own arginase to elude immune reaction. Small-molecule arginase inhibitors are currently described as promising therapeutics for the treatment of several diseases, including allergic asthma, inflammatory bowel disease, ulcerative colitis, cardiovascular diseases (atherosclerosis and hypertension), diseases associated with pathogens (e.g., Helicobacter pylori, Trypanosoma cruzi, Leishmania, Mycobacterium tuberculosis and Salmonella), cancer and induced or spontaneous immune disorders. This article summarizes recent patents in the area of arginase inhibitors and discusses their properties.
Goey, Andrew Kl; Sissung, Tristan M; Peer, Cody J; Figg, William D
The histone deacetylase inhibitor valproic acid (VPA) has been used for many decades in neurology and psychiatry. The more recent introduction of the histone deacetylase inhibitors (HDIs) belinostat, romidepsin and vorinostat for treatment of hematological malignancies indicates the increasing popularity of these agents. Belinostat, romidepsin and vorinostat are metabolized or transported by polymorphic enzymes or drug transporters. Thus, genotype-directed dosing could improve pharmacotherapy by reducing the risk of toxicities or preventing suboptimal treatment. This review provides an overview of clinical studies on the effects of polymorphisms on the pharmacokinetics, efficacy or toxicities of HDIs including belinostat, romidepsin, vorinostat, panobinostat, VPA and a number of novel compounds currently being tested in Phase I and II trials. Although pharmacogenomic studies for HDIs are scarce, available data indicate that therapy with belinostat (UGT1A1), romidepsin (ABCB1), vorinostat (UGT2B17) or VPA (UGT1A6) could be optimized by upfront genotyping.
Skropeta, Danielle; Pastro, Natalie; Zivanovic, Ana
Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included. PMID:22073013
The Food and Drug Administration (FDA) has granted accelerated approval to nelfinavir in both adult and pediatric formulations. Agouron, the manufacturer, used innovative computerized drug design techniques to discover, design, and refine the nelfinavir molecule. Nelfinavir is marketed under the trade name Viracept, and costs $5,000 per year. Early clinical trials find it to be as powerful as the other protease inhibitors, but with a different resistance profile. The drug has relatively few drug indications; however, several compounds have been contraindicated.
Dabhi, Ajay S.; Bhatt, Nikita R.; Shah, Mohit J.
Diabetes Mellitus (DM) is a morbid disease worldwide, with increasing incidence as time passes. It has macro-vascular and micro-vascular complications. The main cause of these complications is poorly controlled postprandial hyperglycaemia. Alpha glucosidase inhibitors, namely acarbose, voglibose and miglitol, are available for therapy. Voglibose is well tolerated and effective in comparable doses among these drugs. This article highlights the important features of voglibose. PMID:24551718
Merino, Pedro; Tejero, Tomás; Delso, Ignacio; Hurtado-Guerrero, Ramon; Gómez-SanJuan, Asier; Sádaba, David
Fucosyltransferases (FucTs) are enzymes that transfer L-fucose from GDP-fucose to a glycoside or a peptide. They have important roles in a variety of diseases including cancer and autoimmune disorders, viral and bacterial infections and inflammatory processes, and thus they represent important drug targets for the development of agents for the treatment of such disorders. This review highlights recent developments regarding carbohydrate mimics as inhibitors of FucTs. The most recent and relevant synthetic strategies are described.
Adams, Jessica L; Greener, Benjamin N; Kashuba, Angela DM
Purpose of Review The purpose of this paper is to review recent and relevant pharmacology data for three HIV integrase inhibitors: raltegravir (marketed), dolutegravir and elvitegravir (both in Phase III drug development). Recent Findings Data from January 2011 to April 2012 were evaluated. These data better characterized integrase inhibitor pharmacokinetics, assessed dosing regimens and investigated previously undescribed drug-drug interactions. Due to formulation challenges, raltegravir inter- and intra-patient pharmacokinetic variability is high. Twice daily 400mg dosing has been shown to be clinically superior to 800mg once daily dosing. A pediatric formulation of raltegravir with less variable pharmacokinetics and greater bioavailability was FDA approved in December 2011. Cobicistat-boosted elvitegravir, and the second generation integrase inhibitor dolutegravir, have lower pharmacokinetic variability and are dosed once daily. Dolutegravir drug interactions are similar to raltegravir, while boosted elvitegravir participates in additional CYP3A mediated interactions. Summary Raltegravir’s potent antiretroviral activity has resulted in widespread use in both treatment naïve and experienced patients. Dolutegravir and cobicistat-boosted elvitegravir have some pharmacokinetic advantages. Pharmacokinetic data in special populations (pregnancy, pediatrics) to optimize dosing are still required. PMID:22789987
Levina, Inna S.
The synthesis and structure-activity relationships of inhibitors of steroid aromatase which catalyses the last stage of a multistep biotransformation of cholesterol into estrogens, viz., aromatisation of C19-steroids into C18-phenolic steroids, are discussed. Compounds of the androstane series which are structurally related to the natural substrate, viz., androst-4-ene-3,17-dione, are the subjects of consideration. The review encompasses problems of synthesis of various substituted androstanes and their aromatase-inhibiting activities and structural requirements for selective specific aromatase inhibitors based on in vitro and in vivo structure-activity studies of compounds synthesised, their biological properties and the results of clinical trials. Special attention is paid to practical applications of aromatase inhibitors in the treatment of hormone-dependent mammary and ovarian tumours as well as benign prostatic tumours. In writing this report, the author has used all the information currently available in the chemical, biochemical, endocrinological and medicinal literature as well as in patents. The bibliography includes 173 references.
