LIPOPROTEIN BINDING TOHUMAN PLATELETS IS LOCATED AT GPIIb/IIIa COMPLEX

1987 ◽  
Author(s):  
E Koller ◽  
F Koller
Keyword(s):  
Specific Binding ◽  
Human Platelets ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Molecular Weights ◽  
Disulfide Reduction ◽  
Specific Binding Sites ◽  
First Results ◽  
Lipoprotein Binding ◽  
High Concentrations

Human Platelets possess specific binding sites for low density lipoproteins (LDL) and high density lipoproteins(HDL)(1). Binding of both classes of plasma lipoproteins, though competitive, has been shown by several groups to facilitate platelet activation.Isolated washed platelets occasionally aggregate upon addition of high concentrations of LDL even in the absence of known platelet activators. The proteins responsible for this binding have been visualized by ligand blotting (2). Both types of ligand specifically bind to two glycoproteins with molecular weights of 135 and 115 kD, respectively. The conditions of binding to these two proteins, however, markedly differ from those known for other lipoprotein receptors.Following extensive purification, these two species are still present at concentrations relative to each other that depend markedly on the conditions of purification. The purified, solubilized receptor was tested under various conditions, including in the absence and presence of calcium, after disulfide-reduction, and following chymotrypsin digestion. In parallel experiments, the same preparations were tested with respect to binding of fibrinogen, different lectins, and thealloantibody anti-PlAI . The results strongly support the assumption, that the two protein bands associated with lipoprotein binding are constituents of the GP-IIb/IIIa complex.These first results may have greatimplications for our understanding ofthe mechanism by which lipoproteins facilitate platelet stimulation.

EFFECT OF Ca2+ and Mg2+ ON PLATELET ACTIVATING FACTOR (PAF) INDUCED AGGREGATION AND SPECIFIC BINDING TO HUMAN PLATELETS

1987 ◽  
Author(s):  
C M Chesney ◽  
D D Pifer
Keyword(s):  
Platelet Aggregation ◽  
Calcium Channel ◽  
Binding Sites ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Human Platelets ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Significant Difference ◽  
Induced Aggregation

Gel filtered human platelets (GFP) collected in Tyrode's buffer containing 0.5 mM Ca+2, ImM Mg+2, and 0.35% albumin exhibit high affinity binding of 3H-PAF with a Kd of 0.109 α 0.029 nM (mean α SD; n=13) and 267 α 70 sites per platelet. When fibrinogen (1.67 mg/ml final concentration) is added to these GFP preparations biphasic aggregation is observed with PAF (4 nM). Normal aggregation is also observed with other platelet agonists including ADP, epinephrine, collagen, arachidonic acid, A23187 and thrombin. If GFP is prepared without added Ca+2 or Mg+2 in the presence of 3mM EDTA, platelets do not aggregate in response to PAF. However the number of specific binding sites remains unchanged (387 per platelet) with some decrease in affinity of binding (Kd = 0.2l4nM). In the presence of ImM Mg+2 there is no significant difference in binding kinetics over a range of Ca+2 concentrations (0-2mM). On the other hand the calcium channel blocker verapamil (5-10uM) exhibits competitive inhibition of 3H-PAF as analyzed by Lineweaver-Burk plots. Specific binding of 3H-PAF to GFP in the presence of ImM Mg+2 and ImM EGTA shows Kd of 0.l66nM but with increase in specific binding sites to 665. Despite increase in number of sites and no change in binding affinity, GFP under these conditions does not exhibit platelet aggregation with PAF in doses up to 80 nM.From these data it appears that external Ca+2 is not necessary for specific binding of 3H-PAF to its high affinity receptor. However, calcium does appear to be necessary for second wave aggregation with PAF. While Mg+2 appears to enhance 3H-PAF binding to platelets Mg+2 cannot substitute for Ca+2 in PAF induced platelet aggregation. Although verapamil appears to competitively inhibit binding of PAF to GFP it is not clear whether the inhibition is due to competition at or near the actual PAF receptor or at a site involving the calcium channel.

