Enzymatic activity is necessary for thrombin-mediated increase in endothelial permeability

1990 ◽  
Vol 259 (4) ◽  
pp. L270-L275 ◽  
Author(s):  
J. L. Aschner ◽  
J. M. Lennon ◽  
J. W. Fenton ◽  
M. Aschner ◽  
A. B. Malik
Keyword(s):  
Active Site ◽  
Endothelial Permeability ◽  
Covalent Bond ◽  
Catalytic Site ◽  
Irreversible Inhibitor ◽  
Chloromethyl Ketone ◽  
High Concentrations ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Fold Excess

alpha-Thrombin causes a dose-dependent increase in endothelial permeability as measured by the clearance rate of 125I-albumin across a monolayer of bovine pulmonary artery endothelial cells. We determined if an active catalytic site is necessary for the thrombin-mediated increase in endothelial permeability. alpha-Thrombin was reacted with 10-fold excess D-phenylalanyl-prolyl-arginine chloromethyl ketone (PPACK), an irreversible inhibitor that forms a covalent bond with thrombin's active site, producing an enzymatically inactive thrombin. PPACK completely inhibited the alpha-thrombin-mediated increase in 125I-albumin permeability. Similar results were obtained with gamma-thrombin, an enzymatically active alpha-thrombin form with an altered fibrinogen recognition domain. PPACK alone and the active site-inhibited PPACK-alpha-thrombin had no effect on permeability. Diisopropylphospho (DIP)-alpha-thrombin was effective only in very high concentrations (10(-6)M), and this effect was abolished by the addition of PPACK. These studies demonstrate that binding alone is insufficient for the thrombin-mediated increase in endothelial monolayer permeability. Thrombin's active catalytic site is a requirement for the increase in transendothelial albumin permeability.

scholarly journals Concentration- and pH-Dependent Oligomerization of the Thrombin-Derived C-Terminal Peptide TCP-25

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1572
Author(s):  
Ganna Petruk ◽  
Jitka Petrlova ◽  
Firdaus Samsudin ◽  
Rita Del Giudice ◽  
Peter J. Bond ◽  
...  
Keyword(s):  
Tryptophan Fluorescence ◽  
Helical Structure ◽  
High Concentration ◽  
Temperature Dependent ◽  
Preclinical Development ◽  
Intrinsic Tryptophan Fluorescence ◽  
Ph Dependent ◽  
High Concentrations ◽  
Dose Dependent Increase ◽  
Dose Dependent

Peptide oligomerization dynamics affects peptide structure, activity, and pharmacodynamic properties. The thrombin C-terminal peptide, TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE), is currently in preclinical development for improved wound healing and infection prevention. It exhibits turbidity when formulated at pH 7.4, particularly at concentrations of 0.3 mM or more. We used biochemical and biophysical approaches to explore whether the peptide self-associates and forms oligomers. The peptide showed a dose-dependent increase in turbidity as well as α-helical structure at pH 7.4, a phenomenon not observed at pH 5.0. By analyzing the intrinsic tryptophan fluorescence, we demonstrate that TCP-25 is more stable at high concentrations (0.3 mM) when exposed to high temperatures or a high concentration of denaturant agents, which is compatible with oligomer formation. The denaturation process was reversible above 100 µM of peptide. Dynamic light scattering demonstrated that TCP-25 oligomerization is sensitive to changes in pH, time, and temperature. Computational modeling with an active 18-mer region of TCP-25 showed that the peptide can form pH-dependent higher-order end-to-end oligomers and micelle-like structures, which is in agreement with the experimental data. Thus, TCP-25 exhibits pH- and temperature-dependent dynamic changes involving helical induction and reversible oligomerization, which explains the observed turbidity of the pharmacologically developed formulation.

Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis

2005 ◽  
Vol 94 (12) ◽  
pp. 1236-1244 ◽  
Author(s):  
Olav Klingenberg ◽  
Reidun Øvstebø ◽  
Gun-Britt Joø ◽  
Åse-Brit Westvik ◽  
Peter Kierulf ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tissue Factor ◽  
Procoagulant Activity ◽  
Clinical Samples ◽  
Meningococcal Infection ◽  
High Concentrations ◽  
Apoptosis And Necrosis ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryTissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.

