scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals Identification of high-affinity (Kd 0.35 mumol/L) and low-affinity (Kd 7.9 mumol/L) platelet binding sites for ADP and competition by ADP analogues

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 110-116 ◽  
Author(s):  
JR Jefferson ◽  
JT Harmon ◽  
GA Jamieson
Keyword(s):  
Platelet Activation ◽  
Dissociation Constants ◽  
Partial Agonist ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Ionophore A23187 ◽  
High Affinity ◽  
Binding Isotherms ◽  
High Affinity Site ◽  
High Concentrations

Abstract Steady-state binding of ADP to blood platelets and isolated membranes has not previously been obtained because of complications arising from metabolism of the ligand and dilution due to its secretion from storage granules. In the present studies, competition binding isotherms (n = 9) using paraformaldehyde-fixed platelets showed that [2–3 H]ADP bound to two sites with a small amount (approximately 5% of total) of nonspecific binding: 410,000 +/- 40,000 sites of low affinity (Kd 7.9 +/- 2.0 mumol/L) and 160,000 +/- 20,000 sites of high affinity (Kd 0.35 +/- 0.04 mumol/L) corresponding to the ADP concentration required for activation in fresh platelets (0.1–0.5 mumol/L). All agonists and antagonists examined were able to compete with ADP at the high-affinity site. The strong platelet agonists 2-methylthio ADP and 2-(3- aminopropylthio)ADP competed with ADP at the high-affinity site with dissociation constant values of 7 mumol/L and 200 mumol/L, respectively. The partial agonist 2′,3′-dialdehyde ADP and the weak agonist GDP also competed at the high-affinity site with Kd values of 5 mumol/L and 49 mumol/L, respectively. The sequence of binding affinities of other adenine nucleotides at the high-affinity site corresponded to their relative activities as known antagonists of platelet activation by ADP; namely, ADP(Kd 0.35 mumol/L) approximately equal to ATP (Kd 0.45 mumol/L) much greater than AMP (Kd 360 mumol/L). Adenosine and 2-chloroadenosine did not compete with ADP. ADP binding to the high-affinity site was inhibited by p-mercuribenzene sulfonate (Ki 250 mumol/L) but only very weakly by 5′-p- fluorosulfonylbenzoyladenosine (Ki 1 mmol/L). All the above nucleotides also competed with ADP at the low-affinity sites but, because of the high concentrations of competing nucleotide required, dissociation constants at this site were obtained only for ATP (21 mumol/L), 2-MeS ADP (200 mumol/L) and 2′,3′-dialdehyde ADP (270 mumol/L). 8-Bromo ADP competed strongly with ADP at the high-affinity site (Kd 0.40 mumol/L) but weakly if at all at the low-affinity site. 8-Bromo ADP inhibited platelet activation induced by ADP (EC50 approximately 100 mumol/L) but not by collagen, thrombin, or ionophore A23187.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals Phencyclidine binds to blood platelets with high affinity and specifically inhibits their activation by adrenaline

1992 ◽  
Vol 285 (1) ◽  
pp. 35-39 ◽  
Author(s):  
G A Jamieson ◽  
A K Agrawal ◽  
N J Greco ◽  
T E Tenner ◽  
G D Jones ◽  
...  
Keyword(s):  
Ion Channel ◽  
Platelet Activation ◽  
Blood Platelets ◽  
Ultrastructural Changes ◽  
Affinity Binding ◽  
Ionophore A23187 ◽  
High Affinity Binding ◽  
High Affinity ◽  
Affinity Probe ◽  
Na Ions

The ion channel probe phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] selectively inhibited aggregation, secretion and ultrastructural changes in platelets induced by adrenaline, but did not affect activation induced by other common platelet agonists such as alpha-thrombin, ADP, collagen or ionophore A23187. [3H]PCP bound to platelets with high affinity (Kd 134 +/- 33 nM; 3600 +/- 1020 sites/platelet), as did the thienyl analogue [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine). PCP binding to platelets was increased 3-4-fold in N-methylglucamine buffer in the absence of Na+ ions. Binding was unaffected by haloperidol and was only weakly inhibited (EC50 10-20 microM), without significant stereoselectivity by the two sets of stereoselective ligands, dexoxadrol/levoxadrol and (+)MK801/(-)MK801. Binding of PCP was not competed for by adrenaline or yohimbine. Only the high-affinity binding of [3H]PCP to platelets was blocked by prior treatment of the platelets with the covalent affinity probe Metaphit, and these platelets no longer aggregated in response to adrenaline although they responded normally to alpha-thrombin, ADP and collagen. These results suggest that platelets contain high-affinity receptors for PCP that can modulate adrenaline-induced platelet activation.

