Plasminogen-Plasmin System IX. Specific Binding of Tranexamic Acid to Plasmin

1975 ◽  
Vol 33 (03) ◽  
pp. 573-585 ◽  
Author(s):  
Masahiro Iwamoto
Keyword(s):  
Tranexamic Acid ◽  
Affinity Chromatography ◽  
Dissociation Constant ◽  
Factor Xiii ◽  
Specific Binding ◽  
The Other ◽  
Inhibitory Mechanism ◽  
Binding Studies ◽  
Similar Affinity ◽  
Fibrin Structure

SummaryInteractions between tranexamic acid and protein were studied in respect of the antifibrinolytic actions of tranexamic acid. Tranexamic acid did neither show any interaction with fibrinogen or fibrin, nor was incorporated into cross-linked fibrin structure by the action of factor XIII. On the other hand, tranexamic acid bound to human plasmin with a dissociation constant of 3.5 × 10−5 M, which was very close to the inhibition constant (3.6 × 10−5 M) for this compound in inhibiting plasmin-induced fibrinolysis. The binding site of tranexamic acid on plasmin was not the catalytic site of plasmin, because TLCK-blocked plasmin also showed a similar affinity to tranexamic acid (the dissociation constant, 2.9–4.8 × 10−5 M).In the binding studies with the highly purified plasminogen and TLCK-plasmin preparations which were obtained by affinity chromatography on lysine-substituted Sepharose, the molar binding ratio was shown to be 1.5–1.6 moles tranexamic acid per one mole protein.On the basis of these and other findings, a model for the inhibitory mechanism of tranexamic acid is presented.

scholarly journals Dissociation between the Effects of Somatostatin (SS) and Octapeptide SS-Analogs on Hormone Release in a Small Subgroup of Pituitary- and Islet Cell Tumors

1997 ◽  
Vol 82 (9) ◽  
pp. 3011-3018 ◽  
Author(s):  
L. J. Hofland ◽  
W. W. de Herder ◽  
H. A. Visser-Wisselaar ◽  
C. van Uffelen ◽  
M. Waaijers ◽  
...  
Keyword(s):  
Specific Binding ◽  
Somatostatin Receptor ◽  
Primary Cultures ◽  
Inhibitory Effect ◽  
The Other ◽  
Receptor Subtype ◽  
Hormone Release ◽  
Binding Studies ◽  
Α Subunit ◽  
Small Subgroup

Abstract The effects of somatostatin (SS-14 and/or SS-28) and of the three octapeptide SS-analogs that are available for clinical use (octreotide, BIM-23014 and RC-160) on hormone release by primary cultures of 15 clinically nonfunctioning pituitary adenomas (NFA), 7 prolactinomas, and 2 insulinomas were investigated. In the pituitary adenoma cultures, a comparison was made with the effects of the dopamine (DA) agonists bromocriptine and/or quinagolide. In 5 NFAs, 2 prolactinomas and 1 insulinoma somatostatin receptor (subtype) expression was determined by ligand binding studies and by in situ hybridization to detect sst1, sst2, and sst3 messenger RNAs (mRNAs). Four NFA cultures did not secrete detectable amounts of α-subunit, FSH, and/or LH. In the other cultures, hormone and/or subunit release was inhibited by DA-agonists (10 nm) in 9 of 11, by SS (10 nm) in 7 of 11, and by octapeptide SS-analogs (10 nm) in 3 of 10 cultures. In three NFA cultures, hormone release was sensitive to SS but not to SS-analogs. In all cultures, except for one, DA-agonists were the most effective in inhibiting hormone release. In the prolactinoma cultures, PRL release was inhibited by DA-agonists (10 nm) in 7 of 7, by SS in 4 of 4, and by octapeptide SS-analogs in 3 of 7 cultures. A dissociation between the effects of SS and SS-analogs was found in 3 cases. In the cultures sensitive to both bromocriptine and SS-28, bromocriptine was the most potent compound in 2 out of 4 cultures. In the 2 other cultures, both compounds were equally effective. In 2 insulinoma cultures, insulin release was inhibited by SS, and by octapeptide SS-analogs in only one. The presence or absence of an inhibitory effect by octreotide was in all cases in parallel with the presence or absence of the inhibitory effect by BIM-23014 and RC-160. Autoradiographic studies using[ 125I-Tyr0]SS28 showed specific binding in 4 of 5 NFAs, 1 of 2 prolactinomas, and 1 of 1 insulinoma. Specific[ 125I-Tyr3]octreotide binding was found in 2 of 5 NFAs, in 1 of 2 prolactinomas, and in the insulinoma. Two NFAs showed binding of SS28, but not of the sst2,5 specific ligand octreotide. The tumors showed variable sst1 and/or sst3 mRNA expression, whereas no sst2 expression was found. In conclusion, a dissociation between the inhibitory effects of SS on the one hand and of the octapeptide SS-analogs octreotide, BIM-23014 and RC-160 on the other hand, is observed in a small subgroup of NFAs, prolactinomas, and insulinomas, suggesting that novel sst subtype specific SS-analogs might be of benefit in the treatment of selected patients with somatostatin receptor positive secreting tumors not responding to octapeptide SS-analogs. However, in the majority of NFAs and prolactinomas, DA-agonists were equally or more effective than SS in the suppression of tumoral secretion products.

