Exogenous Activator Research Articles

KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1pM to 10μM). The resulting elevated alkaline phosphatase (ALP) levels measured 9days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Activation of heme oxygenase-1 by Ginkgo biloba extract differentially modulates endothelial and smooth muscle-like progenitor cells for vascular repair

Vascular progenitors such as endothelial progenitor cells (EPCs) and smooth muscle-like progenitor cells (SMPCs) may play different roles in vascular repair. Ginkgo biloba extract (GBE) is an exogenous activator of heme oxygenase (HO)-1, which has been suggested to improve vascular repair; however, the detailed mechanisms have yet to be elucidated. This study aimed to investigate whether GBE can modulate different vascular progenitor cells by activating HO-1 for vascular repair. A bone marrow transplantation mouse model was used to evaluate the in vivo effects of GBE treatment on wire-injury induced neointimal hyperplasia, which is representative of impaired vascular repair. On day 14 of GBE treatment, the mice were subjected to wire injury of the femoral artery to identify vascular reendothelialization. Compared to the mice without treatment, neointimal hyperplasia was reduced in the mice that received GBE treatment for 28 days in a dose-dependent manner. Furthermore, GBE treatment increased bone marrow-derived EPCs, accelerated endothelial recovery, and reduced the number of SMPCs attached to vascular injury sites. The effects of GBE treatment on neointimal hyperplasia could be abolished by co-treatment with zinc protoporphyrin IX, an HO-1 inhibitor, suggesting the in vivo role of HO-1. In this in vitro study, treatment with GBE activated human early and late EPCs and suppressed SMPC migration. These effects were abolished by HO-1 siRNA and an HO-1 inhibitor. Furthermore, GBE induced the expression of HO-1 by activating PI3K/Akt/eNOS signaling in human late EPCs and via p38 pathways in SMPCs, suggesting that GBE can induce HO-1 in vitro through different molecular mechanisms in different vascular progenitor cells. Accordingly, GBE could activate early and late EPCs, suppress the migration of SMPCs, and improve in vivo vascular repair after mechanical injury by activating HO-1, suggesting the potential role of pharmacological HO-1 activators, such as GBE, for vascular protection in atherosclerotic diseases.

Notch1 Signaling Pathway Promotes Proliferation and Mediates Differentiation Direction in Hippocampus of Streptococcus pneumonia Meningitis Rats.

Streptococcus pneumonia meningitis (PM) is a major cause of childhood neurological deficits. Although the Notch1 signaling pathway regulates neurogenesis and neuroinflammation, we know little about its expression or influence on hippocampal neurogenesis and gliogenesis during PM. We used immunofluorescence and Western blots to detect Notch1 signaling expression during experimental PM. Through double-labeling immunofluorescence, we investigated proliferation and differentiation in the dentate gyrus (DG) in PM before and after treatment with exogenous Notch1 activator (Jagged1) and inhibitor (IMR-1). Our results showed that Notch1 was activated after 24 hours in PM. Compared with the phosphate-buffered saline (PBS) control, Jagged1 increased the proliferation of neural stem cells and progenitor cells (NS/PCs) in DG. After 14 and 28 days of meningitis, astrocyte differentiation increased compared with control. Astrocyte differentiation was higher in the Jagged1 versus the PBS group. In contrast, IMR-1 increased neuronal differentiation but decreased astrocyte differentiation compared with dimethyl sulfoxide treatment. Under PM, Notch1 signaling promotes NS/PC proliferation and astrocyte differentiation in DG, while decreasing neuronal differentiation. Transient activation of the Notch1 signaling pathway explains the reactive gliogenesis and limited neuronal differentiation observed in PM.

Clot activators do not expedite the time to predict massive transfusion in trauma patients analyzed with tissue plasminogen activator thrombelastography

