scholarly journals Determination of the structure of two novel echistatin variants and comparison of the ability of echistatin variants to inhibit aggregation of platelets from different species

1995 ◽  
Vol 305 (2) ◽  
pp. 513-520 ◽  
Author(s):  
Y L Chen ◽  
T F Huang ◽  
S W Chen ◽  
I H Tsai
Keyword(s):  
Platelet Glycoprotein ◽  
Medium Size ◽  
Recognition Sequence ◽  
High Affinity ◽  
Cnbr Cleavage ◽  
Echis Carinatus ◽  
Fluorescent Spectra ◽  
Methionine Residues ◽  
Aggregation Of Platelets

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD loop, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues flanking both sides of the ARGDDM motif in echistatin gamma, we deleted this hexapeptide by CNBr cleavage to produce des-(23-28)-echistatin gamma. The modified protein showed c.d. and fluorescent spectra grossly similar to the intact echistatin but its antiplatelet potency decreased more than 200-fold. We thus propose that a favourable conformation of the RGD region is responsible mainly for the high-affinity binding of echistatin to the platelet glycoprotein IIb-IIIa as shown previously for the binding of medium-size disintegrin.

Interaction of Echicetin with a High Affinity Thrombin Binding Site on Platelet Glycoprotein GPIb

1995 ◽  
Vol 74 (03) ◽  
pp. 954-957 ◽  
Author(s):  
Manling Peng ◽  
Frances A Emig ◽  
Ahua Mao ◽  
Weiqi Lu ◽  
Edward P Kirby ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Snake Venom ◽  
Platelet Glycoprotein ◽  
High Affinity ◽  
Low Concentrations ◽  
High Affinity Site ◽  
Echis Carinatus

SummaryEchicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin (<0.2 U/ml), by binding to platelet glycoprotein lb (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin (>0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.

A Monoclonal Antibody to the Human Platelet Glycoprotein II b/III a Complex Induces Platelet Activation

1988 ◽  
Vol 60 (01) ◽  
pp. 068-074 ◽  
Author(s):  
Piet W Modderman ◽  
Han G Huisman ◽  
Jan A van Mourik ◽  
Albert E G Kr von dem Borne
Keyword(s):  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Dense Granule ◽  
Platelet Glycoprotein ◽  
Activated Platelets ◽  
Complex Functions ◽  
Slow Aggregation ◽  
Irreversible Aggregation ◽  
Platelet Release ◽  
Aggregation Of Platelets

SummaryThe platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex. Whereas C17 inhibited the binding of fibrinogen to platelets and platelet aggregation induced by adenosine diphosphate (ADP) or collagen, 6C9 caused irreversible aggregation of platelets, both in the presence and absence of extracellular fibrinogen. When incubated with unstirred (nonaggregating) platelets, 6C9 induced release of alpha and dense granule-constituents as well as binding of 125I-fibrinogen to platelets. The latter was evidently mediated in part by platelet-derived ADP, since it was inhibited to a large extent by apyrase, the ADP-hydrolyzing enzyme. F(ab’)2 fragments of 6C9 did not induce platelet-release reactions but caused (slow) aggregation of platelets in the presence of extracellular fibrinogen.These results indicate that binding of an antibody to a specific site on the platelet GPIIb/IIIa complex may cause fibrinogen-mediated aggregation. The Fc part of the platelet-bound antibody appears to be involved in the induction of platelet release.

On The Mechanism Of Heparin-Induced Potentiation Of Platelet Aggregation

1981 ◽  
Author(s):  
M Yamamoto ◽  
K Watanabe ◽  
Y Ando ◽  
H Iri ◽  
N Fujiyama ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Coagulation Factors ◽  
Marked Enhancement ◽  
Matrix Bound ◽  
Induced Aggregation ◽  
Aggregation Of Platelets ◽  
Plasma Heparin