McKenna, Robert; Supuran, Claudiu T
Inhibition of the metalloenzyme carbonic anhydrase (CA, EC 188.8.131.52) has pharmacologic applications in the field of antiglaucoma, anticonvulsant, antiobesity, and anticancer agents but is also emerging for designing anti-infectives (antifungal and antibacterial agents) with a novel mechanism of action. As a consequence, the drug design of CA inhibitors (CAIs) is a very dynamic field. Sulfonamides and their isosteres (sulfamates/sulfamides) constitute the main class of CAIs which bind to the metal ion in the enzyme active site. Recently the dithiocarbamates, possessing a similar mechanism of action, were reported as a new class of inhibitors. Other families of CAIs possess a distinct mechanism of action: phenols, polyamines, some carboxylates, and sulfocoumarins anchor to the zinc-coordinated water molecule. Coumarins and five/six-membered lactones are prodrug inhibitors, binding in hydrolyzed form at the entrance of the active site cavity. Novel drug design strategies have been reported principally based on the tail approach for obtaining all these types of CAIs, which exploit more external binding regions within the enzyme active site (in addition to coordination to the metal ion), leading thus to isoform-selective compounds. Sugar-based tails as well as click chemistry were the most fruitful developments of the tail approach. Promising compounds that inhibit CAs from bacterial and fungal pathogens, of the dithiocarbamate, phenol and carboxylate types have also been reported.
Saccoccia, Fulvio; Angelucci, Francesco; Boumis, Giovanna; Carotti, Daniela; Desiato, Gianni; Miele, Adriana E; Bellelli, Andrea
Thioredoxin plays a crucial role in a wide number of physiological processes, which span from reduction of nucleotides to deoxyriboucleotides to the detoxification from xenobiotics, oxidants and radicals. The redox function of Thioredoxin is critically dependent on the enzyme Thioredoxin NADPH Reductase (TrxR). In view of its indirect involvement in the above mentioned physio/pathological processes, inhibition of TrxR is an important clinical goal. As a general rule, the affinities and mechanisms of binding of TrxR inhibitors to the target enzyme are known with scarce precision and conflicting results abound in the literature. A relevant analysis of published results as well as the experimental procedures is therefore needed, also in view of the critical interest of TrxR inhibitors. We review the inhibitors of TrxR and related flavoreductases and the classical treatment of reversible, competitive, non competitive and uncompetitive inhibition with respect to TrxR, and in some cases we are able to reconcile contradictory results generated by oversimplified data analysis. PMID:24875642
... Cancer Risk and Prevention Aromatase Inhibitors for Lowering Breast Cancer Risk Aromatase inhibitors (drugs that lower estrogen levels) ... day. Can aromatase inhibitors lower the risk of breast cancer? Aromatase inhibitors are used mainly to treat hormone ...
... mortality in the United States. American Journal of Hematology. 2015; 90:400-405. Miller CH, Benson J, ... A: rationale and latest evidence. Therapeutic Advances in Hematology. 2013; 4(1):59-72. Hay CR, DiMichele ...
Day, Joshua A; Cohen, Seth M
The inhibitory activity of a broad group of known metalloenzyme inhibitors against a panel of metalloenzymes was evaluated. Clinically approved inhibitors were selected as well as several other reported metalloprotein inhibitors in order to represent a broad range of metal binding groups (MBGs), including hydroxamic acid, carboxylate, hydroxypyridinonate, thiol, and N-hydroxyurea functional groups. A panel of metalloenzymes, including carbonic anhydrase (hCAII), several matrix metalloproteinases (MMPs), angiotensin converting enzyme (ACE), histone deacetylase (HDAC-2), and tyrosinase (TY), was selected based on their clinical importance for a range of pathologies. In addition, each inhibitor was evaluated for its ability to remove Fe(3+) from holo-transferrin to gauge the ability of the inhibitors to access Fe(3+) from a primary transport protein. The results show that the metalloenzyme inhibitors are quite selective for their intended targets, suggesting that despite their ability to bind metal ions, metalloprotein inhibitors are not prone to widespread off-target enzyme inhibition activity.
Bern, Murray M; McCarthy, Nancy
Plasminogen activator Inhibitor 1 (PAI-1) inhibits plasminogen activators leading to decreased fibrinolysis and increased risk of thromboembolic disease (TED). Shifts in PAI-1 promoter genome from normal 5G>5G to 4G>5G or 4G>4G alleles are associated with overexpression of PAI-1. In this study patients with residual venous thrombi were observed to have increased PAI-1 levels and more frequent shifts to 4G alleles. Of the 26, 20 (76.9%) patients with unresolved thrombus had elevated PAI-1 values. 4G genomic shifts were found in 92.9% patients studied. Normal PAI-1 levels were found in 5 patients with 4G polymorphisms. Thus, PAI-1 is often elevated among patients with residual thrombus, with an unexpectedly high prevalence of the 4G polymorphism of the promoter genome. Patients with persistent thrombus should be considered at risk of having constituently increased PAI-1 due to genomic changes in the PAI-1 promoter genome. Hypotheses are proposed to explain those with normal PAI-1, despite having 4G polymorphisms.
Montagner, A.F.; Sarkis-Onofre, R.; Pereira-Cenci, T.; Cenci, M.S.