BackgroundTrauma patients with hypersensitivity to tissue plasminogen activator mediated fibrinolysis quantified by tissue plasminogen activator thromboelastography are at increased risk of massive transfusion. The tissue plasminogen activator thromboelastography assay has been tested in trauma patients using native thromboelastography with no exogenous activator. We hypothesize that adding an activator will expedite the time to results. MethodsHealthy whole blood was assayed with and without exogenous plasmin, which acts to deplete inhibitors of fibrinolysis, mimicking trauma blood. Samples were assessed using native, kaolin, and rapid thromboelastography with and without tissue plasminogen activator. The tissue plasminogen activator thromboelastography indices of time to maximum amplitude and lysis at 30 minutes were contrasted between healthy blood with and without plasmin using the three different activators. The activators were then used with a tissue plasminogen activator thromboelastography in 100 trauma patients to assess performance in predicting massive transfusion. ResultsIn healthy blood, regardless of activator, lysis at 30 minutes did not increase with plasmin alone, but did increase with tissue plasminogen activator (P = .012). Adding tissue plasminogen activator and plasmin increased lysis at 30 minutes (P = .036). Time to maximum amplitude was reduced with tissue plasminogen activator and plasmin compared with tissue plasminogen activator alone (P = .012). Activated thromboelastographies had increased lysis at 30 minutes (P = .002), but no difference in time to maximum amplitude compared with native thromboelastographies. In trauma patients, native tissue plasminogen activator thromboelastography had greater performance in predicting massive transfusion than activated tissue plasminogen activator thromboelastographies with no difference in time to maximum amplitude. ConclusionAdding an activator to tissue plasminogen activator thromboelastography does not expedite time to maximum amplitude in healthy blood depleted of fibrinolysis inhibitors. Activated tissue plasminogen activator thromboelastographies are inferior to native tissue plasminogen activator thromboelastography for predicting massive transfusion and do not reduce the time to results.

Molecular docking, dynamics, and pharmacology studies on bexarotene as an agonist of ligand-activated transcription factors, retinoid X receptors.

Retinoid X receptors (RXRs) belong to the nuclear receptor superfamily, and upon ligand activation, these receptors control gene transcription via either homodimerization with themselves or heterodimerization with the partner-nuclear receptor. The protective effects of RXRs and RXR agonists have been reported in several neurodegenerative diseases, including in the retina. This study was aimed to prioritize compounds from natural and synthetic origin retinoids as potential RXR agonists by molecular docking and molecular dynamic simulation strategies. The docking studies indicated bexarotene as a lead compound that can activate various RXR receptor isoforms (α, β, and γ) and has a strong binding affinity to the receptor protein than retinoic acid, which is known as a natural endogenous RXR agonist. Dynamic simulation studies confirmed that the hydrogen bonding and hydrophobic interactions were highly stable in all the three isoforms of the RXR-bexarotene complex. To further validate the significance of the RXR receptor in neurons, in vitro pharmacological treatment of neuronal SH-SY5Y cells with bexarotene was performed. In vitro data from SH-SY5Y cells confirmed that bexarotene activated RXR-simulated neurite outgrowth significantly. We conclude that bexarotene could be potentially used as an exogenous activator of RXRs and emerge as a good drug target for several neurodegenerative disorders.

Expression, Localization, and Activity of the Aryl Hydrocarbon Receptor in the Human Placenta.

The human placenta is an organ between the blood of the mother and the fetus, which is essential for fetal development. It also plays a role as a selective barrier against environmental pollutants that may bypass epithelial barriers and reach the placenta, with implications for the outcome of pregnancy. The aryl hydrocarbon receptor (AhR) is one of the most important environmental-sensor transcription factors and mediates the metabolism of a wide variety of xenobiotics. Nevertheless, the identification of dietary and endogenous ligands of AhR suggest that it may also fulfil physiological functions with which pollutants may interfere. Placental AhR expression and activity is largely unknown. We established the cartography of AhR expression at transcript and protein levels, its cellular distribution, and its transcriptional activity toward the expression of its main target genes. We studied the profile of AhR expression and activity during different pregnancy periods, during trophoblasts differentiation in vitro, and in a trophoblast cell line. Using diverse methods, such as cell fractionation and immunofluorescence microscopy, we found a constitutive nuclear localization of AhR in every placental model, in the absence of any voluntarily-added exogenous activator. Our data suggest an intrinsic activation of AhR due to the presence of endogenous placental ligands.