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

Determination of Factor XIII Activity and of Factor XIII Inhibitors Using an Ammonium-Sensitive Electrode

1983 ◽  
Vol 50 (02) ◽  
pp. 563-566 ◽  
Author(s):  
P Hellstern ◽  
K Schilz ◽  
G von Blohn ◽  
E Wenzel
Keyword(s):  
Factor Xiii ◽  
Normal Subjects ◽  
The Other ◽  
Activity Measurement ◽  
Factor Xiii Deficiency ◽  
Assay Procedure ◽  
Stabilization Factor ◽  
Release Method ◽  
Sensitive Electrode

SummaryAn assay for rapid factor XIII activity measurement has been developed based on the determination of the ammonium released during fibrin stabilization. Factor XIII was activated by thrombin and calcium. Ammonium was measured by an ammonium-sensitive electrode. It was demonstrated that the assay procedure yields accurate and precise results and that factor XIII-catalyzed fibrin stabilization can be measured kinetically. The amount of ammonium released during the first 90 min of fibrin stabilization was found to be 7.8 ± 0.5 moles per mole fibrinogen, which is in agreement with the findings of other authors. In 15 normal subjects and in 15 patients suffering from diseases with suspected factor XIII deficiency there was a satisfactory correlation between the results obtained by the “ammonium-release-method”, Bohn’s method, and the immunological assay (r1 = 0.65; r2= 0.70; p<0.01). In 3 of 5 patients with paraproteinemias the values of factor XIII activity determined by the ammonium-release method were markedly lower than those estimated by the other methods. It could be shown that inhibitor mechanisms were responsible for these discrepancies.

scholarly journals Ultrafiltration Method for Plasma Protein Binding Studies and Its Limitations

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 382
Author(s):  
Camelia-Maria Toma ◽  
Silvia Imre ◽  
Camil-Eugen Vari ◽  
Daniela-Lucia Muntean ◽  
Amelia Tero-Vescan
Keyword(s):  
Protein Binding ◽  
Plasma Protein Binding ◽  
Plasma Protein ◽  
Specific Binding ◽  
Critical Role ◽  
Volume Ratio ◽  
Binding Studies ◽  
Experimental Conditions ◽  
Key Aspects

Plasma protein binding plays a critical role in drug therapy, being a key part in the characterization of any compound. Among other methods, this process is largely studied by ultrafiltration based on its advantages. However, the method also has some limitations that could negatively influence the experimental results. The aim of this study was to underline key aspects regarding the limitations of the ultrafiltration method, and the potential ways to overcome them. The main limitations are given by the non-specific binding of the substances, the effect of the volume ratio obtained, and the need of a rigorous control of the experimental conditions, especially pH and temperature. This review presents a variety of methods that can hypothetically reduce the limitations, and concludes that ultrafiltration remains a reliable method for the study of protein binding. However, the methodology of the study should be carefully chosen.

scholarly journals Preclinical Evaluation of [18F]FACH in Healthy Mice and Piglets: An 18F-Labeled Ligand for Imaging of Monocarboxylate Transporters with PET

2021 ◽  
Vol 22 (4) ◽  
pp. 1645
Author(s):  
Daniel Gündel ◽  
Masoud Sadeghzadeh ◽  
Winnie Deuther-Conrad ◽  
Barbara Wenzel ◽  
Paul Cumming ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Ex Vivo ◽  
Specific Binding ◽  
Monocarboxylate Transporters ◽  
Binding Potential ◽  
Kidney Cortex ◽  
Binding Studies ◽  
Imaging Tool ◽  
Pre Treatment