The aim of this study was to systematically review the literature for in vitro and ex vivo studies that evaluated the effect of matrix metalloproteinase (MMP) inhibitors during the adhesive procedure on the immediate and long-term resin-dentin bond strength. The search was conducted in 6 databases with no publication year or language limits, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. From 1,336 potentially eligible studies, 48 were selected for full-text analysis, and 30 were included for review, with 17 considered in the meta-analysis. Two reviewers independently selected the studies, extracted the data, and assessed the risk of bias. Pooled effect estimates were expressed as the weighted mean difference between groups. The most used MMP inhibitor was chlorhexidine (CHX). Immediate bond strength results showed no difference between 2% CHX and control; however, a difference was found between 0.2% CHX and control at baseline. After aging, CHX presented higher bond strength values compared to control groups (p < .05). However, this was not observed for longer periods of aging. High heterogeneity was found in some comparisons, especially for the water storage aging subgroup. Subgroup analyses showed that self-etching and etch-and-rinse adhesives are benefited by the CHX use. From the studies included, only 1 presented low risk of bias, while the others showed medium or high risk of bias. The use of MMP inhibitors did not affect the immediate bond strength overall, while it influenced the aged bond strength. Aging procedures influenced bond strength values of the dentin adhesion stability. PMID:24935066
Ferreira, Cláudia N; Carvalho, Maria G; Gomes, Karina B; Reis, Helton J; Fernandes, Ana-Paula; Sousa, Marinez O
Apolipoprotein gene polymorphism has an important role in lipid metabolism and in the development of cerebro- and cardio-vascular disease (CCVD), including dementia. Dyslipidemia and hemostatic abnormalities are key risk factors associated with athero-sclerotic events preceding CCVD. The aim of this study was to evaluate the possible relationships of various apolipoprotein-species with hemostatic parameters and cognitive function. Lipid profile, gene polymorphism, coagulation markers, and mini-mental state examination (MMSE) scores were assessed in 109 dys-lipidemic subjects and in 107 healthy control volunteers. Thrombin-activatable fibrinolysis inhibitor (TAFI) plasma levels were significantly higher in apolipoprotein-E2 (apoE2) patients when compared to other apoE forms. The apoA5 -1131T>C polymorphism was associated with elevated D-dimer concentration in dyslipidemic TT homozygous individuals. MMSE did not correlate with lipid or coagulation profile. These data suggest that apoE and apoA5 variants have an effect on hemostatic parameters, but they neither influence nor predict cognitive performance in non-demented individuals. PMID:25073959
Antovic, Aleksandra; Mikovic, Danijela; Elezovic, Ivo; Zabczyk, Michael; Hutenby, Kjell; Antovic, Jovan P
Patients with haemophilia A have seriously impaired thrombin generation due to an inherited deficiency of factor (F)VIII, making them form unstable fibrin clots that are unable to maintain haemostasis. Data on fibrin structure in haemophilia patients remain limited. Fibrin permeability, assessed by a flow measurement technique, was investigated in plasma from 20 patients with severe haemophilia A treated on demand, before and 30 minutes after FVIII injection. The results were correlated with concentrations of fibrinogen, FVIII and thrombin-activatable fibrinolysis inhibitor (TAFI), and global haemostatic markers: endogenous thrombin potential (ETP) and overall haemostatic potential (OHP). Fibrin structure was visualised using scanning electron microscopy (SEM). The permeability coefficient Ks decreased significantly after FVIII treatment. Ks correlated significantly with FVIII levels and dosage, and with ETP, OHP and levels of TAFI. SEM images revealed irregular, porous fibrin clots composed of thick and short fibers before FVIII treatment. The clots had recovered after FVIII replacement almost to levels in control samples, revealing compact fibrin with smaller intrinsic pores. To the best of our knowledge, this is the first description of fibrin porosity and structure before and after FVIII treatment of selected haemophilia patients. It seems that thrombin generation is the main determinant of fibrin structure in haemophilic plasma.
Remick, Ronald A.; Froese, Colleen
Monoamine oxidase inhibitors (MAOIs) are effective antidepressant agents. They are increasingly and effectively used in a number of other psychiatric and non-psychiatric medical syndromes. Their potential for serious toxicity (i.e., hypertensive reaction) is far less than original reports suggest, and newer reversible substrate-specific MAOIs may offer even less toxicity. The author reviews the pharmacology, mechanism of action, clinical indications, and dosing strategies of MAOIs. The common MAOI side-effects (hypotension, weight gain, sexual dysfunction, insomnia, daytime sedation, myoclonus, and hypertensive episodes) are described and management techniques suggested. Recent clinical developments involving MAOIs are outlined. PMID:21233984
Osterman, Ilya A.; Bogdanov, Alexey A.; Dontsova, Olga A.; Sergiev, Petr V.
The machinery of translation is one of the most common targets of antibiotics. The development and screening of new antibiotics usually proceeds by testing antimicrobial activity followed by laborious studies of the mechanism of action. High-throughput methods for new antibiotic screening based on antimicrobial activity have become routine; however, identification of molecular targets is usually a challenge. Therefore, it is highly beneficial to combine primary screening with the identification of the mechanism of action. In this review, we describe a collection of methods for screening translation inhibitors, with a special emphasis on methods which can be performed in a high-throughput manner. PMID:27348012
Westling et al., 2002). 3) Cleavage of TSP-I is not shared by ADAMTS-4. ADAMTS-1 and ADAMTS-4 display high sequence homology and more importantly, both...Manzaneque JC, Westling J, Thai SN, Luque A, Knauper V, Murphy G, Sandy JD, Iruela-Arispe ML. ADAMTS1 cleaves aggrecan at multiple sites and is...differentially inhibited by metalloproteinase inhibitors. Biochem Biophys Res Commun. 2002 Apr 26;293(1):501-8. * Sandy JD, Westling J, Kenagy RD, Iruela-Arispe
Balunas, Marcy J; Su, Bin; Brueggemeier, Robert W; Kinghorn, A Douglas
With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always available through current synthetic chemistry techniques. Natural products that have been used traditionally for nutritional or medicinal purposes (e.g., botanical dietary supplements) may also afford AIs with reduced side effects. A thorough review of the literature regarding natural product extracts and secondary metabolites of plant, microbial, and marine origin that have been shown to exhibit aromatase inhibitory activity is presented herein.
Osterman, Ilya A; Bogdanov, Alexey A; Dontsova, Olga A; Sergiev, Petr V
The machinery of translation is one of the most common targets of antibiotics. The development and screening of new antibiotics usually proceeds by testing antimicrobial activity followed by laborious studies of the mechanism of action. High-throughput methods for new antibiotic screening based on antimicrobial activity have become routine; however, identification of molecular targets is usually a challenge. Therefore, it is highly beneficial to combine primary screening with the identification of the mechanism of action. In this review, we describe a collection of methods for screening translation inhibitors, with a special emphasis on methods which can be performed in a high-throughput manner.