Analysis of Autoinduction, Inhibition, and Autoinhibition in a Rh-Catalyzed C–C Cleavage: Mechanism of Decyanative Aryl Silylation

The mechanism the Chatani–Tobisu rhodium-catalyzed decyanative silylation of aryl nitriles by hexamethyldisilane (Me3Si-SiMe3) has been investigated by in situ NMR spectroscopy. The production of Ar-SiMe3 evolves in three distinct phases: slow catalyst induction is followed by a period of rapidly accelerating turnover and then, after approximately three catalyst turnovers, the onset of progressive inhibition. The processes giving rise to these phenomena have been elucidated by isotopic labeling (13C/15N) and kinetic analysis, and it is shown that, in addition to facilitating catalyst turnover to generate Ar-SiMe3, the reactants serve other roles. Me3Si-SiMe3 functions as a slow exogenous precatalyst activator and as a moderately powerful catalyst inhibitor. In contrast, ArCN acts as a precatalyst inhibitor. Moreover, the coproduct from the reaction (trimethylsilyl cyanide, Me3SiCN) acts as a powerful endogenous precatalyst activator and catalyst inhibitor, together giving rise to sigmoidal temporal concen...

Oridonin inhibits metastasis of human ovarian cancer cells by suppressing the mTOR pathway.

IntroductionOridonin, which is isolated from the Chinese herb Rabdosia rubescens, has been reported to exhibit an anti-tumorous effect on different cancers. In this study, we investigated the molecular mechanism by which oridonin suppresses human ovarian cancer.Material and methodsThe inhibition of oridonin on cell proliferation was assessed by CCK8 assay. Cell cycle and apoptosis were analyzed by flow cytometry, staining with propidium iodide (PI) or annexin-V/PI respectively. The metastasis rate was evaluated using a transwell migration assay. The expression of metastasis-associated genes and mTOR pathway related genes were detected by western blot.ResultsWe demonstrated that oridonin suppressed the proliferation and blocked the cell cycle in G1/S phage and induced apoptosis in SKOV3 and A2780 cells (p < 0.01). We further found that the mTOR signaling pathway was suppressed by the treatment with oridonin, and the activation of the mTOR pathway attenuated the anti-tumorous effect of oridonin in human ovarian cancer cells, suggesting that the mTOR pathway was involved in the anti-tumorous process of oridonin. Additionally, the activation of the mTOR pathway by an exogenous activator reduced the expression level of FOXP3 (p < 0.01), thus providing evidence that FOXP3 is a factor that is necessary for the anti-tumorous effect of oridonin, and is negatively regulated by the mTOR pathway.ConclusionsThese results suggested that oridonin suppressed the mTOR signaling pathway, up-regulated the FOXP3 level, and inhibited metastasis of human ovarian cancer cells.

A multiplexable TALE-based binary expression system for in vivo cellular interaction studies

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE–VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

Breast cancer-associated gene 3 interacts with Rac1 and augments NF-κB signaling in vitro, but has no effect on RANKL-induced bone resorption in vivo.

Breast cancer-associated gene 3 (BCA3) is a recently identified adaptor protein whose functions are still being defined. BCA3 has been reported to be an important regulator of nuclear factor-κB (NF-κB) signaling. It has also been reported to interact with the small GTPase, Rac1. Consistent with that observation, in the present study, BCA3 was found to interact with nuclear Rac1 in 293 cells and influence NF-κB signaling. Additional experiments revealed that depending on cell type, BCA3 augmented, attenuated or had no effect on NF-κB signaling in vitro. Since canonical NF-κB signaling is a critical downstream target from activated receptor activator of nuclear factor κB (RANK) that is required for mature osteoclast formation and function, BCA3 was selectively overexpressed in osteoclasts in vivo using the cathepsin K promoter and the response to exogenous receptor activator of nuclear factor κB ligand (RANKL) administration was examined. Despite its ability to augment NF-κB signaling in other cells, transgenic animals injected with high-dose RANKL had the same hypercalcemic response as their wild-type littermates. Furthermore, the degree of bone loss induced by a 2-week infusion of low-dose RANKL was the same in both groups. Combined with earlier studies, the data from our study data indicate that BCA3 can affect NF-κB signaling and that BCA3 plays a cell-type dependent role in this process. The significance of the BCA3/NF-κB interaction in vivo in bone remains to be determined.