scholarly journals Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein)

1996 ◽  
Vol 318 (3) ◽  
pp. 923-929 ◽  
Author(s):  
Engelbert BUXBAUM ◽  
Philip G WOODMAN
Keyword(s):  
Heat Shock ◽  
Temperature Dependence ◽  
Mung Bean ◽  
Dissociation Constants ◽  
High Affinity ◽  
Pig Brain ◽  
High Affinity Site ◽  
Heat Shock Cognate Protein ◽  
Atp Analogues ◽  
Cognate Protein

Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1±0.1 µM and 1.4±0.7 mM respectively at 25 °C. This corresponds to ΔG0´ = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, ΔH0´ was calculated to -36±2 kJ/mol. This gives ΔS0´ = 6.7 J/mol per K. Adenosine 5´-[γ-thio]triphosphate, ADP, adenosine 5´-[β,γ-imino]triphosphate and adenosine 5´-[β,γ-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 µM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.

Dihydropyridine and ryanodine binding in ventricles from rat, trout, dogfish and hagfish.

1996 ◽  
Vol 199 (9) ◽  
pp. 1999-2009
Author(s):  
M J Thomas ◽  
B N Hamman ◽  
G F Tibbits
Keyword(s):  
Dissociation Constants ◽  
Indirect Evidence ◽  
Active Species ◽  
Apparent Difference ◽  
Release Channel ◽  
Quantitative Expression ◽  
High Affinity ◽  
Lower Vertebrates ◽  
High Affinity Site ◽  
Elasmobranch Species

In the adult mammalian heart, the majority of Ca2+ required for contraction is released from the sarcoplasmic reticulum (SR) via the Ca2+-release channel or ryanodine receptor (RyR). Such release is dependent upon a relatively small influx of Ca2+ entering the cell across the sarcolemma (SL) by means of the L-type Ca2+ channel or the dihydropyridine receptor (DHPR). In lower vertebrates, there is indirect evidence suggesting that Ca2+ influx across the SL may be sufficient to support contraction in the absence of Ca2+ release from the SR. This apparent difference in myocardial excitation-contraction (E-C) coupling was investigated further by determining DHPR and RyR densities in ventricular homogenate preparations from rat, trout, dogfish and hagfish. DHPR Bmax values (means +/- S.E.M.) were highest in rat (0.30 +/- 0.01 pmol mg-1), lower in trout (0.16 +/- 0.01 pmol mg-1) and dogfish (0.27 +/- 0.03 pmol mg-1), and slightly above the level of detection in hagfish (0.03 +/- 0.01 pmol mg-1). The DHPR dissociation constants (Kd) of 40-70 pmoll-1 in these three species were of similar magnitude. RyR binding revealed both high- and low-affinity sites in all species. RyR Bmax for the high-affinity site was greatest in the rat (0.68 pmol mg-1), lower in trout (0.19 pmol mg-1) and dogfish (0.07 pmol mg-1) and lowest in hagfish (0.01 pmol mg-1). The RyR Kd1 values for the high-affinity sites were comparable in all preparations (range 12-87 nmoll-1). The quantitative expression of RyRs in these species is consistent with the relative amount of SR present as indicated in physiological experiments and electron micrographs. Taking into consideration myocyte morphology of teleost and elasmobranch species, the data are consistent with a greater reliance on Ca2+ influx across the SL during E-C coupling in lower vertebrates, although a functional role for Ca2+ release from the SR in the more active species await further investigation.

Glucagon binding to hepatocytes isolated from two teleost fishes, the American eel and the brown bullhead

1994 ◽  
Vol 140 (2) ◽  
pp. 217-227 ◽  
Author(s):  
I Navarro ◽  
T W Moon
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Insulin Binding ◽  
American Eel ◽  
Brown Bullhead ◽  
Teleost Species ◽  
High Affinity ◽  
Glucagon Like Peptide 1 ◽  
High Concentrations