Khan, Anwar A.; Zeng, Guang-Wen
`Grand Rapids' lettuce Lactuca sativa L. seeds germinate readily at 15°C but poorly at 25°C in darkness. When held in dark at 25°C for an extended period, the ungerminated seeds become dormant as shown by their inability to germinate or transfer to 15°C in darkness. Induction of dormancy at 25°C was prevented by exposure to CN−, azide, salicylhydroxamic acid (SHAM), dinitrophenol, and pure N2 as determined by subsequent germination at 15°C on removal of inhibitors. The effectiveness of inhibitors to break dormancy declined as dormancy intensified. At relatively low levels, CN−, SHAM, and azide promoted dark germination at 25°C while at high levels they were inhibitory. Uptake of O2 by seeds held at 25°C for 4 days in 1.0 millimolar KCN was inhibited by 67% but was promoted 61% when KCN was removed. Correspondingly greater inhibition (79%) and promotion (148%) occurred when 1.0 millimolar SHAM was added to KCN solution. When applied alone, SHAM had little effect on O2 uptake. These data indicate that Cyt pathway of respiration plays a dominant role in the control of both dormancy induction and germination of lettuce seeds, and `alternative pathway' is effectively engaged in presence of CN−. The channeling of respiratory energy use for processes governing germination or dormancy is subject to control by physical and chemical factors. A scheme is proposed that illustrates compensatory use of energy for processes controlling dormancy induction and germination. A block of germination, e.g. by low water potential polyethylene glycol solution or a supraoptimal temperature spares energy to be utilized for dormancy induction while a block of dormancy induction by low levels of CN− (similar to GA and light effects) drives germination. Blocking both processes by inhibitors (e.g. CN−, CN− + SHAM) presumably leads to accumulation of `reducing power' with consequent improvement in O2 uptake and oxidation rates of processes controlling germination or dormancy
Baldisserotto, Anna; Marastoni, Mauro; Gavioli, Riccardo; Tomatis, Roberto
Here we report the study of a new series of vinyl ester cyclopeptide analogues synthesized on the basis of our previous development of a class of cyclopeptides derived from our linear prototype inhibitors. In these compounds, the exocyclic pharmacophoric unit Leu-VE was linked to the gamma-carboxyl group of the glutamic acid residue at the C-terminal. The best analogues of the series have been shown to inhibit the caspase-like activity of the proteasome at nanomolar concentrations and have also demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.
Ververis, Katherine; Hiong, Alison; Karagiannis, Tom C; Licciardi, Paul V
Histone deacetylase (HDAC) inhibitors are an emerging class of therapeutics with potential as anticancer drugs. The rationale for developing HDAC inhibitors (and other chromatin-modifying agents) as anticancer therapies arose from the understanding that in addition to genetic mutations, epigenetic changes such as dysregulation of HDAC enzymes can alter phenotype and gene expression, disturb homeostasis, and contribute to neoplastic growth. The family of HDAC inhibitors is large and diverse. It includes a range of naturally occurring and synthetic compounds that differ in terms of structure, function, and specificity. HDAC inhibitors have multiple cell type-specific effects in vitro and in vivo, such as growth arrest, cell differentiation, and apoptosis in malignant cells. HDAC inhibitors have the potential to be used as monotherapies or in combination with other anticancer therapies. Currently, there are two HDAC inhibitors that have received approval from the US FDA for the treatment of cutaneous T-cell lymphoma: vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax). More recently, depsipeptide has also gained FDA approval for the treatment of peripheral T-cell lymphoma. Many more clinical trials assessing the effects of various HDAC inhibitors on hematological and solid malignancies are currently being conducted. Despite the proven anticancer effects of particular HDAC inhibitors against certain cancers, many aspects of HDAC enzymes and HDAC inhibitors are still not fully understood. Increasing our understanding of the effects of HDAC inhibitors, their targets and mechanisms of action will be critical for the advancement of these drugs, especially to facilitate the rational design of HDAC inhibitors that are effective as antineoplastic agents. This review will discuss the use of HDAC inhibitors as multitargeted therapies for malignancy. Further, we outline the pharmacology and mechanisms of action of HDAC inhibitors while
Bridge, Katherine; Revill, Charlotte; Macrae, Fraser; Bailey, Marc; Yuldasheva, Nadira; Wheatcroft, Stephen; Butlin, Roger; Foster, Richard; Scott, D Julian; Gils, Ann; Ariens, Robert
Thrombin-activatable fibrinolysis inhibitor (TAFI) reduces the breakdown of fibrin clots through its action as an indirect inhibitor of plasmin. Studies in TAFI-deficient mice have implicated a potential role for TAFI in Abdominal Aortic Aneurysm (AAA) disease. The role of TAFI inhibition on AAA formation in adult ApoE-/- mice is unknown. The aim of this paper was to investigate the effects of TAFI inhibition on AAA development and progression. Using the Angiotensin II model of AAA, male ApoE-/- mice were infused with Angiotensin II 750ng/kg/min with or without a monoclonal antibody inhibitor of plasmin-mediated activation of TAFI, MA-TCK26D6, or a competitive small molecule inhibitor of TAFI, UK-396082. Inhibition of TAFI in the Angiotensin II model resulted in a decrease in the mortality associated with AAA rupture (from 40.0% to 16.6% with MA-TCK26D6 (log-rank Mantel Cox test p = 0.16), and 8.3% with UK-396082 (log-rank Mantel Cox test p = 0.05)). Inhibition of plasmin-mediated TAFI activation reduced the incidence of AAA from 52.4% to 30.0%. However, late treatment with MA-TCK26D6 once AAA were already established had no effect on the progression of AAA in this model. The formation of intra-mural thrombus is responsible for the dissection and early rupture in the angiotensin II model of AAA, and this process can be prevented through inhibition of TAFI. Late treatment with a TAFI inhibitor does not prevent AAA progression. These data may indicate a role for inhibition of plasmin-mediated TAFI activation in the early stages of AAA development, but not in its progression.