New insights in oxybutynin chemical stability: Identification in transdermal patches of a new impurity arising from oxybutynin N-oxide rearrangement

Oxybutynin hydrochloride (Oxy), the first choice drug used for the management of urinary incontinence, is available in different types of formulations. However, due to its better lipophylicity and permeability, Oxyfree base was used in the new topical formulations such as transdermal patch and gel. The presence of an unprecedented impurity (Oxy-EK) in transdermal patches led to reinvestigate the chemical stability of Oxyfree base in oxidative conditions assigning, to Oxy-EK, the structure of (3E)-4-(N,N-diethylamino)-2-oxo-3-buten-1-yl 1-cyclohexyl-1-phenylglycolate. Oxy-EK arises from the prototropic rearrangement of oxybutynin N-oxides leading to the formation of an enamino ketone function which shows a long-wavelength UV-absorption. The total synthesis of Oxy-EK was performed, allowing to propose it as the indicator of stability for oxidative degradation of Oxy free base in transdermal formulations. The presence in the structure of Oxy-EK of an α,β-unsaturated carbonyl function, a potential Michael acceptor, suggested the need of evaluating its possible mutagenic power. Accordingly, the Ames test was performed: at nontoxic concentrations, Oxy-EK did not increase the number of revertant colonies in all strains tested both in the absence and presence of the exogenous metabolic activator S9.

A synthetic heparin antidote with negligible effect on fibrin(ogen), clotting and clot morphology

Event Abstract Back to Event A synthetic heparin antidote with negligible effect on fibrin(ogen), clotting and clot morphology Manu Thomas Kalathottukaren1*, Benjamin F. Lai1*, Libin Abraham2*, Fredrico Rosell1*, Edward L. Pryzdial1, Charles A. Haynes3* and Jayachandran N. Kizhakkedathu1, 4* 1 University of British Columbia, Pathology and laboratory Medicine, Canada 2 University of British Columbia, Department of Immunolgy and Microbiology, Canada 3 University of British Columbia, Department of Chemical and Biological Engineering, Canada 4 University of British Columbia, Chemistry, Canada Background and Objective: Heparin anticoagulants are used to treat thrombosis; the leading cause of deaths worldwide. However, anticoagulation associated haemorrhage is a major concern. Hence, antidote is required to counteract anticoagulation and to restore hemostasis. Protamine sulphate (PS), a cationic polypeptide is the only clinically approved antidote for unfractionated heparin. PS is unable to completely reverse low molecular weight heparins and fondaparinux due to its low binding affinity to these drugs. In addition, PS exhibits non-specific interaction with clotting proteins such as fibrinogen to form aggregates and alters fibrin clot morphology. This often leads to hematological complications. To address these challenges, we developed a synthetic universal heparin reversal agent (UHRA) with high binding affinity to heparins, thereby completely reversing the activity of all clinically available parenteral anticoagulants1.This study aims to demonstrate and corroborate the nontoxic nature of UHRA by assessing its influence on fibrinogen, clotting, fibrin/blood clot architecture and stability. Methods: UHRA was developed by incorporating tertiary amine based heparin binding groups on a dendritic hyperbranched polyglycerol scaffold and capping it with methoxy polyethylene glycol chains. Recalcification and tissue factor (TF) initiated plasma clotting assays was performed to understand the impact of UHRA on clotting system. The interaction of UHRA on fibrinogen was investigated by fibrin polymerization assay and by spectroscopic analysis (fluorescence and circular dichroism (CD)). The influence of UHRA on fibrin/blood clot architecture was evaluated by scanning electron microscopy (SEM). Confocal microscopy was used to investigate the incorporation of Alexa-488 tagged UHRA into fibrin clots. The lysis of TF-induced plasma clot containing UHRA or PS exposed to exogenous tissue plasminogen activator (t-PA) was studied by turbidimetric assay. Results and discussion: Clotting assays revealed that UHRA did not alter clotting parameters (lag time and maximum absorbance) compared to PS. This suggests that UHRA has no effect on clotting cascade. Unlike protamine, the fibrinogen aggregation and fibrin polymerization assay was not influenced by UHRA over a broad range of concentrations from 0.05 mg/mL to 1 mg/mL. Tryptophan fluorescence quenching and fibrinogen secondary structure measurements, corroborates that UHRA is not interacting with fibrinogen. However, PS and other synthetic cationic polymers interact with fibrinogen, eliciting aggregation and conformational changes. Fibrin clots generated in presence of UHRA (even at 0.5 mg/mL) showed similar structure and fiber size remains same as normal fibrin clot (control vs UHRA 0.5 mg/mL clot, p= 0.12) (Figure 1). On the other hand, fibrin clots formed in the presence of 0.05mg/mL PS (clinical dose) increased the fiber size and changed the clot structure dramatically (control vs PS 0.05mg/mL clot, p< 0.0001). Confocal images of fibrin clots show no incorporation of UHRA into clots, compared to PS (Figure 2). Clot lysis studies in the presence of exogenous t-PA demonstrate that UHRA did not enhance clot degradation. Conclusion and significance: Studies demonstrate that, UHRA, has negligible impact on fibrinogen, fibrin polymerization, clot structure, clot degradation, coagulation system and shows no binding to clot structures, revealing their excellent hemocompatibility compared to protamine. Our results support the fact that UHRA could be an ideal antidote to restore hemostasis following invasive surgical procedures and to address bleeding complications by heparin based anticoagulants. [1] Canadian Institutes of Health Research (CIHR); Natural Science and Engineering Research Council (NSERC)