Chemoselective cluster labelling of biomolecules

1987 ◽  
Vol 45 ◽  
pp. 760-761
Author(s):  
C. J. Foley ◽  
L. E. Maelia ◽  
J. F. Hainfeld ◽  
J. S. Wall
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Heavy Atom ◽  
The Other ◽  
Biological Structure ◽  
Heavy Atoms ◽  
Specific Binding Sites ◽  
Lower Beam ◽  
High Concentrations ◽  
Beam Dose

The Brookhaven STEM is capable of visualizing single heavy atoms at a beam dose of >103 el/Å2. Heteropolytungstate clusters, including W12PO403, have been found to incorporate several desirable properties as labels for biological specimens. They may be resolved at much lower beam doses due to their high concentrations of multiple heavy atoms and are directly visible labels. A lower beam dose also helps to preserve the biological structure of the specimens. Furthermore, they are extremely stable in the electron beam. Lastly, they are capable of being derivatized as chemoselective reagents for specific binding sites on biomolecules, as in the previously reported undecagold compound.Two new classes of heavy atom labels, one specific for sulfhydryl and the other specific for both amino and sulfhydryl binding sites on proteins, have been synthesized by reactions analogous to those illustrated in Scheme 1.

Expression of follicle-stimulating hormone and luteinising hormone binding sites in the bitch ovary during the follicular phase

2008 ◽  
Vol 20 (8) ◽  
pp. 925 ◽  
Author(s):  
Marie Saint-Dizier ◽  
Nina Jaffré ◽  
Karine Reynaud ◽  
Benoît Remy ◽  
Sandra Thoumire ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Follicular Phase ◽  
Follicular Growth ◽  
Physiological Control ◽  
Lh Surge ◽  
Specific Binding Sites ◽  
Follicle Diameter ◽  
High Concentrations

In the female dog, in contrast with most mammals, the growing follicle starts to luteinise several days before ovulation. Little is known about the physiological control of the final follicular growth in this species. In order to better understand the pituitary regulation of follicular growth, specific binding sites for FSH and LH were localised and quantified by autoradiography using [125I]-porcine (p) gonadotrophins on ovarian sections (7 μm) from adult Beagle bitches during the follicular phase. Follicles were analysed either before the LH surge (n = 4 bitches; n = 117 follicles) or after the LH surge and before ovulation (n = 5 bitches; n = 110 follicles). FSH binding sites were specifically and homogeneously expressed at high levels on granulosa cells of all healthy follicles from the preantral stage onwards. In contrast, LH binding sites were detected homogeneously and at high levels only on granulosa cells of follicles larger than 1 mm in diameter, including luteinised follicles. Theca binding of LH (but not FSH) was also observed, but only when using high concentrations of [125I]-pLH. The overall incidence of atresia was 45.8% and was dependent upon follicular diameter. Quantitative analysis of labelling showed that atretic follicles had reduced levels of both FSH and LH binding sites compared with healthy follicles. In healthy follicles, levels of both FSH and LH binding sites changed with follicle diameter. Compared with other mammals, the acquisition of LH binding on canine granulosa cells occurs in smaller sized follicles relative to the size of ovulation.

scholarly journals Interaction of plasma lipoproteins with human platelets

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 365-374 ◽  
Author(s):  
LK Curtiss ◽  
EF Plow
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ldl Receptor ◽  
Human Platelets ◽  
Plasma Lipoproteins ◽  
High Density Lipoproteins ◽  
Interaction Sites ◽  
Ldl Particles ◽  
Ldl Binding