Abstract We have characterized the specific binding of glucagon in hepatocytes isolated from two teleost species, the American eel (Anguilla rostrata) and the brown bullhead (Ictalurus nebulosus). Specific glucagon binding was 9·3 and 10·7% in bullhead and eel hepatocytes respectively, after a 2-h incubation at 12 °C. Curvilinear Scatchard plots suggest the presence of two classes of binding sites with apparent dissociation constants (Kd) of 1·97 nm (high affinity) and 17·3 nm (low affinity) for bullhead and 2·68 and 22·9 nm for eel cells. The number of high-affinity binding sites per cell was significantly higher in the eel (10 413) than in the bullhead (3811). The number of high-affinity insulin-binding sites was approximately two times higher than that for glucagon in bullheads and the opposite in the eel hepatocytes. In competition experiments, insulin did not displace 125I-labelled glucagon binding in the hepatocytes of either species, while glucagon-like peptide-1(7–37) (GLP-1) displaced glucagon but only at high concentrations, suggesting separate glucagon- and GLP-1-binding sites. The rate of dissociation of hepatocyte-bound 125I-labelled glucagon was similar for both species. Preincubation of hepatocytes in 100 nm glucagon decreased the number of high-affinity glucagon-binding sites by approximately 55% in both species, while the Kd values remained unchanged. Glucagon bound to the cell surface is internalized by fish hepatocytes. These properties indicate that the glucagon binding to hepatocytes of these two teleost species is similar to that reported for mammalian hepatocytes. Journal of Endocrinology (1994) 140, 217–227

scholarly journals Effect of Mg2+ on the binding of oxytocin to sheep myometrial cells

1991 ◽  
Vol 277 (1) ◽  
pp. 97-101 ◽  
Author(s):  
V Pliška ◽  
H Kohlhauf Albertin
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Binding Capacity ◽  
Hill Coefficient ◽  
Receptor Interaction ◽  
Short Term ◽  
Free Concentration ◽  
Myometrial Cells ◽  
High Affinity ◽  
Binding Isotherms

Binding isotherms of oxytocin to sheep myometrial cells in a short-term cell culture were investigated in the presence of Mg2+ in the concentration range 0-10 mM. The occurrence of at least three binding sites had been demonstrated earlier. Mg2+ influences individual sites and individual features of the binding isotherm differently. Dissociation constants (Kd) of the high- and medium-affinity sites attain a minimum of 6.8 x 10(-10) and 4.2 x 10(-8) mol/l respectively at 2.75 mM-Mg2+. The two sites display the highest binding capacity (B) at 1 mM-Mg2+ (ratio of high affinity/medium affinity is 1:16). The B/Kd quotients reflecting relative binding (bound-to-free concentration) at the half-saturation of binding sites also have their maxima at 1 mM-Mg2+. The high-affinity site displays a strong positive co-operativity (Hill coefficient at 4 mM-Mg2+ of 2.4), which is amplified in the presence of Mg2+. Positive co-operativity of the medium-affinity site is markedly lower (Hill coefficient at 4 mM-Mg2+ of 1.5) and shows less dramatic Mg(2+)-dependence. Low-affinity sites are not co-operative at any Mg2+ concentration. It is concluded that Mg2+ may display its effect upon the oxytocin-receptor interaction predominantly by influencing positive co-operativity.

scholarly journals Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes

1979 ◽  
Vol 181 (2) ◽  
pp. 309-320 ◽  
Author(s):  
N G Oikonomakos ◽  
T G Sotiroudis ◽  
A E Evangelopoulos
Keyword(s):  
Binding Sites ◽  
Dissociation Constants ◽  
Protein Molecule ◽  
Optical Probe ◽  
Rabbit Muscle ◽  
High Affinity ◽  
High Affinity Site ◽  
Dye Absorption ◽  
Competitive Model ◽  
The Difference

The interactions of rabbit muscle glycogen phosphorylase b with Eosin (2′,4′,5′,7′-tetrabromofluorescein) was studied. Eosin was found to be an effective inhibitor of the enzyme. The inhibition constants for the dye were estimated to be approx. 36 and 60 microM with respect to AMP and glucose 1-phosphate respectively. The binding of Eosin to phosphorylase b is accompanied by a red-shift of about 12 nm in the dye absorption-spectrum maximum, indicating low-polarity binding sites on the enzyme molecule for the dye. The absorbance in the difference absorption maximum at 537 nm was utilized to follow the conjugation of phosphorylase b with Eosin. Scatchard plots of the titration data revealed the existence of at least two classes of binding sites on the protein molecule for Eosin, and the dissociation constants measured in Tris/HCl buffer, pH 7.0 (IO.091), were 7.7 and 41.7 microM respectively. The influence of the substrates and effectors on Eosin-enzymes complexes was used to study the ligand-phosphorylase b interactions. IMP displaced the dye completely from the enzyme, indicating that there are two IMP-binding sites per phosphorylase b monomer. AMP binding to the enzyme with respect to Eosin concentration is of two types: a non-competitive one for the high-affinity site for AMP and a competitive one for the low-affinity site for the activator. The effects of glucose 6-phosphate, ATP, Pi and glycerol 2-phosphate in the system are in according dance with a partially competitive model. Glucoes 1-phosphate and UDP-glucose appear to affect only the high-affinity site for Eosin, whereas glucose and glycogen have no effect on Eosin-phosphorylase b complexes. Our results suggest that Eosin can be used as an efficient optical probe for studying the phosphorylase b system.