Dabigatran but not rivaroxaban or apixaban treatment decreases fibrinolytic resistance in patients with atrial fibrillation

IntroductionMost anticoagulants stimulate fibrinolysis in vitro through mechanisms dependent on and independent of thrombin activatable fibrinolysis inhibitor (TAFI). We evaluated the effect of dabigatran, rivaroxaban and apixaban treatment on plasma fibrinolysis in patients with non-valvular atrial fibrillation. Methods and resultsPatients treated with dabigatran etexilate (n=22), rivaroxaban (n=24) or apixaban (n=22) were studied. Plasma was obtained before (trough) and 2h after drug intake (peak). Fibrinolytic resistance of clots exposed to exogenous tissue plasminogen activator was significantly lower in peak than in trough samples and correlated with drug concentration only in dabigatran group. Moreover, fibrinolytic resistance at peak was lower in dabigatran than in rivaroxaban and apixaban groups. This difference disappeared if the TAFI pathway was inhibited. Thrombin generation and TAFI activation were markedly lower in peak than in trough samples in all three groups. However, TAFIa levels in trough and peak samples were significantly lower in dabigatran group than in rivaroxaban and apixaban groups. Circulating levels of prothrombin fragment F1+2 (reflecting in vivo thrombin generation) and plasmin-antiplasmin complex (reflecting plasmin generation) were not or barely influenced by drug levels in all groups. ConclusionsOur data suggest that dabigatran, contrary to rivaroxaban and apixaban, reduces fibrinolytic resistance by virtue of its greater impact on TAFI activation. The profibrinolytic effect of dabigatran may play a role locally, at sites of fibrin formation, by making the nascent thrombus more susceptible to plasminogen-dependent degradation.

Ex vivo recapitulation of trauma-induced coagulopathy and preliminary assessment of trauma patient platelet function under flow using microfluidic technology.

Relevant to trauma-induced coagulopathy diagnostics, microfluidic assays allow controlled hemodynamics for testing of platelet and coagulation function using whole blood. Hemodilution or hyperfibrinolysis was studied under flow with modified healthy whole blood. Furthermore, platelet function was also measured using whole blood from trauma patients admitted to a Level I trauma center. Platelet deposition was measured with PPACK-inhibited blood perfused over collagen surfaces at a wall shear rate of 200 s, whereas platelet/fibrin deposition was measured with corn trypsin inhibitor-treated blood perfused over tissue factor (TF)/collagen. In hemodilution studies, PPACK-treated blood displayed almost no platelet deposition when diluted to 10% hematocrit with saline, platelet-poor plasma, or platelet-rich plasma. Using similar dilutions, platelet/fibrin deposition was essentially absent for corn trypsin inhibitor-treated blood perfused over TF/collagen. To mimic hyperfibrinolysis during trauma, exogenous tissue plasminogen activator (50 nM) was added to blood before perfusion over TF/collagen. At both venous and arterial flows, the generation and subsequent lysis of fibrin were detectable within 6 minutes, with lysis blocked by addition of the plasmin inhibitor, ε-aminocaproic acid. Microfluidic assay of PPACK-inhibited whole blood from trauma patients revealed striking defects in collagen response and secondary platelet aggregation in 14 of 21 patients, whereas platelet hyperfunction was detected in three of 20 patients. Rapid microfluidic detection of (1) hemodilution-dependent impairment of clotting, (2) clot instability because of lysis, (3) blockade of fibrinolysis, or (4) platelet dysfunction during trauma may provide novel diagnostic opportunities to predict trauma-induced coagulopathy risk.