Calcium entry and myogenic phenomena in skeletal muscle arterioles

1994 ◽  
Vol 267 (3) ◽  
pp. H1085-H1092 ◽  
Author(s):  
M. A. Hill ◽  
G. A. Meininger
Keyword(s):  
Bay K 8644 ◽  
In Vivo Studies ◽  
Basal Tone ◽  
Biphasic Effect ◽  
Prime Determinant ◽  
High Concentrations ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Intravascular Pressure

Studies were conducted to examine Ca2+ entry in several vasomotor situations related to myogenic properties of arterioles (basal tone, vasomotion, and responsiveness to alterations in intravascular pressure). In vivo studies were performed on small cremaster muscle arterioles of anesthetized rats. Intravascular pressure was increased using the pressure-box technique. Voltage-operated Ca2+ channel (VOC) activity was inhibited by nifedipine or methoxyverapamil and was stimulated with BAY K 8644. To examine the effect of hyperpolarization, studies were performed in the presence of pinacidil. Nifedipine and methoxyverapamil exhibited a trend toward dose-dependent dilation; however, neither agent caused dilation comparable to adenosine (10(-4) M). BAY K 8644 produced a biphasic effect, constriction below 10(-8) M, and dilation at higher levels. These data indicate that basal tone can be modulated by agents acting on VOCs; however, as high concentrations of nifedipine do not abolish tone, other mechanisms contribute. At similar concentrations, the Ca2+ channel antagonist significantly inhibited vasomotion, abolishing vasomotion at concentrations above 5 x 10(-6) M. In contrast, BAY K 8644 caused a dose-dependent increase in vasomotion amplitude (e.g., 269 +/- 52% of basal at 10(-7) M). Thus vasomotion appears highly dependent on VOCs. Experiments performed in the presence of the antagonists/agonists indicated that VOCs are not the prime determinant of constrictor responses to acute increases in intravascular pressure. Exposure to pinacidil resulted in dose-dependent vasodilatation and inhibition of vasomotion while showing little effect on acute myogenic responses. Similar effects of pinacidil were observed in isolated, cannulated, cremaster arterioles.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inactivation of TEM-1 β-lactamase by 6-acetylmethylenepenicillanic acid

1983 ◽  
Vol 209 (3) ◽  
pp. 609-615 ◽  
Author(s):  
M Arisawa ◽  
R Then
Keyword(s):  
Rate Constant ◽  
Active Site ◽  
Native Enzyme ◽  
Stable Form ◽  
Beta Lactamase ◽  
Irreversible Inhibitor ◽  
Beta Lactamases ◽  
Inactivation Constant ◽  
Inactivation Process ◽  
Fold Excess

6-Acetylmethylenepenicillanic acid is a new kinetically irreversible inhibitor of various beta-lactamases. Interaction between 6-acetylmethylenepenicillanate and purified TEM-1 beta-lactamase during the inactivation process was investigated. 6-Acetylmethylenepenicillanate inhibited the enzyme in a second-order fashion with a rate constant of 0.61 microM-1 . S-1. The apparent inactivation constant decreased in the presence of increasing concentrations of the substrate benzylpenicillin. Native enzyme (pI 5.4) was converted into two inactive forms with pI 5.25 and 5.15, the latter form being transient and readily converted into the more stable form with pI 5.15. Even a 50-fold excess of inhibitor over enzyme did not produce any other inactivated species of the enzyme. All the results obtained suggest that 6-acetylmethylenepenicillanate is a potent irreversible and active-site-directed inhibitor of TEM-1 beta-lactamase.

Increased Expression of u-PA and u-PAR on Monocytes by LDL and Lp(a) Lipoproteins – Consequences for Plasmin Generation and Monocyte Adhesion

1999 ◽  
Vol 81 (04) ◽  
pp. 594-560 ◽  
Author(s):  
Florence Ganné ◽  
Marc Vasse ◽  
Jean-Louis Beaudeu ◽  
Jacqueline Peynet ◽  
Arnaud François ◽  
...  
Keyword(s):  
Fold Increase ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Atheromatous Plaque ◽  
Monocyte Adhesion ◽  
Blood Monocytes ◽  
Proteolytic Cascade ◽  
Ecm Degradation ◽  
Dose Dependent Increase ◽  
Dose Dependent