Bridge, Katherine; Revill, Charlotte; Macrae, Fraser; Bailey, Marc; Yuldasheva, Nadira; Wheatcroft, Stephen; Butlin, Roger; Foster, Richard; Scott, D. Julian; Gils, Ann; Ariёns, Robert
Objective Thrombin-activatable fibrinolysis inhibitor (TAFI) reduces the breakdown of fibrin clots through its action as an indirect inhibitor of plasmin. Studies in TAFI-deficient mice have implicated a potential role for TAFI in Abdominal Aortic Aneurysm (AAA) disease. The role of TAFI inhibition on AAA formation in adult ApoE-/- mice is unknown. The aim of this paper was to investigate the effects of TAFI inhibition on AAA development and progression. Methods Using the Angiotensin II model of AAA, male ApoE-/- mice were infused with Angiotensin II 750ng/kg/min with or without a monoclonal antibody inhibitor of plasmin-mediated activation of TAFI, MA-TCK26D6, or a competitive small molecule inhibitor of TAFI, UK-396082. Results Inhibition of TAFI in the Angiotensin II model resulted in a decrease in the mortality associated with AAA rupture (from 40.0% to 16.6% with MA-TCK26D6 (log-rank Mantel Cox test p = 0.16), and 8.3% with UK-396082 (log-rank Mantel Cox test p = 0.05)). Inhibition of plasmin-mediated TAFI activation reduced the incidence of AAA from 52.4% to 30.0%. However, late treatment with MA-TCK26D6 once AAA were already established had no effect on the progression of AAA in this model. Conclusions The formation of intra-mural thrombus is responsible for the dissection and early rupture in the angiotensin II model of AAA, and this process can be prevented through inhibition of TAFI. Late treatment with a TAFI inhibitor does not prevent AAA progression. These data may indicate a role for inhibition of plasmin-mediated TAFI activation in the early stages of AAA development, but not in its progression. PMID:28472123
Nepali, Kunal; Ojha, Ritu; Sharma, Sahil; Bedi, Preet M S; Dhar, Kanaya L
Tubulin is one of the most useful and strategic molecular targets for anticancer drugs. The dynamic process of microtubule assembly and disassembly can be blocked by various agents that bind to distinct sites in the β-tubulin subunit. By interfering with microtubule function in vitro, these agents arrest cells in mitosis, eventually leading to cell death, by both apoptosis and necrosis. So far, three binding domains have been identified a) the colchicine site close to the α/β interface, b) the area where the vinca alkaloids bind, and c) the taxane-binding pocket. This review compiles the patent literature up to 2013 and offers a detailed account of all the advances on Tubulin inhibitors (lead molecules) along with in depth knowledge about the number of novel scaffolds, modified analogs and derivatives of the lead molecules. Colchicine binding site remains the most explored site indicated by the patent survey as majority of the patents revolves around phenstatin and combretastatin based molecules where the key structural feature for tubulin inhibition is an appropriate arrangement of the two aromatic rings at an appropriate distance and optimal dihedral angle maximizing interactions with tubulin. A brief account of promising tubulin inhibitors in stages of clinical development and some strategies for the development of potent molecules overcoming the problem of drug resistance have also been discussed.
PEREZ, EDITH A.; M., Serene; Durling, Frances C.; WEILBAECHER, KATHERINE
The aromatase inhibitors (AIs) anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin) are significantly more effective than the selective estrogen-receptor modulator (SERM) tamoxifen in preventing recurrence in estrogen receptor–positive early breast cancer. Aromatase inhibitors are likely to replace SERMs as first-line adjuvant therapy for many patients. However, AIs are associated with significantly more osteoporotic fractures and greater bone mineral loss. As antiresorptive agents, oral and intravenous bisphosphonates such as alendronate (Fosamax), risedronate (Actonel), ibandronate (Boniva), pamidronate (Aredia), and zoledronic acid (Zometa) have efficacy in preventing postmenopausal osteoporosis, cancer treatment–related bone loss, or skeletal complications of metastatic disease. Clinical practice guidelines recommend baseline and annual follow-up bone density monitoring for all patients initiating AI therapy. Bisphosphonate therapy should be prescribed for patients with osteoporosis (T score < −2.5) and considered on an individual basis for those with osteopenia (T score < −1). Modifiable lifestyle behaviors including adequate calcium and vitamin D intake, weight-bearing exercise, and smoking cessation should be addressed. Adverse events associated with bisphosphonates include gastrointestinal toxicity, renal toxicity, and osteonecrosis of the jaw. These safety concerns should be balanced with the potential of bisphosphonates to minimize or prevent the debilitating effects of AI-associated bone loss in patients with early, hormone receptor–positive breast cancer. PMID:16986348
Waghorn, Philip A; Jackson, Mark R; Gouverneur, Veronique; Vallis, Katherine A
The expression of telomerase in approximately 85% of cancers and its absence in the majority of normal cells makes it an attractive target for cancer therapy. However the lag period between initiation of telomerase inhibition and growth arrest makes direct inhibition alone an insufficient method of treatment. However, telomerase inhibition has been shown to enhance cancer cell radiosensitivity. To investigate the strategy of simultaneously inhibiting telomerase while delivering targeted radionuclide therapy to cancer cells, (123)I-radiolabeled inhibitors of telomerase were synthesized and their effects on cancer cell survival studied. An (123)I-labeled analogue of the telomerase inhibitor MST-312 inhibited telomerase with an IC50 of 1.58 μM (MST-312 IC50: 0.23 μM). Clonogenic assays showed a dose dependant effect of (123)I-MST-312 on cell survival in a telomerase positive cell line, MDA-MB-435. Copyright Â© 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.
Metcalf, Rainer; Scott, Latanya M; Daniel, Kenyon G; Dou, Q Ping
Over the past 3 years, numerous patents and patent applications have been submitted and published involving compounds designed to inhibit the proteasome. Proteasome inhibition has been of great interest in cancer research since disruption of proteolysis leads to a significant buildup of cytotoxic proteins and activation of apoptotic pathways, particularly in rapidly proliferating cells. The current standards in proteasome inhibition are the only FDA-approved inhibitors, bortezomib and carfilzomib. Although these drugs are quite effective in treating multiple myeloma and other blood tumors, there are shortcomings, including toxicities and resistance. Most of the current patents attempt to improve on existing compounds, by increasing bioavailability and selectivity, while attempting to reduce toxicity. A general categorization of similar compounds was employed to evaluate and compare drug design strategies. This review focuses on novel compounds and subsequent analogs developed for proteasome inhibition, used in preventing and treating human cancers. A comprehensive description and categorization of patents related to each type of compound and its derivatives, as well as their uses and efficacies as anticancer agents is included. A review of combination therapy patents has also been included. Although there are many diverse chemical scaffolds being published, there are few patented proteasome inhibitors whose method of inhibition is genuinely novel. Most patents utilize a destructive chemical warhead to attack the catalytic threonine residue of the proteasome active sites. Few patents try to depart from this, emphasizing the need for developing new mechanisms of action and specific targeting.