Abstract Human plasma low density lipoproteins (LDL) and high density lipoproteins (HDL) were radioiodinated and their interaction with washed human platelets was assessed. Both ligands were bound by the platelet at 37 degrees C, and an apparent equilibrium was attained within two hours. Minimal binding was observed at 22 degrees C or 4 degrees C. The specificity of these interactions was indicated by the observations that: (a) labeled and nonlabeled lipoproteins interacted with the platelet with the same apparent affinity; (b) nonlabeled lipoproteins inhibited binding, whereas unrelated plasma proteins did not; and (c) the platelet-bound ligands exhibited the appropriate apoprotein chain compositions when analyzed by polyacrylamide gel electrophoresis. Binding of HDL and LDL was found to be independent of the state of platelet activation and did not require divalent ions. Binding of HDL to the platelet was saturable, and a class of sites that maximally bound 1,585 +/- 390 HDL particles, with a dissociation constant of 3.1 X 10(-8) mol/L, was identified. Binding of LDL to the platelet was more complex, but evidence for a class of sites that bound 7,075 +/- 4,800 LDL particles, with a dissociation constant of 4 X 10(- 8) mol/L, was found. LDL was a poor inhibitor of 125I-HDL binding to the platelet, whereas HDL was an effective inhibitor of 125I-LDL binding. The capacity of HDL to bind or inhibit LDL binding was not dependent on its apoprotein E content. These results are most readily interpreted in terms of two types of lipoprotein interaction sites on platelets: (1) an HDL binding site that does not bind or interacts poorly with LDL, and (2) an LDL binding site that recognizes or is otherwise altered by HDL. The HDL site may be similar to the HDL receptor expressed by steroidogenic tissues in terms of apoprotein specificity. The LDL site is not the same as the LDL receptor of most extrahepatic cells.

scholarly journals Species- and substrate-specific stimulation of human plasma paraoxonase 1 (PON1) activity by high chloride concentration.

2002 ◽  
Vol 49 (4) ◽  
pp. 927-936 ◽  
Author(s):  
Jerzy Bełtowski ◽  
Grazyna Wójcicka ◽  
Andrzej Marciniak
Keyword(s):  
Mammalian Species ◽  
Chloride Concentration ◽  
Plasma Lipoproteins ◽  
Paraoxonase 1 ◽  
High Density Lipoproteins ◽  
Pon1 Activity ◽  
Phenyl Acetate ◽  
Halide Anion ◽  
Plasma High Density ◽  
High Concentrations

Paraoxonase 1 (PON1), contained in plasma high-density lipoproteins, plays an important role in the protection of plasma lipoproteins and cell membranes from oxidative damage. Previous studies indicate that human PON1 is stimulated by high NaCl concentrations. The aim of this study was to characterize in more detail the effect of salts on serum PON1. Paraoxon-hydrolyzing activity of human serum was stimulated by 81.6% following the addition of 1 M NaCl. The effect of NaCl was dose-dependent between 0.5 and 2 M. PON1 activity toward phenyl acetate was reduced by 1 M NaCl by 55.2%. Both the paraoxon- and phenyl acetate-hydrolysing activity was slightly lower in heparinized plasma than in serum, but NaCl had similar stimulatory and inhibitory effects on these activities, respectively. In rat, rabbit, and mouse, NaCl reduced PON1 activity. KCl had a similar effect on human PON1 as NaCl. Sodium nitrite also stimulated human PON1 but much less effectively than chloride salts. In contrast, sucrose, sodium acetate and sodium lactate had no significant effect. NaBr was a less effective PON1 activator than NaCl, whereas the effect of NaJ was non-significant. The activity of human PON1 toward homogentisic acid lactone and gamma-decanolactone was unaltered by NaCl. These data indicate that: 1) high concentrations of chlorides stimulate human PON1 activity toward paraoxon but not other substrates, 2) PON1 is inhibited by Cl(-) in other mammalian species, 3) the potency of human PON1 activation by halogene salts increases with decreasing atomic mass of the halide anion.

scholarly journals Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membranes of human platelets