It has been suggested that heparin caused potentiation of aggregation induced by ADP or epinephrine. The exact mechanism of heparin-induced platelet activation, however, remained unknown. In this paper, we have investigated the role of anti-thrombin III ( AT ) in heparin-induced platelet activation using purified AT and AT depleted plasma. When ADP or epinephrine was added to citrated PRP one minute after addition of heparin ( 1 u/ml, porcine intestinal mucosal heparin, Sigma Co. USA ), marked enhancement of platelet aggregation was observed, compared with the degree of aggregation in the absence of heparin. However, in platelet suspensions prepared in modified Tyrode’s solution, heparin exhibited no potentiating effect on platelet aggregation induced by epinephrine or ADP. Potentiation of epinephrine- or ADP-induced platelet aggregation by heparin was demonstrated when purified AT was added to platelet suspensions at a concentration of 20 μg/ml. AT depleted plasma, which was prepared by immunosorption using matrix-bound antibodies to AT, retained no AT, while determination of α1-antitrypsinα2- macroglobulin and fibrinogen in AT depleted plasma produced values which corresponded to those of the original plasma when dilution factor was taken into account. The activities of coagulation factors were also comparable to those of the original plasma. Heparin exhibited potentiating effect on ADP- or epinephrine-induced aggregation of platelets in original plasma, but no effect in AT depleted plasma. When purified AT was added back to AT depleted plasma at a concentration of 20 μg/ml, potentiation of platelet aggregation by heparin was clearly demonstrated.Our results suggest that effect of heparin on platelet aggregation is also mediated by anti-thrombin III.

scholarly journals Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Avital Shushan ◽  
Mickey Kosloff
Keyword(s):  
Protein Interactions ◽  
Structural Elements ◽  
Protein Protein Interactions ◽  
Immunity Protein ◽  
Rational Engineering ◽  
High Affinity ◽  
Comparative Structure ◽  
Polar Interactions ◽  
Exquisite Specificity

AbstractThe interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein–protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1–α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1–α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein–protein interactions, with implications for drug development and rational engineering of these interfaces.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a &gt;200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals Determination of the molecular mass of apolipoprotein B-100 A chemical approach

1986 ◽  
Vol 239 (3) ◽  
pp. 777-780 ◽  
Author(s):  
C Y Yang ◽  
F S Lee ◽  
L Chan ◽  
D A Sparrow ◽  
J T Sparrow ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Apolipoprotein B ◽  
Cell Surface Receptor ◽  
Specific Cell ◽  
Apo B ◽  
Chemical Approach ◽  
Surface Receptor ◽  
Cnbr Cleavage ◽  
Specific Cell Surface

Apolipoprotein B-100 (apo B-100) is the protein ligand in low-density lipoproteins that binds to a specific cell-surface receptor. Its molecular mass has been a subject of controversy. We have determined the molecular mass of the protein by a chemical approach. After complete CNBr cleavage, the C-terminal fragment of apo B-100 was purified by reverse-phase h.p.l.c. Amino acid N- and C-terminal analyses confirm that this peptide represents the C-terminal peptide as deduced from the DNA sequence of a human apo B-100 cDNA clone. A chemically synthesized peptide was used to determine the recovery of the peptide (74.72%). On the basis of these data, the molecular mass of apo B-100 was determined to be 496.82 +/- 24.84 kDa.

scholarly journals Hemoglobin Non-equilibrium Oxygen Dissociation Curve

2020 ◽  
Author(s):  
Rosella Scrima ◽  
Sabino Fugetto ◽  
Nazzareno Capitanio ◽  
Domenico L. Gatti
Keyword(s):  
Partial Pressure ◽  
Correct Diagnosis ◽  
Oxygen Affinity ◽  
Oxygen Dissociation Curve ◽  
Dissociation Curve ◽  
High Affinity ◽  
Oxygen Dissociation ◽  
Sequential Binding ◽  
Non Equilibrium

AbstractAbnormal hemoglobins can have major consequences for tissue delivery of oxygen. Correct diagnosis of hemoglobinopathies with altered oxygen affinity requires a determination of hemoglobin oxygen dissociation curve (ODC), which relates the hemoglobin oxygen saturation to the partial pressure of oxygen in the blood. Determination of the ODC of human hemoglobin is typically carried out under conditions in which hemoglobin is in equilibrium with O2 at each partial pressure. However, in the human body due to the fast transit of RBCs through tissues hemoglobin oxygen exchanges occur under non-equilibrium conditions. We describe the determination of non-equilibrium ODC, and show that under these conditions Hb cooperativity has two apparent components in the Adair, Perutz, and MWC models of Hb. The first component, which we call sequential cooperativity, accounts for ∼70% of Hb cooperativity, and emerges from the constraint of sequential binding that is shared by the three models. The second component, which we call conformational cooperativity, accounts for ∼30% of Hb cooperativity, and is due either to a conformational equilibrium between low affinity and high affinity tetramers (as in the MWC model), or to a conformational change from low to high affinity once two of the tetramer sites are occupied (Perutz model).

scholarly journals Clay-Based Affinity Probes for Selective Cleanup and Determination of Aflatoxin B1 Using Nanostructured Montmorillonite on Quartz