Perez, Edith A; Weilbaecher, Katherine
The aromatase inhibitors (AIs) anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin) are significantly more effective than the selective estrogen-receptor modulator (SERM) tamoxifen in preventing recurrence in estrogen receptor-positive early breast cancer. Aromatase inhibitors are likely to replace SERMs as first-line adjuvant therapy for many patients. However, AIs are associated with significantly more osteoporotic fractures and greater bone mineral loss. As antiresorptive agents, oral and intravenous bisphosphonates such as alendronate (Fosamax), risedronate (Actonel), ibandronate (Boniva), pamidronate (Aredia), and zoledronic acid (Zometa) have efficacy in preventing postmenopausal osteoporosis, cancer treatment-related bone loss, or skeletal complications of metastatic disease. Clinical practice guidelines recommend baseline and annual follow-up bone density monitoring for all patients initiating AI therapy. Bisphosphonate therapy should be prescribed for patients with osteoporosis (T score < -2.5) and considered on an individual basis for those with osteopenia (T score < -1). Modifiable lifestyle behaviors including adequate calcium and vitamin D intake, weight-bearing exercise, and smoking cessation should be addressed. Adverse events associated with bisphosphonates include gastrointestinal toxicity, renal toxicity, and osteonecrosis of the jaw. These safety concerns should be balanced with the potential of bisphosphonates to minimize or prevent the debilitating effects of AI-associated bone loss in patients with early, hormone receptor-positive breast cancer.
de los Ríos, Cristóbal
Cholinesterase inhibitors participate in the maintenance of the levels of the neurotransmitter acetylcholine by inhibiting the enzymes implicated in its degradation, namely, butyrylcholinesterase and acetylcholinesterase. This pharmacological action has an important role in several diseases, including neurodegenerative diseases such as Alzheimer's. This article reviews recent advances in the development of cholinesterase enzyme inhibitors, covering the development of new chemical entities, new pharmaceutical formulations with known inhibitors or treatments in combination with other drug families. The development of cholinesterase inhibitors has to face several issues, including the fact that the principal indication for these drugs, Alzheimer's disease, is not currently believed to derivate from a cholinergic deficiency, although most of the drugs clinically used for these disease are cholinesterase inhibitors. Moreover, the adverse effects found when administering cholinesterase inhibitors limit their use in other diseases, such as gastrointestinal diseases, glaucoma, or analgesia.
Varga, Lilian; Dobó, József
C1 inhibitor is a multipotent serpin capable of inhibiting the classical and the lectin pathways of complement, the fibrinolytic system, and contact/kinin system of coagulation. Deficiency of C1 inhibitor manifest as hereditary angioedema (HAE), an autosomal dominant hereditary disease. Measuring the C1 inhibitor level is of vital importance for the diagnosis of HAE and also for monitoring patients receiving C1 inhibitor for therapy. Determination of the antigenic C1 inhibitor level by the radial immunodiffusion (RID) technique is described in detail in this chapter. The presented purification method of plasma C1 inhibitor is primarily based on its high carbohydrate content and its affinity to the lectin jacalin.
REPORT Final Report for Plant Biofilm Inhibitors to Discover Biofilm Genes 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: To control biofilms , we have...synthesized the natural biofilm inhibitor (5Z)-4-bromo-5-(bromomethylene) -3-butyl-2(5H)-furanone from the red alga Delisea pulchra and determined that...Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS biofilms , biofilm inhibitors Thomas K. Wood Texas Engineering
Dawson, R M; Hemington, N
1. Bovine, dog and human saliva contain substances which inhibit the soluble phospholipase D present in grass leaf or celery stalk. 2. The inhibitor in bovine saliva is of high molecular weight and exhibits considerable stability to heat, acids and alkalis. 3. The inhibitor has been purified free from salivary mucoprotein. 4. It is suggested that the inhibitor could protect the upper alimentary tract of a herbage-eating animal from the necrotic action of phospholipase D.
Karoulia, Zoi; Wu, Yang; Ahmed, Tamer A; Xin, Qisheng; Bollard, Julien; Krepler, Clemens; Wu, Xuewei; Zhang, Chao; Bollag, Gideon; Herlyn, Meenhard; Fagin, James A; Lujambio, Amaia; Gavathiotis, Evripidis; Poulikakos, Poulikos I
The complex biochemical effects of RAF inhibitors account for both the effectiveness and mechanisms of resistance to these drugs, but a unified mechanistic model has been lacking. Here we show that RAF inhibitors exert their effects via two distinct allosteric mechanisms. Drug resistance due to dimerization is determined by the position of the αC helix stabilized by inhibitor, whereas inhibitor-induced RAF priming and dimerization are the result of inhibitor-induced formation of the RAF/RAS-GTP complex. The biochemical effect of RAF inhibitor in cells is the combined outcome of the two mechanisms. Therapeutic strategies including αC-helix-IN inhibitors are more effective in multiple mutant BRAF-driven tumor models, including colorectal and thyroid BRAF(V600E) cancers, in which first-generation RAF inhibitors have been ineffective.
Miyazaki, A; Horiuchi, S
Development of new antiatherosclerotic agents were reviewed focusing on ACAT inhibitors and CETP inhibitors. ACAT inhibitors enhance intracellular degradation of VLDL in hepatocytes. Cholesterol absorption in small intestine is inhibited by ACAT inhibitors. Thus, ACAT inhibitors reduce plasma cholesterol levels. In atherosclerotic lesions, ACAT inhibitors suppress foam cell formation (cholesteryl ester accumulation) in macrophages. Since ACAT inhibitors have multiple anti-atherogenic effects, they are considered future drugs controlling hypercholesterolemia and atherosclerosis. CETP inhibitors are expected to increase HDL and decrease LDL. Although the patients with CETP deficiency show high level of HDL, recent studies showed that they are not necessarily resistant to atherosclerosis. The strategy to inhibit CETP for suppressing atherosclerosis has not been established.