SummaryMonocyte-derived foam cells figure prominently in rupture-prone regions of atherosclerotic plaque. As urokinase/urokinase-receptor (u-PA/u-PAR) is the trigger of a proteolytic cascade responsible for ECM degradation, we have examined the effect of atherogenic lipoproteins on monocyte surface expression of u-PAR and u-PA. Peripheral blood monocytes, isolated from 10 healthy volunteers, were incubated with 10 to 200 µg/ml of native or oxidised (ox-) atherogenous lipoproteins for 18 h and cell surface expression of u-PA and u-PAR was analysed by flow cytometry. Both LDL and Lp(a) induced a dose-dependent increase in u-PA (1.6-fold increase with 200 μg/ml of ox-LDL) and u-PAR [1.7-fold increase with 200 μg/ml of ox-Lp(a)]. There is a great variability of the response among the donors, some of them remaining non-responders (absence of increase of u-PA or u-PAR) even at 200 μg/ml of lipoproteins. In positive responders, enhanced u-PA/u-PAR is associated with a significant increase of plasmin generation (1.9-fold increase with 200 μg/ml of ox-LDL), as determined by an amidolytic assay. Furthermore, monocyte adhesion to vitronectin and fibrinogen was significantly enhanced by the lipoproteins [respectively 2-fold and 1.7-fold increase with 200 μg/ml of ox-Lp(a)], due to the increase of u-PAR and ICAM-1, which are receptors for vitronectin and fibrinogen. These data suggest that atherogenous lipoproteins could contribute to the development of atheromatous plaque by increasing monocyte adhesion and trigger plaque weakening by inducing ECM degradation.

The Ability of Thrombin Inhibitors to Reduce the Thrombin Activity Generated in Plasma on Extrinsic and Intrinsic Activation

1997 ◽  
Vol 77 (03) ◽  
pp. 498-503 ◽  
Author(s):  
D Prasa ◽  
L Svendsen ◽  
J Stürzebecher
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Anion Binding ◽  
Thrombin Inhibitors ◽  
Coagulation System ◽  
Thrombin Activity ◽  
Continuous Registration ◽  
High Concentrations ◽  
20 Nm ◽  
Generation Test

SummaryIn a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action to influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced.By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki ≤ 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1 - 0.6 μM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.

LH-RH TEST IN 100 PATIENTS WITH OVARIAN INSUFFICIENCY

1974 ◽  
Vol 75 (3) ◽  
pp. 428-434 ◽  
Author(s):  
P.-J. Czygan ◽  
M. Breckwoldt ◽  
F. Lehmann ◽  
R. Langefeld ◽  
G. Bettendorf
Keyword(s):  
Intravenous Injection ◽  
Functional State ◽  
Clear Correlation ◽  
Normal Range ◽  
Ovarian Insufficiency ◽  
Lh Rh ◽  
Dose Dependent Increase ◽  
Dose Dependent

ABSTRACT The effect of synthetic LH-RH was studied in 100 patients with various types of ovarian insufficiency by following up the FSH- and LH-levels in plasma. LH-RH was administered in doses of 12.5, 25 and 100 μg as a rapid intravenous injection. The patients were classified according to the endocrine state of the pituitary as evidenced by the urinary gonadotrophin levels. A clear correlation between the functional state of the pituitary and its responsiveness to exogenous LH-RH was demonstrated. Most of the patients with undetectable low urinary gonadotrophin levels failed to respond. The majority of patients with gonadotrophin excretion in the normal range and those with elevated levels reacted with a dose dependent increase in circulating LH. The amount of liberated FSH however was related to the injected dose only in patients with high gonadotrophic excretion. The present study indicates that synthetic LH-RH provides a useful tool in the evaluation of the pitutiary function particularly in patients with low and with undetectable gonadotrophin excretion. The data presented in this paper also demonstrate that the functional state of the pituitary is clearly reflected by the urinary gonadotrophin levels.