Nuclear magnetic resonance studies of blood platelets

1980 ◽  
Vol 289 (1037) ◽  
pp. 413-423 ◽  
Keyword(s):  
Molecular Weight ◽  
Metal Ions ◽  
High Molecular Weight ◽  
Human Platelet ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Biological Significance ◽  
Divalent Metal Ions ◽  
Motional State ◽  
High Concentrations

Blood platelets contain membrane-enclosed granules which have inside them high concentrations of 5-hydroxytryptamine (serotonin) along with adenine nucleotides and divalent metal ions. 19 F n.m.r. of fluorinated serotonin incorporated into the granules of both human and pig intact platelets has shown that the motional state of the serotonin is restricted. Comparison with 31 P n.m.r. experiments indicates that this restriction of motion is a consequence of high molecular weight aggregates formed by the adenine nucleotides and metal ions, and that it varies with the species from which the platelets are obtained. In the case of human platelet granules, at least, these high molecular weight aggregates are present in the absence as well as in the presence of serotonin. The biological significance of these data is briefly discussed.

Brain-derived Neurotrophic Factor Is Stored in Human Platelets and Released by Agonist Stimulation

2002 ◽  
Vol 87 (04) ◽  
pp. 728-734 ◽  
Author(s):  
Hironobu Fujimura ◽  
Ruoyan Chen ◽  
Takashi Nakamura ◽  
Takeshi Nakahashi ◽  
Jun-ichi Kambayashi ◽  
...  
Keyword(s):  
Pituitary Gland ◽  
Cell Lines ◽  
Neurotrophic Factor ◽  
Brain Derived Neurotrophic Factor ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Platelet Surface ◽  
High Affinity ◽  
Agonist Stimulation ◽  
High Affinity Site

SummaryBrain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays critical roles in the survival, growth, and maintenance of brain and peripheral neurons. We report the presence of BDNF protein in human platelets and its release upon agonist stimulation. The BDNF content of washed platelets varied widely, from 3.5 to 67 ng/ 4 X 108 platelets, averaging 25.2 ± 21.2 ng/4 X 108 platelets (mean ± SD). The BDNF concentration in platelet-poor plasma was low (1.7 ± 1.7 ng/ml, n = 11). Thrombin, collagen, the Ca++ ionophore A23187, and shear stress each induced a rapid release of BDNF from platelets. Up to only half of platelet BDNF was secreted upon agonist stimulation, suggesting that platelets may have a non-releasable pool of BDNF, or that the released BDNF binds to a recognition site on the platelet surface and is internalized, as occurs with serotonin. However, the cognate BDNF receptor, TrkB, was not detected in platelets. Nevertheless, the ability of BDNF to bind washed platelets was shown by FACS analysis confocal microscopy and by the binding and apparent internalization of [125I]-BDNF by platelets. A very high affinity site (Kd = 130 X 10−15 M, ∼80 sites/platelet) and a moderately high affinity site (Kd = 20 nM, ∼3750 sites/platelet) were identified. The BDNF content in two mega-karyocytic cell lines, DAMI and Meg-01, was only 0.1% of the content measured in platelets. No BDNF mRNA was detected by Northern blotting in these cell lines or in platelets. The pituitary gland was also ruled out as a source for platelet BDNF, since the BDNF content of rat platelets did not decrease 2 weeks after hypophysectomy. Thus, platelet BDNF is not acquired from the megakaryocyte or pituitary gland, but is probably acquired from other sources via the blood circulation. Platelets appear to bind, store and release BDNF upon activation at the site of traumatic injury to facilitate the repair of peripheral nerves or other tissues that contain TrkB.

scholarly journals Characterization of calcium binding to brush-border membranes from rat duodenum

1982 ◽  
Vol 208 (3) ◽  
pp. 773-781 ◽  
Author(s):  
A Miller ◽  
S T Li ◽  
F Bronner
Keyword(s):  
Brush Border ◽  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Relative Effectiveness ◽  
Brush Border Membranes ◽  
High Affinity ◽  
High Affinity Site ◽  
Rat Duodenum ◽  
High Concentrations

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.

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