Jeffers, Ann; Alvarez, Alexia; Owens, Shuzi; Koenig, Kathleen; Quaid, Brandon; Komissarov, Andrey A.; Florova, Galina; Kothari, Hema; Pendurthi, Usha; Mohan Rao, L. Vijaya; Idell, Steven
Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1−/− mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1−/− mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1−/− mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial–mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1−/− mice, D-dimer and thrombin–antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction. PMID:24024554
Ando, Hitoshi; Otoda, Toshiki; Ookami, Hitoshi; Nagai, Yukihiro; Inano, Akihiro; Takamura, Toshinari; Ushijima, Kentarou; Hosohata, Keiko; Matsushita, Eiki; Saito, Tetsuo; Kaneko, Shuichi; Fujimura, Akio
Raloxifene, a selective oestrogen receptor modulator commonly used for the treatment of post-menopausal osteoporosis, affects the coagulation and fibrinolytic systems and consequently increases the risk of venous thromboembolism. Because both the coagulation and fibrinolytic systems exhibit circadian rhythms, the aim of the present study was to investigate the effects of dosing time of raloxifene on markers of coagulation and fibrinolysis, as well as on markers of bone metabolism. Thirty-nine post-menopausal patients with osteoporosis were randomly allocated to two groups: one received 60 mg raloxifene once daily in the morning, whereas the other received 60 mg raloxifene once daily in the evening, for 12 months. In both groups, the activity of coagulation Factors IX and XII was increased significantly after 12 months treatment compared with baseline. The activity of coagulation Factors II and V and levels of markers of bone metabolism (i.e. bone alkaline phosphatase and tartrate-resistant acid phosphatase 5b) decreased in both groups. The changes in these markers did not differ between the two groups. In contrast, the plasma concentration of plasminogen activator inhibitor (PAI)-1 increased in the group receiving the morning dose (mean change 40.9%; 95% confidence interval (CI) 9.4, 72.5), but not in the groups receiving the evening dose (mean change -0.3%; 95% CI -31.5, 30.9); these percentage changes differed significantly (P < 0.05). Because an elevated concentration of PAI-1 is known to be associated with the risk of venous thromboembolism, the findings of the present study suggest that the dosing time of raloxifene influences its safety. Further larger-scale studies are needed to determine the clinical usefulness of chronotherapy with raloxifene.
Espino, Alberto; Villagrán, Andrea; Vollrath, Valeska; Hanckes, Paulina; Salas, Roberto; Farah, Andrea; Solís, Nancy; Pizarro, Margarita; Escalona, Alex; Boza, Camilo; Pérez, Gustavo; Carrasco, Gonzalo; Padilla, Oslando; Miquel, Juan Francisco; Nervi, Flavio; Chavez-Tapia, Norberto C; Arab, Juan Pablo; Alvarez-Lobos, Manuel; Arrese, Marco; Riquelme, Arnoldo
The plasminogen activator inhibitor type-1 (PAI-1) has been implicated in the regulation of fibrinolysis and extracellular matrix components. The single base pair guanine insertion/deletion polymorphism (4G/5G) within the promoter region of the PAI-1 gene influences PAI-1 synthesis and may modulate hepatic fibrogenesis. To evaluate the influence of PAI-1 serum levels and 4G/5G polymorphism on the risk of liver fibrosis associated to non-alcoholic fatty liver disease (NAFLD) in morbidly obese patients. Case-control study of 50 obese patients undergoing bariatric surgery and 71 non-obese subjects matched by age and sex. Anthropometric and biochemical measurements were performed, including PAI-1 serum levels. Genomic DNA was obtained to assess the presence of 4G/5G polymorphism. BMI, insulinemia, triglycerides, HOMA-IR, hypertension and diabetes were significantly higher in obese patients compared to control subjects. PAI-1 serum levels observed in obese patients were significantly lower (10.63 ± 4.82) compared to controls (14.26 ± 11.4; p < 0.05). No differences were observed in the PAI-1 4G/5G promoter genotypes frequencies (p = 0.12). No differences were observed in PAI-1 plasma levels among obese patients with liver fibrosis (10.64 ± 4.35) compared to patients without liver fibrosis (10.61 ± 5.2; p = 0.985). PAI-1 4G/5G promoter genotypes frequencies were similar in patients with or without liver fibrosis associated to NASH (p = 0.6). Morbidly obese patients had significantly lower PAI-1 serum levels with similar PAI-1 4G/5G genotypes frequencies compared to non-obese subjects. The frequency of 4G/5G genotypes in Chilean Hispanic healthy subjects was similar to that described in other populations. No association was found between PAI-1 serum levels or 4G/5G genotype with liver fibrosis in obese patients.
Gilfillan, Robert; Kerr, Jennifer; Walker, Glenn; Wishart, Grant
Glycine plays a ubiquitous role in many biological processes. In the central nervous system it serves as an important neurotransmitter acting as an agonist at strychnine-sensitive glycine receptors and as an essential co-agonist with glutamate at the NMDA receptor complex. Control of glycine concentrations in the vicinity of these receptors is mediated by the specific glycine transporters, GlyT1 and GlyT2. Inhibition of these transporters has been postulated to be of potential benefit in several therapeutic indications including schizophrenia and pain. In this review we discuss our current knowledge of glycine transporters and focus on recent advances in the medicinal chemistry of GlyT1 and GlyT2 inhibitors.
Johnson, Victoria A.; Singh, Erinprit K.; Nazarova, Lidia A.; Alexander, Leslie D.; McAlpine, Shelli R.
