scholarly journals Binding kinetics of PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human platelets

1984 ◽  
Vol 223 (3) ◽  
pp. 901-909 ◽  
Author(s):  
E Kloprogge ◽  
J W N Akkerman
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Human Platelets ◽  
Equilibrium Analysis ◽  
Binding Studies ◽  
Prolonged Incubation ◽  
Fibrinogen Receptor ◽  
Receptor Aggregation ◽  
Specific Receptors ◽  
High Doses

The binding of [3H]PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to intact human gel-filtered platelets was measured at 22 degrees C. Specific binding reached saturation within 15 min at high doses of [3H]PAF-acether (0.5-0.9 nM), whereas about 90 min were required when low doses (0.02-0.5 nM) were used. Above 1 nM, [3H]PAF-acether non-specific binding increased progressively, which together with the demonstration of a 3H-labelled metabolite suggested uptake and metabolism of [3H]PAF-acether. Equilibrium analysis revealed one class of specific receptors with a Ka of 18.86 +/- 4.82×10(9) M-1 and 242 +/- 64 binding sites per platelet. Non-equilibrium binding revealed a similar Ka (16.87×10(9) M-1). Specific binding became irreversible after prolonged incubation, a process that was enhanced at increasing concentrations of [3H]PAF-acether. Platelets made desensitized to PAF-acether by prior incubation with unlabelled PAF-acether failed to bind a second dose of PAF-acether (3H-labelled), suggesting that desensitization resulted from loss of available binding sites. Under the conditions of the binding studies, PAF-acether induced exposure of the fibrinogen receptor, aggregation (in a stirred suspension) and alterations in (poly)-phosphatidylinositides. These results suggest that PAF-acether initiates platelet responses via receptor-mediated processes.

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.

A New Method for the Assay of Exposed Platelet Fibrinogen Receptor Using a Chemiluminescent Label

1995 ◽  
Vol 74 (06) ◽  
pp. 1546-1550 ◽  
Author(s):  
Makoto Katoh ◽  
Susumu Chishima ◽  
Nobukazu Kiuchi ◽  
Tomihiro Ikeo ◽  
yasuhiko Sasaki
Keyword(s):  
Binding Sites ◽  
High Selectivity ◽  
Binding Assay ◽  
Specific Binding ◽  
Human Platelets ◽  
Assay System ◽  
Human Fibrinogen ◽  
Fibrinogen Receptor ◽  
Plot Analysis ◽  
Fibrinogen Binding

SummaryAssay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative I-labeled fibrinogen (acridinium-fibrinogen).Human fibrinogen in saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa was assayed by measuring chemiluminescence emitted on addition of 0.1 N NaOH containing 0.06% H202 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with a Kd of 326 nM and Bmax of 7.8 pmol/108 platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen γ-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 μM and 31.3μM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotop.

Detection Of High Affinity Fibrinogen Binding Sites On Human Platelets Stimulated By ADP: Comparison Of Two Methods Of Platelet Separation

1981 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Thomas A Morinelli ◽  
Elizabeth Kornecki
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Gel Filtration ◽  
Human Platelets ◽  
Binding Studies ◽  
Test Analysis ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
Specific Receptors

Binding of fibrinogen to specific receptors on human platelets exposed by ADP results in platelet aggregation. There are controversial data regarding classes and number of fibrinogen receptors, the values range from one to two classes and 1,000-80,000 receptors per platelet as reported in the literature. We have studied the interaction of fibrinogen with a) platelets washed by differential centrifugation according to Mustard and colleagues (washed platelets - WP) and with b) gel-filtered platelets (GFP). Platelet aggregation was studied with 100 μM ADP and with various concentration of fibrinogen. Maximal velocities of aggregation for WP and GFP were 81 and 47 units per min, respectively, and the Km values for fibrinogen calculated from the rate of aggregation were 0.9 × 10-7M for WP and 5.8 × 10-7M for GFP. The level of platelet fibrinogen released into the suspension from WP and GFP amounted to 2.4 μg and 15.0 μg per 10 9 platelets/ml, respectively, as measured by the staphylococcal clumping test. Analysis of 125I-fibrinogen binding data by the method of Scatchard and Feldman revealed 1,300 high affinity receptors (KD 3.2 × 10-8M) and 80,000 low affinity receptors (KD 5.6 × 10-5M) for WP. The binding of 125I-fibrinogen to GFP was greatly diminished. The number of fibrinogen receptors exposed by ADP on GFP and their binding affinity are under investigation in our laboratory. In conclusion, GFP were less sensitive to fibrinogen than were WP as shown in the aggregation and 125I-fibrinogen binding studies. It appears that the method of platelet separation is critical for the assessment of fibrinogen binding. Platelet activation and release of intact platelet fibrinogen during gel-filtration may interfere with the detection of high affinity fibrinogen binding sites.

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.