Histone H4-Induced Thrombin Generation in Fresh and "Reconstituted" Blood

Background. Histones are components of nucleosomes and can be released from activated immune cells as well as from necrotic and apoptotic cells. The release of histones is associated with an increased prothrombotic activity. It has been suggested that this activity is caused by the stimulation of platelets, interference with the protein C pathway, changes in red blood cell (RBC) function, etc. Studies published so far were done in plasma or using purified cells and proteins. We extended these studies by evaluating thrombin (FIIa) generation in fresh and "reconstituted" blood triggered with histone H4 and examined contribution of several proteins on this process.Methods. Blood from 8 healthy donors (4 male and 4 female, none of them on oral contraceptives; one male and one female donor were drawn twice) was drawn into 3.2% sodium citrate, 0.1 mg/mL corn trypsin inhibitor (CTI; prevents contact pathway activation) and 20 µg/mL H4 were added followed by recalcification. FIIa generation (TG) was monitored in a fluorogenic assay derived from the CAT method. When desired, either monoclonal inhibitory antibodies to factor (F)XIa, FIXa and tissue factor (TF) (αFXIa-2, αFIX-91 and αTF-5, respectively; all at 0.1 mg/mL) or kallikrein inhibitor aprotinin (10 µM) were added to blood. In parallel, TG in washed RBC reconstituted (50/50) either with platelet poor (PPP) or platelet rich (PRP) plasma ("reconstituted blood") was analyzed at the same conditions. FIIa generation in an autologous plasma was also analyzed.Results.A. Whole blood. In the absence of any exogenous activator, the lag phase and peak value of TG in CTI blood varied in a wide range (see the table). An addition of H4 to the blood shortened the lag phase and increased peak FIIa concentration for all donors. An addition of αFXIa-2 slightly prolonged the lag phase and slightly suppressed other parameters of FIIa generation. Aprotinin had a somewhat more pronounced effect on the lag phase but only marginally affected other parameters of FIIa generation. αFIX-91 almost completely suppressed H4-initiated FIIa generation, whereas αTF-5 had almost no effect on the process. Table 1ConditionsLag (min)Max FIIa (nM)Max Rate (nM/min)AUC (nM*min)RangeAverage ± SDRangeAverage ± SDRangeAverage ± SDRangeAverage ± SDCTI(n=10)9 - 2313.4 ± 4.236.6 - 109.563.7 ± 25.114.5 - 92.648.9 ± 26.6309.3 - 687.8487.6 ± 130.1CTI + H4(n=10)2.6 - 7.44.7 ± 1.466.3 - 134.799.4 ± 22.436.8 - 83.357.9 ± 15.4417.5 - 865.8571.1 ± 145CTI + H4 + αFXIa-2(n=9)2.4 - 8.55.2 ± 1.956.6 - 103.983.6 ± 13.222.9 - 69. 444.9 ± 13.2409.9 - 680.3522 ± 90.4CTI + H4 + aprotinin(n=9)3.3 - 8.15.5 ± 1.662.3 - 144.295.1 ± 25.736 - 94.458.1 ± 19.4409.2 - 1056.2599.5 ± 203.6CTI + H4 + αFIXa-91(n=6)NQNQNQNQNQNQNQNQCTI + H4 + αTF-2(n=6)3.2 - 7.44.9 ± 1.765.1 - 128.490.1 ± 21.843.4 - 16973.9 ± 47.6434.1 - 867.7543.1 ± 166*NQ = Not Quantifiable (in 4 of 6 experiments no FIIa generation was observed and in 2 experiments it was negligible).B. RBC. In the absence of any exogenous activator, no FIIa generation was observed in 50 min. in RBC reconstituted with PPP. An addition of H4 led to FIIa generation after a lag phase of 17.7±4.3 min with the maximum active FIIa concentration (66.5±12.4 nM) lower than that observed in whole blood. Additions of either αFXIa-2 or aprotinin had no pronounced effect on FIIa generation, whereas no FIIa was observed in the presence of αFIX-91. When RBC were reconstituted with PRP, H4-induced FIIa generation parameters became similar to those observed in whole blood.C. Autologous PPP. No FIIa generation was observed in CTI PPP in the absence of any exogenous activator. An addition of H4 led to a delayed (25-30 min lag phase) and inefficient FIIa generation (maximum 10-15 nM). αFXIa-2 had no effect on FIIa generation, αFIX-91 completely suppressed it and aprotinin had an intermediate effect.Conclusions. 1). H4 has a pronounced effect in enhancing FIIa generation in fresh blood and washed RBC reconstituted with PPP and PRP; 2). Inhibition of contact pathway proteins has minor effect on H4-induced FIIa generation; 3). Inhibition of FIXa almost completely abolishes H4-induced FIIa generation; 4). RBC reconstituted with PPP can support H4-induced FIIa generation and platelets enhance this process; 5). H4-induced FIIa generation in PPP is negligible. DisclosuresMann:Haematologic Technologies, Inc; Baxter, Diagnostica Stago, Bayer, CSL Behring, Alnylam.: Consultancy, Equity Ownership.