1994 ◽  
Vol 298 (3) ◽  
pp. 739-742 ◽  
Author(s):  
P J Cullen ◽  
Y Patel ◽  
V V Kakkar ◽  
R F Irvine ◽  
K S Authi
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Specific Binding ◽  
Human Platelets ◽  
Intracellular Membranes ◽  
Specific Binding Sites ◽  
Carrier Free ◽  
Membrane Location

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

Arachidonate Transfer Between Platelets and Lipoproteins

1992 ◽  
Vol 68 (06) ◽  
pp. 719-726 ◽  
Author(s):  
Ingrid I Surya ◽  
Gertie Gorter ◽  
Jan Willem N Akkerman
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Human Platelets ◽  
Temperature Sensitive ◽  
High Concentration ◽  
Prolonged Incubation ◽  
Specific Binding Sites ◽  
Lipid Exchange ◽  
Exchange Processes ◽  
Platelet Functions

SummaryAlthough platelets have specific bindingsites for LDL and HDL, it is doubtful whether lipoproteins modulate platelet functions via receptor-mediated processes. We investigated platelet-lipoprotein interaction during prolonged incubation with concentrations of LDL and HDL that saturate the bindingsites within a few minutes. When [3H]arachidonate-labeled human platelets were incubated for 4 h with lipoproteins, part of the 3H-radioactivity transferred to LDL and to a lesser extent to HDL. The transfer was temperature-sensitive, unaffected by modification of lysine in LDL or indomethacin treatment of the platelets, and almost irreversible. [3H]arachidonate transfer to lipoproteins could be mimicked by incubating platelets with a high concentration of fatty acid free albumin. This showed, that the loss of 3H-radioactivity reflected a decrease in endogenous arachidonate, leading to impaired aggregation, secretion and thromboxane B2 formation in platelets after stimulation with thrombin but not with arachidonate. Thus, the decrease in platelet functions seen after long incubation with HDL is caused by depletion of platelet arachidonate. Despite an even stronger arachidonate depletion by LDL, this lipoprotein initiated arachidonate metabolism and secretion independent of specific binding sites for LDL on the platelet. Surprisingly, the major part of the secretion was preserved when the formation of prostaglandin endoperoxides/ thromboxane A2 was inhibited with indomethacin. These findings argue against a role for LDL and HDL receptors in the modulation of platelet functions and are more in favor of lipid exchange processes between platelets and lipoproteins.

scholarly journals The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin

1983 ◽  
Vol 216 (1) ◽  
pp. 43-49 ◽  
Author(s):  
D G Hassall ◽  
J S Owen ◽  
K R Bruckdorfer
Keyword(s):  
Platelet Aggregation ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
High Concentrations ◽  
Very High

Addition of prostacyclin (PGI2) temporarily inhibits platelet aggregation and permits the isolation of platelets free from plasma proteins, which have the same sensitivity as those in plasma [Moncada, Radomski & Vargas (1982) Br. J. Pharmacol. 75, 165P]. By using a modification of this technique we have established that platelets isolated from normal subjects aggregate more readily in response to ADP and adrenaline when physiological concentrations of low-density lipoproteins (LDL) are present. At high LDL concentrations spontaneous aggregation occurs. High-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) had no effect on agonist-induced platelet aggregation at normal concentrations, but HDL sensitized at higher concentrations. These effects by lipoproteins are not accompanied by changes in platelet lipid content. Cyclohexanedione treatment of LDL to modify apolipoproteins appeared to abolish the sensitization effect, indicating that binding to receptors was essential for the effects of LDL. LDL, but not HDL, overcame the inhibitory effect of PGI2 on platelet aggregation, except at very high concentrations of PGI2. PGI2 raised the cyclic AMP content of isolated platelets, but LDL only partially prevented this rise. These results suggest that LDL may have a greater role in platelet aggregation than previously recognized and may also regulate effects of PGI2. These findings may be of relevance to an understanding of cardiovascular diseases.

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