2003 ◽  
Vol 86 (3) ◽  
pp. 534-539 ◽  
Author(s):  
Henry J Huebner ◽  
Timothy D Phillips
Keyword(s):  
Aflatoxin B1 ◽  
Composite Media ◽  
Enthalpy Of Adsorption ◽  
Composite Columns ◽  
High Affinity ◽  
Composite Adsorbents ◽  
Immunoaffinity Columns ◽  
Affinity Probes ◽  
Linear Trends

Abstract A study was conducted to investigate the selective cleanup and determination of aflatoxin B1 (AfB1) from contaminated media. Composite adsorbents were formulated from calcium montmorillonite clay, which possesses a high affinity and enthalpy of adsorption for AfB1. Nanostructuring techniques were used to construct various formulations of the clay-based composite media. In AfB1 adsorption studies with prototypical affinity columns, these composites offered narrowly defined, reproducible capacity ranges. Composite recoveries of AfB1 from spiked grains exhibited linear trends that correlated well with the range of spike levels. Composite columns provided lower recoveries of AfB1 from naturally contaminated corn than did immunoaffinity columns; however, recoveries were consistent and purified extracts were free of inter-fering compounds, as determined by liquid chro-matography with fluorescence detection.

scholarly journals New perspective in the assessment of total intracellular magnesium

2014 ◽  
Vol 87 (1) ◽  
Author(s):  
Azzurra Sargenti ◽  
Lucia Merolle ◽  
Giulia Andreani ◽  
Concettina Cappadone ◽  
Giovanna Farruggia ◽  
...  
Keyword(s):  
Quantitative Determination ◽  
Atomic Absorption Spectroscopy ◽  
Biological Processes ◽  
Intracellular Magnesium ◽  
Qualitative And Quantitative ◽  
High Affinity ◽  
New Family ◽  
New Perspective ◽  
Good Agreement

Magnesium (Mg) is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ), that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

DETERMINATION OF SOLUBLE FIBRIN IN PLASMA WITH A CHR0M0GENIC KIT METHOD UTILIZING THE HIGH AFFINITY PLASMIN SUBSTRATE S-2390

1987 ◽  
Author(s):  
E Ersdel ◽  
M Andersson ◽  
S Rosen
Keyword(s):  
Reagent Blank ◽  
Soluble Fibrin ◽  
Standard Curve ◽  
High Affinity ◽  
Absorbance Unit ◽  
Assay Procedure ◽  
Bovine Plasma ◽  
Fresh Frozen ◽  
Reliable Differentiation

A sensitive and quantitative assay of soluble fibrin is of clinically diagnostic relevance in an early thrombotic state where there is a risk for development of DIC. Recently Wiman and Ranby (Thromb. Haemostas 55, 189-193 (1986)) published a spectro-photometric assay which met these criterions. The single-stage assay procedure is based upon activation of Glu-Plasminogen to Plasmin by t-PA in the presence of soluble fibrin and hydrolysis of the chromogenic plasmin substrate S-2390, H-D-Val-Phe-Lys-pNA, which has a high affinity for plasmin. The rate of plasmin generation is correlated to the amount of soluble fibrin monomers present in the sample.A complete kit containing optimized, stable reagents has now been developed which allows a quantitative determination of soluble fibrin in the range 30-200 nmol/1 within 30 min. at room temperature (20-25°C). The assay procedure is straightforward involving addition of 200 pi diluted plasma sample to 200 pi Glu-Plasminogen and 100 ul of a t-PA/S-2390-reagent.The results show a high resolution of the standard curve as illustrated by a AA405 amounting to about one absorbance unit between a 200 nmol/1 sample of soluble fibrin and the reagent blank, some variation, ±0.1 absorbance unit, being caused mainly by differences in temperature. In combination with an intra-assay variation coefficient = 6.3% and 5.0% at 150 and 50 nmol/1, respectively, this will allow safe and reliable differentiation of pathological levels of soluble fibrin from levels found in healthy subjects (below 10 nmol/1). A similar precision is also obtained when the assay is performed in microplates.In the original procedure fresh frozen human plasma was utilized as a dilution medium for soluble fibrin. Comparisons with carefully collected bovine plasma proved this source to be a convenient substitute. Furthermore, lyophilization of the bovine plasma did not produce any significant degradation of fibrinogen which otherwise might interfere in the assay. This simple kit procedure should make it a suitable tool in early determinations of soluble fibrin in a number of pathological states which may result in severe haemostatic disturbances.

Sign in / Sign up

Export Citation Format

Share Document