Dual-Function, Light Switchable Cobalt Catalysis via Active Site Metamorphosis

2019 ◽  
Author(s):  
Enrico Bergamaschi ◽  
Frédéric Beltran ◽  
Christopher Teskey
Keyword(s):  
Visible Light ◽  
Active Site ◽  
Catalytic Site ◽  
Dual Function ◽  
Catalytic Systems ◽  
Hydride Complex ◽  
Dual Functional ◽  
Multiple Processes ◽  
Olefin Migration

<p></p><p></p><p>Switchable catalysis offers opportunities to control the rate or selectivity of a reaction <i>via</i> a stimulus such as pH or light. However, few examples of switchable catalytic systems that can facilitate multiple processes exist. Here we report a rare example of such dual-functional, switchable catalysis. Featuring an easily prepared, bench-stable cobalt(I) hydride complex in conjunction with pinacolborane, we can completely alter the reaction outcome between two widely employed transformations – olefin migration and hydroboration – with visible light as the sole trigger. This dichotomy arises from ligand photodissociation which leads to metamorphosis of the active catalytic site, resulting in divergent mechanistic pathways.</p><p></p><p></p>

Evaluation of the Laxative activity of Saponin Enriched Hydroethanolic Pericarp extract of Sapindus emarginatus in Animal models

2020 ◽  
Vol 16 ◽  
Author(s):  
Lalitha Vivekanandan ◽  
Roxanne Gekonge Mandere ◽  
Sivakumar Thangavel
Keyword(s):  
Animal Models ◽  
Gravimetric Analysis ◽  
Triterpene Saponins ◽  
Laxative Effect ◽  
Saponin Content ◽  
The Family ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Efficacious Drug

Background: Constipation is a common, predominant, chronic gastrointestinal functional disorder. The drugs available to treat constipation are limited because of their side effects in long term use. So we need of efficacious drug to treat constipation. Sapindus emarginatus Vahl belongs to the family Sapindaceae, commonly known as soapnut. Traditionally used for the antipruritic, antifertility, constipation, and anti-inflammatory agents. Objective: The present study was undertaken to evaluate the laxative activity of hydroethanolic pericarp extract of Sapindus emarginatus (HESE) in animal models. Methods: The saponin content in extract was measured by gravimetric analysis. The laxative activity of hydroethanolic pericarp extract of Sapindus emarginatus is evaluated by the weight of feces matter, charcoal meal hyperperistalsis test, and loperamide induced constipation model. Results: The saponin content of the soapnut pericarp was 13.48 % and the extract was found to be 11.92 %. The results obtained from these models showed a significant dose-dependent increase in fecal weight, peristalsis index, and moisture content compared to control animals. Conclusion: The present study concluded that the oral administration of HESE showed a significant laxative activity by using different animal models. The presence of triterpene saponins is responsible for this activity. Further studies are needed to confirm their mechanism behind the laxative effect. The administration of extract was found to be a valid candidate in constipation therapy.

Hepatic uptake of intact glutathione S-conjugate, inhibition by organic anions, and sinusoidal catabolism

1993 ◽  
Vol 265 (3) ◽  
pp. G547-G554
Author(s):  
C. A. Hinchman ◽  
A. T. Truong ◽  
N. Ballatori
Keyword(s):  
Guinea Pig ◽  
Rat Liver ◽  
Marked Decrease ◽  
Hepatic Uptake ◽  
Half Time ◽  
High Concentrations ◽  
Rat Livers ◽  
Dose Dependent ◽  
Uptake And Metabolism ◽  
Guinea Pig Liver

To identify potential mechanisms for hepatic removal of circulating glutathione (GSH) conjugates, uptake and metabolism of S-2,4-dinitrophenylglutathione (DNP-SG) were examined in isolated perfused livers from rat and guinea pig. Guinea pig livers perfused with 5 mumol of DNP-SG in a recirculating system (50 microM initial concn) rapidly cleared the conjugate from the perfusate (half time 3.7 min), whereas clearance was considerably slower in rat liver (half time 35 min). Disappearance of DNP-SG from the perfusate was accompanied by a simultaneous appearance of DNP-SG and its metabolites in bile. Addition of acivicin, an inhibitor of gamma-glutamyltransferase (gamma-GT), to the perfusate resulted in a marked decrease in DNP-SG clearance by guinea pig liver but had no effect in rat liver, suggesting that in the guinea pig this process is largely dependent on sinusoidal gamma-GT activity. However, even in the presence of acivicin, rat and guinea pig livers removed nearly one-half of the administered DNP-SG from the recirculating perfusate over 30 min. High concentrations of DNP-SG were found in bile (up to 3.7 mM), indicating that the liver is capable of transporting the intact conjugate from the circulation. When rat livers were perfused with higher concentrations of DNP-SG (100 and 250 microM), biliary excretion of DNP-SG increased dose dependently, with concentrations in bile reaching 10 mM at the higher dose. This was accompanied by a dose-dependent choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

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