Heat shock proteins (HSP) are a family of highly conserved proteins, whose expression increases in response to stresses that may threaten cell survival. Over the past decade, heat shock protein 90 (Hsp90) has emerged as a potential therapeutic target for cancer as it plays a vital role in normal cell maturation and acts as a molecular chaperone for proper folding, assembly, and stabilization of many oncogenic proteins. To date, a majority of Hsp90 inhibitors that have been discovered are macrocycles. The relatively rigid conformation provided by the macrocyclic scaffold allows for a selective interaction with a biological target such as Hsp90. This review highlights the discovery and development of nine macro-cycles that inhibit the function of Hsp90, detailing their potency and the client proteins affected by Hsp90 inhibition. PMID:20536417
Jezeršek Novaković, Barbara
Hodgkin's lymphoma is unusual among cancers in that it consists of a small number of malignant Hodgkin/Reed-Sternberg cells in a sea of immune system cells, including T cells. Most of these T cells are reversibly inactivated in different ways and their reactivation may induce a very strong immune response to cancer cells. One way of reactivation of T cells is with antibodies blocking the CTLA-4 and especially with antibodies directed against PD-1 or the PD-L1 ligand thereby reversing the tumor-induced downregulation of T-cell function and augmenting antitumor immune activity at the priming (CTLA-4) or tissue effector (PD-1) phase. Immune checkpoint inhibitors have been evidenced as an additional treatment option with substantial effectiveness and acceptable toxicity in heavily pretreated patients with Hodgkin's lymphoma. Particularly, PD-1 blockade with nivolumab and pembrolizumab has demonstrated significant single-agent activity in this select population.
Balasubramanian, Gopalan; Kilambi, Narasimhan; Rathinasamy, Suresh; Rajendran, Praveen; Narayanan, Shridhar; Rajagopal, Sridharan
HDAC inhibitors emerged as promising drug candidates in combating wide variety of cancers. At present, two of the compounds SAHA and Romidepsin were approved by FDA for cutaneous T-cell lymphoma and many are in various clinical phases. A new quinolone cap structure was explored with hydroxamic acid as zinc-binding group (ZBG). The pan HDAC inhibitory and antiproliferative activities against three human cancer cell lines HCT-116 (colon), NCI-H460 (lung) and U251 (glioblastoma) of the compounds (4a-4w) were evaluated. Introduction of heterocyclic amines in CAP region increased the enzyme inhibitory and antiproliferative activities and few of the compounds tested are metabolically stable in both MLM and HLM.
Meta, Margarita; Yang, Shao H; Bergo, Martin O; Fong, Loren G; Young, Stephen G
Genetic mutations that lead to an accumulation of farnesyl-prelamin A cause progeroid syndromes, including Hutchinson-Gilford progeria syndrome. It seemed possible that the farnesylated form of prelamin A might be toxic to mammalian cells, accounting for all the disease phenotypes that are characteristic of progeria. This concept led to the hypothesis that protein farnesyltransferase inhibitors (FTIs) might ameliorate the disease phenotypes of progeria in mouse models. Thus far, two different mouse models of progeria have been examined. In both models, FTIs improved progeria-like disease phenotypes. Here, prelamin A post-translational processing is discussed and several mutations underlying human progeroid syndromes are described. In addition, recent data showing that FTIs ameliorate disease phenotypes in a pair of mouse models of progeria are discussed.
United Energy Corporation (UNRG) and the U.S. Department of Energy personnel tested KH-30 at the Rocky Mountain Oilfield Testing Center (RMOTC) outside Casper, Wyoming on two separate occasions. KH-30 is a non-toxic, non-hazardous product, which combines the functions of a solvent dispersant, crystal modifier and inhibitor into a single solution. The first test was held in March of 2001, wherein five wells were treated with a mixture of KH-30 and brine water, heated to 180 degrees F. No increase in production was attained in these tests. In June, 2001, three shallow, low pressure RMOTC wells with 30 years of production were treated with a mixture of 40% KH-30 and 60% diesel. Increases were seen in three wells. The wells then returned to their original rates.
Kalia, Vipin Chandra
Excessive and indiscriminate use of antibiotics to treat bacterial infections has lead to the emergence of multiple drug resistant strains. Most infectious diseases are caused by bacteria which proliferate within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. These molecules act primarily by quenching the QS system. The phenomenon is also termed as quorum quenching (QQ). In addition, synthetic compounds have also been found to be effective in QQ. This review focuses primarily on natural and synthetic quorum sensing inhibitors (QSIs) with the potential for treating bacterial infections. It has been opined that the most versatile prokaryotes to produce QSI are likely to be those, which are generally regarded as safe. Among the eukaryotes, certain legumes and traditional medicinal plants are likely to act as QSIs. Such findings are likely to lead to efficient treatments with much lower doses of drugs especially antibiotics than required at present.
Patel, Jayendra Z; Ahenkorah, Stephen; Vaara, Miia; Staszewski, Marek; Adams, Yahaya; Laitinen, Tuomo; Navia-Paldanius, Dina; Parkkari, Teija; Savinainen, Juha R; Walczyński, Krzysztof; Laitinen, Jarmo T; Nevalainen, Tapio J
Compound 12a (JZP-361) acted as a potent and reversible inhibitor of human recombinant MAGL (hMAGL, IC50=46 nM), and was found to have almost 150-fold higher selectivity over human recombinant fatty acid amide hydrolase (hFAAH, IC50=7.24 μM) and 35-fold higher selectivity over human α/β-hydrolase-6 (hABHD6, IC50=1.79 μM). Additionally, compound 12a retained H1 antagonistic affinity (pA2=6.81) but did not show cannabinoid receptor activity, when tested at concentrations ⩽ 10 μM. Hence, compound 12a represents a novel dual-acting pharmacological tool possessing both MAGL-inhibitory and antihistaminergic activities.
Balunas, Marcy J.; Su, Bin; Brueggemeier, Robert W.; Kinghorn, A. Douglas
With the clinical success of several synthetic aromatase inhibitors (AIs) in the treatment of postmenopausal estrogen receptor-positive breast cancer, researchers have also been investigating also the potential of natural products as AIs. Natural products from terrestrial and marine organisms provide a chemically diverse array of compounds not always