The expression of monocarboxylate transporters (MCTs) is linked to pathophysiological changes in diseases, including cancer, such that MCTs could potentially serve as diagnostic markers or therapeutic targets. We recently developed [18F]FACH as a radiotracer for non-invasive molecular imaging of MCTs by positron emission tomography (PET). The aim of this study was to evaluate further the specificity, metabolic stability, and pharmacokinetics of [18F]FACH in healthy mice and piglets. We measured the [18F]FACH plasma protein binding fractions in mice and piglets and the specific binding in cryosections of murine kidney and lung. The biodistribution of [18F]FACH was evaluated by tissue sampling ex vivo and by dynamic PET/MRI in vivo, with and without pre-treatment by the MCT inhibitor α-CCA-Na or the reference compound, FACH-Na. Additionally, we performed compartmental modelling of the PET signal in kidney cortex and liver. Saturation binding studies in kidney cortex cryosections indicated a KD of 118 ± 12 nM and Bmax of 6.0 pmol/mg wet weight. The specificity of [18F]FACH uptake in the kidney cortex was confirmed in vivo by reductions in AUC0–60min after pre-treatment with α-CCA-Na in mice (−47%) and in piglets (−66%). [18F]FACH was metabolically stable in mouse, but polar radio-metabolites were present in plasma and tissues of piglets. The [18F]FACH binding potential (BPND) in the kidney cortex was approximately 1.3 in mice. The MCT1 specificity of [18F]FACH uptake was confirmed by displacement studies in 4T1 cells. [18F]FACH has suitable properties for the detection of the MCTs in kidney, and thus has potential as a molecular imaging tool for MCT-related pathologies, which should next be assessed in relevant disease models.

Structure, function and brain localization of neurotransmitter transporters.

1994 ◽  
Vol 196 (1) ◽  
pp. 283-295 ◽  
Author(s):  
F Jursky ◽  
S Tamura ◽  
A Tamura ◽  
S Mandiyan ◽  
H Nelson ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Specific Binding ◽  
Site Directed Mutagenesis ◽  
Binding Studies ◽  
Inhibitory System ◽  
Cytochemical Localization ◽  
Gaba Transporters ◽  
Glycine Transporter ◽  
Neurotransmitter Transporters ◽  
Glycine Transporters

We studied four different cDNAs encoding GABA transporters and three different cDNAs encoding glycine transporters in mouse and rat brains. A genomic clone of two of the glycine transporters (GLYT1a and GLYT1b) revealed that they derive from differential splicing of a single gene. The third glycine transporter (GLYT2) is encoded by a separate gene. Antibodies were raised against seven of these neurotransmitter transporters and their cytochemical localization in the mouse brain was studied. In general, we observed a deviation from the classical separation of neuronal and glial transporters. It seems that each of the neurotransmitter transporters is present in specific places in the brain and is expressed in a different way in very specific areas. For example, the GABA transporter GAT4, which also transports beta-alanine, was localized to neurons. However, GAT1, which is specific for GABA, was localized not only to neurons but also to glial cells. The recently discovered glycine transporter GLYT2 was of particular interest because of its deviation from the general structure by a very extended N terminus containing multiple potential phosphorylation sites. Western analysis and immunocytochemistry in frozen sections of mouse brain demonstrated a clear caudal-rostral gradient of GLYT2 distribution, with massive accumulation in the spinal cord and brainstem and less in the cerebellum. Its distribution is typically neuronal and it is present in processes with varicosities. A correlation as observed between the pattern we obtained and that observed previously from strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission at the classical inhibitory system in the hindbrain. The availability of four different GABA transporters made it possible to look for specific binding sites upon the neurotransmitter transporters. An extensive program of site-directed mutagenesis led us to identify a potential neurotransmitter binding site on the GABA transporters.