ACTIVATION OF PROTEIN C IN THE IN VITRO THROMBOLYSIS

Physiological conditions of formation and subsequent lysis of thrombus were reconstituted in vitro in our research. Thrombus formation was initiated either by addition of exogenous thrombin or by contact of blood with anionic surface, which stimulates spontaneous coagulation of blood. Tissue plasminogen activator and/or protein C were previously added in the blood sample. The time of the beginning and total degradation of formed thrombi as well as the level of PC in lysates was controlled then. Only an addition of protein C alone or in combination with tissue plasminogen activator led to the most effective lysis of thrombi: their residual weight was 18% and 5% comparing to control. Addition of exogenous tissue plasminogen activator alone or in combination with protein C caused a 83% and 74% decrease of PC level in lysates of spontaneously formed thrombi, and 72% and 56% decrease for thrombi formed by thrombin, respectively. Without an addition of tissue plasminogen activator protein C level in lysates of thrombi formed by thrombin was 54% down on spontaneously formed thrombi. Thus, changes of PC concentration in isolated volume of clot seem to be controlled by thrombin at the stage of thrombus formation and by fibrinolytic system at the stage of fibrinolysis. Concentration of PC in lysates from clots formed by exogenous thrombin was decreasing over the next 10 hours of thrombolysis, which can also be the evidence of the interaction between the fibrinolytic and PC activation systems. A hypothesis is. formulated about an existence of endothelium-independent mechanism of PC activation in blood plasma with blood cells participation, which effectiveness increases in the process of thrombolysis.

Fibrinolysis shutdown phenotype masks changes in rodent coagulation in tissue injury versus hemorrhagic shock

Systemic hyperfibrinolysis (accelerated clot degradation) and fibrinolysis shutdown (impaired clot degradation) are associated with increased mortality compared with physiologic fibrinolysis after trauma. Animal models have not reproduced these changes. We hypothesize rodents have a shutdown phenotype that require an exogenous profibrinolytic to differentiate mechanisms that promote or inhibit fibrinolysis. Fibrinolysis resistance was assessed by thrombelastography (TEG) using exogenous tissue plasminogen activator (tPA) titrations in whole blood. There were 3 experimental groups: (1) tissue injury (laparotomy/bowel crush), (2) shock (hemorrhage to mean arterial pressure of 20 mmHg), and (3) control (arterial cannulation and tracheostomy). Baseline and 30-minute postintervention blood samples were collected, and assayed with TEG challenged with taurocholic acid (TUCA). Rats were resistant to exogenous tPA; the percent clot remaining 30 minutes after maximum amplitude (CL30) at 150 ng/mL (P = .511) and 300 ng/mL (P = .931) was similar to baseline, whereas 600 ng/mL (P = .046) provoked fibrinolysis. Using the TUCA challenge, the percent change in CL30 from baseline was increased in tissue injury compared with control (P = .048.), whereas CL30 decreased in shock versus control (P = .048). tPA increased in the shock group compared with tissue injury (P = .009) and control (P = .012). Rats have an innate fibrinolysis shutdown phenotype. The TEG TUCA challenge is capable of differentiating changes in clot stability with rats undergoing different procedures. Tissue injury inhibits fibrinolysis, whereas shock promotes tPA-mediated fibrinolysis.