Adrenergic receptors on cerebral microvessels: pericyte contribution

1989 ◽  
Vol 256 (1) ◽  
pp. R224-R230 ◽  
Author(s):  
R. M. Elfont ◽  
P. R. Sundaresan ◽  
C. D. Sladek
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Adrenergic Receptors ◽  
Radioligand Binding ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Binding Studies ◽  
Cerebral Microvessels ◽  
Beta Adrenoceptor ◽  
Number Of Binding Sites

R224-R230, 1989.--[125I]iodocyanopindolol ([125I]ICYP) and [3H]rauwolscine were used to quantitate, respectively, the beta- and alpha 2-adrenergic receptors in freshly isolated bovine cerebral microvessels and in pericyte cultures derived from these microvessels. Morphological and immunocytochemical criteria distinguished the pericytes from endothelial cells. Competitive binding studies established the specificity of the radioligand binding. The maximal number of binding sites (Bmax) for [125I]ICYP in the pericytes constituted only 8% of that in the microvessels (3.5 +/- 1.3 vs. 44.4 +/- 6.6 fmol/mg protein). In contrast, the Bmax for [3H]rauwolscine in the pericytes was 50% of that in the microvessels (55.4 +/- 11.8 vs. 111.1 +/- 9.5 fmol/mg protein). The dissociation constants for both [125I]ICYP and [3H]rauwolscine were similar in the two preparations. No alpha 1-adrenergic receptors, as defined by the specific binding of [3H]prazosin, were identified either in the pericytes or microvessels. Overall, our results suggest that pericytes contribute minimally to the total beta-adrenoceptor number of cerebral microvessels, and thus the beta-adrenoceptors must be located predominantly on endothelial cells. However, the contribution of pericytes to the total alpha 2-adrenoceptor number of the microvessels may be substantial.

scholarly journals Internalization and down-regulation of the prostacyclin receptor in human platelets

1997 ◽  
Vol 325 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Serenella GIOVANAZZI ◽  
Maria R. ACCOMAZZO ◽  
Ornella LETARI ◽  
Daniela OLIVA ◽  
Simonetta NICOSIA
Keyword(s):  
Binding Sites ◽  
Organic Anion ◽  
Subcellular Fractionation ◽  
Human Platelets ◽  
Organic Anion Transporters ◽  
Camp Production ◽  
Binding Studies ◽  
Anion Transporters ◽  
Prostacyclin Receptor ◽  
High Affinity Binding Site

The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 °C, addition of unlabelled ligand at either 37 °C or 4 °C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 °C or 4 °C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 °C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 °C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 °C, two classes of binding sites were present, whereas after preincubation at 37 °C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.

The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Binding Constant ◽  
Specific Binding ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

Autoradiographic detection of kinin receptors in the human medulla of control, hypertensive, and diabetic donors

2002 ◽  
Vol 80 (4) ◽  
pp. 249-257 ◽  
Author(s):  
Hudson de Sousa Buck ◽  
Brice Ongali ◽  
Gaétan Thibault ◽  
Charles J Lindsey ◽  
Réjean Couture
Keyword(s):  
Receptor Binding ◽  
Binding Sites ◽  
Specific Binding ◽  
Inferior Olivary Nucleus ◽  
Binding Studies ◽  
Specific Binding Sites ◽  
Kinin Receptors ◽  
Medullary Nuclei ◽  
Inferior Olivary

Kinins have been elected to the status of central neuromediators. Their effects are mediated through the activation of two G-protein-coupled receptors, denoted B1 and B2. Functional and binding studies suggested that B1 and B2 receptors are upregulated in the medulla and spinal cord of hypertensive and diabetic rats. The aim of this study was to localize and quantify kinin receptors in post-mortem human medulla obtained from normotensive, hypertensive, and diabetic subjects, using in vitro receptor autoradiography with the radioligands [125I]HPP-HOE140 (B2 receptor) and [125I]HPP[des-Arg10]-HOE140 (B1 receptor). Data showed specific binding sites for B2 receptor (0.4–1.5 fmol/mg tissue) in 11 medullary nuclei from 4 control specimens (paratrigeminal > ambiguus > cuneate, gelatinous layer of the caudal spinal trigeminal nucleus > caudal and interpolar spinal trigeminal, external cuneate, solitary tract > hypoglossal > gracile > inferior olivary nuclei). Increased density of B2 receptor binding sites was observed in seven medullary nuclei of four hypertensive specimens (paratrigeminal > external cuneate > interpolar and caudal spinal trigeminal, gracile, inferior olivary > hypoglossal nuclei). B2 receptor binding sites were seemingly increased in the same medullary nuclei of two diabetic specimens. Specific binding sites for B1 receptor (1.05 and 1.36 fmol/mg tissue) were seen only in the inferior olivary nucleus in two out of the ten studied specimens. The present results support a putative role for kinins in the regulation of autonomic, nociceptive, and motor functions at the level of the human medulla. Evidence is also provided that B2 receptors are upregulated in medullary cardiovascular centers of subjects afflicted of cardiovascular diseases.Key words: bradykinin, hypertension, diabetes, human brain.

Development and regulation of the prostaglandin F2α receptor in the rat ovary

1981 ◽  
Vol 98 (3) ◽  
pp. 441-445 ◽  
Author(s):  
Ulrich Müller ◽  
Th. Bauknecht ◽  
Jan Willem Siebers
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Prostaglandin F2α ◽  
Cell Type ◽  
Adult Rats ◽  
Endogenous Prostaglandin ◽  
Prostaglandin F ◽  
Inhibition Of Prostaglandin Synthesis ◽  
Specific Receptors ◽  
Rat Ovary

Abstract. Ovaries of adult rats specifically bind PGF2α while those of immature animals do not. Induction of luteinization by hCG in juvenile animals, however, results in specific binding of PGF2α It is suggested that luteal cells are the only cell type of the ovary, which is endowed with specific receptors for PGF2α The number of PGF2α binding sites varies during the ovarian cycle. Most free receptors are detectable in early pro-oestrus, least in the oestrus stage. The oscillation of receptors disappears after inhibition of prostaglandin synthesis by indomethacin. Therefore the apparent cyclic variation of prostaglandin receptors must be ascribed to occupancy of the receptors by varying amounts of endogenous prostaglandin F2α.

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