The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors

1993 ◽  
Vol 13 (8) ◽  
pp. 4494-4504
Author(s):  
D Nitsch ◽  
G Schütz
Keyword(s):  
Thymidine Kinase ◽  
Specific Binding ◽  
Regulatory Element ◽  
Hepatoma Cell ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Distal Enhancer ◽  
Binding Studies ◽  
Enhancer Sequences

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

Pneumococcal phosphoglycerate kinase interacts with plasminogen and its tissue activator

2014 ◽  
Vol 111 (03) ◽  
pp. 401-416 ◽  
Author(s):  
Manfred Rohde ◽  
Nadine Nachtigall ◽  
Ronald Frank ◽  
Klaus Preissner ◽  
Javier Klett ◽  
...  
Keyword(s):  
Specific Binding ◽  
Phosphoglycerate Kinase ◽  
Analysis Data ◽  
Glycolytic Enzyme ◽  
Potential Interaction ◽  
Plasmin Activity ◽  
Binding Studies ◽  
Kringle Domains ◽  
Life Threatening ◽  
Affinity Interaction

SummaryStreptococcus pneumoniae is not only a commensal of the nasopharyngeal epithelium, but may also cause life-threatening diseases. Immune-electron microscopy studies revealed that the bacterial glycolytic enzyme, phosphoglycerate kinase (PGK), is localised on the pneumococcal surface of both capsulated and non-capsulated strains and colocalises with plasminogen. Since pneumococci may concentrate host plasminogen (PLG) together with its activators on the bacterial cell surface to facilitate the formation of plasmin, the involvement of PGK in this process was studied. Specific binding of human or murine PLG to strain-independent PGK was documented, and surface plasmon resonance analyses indicated a high affinity interaction with the kringle domains 1–4 of PLG. Crystal structure determination of pneumococcal PGK together with peptide array analysis revealed localisation of PLG-binding site in the N-terminal region and provided structural motifs for the interaction with PLG. Based on structural analysis data, a potential interaction of PGK with tissue plasminogen activator (tPA) was proposed and experimentally confirmed by binding studies, plasmin activity assays and thrombus degradation analyses.

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

2020 ◽  
Author(s):  
Cecy Xi ◽  
Arianna Arianna Di Fazio ◽  
Naveed Nadvi ◽  
Karishma Patel ◽  
Michelle Xiang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Specific Activity ◽  
Specific Binding ◽  
Exchange Chromatography ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure ◽  
Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity &gt;30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

scholarly journals Rationale design and synthesis of some novel imidazole linked thiazolidinone hybrid molecules as DNA minor groove binders

2020 ◽  
Vol 11 (2) ◽  
pp. 120-132
Author(s):  
Javeed Ahmad War ◽  
Santosh Kumar Srivastava
Keyword(s):  
Molecular Docking ◽  
Binding Affinity ◽  
Specific Binding ◽  
Minor Groove ◽  
Stable Complex ◽  
Binding Studies ◽  
In Vitro Susceptibility ◽  
Reference Drug ◽  
Hybrid Molecules

A new series of imidazole linked thiazolidinone hybrid molecules was designed and subsequently synthesized through a feasible, three step reaction protocol. The structures of these molecules were established using FT-IR, 1H NMR, 13C NMR and HRMS techniques. In vitro susceptibility tests against some Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram negative bacteria (Escherichia coli and Pseudomonas aeruginosa) exhibited broad spectrum potency of the molecules. The most potent molecule (S2A7) amongst the screened molecules, showed minimum inhibitory concentration (MIC) value not less than 2.0 µg/mL which was at par with the reference drug Streptomycin. Structure activity relationships revealed nitro and chloro groups being crucial for bioactivity when present at meta position of arylidene ring in 3-(3-(imidazol-1-yl)propyl)-5-(benzylidene)-2-(phenylimino)thiazolidin-4-one. Deoxyribonucleic acid (DNA)and bovine serum albumin (BSA) binding studies for S2A7 under simulated physiological pH were probed using UV-Visible, fluorescence quenching, gel electrophoresis and molecular docking techniques. These studies established that S2A7 has strong binding affinity towards DNA and binds at the minor groove of DNA with binding constant (Kb) of 0.1287×102 L/mol. Molecular docking simulations of S2A7 with DNA and BSA predicted binding affinity of -9.2 and -7.2 kcal/mol, respectively. Van der Waals forces and hydrogen bonding interactions were predicted as the main forces of interaction. With DNA, S2A7 exhibited specific binding affinity towards adenine-thiamine base pairs. The compound S2A7 forms a stable complex with BSA by binding at subdomain IIIA implying high bio-distribution of the compound.

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