The Reversal of the Anticoagulant and Anti‐hemostatic Effect of LMWH by rLopap

Lopap, found in the bristles of the caterpillar&lt;em style=“mso‐bidi‐font‐style: normal;”&gt; Lonomia obliqua, is the first exogenous prothrombin activator that shows serineprotease‐like activity and only lipocalin reported to interfere with hemostasis mechanisms. The aim was to assess the effect of recombinant Lopap, obtained using an E. coli expression system, as an exogenous hemostatic factor in reversing bleeding induced by LMWH .The experimental protocol was approved by the Ethics Committee for the Use of Animals of the Teaching and Research Institute, Hospital Sírio‐Libanês. Briefly, anesthesia was induced with ketamine HCl (40 mg/kg, im) and xylazine (5 mg/kg, im) and was maintained by intermittent injections of the same mixture. Rabbits were allocated to 4 groups and received 1800IU/kg LMWH iv over 2 min, followed by iv administration of saline (SG) or rLopap at a dose of 1 μg/kg (LG1) or 10 μg/kg (LG10), 10 min after the injection of LMWH. The reversal of the anticoagulant and anti‐hemostatic effect of LMWH by rLopap were assessed in terms of bleeding time (BT) and laboratory tests; activated partial thromboplastin time (aPTT), prothrombin time (TP), prothrombin fragment F1+2, Thrombin generation, Thrombin‐Antithrombin complex (TAT) and fibrinogen. LG10 animals showed a decreased BT near normal baseline. The levels of prothrombin fragment F1+2 and measured thrombin were inversely proportional to BT. rLopap can be a possible template for the reversal of bleeding and anticoagulant events.Supported by FAPESP

Mutagenicity of cigarette butt waste in the bacterial reverse mutation assay: The protective effects of β-caryophyllene and β-caryophyllene oxide.

Cigarette filters pose a serious litter and toxic waste disposal problem, because of their not biodegradability and to the leaching of toxins in the environment. Therefore, cigarette butts need to be manipulated as special waste, with potential risks to human health and environment. In the present study, the genotoxic potential of a methanol extract from commonly discharged cigarette butts (CBE) was evaluated in the bacterial reverse mutation assay on Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2uvrA strains, both in the absence and presence of the S9 exogenous metabolic activator. Furthermore, the ability of the natural sesquiterpenes β-caryophyllene (CRY) and β-caryophyllene oxide (CRYO) to inhibit the mutagenicity of CBE was studied as a possible preventive strategy. In order to identify the potential antimutagenic mechanisms, three different protocols (pretreatment, cotreatment, and posttreatment) were applied. CBE showed to increase the number of revertant colonies in all the strains tested in presence of S9, so resulting mutagenic. In the antimutagenicity assay, both CRY and CRYO significantly reduced the revertant colonies induced by CBE, although with different potency and specificity. For both sesquiterpenes, the antimutagenicity was strong in all experimental conditions, except for the cotreatment of CRY with CBE in WP2uvrA, which produced a moderate inhibition. Both desmutagenic and bioantimutagenic mechanisms seem to be involved in the antimutagenicity of the test substances. Taking into account the potential genotoxicity of cigarette butts, CRY and CRYO appear as possible further candidates as environmental decontaminants against this hazardous waste. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1319-1328, 2016.

Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C

IntroductionCell surface thiol isomerase enzymes, principally protein disulphide isomerase (PDI), have emerged as important regulators of platelet function and tissue factor activation via their action on allosteric disulphide bonds. Allosteric disulphides are present in other haemostasis-related proteins, and we have therefore investigated whether thiol isomerase inhibition has any influence on two endothelial activities relevant to haemostatic regulation, namely activation of protein C and activation of plasminogen, with subsequent fibrinolysis. Materials and MethodsThe study was performed using the human microvascular endothelial cell line HMEC-1. Thiol isomerase gene expression was measured by RT-PCR and activation of protein C and plasminogen by cell-based assays using chromogenic substrates S2366 and S2251, respectively. Cell mediated fibrinolysis was measured by monitoring absorbance at 405nm following fibrin clot formation on the surface of HMEC-1 monolayers. Results and ConclusionsA variety of thiol isomerase enzymes, including PDI, were expressed by HMEC-1 cells and thiol reductase activity detectable on the cell surface was inhibited by both RL90 anti-PDI antibody and by the PDI inhibitor quercetin-3-rutinoside (rutin). In cell-based assays, activation of plasminogen, but not of protein C, was inhibited by RL90 antibody and, to a lesser extent, by rutin. Fibrin clot lysis occurring on a HMEC-1 monolayer was also significantly slowed by RL90 antibody and by rutin, but RL90-mediated inhibition was abolished in the presence of exogenous tissue plasminogen activator (tPA). We conclude that thiol isomerases, including PDI, are involved in fibrinolytic regulation at the endothelial surface, although not via a direct action on tPA. These findings broaden understanding of haemostatic regulation by PDI, and may aid in development of novel anti-thrombotic therapeutic strategies targeted via the fibrinolysis system.