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.

scholarly journals Binding of Calcium to Fibrinogen: Some Related Properties

1977 ◽  
Author(s):  
G. Marguerie
Keyword(s):  
Binding Sites ◽  
Calcium Binding ◽  
Specific Binding ◽  
Equilibrium Dialysis ◽  
Structural Features ◽  
Salt Bridges ◽  
High Affinity ◽  
Direct Titration ◽  
Specific Binding Sites ◽  
Calcium Binding Sites

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.

Interaction of β2-Glycoprotein-I with Human Blood Platelets: Influence Upon the ADP-Induced Aggregation

1985 ◽  
Vol 54 (02) ◽  
pp. 397-401 ◽  
Author(s):  
Johannes Nimpf ◽  
Helmut Wurm ◽  
Gerhard M Kostner
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Human Platelets ◽  
Glycoprotein I ◽  
Unspecific Binding ◽  
Induced Aggregation ◽  
Human Blood Platelets

SummaryThe interaction of β2-glycoprotein-I (β2-G-I), a plasma constituent of unknown function, with blood platelets was studied. The following results were obtained: 1) β2-G-I binds to washed human platelets isolated by centrifugation (WP) at one kind of specific, saturable binding sites. The dissociation constant was found to be approx. 1 × 10−6M.2) In the presence of physiological concentrations of Ca++ (2.5 mM), this specific binding is markedly reduced. Unspecific binding of β2-G-I to platelets, however, is not influenced by Ca++.3) Platelets prepared by gel filtration (GFP), differing in their in vitro aggregability from WP, exhibit no specific binding of β2-G-I. Binding to GFP is also not induced by activation with thrombin, collagen or ADP.4) β2-G-I causes significant alteration of the ADP-induced aggregation of GFP. Aggregation induced by thrombin, collagen, arachidonic acid or PAF-acether, however is not altered by β2G-I.It is suggested, that pelleting during centrifugation causes irreversible rearrangements in the membrane of platelets.

Evidence for Presynaptic High Affinity Imidazoline-Specific Binding Sites in the Bovine Brainstem

1999 ◽  
Vol 881 (1 IMIDAZOLINE R) ◽  
pp. 185-188 ◽  
Author(s):  
F. M. J. HEEMSKERK ◽  
M. DONTENWILL ◽  
H. GRENEY ◽  
C. VONTHRON ◽  
P. BOUSQUET
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
High Affinity ◽  
Specific Binding Sites

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.

Interactions of some partial agonists with high and low affinity binding sites in β-adrenoceptors

1987 ◽  
Vol 65 (1) ◽  
pp. 18-22 ◽  
Author(s):  
I. Takayanagi ◽  
K. Koike ◽  
A. Nakagoshi
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Specific Binding ◽  
The Other ◽  
Affinity Binding ◽  
High Affinity ◽  
Partial Agonists ◽  
Significant Difference ◽  
High Affinity Site ◽  
Derivatives Of

Interactions of derivatives of befunolol (BFE-37, BFE-55, and BFE-61), carteolol, and pindolol with β-adrenoceptors were tested in guinea pig isolated taenia caecum. All the drugs used acted as partial agonists on the β-adrenoceptors when compared with isoprenaline, a full agonist. The pA2 values of BFE-61, carteolol, and pindolol were significantly larger than their pD2 values, while there was no significant difference between the pA2 and pD2 values for BFE-37 and BFE-55. The specific binding of [3H]befunolol to microsomal fractions from the guinea pig taenia caecum distinguished two binding sites, high affinity and low affinity sites. Both sites are considered to be bound by 50 nM of [3H]befunolol. Specific 3H binding was displaced by BFE-61, carteolol, and pindolol in a biphasic manner but in a monophasic manner by BFE-37 and BFE-55. Furthermore, [3H]befunolol binding was only partially displaced by BFE-55 but completely displaced by the other drugs used. These results, together with our previous findings, suggest that BFE-61, carteolol, and pindolol discriminate between the two affinity binding sites in the β-adrenoceptors, which are not discriminated between by BFE-37, and further that BFE-55 may bind with only the high affinity site.

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.

Thrombin Interaction with Human Platelets Potentiation of Thrombin-induced Aggregation and Release by Inactivated Thrombin

1974 ◽  
Vol 32 (01) ◽  
pp. 207-215 ◽  
Author(s):  
David R. Phillips
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Induce Platelet Aggregation ◽  
Release Reaction ◽  
Proteolytic Site ◽  
Induced Aggregation ◽  
Inhibited Enzyme

SummaryThe possibility that thrombin acts on platelets by a mechanism other than proteolysis was investigated. The proteolytic site of thrombin was modified with phenylmethylsulfonyl fluoride (PMSF). This modified enzyme did not induce platelet aggregation or the platelet release reaction. Platelets were then incubated with the inactivated enzyme (PMS-thrombin) and later with active thrombin. In this sequence of incubation, PMS-thrombin enhanced not only platelet aggregation induced by active thrombin but also the thrombin-induced release reaction. Preincubation with PMS-thrombin was essential for this enhancement as the inhibited enzyme did not affect aggregation if added after active thrombin. The effect of PMS-thrombin was limited to thrombin-induced reactions of the platelet. The inhibited enzyme had no effect on aggregation induced by adenosine diphosphate or collagen, or on thrombininduced coagulation of fibrinogen. These results suggest (1) that both proteolytic and binding sites for thrombin are present on the human platelet plasma membrane ; and (2) that interaction of thrombin with the binding site potentiates the activity of the proteolytic site.

The High Affinity Calcium Ion Bending Sites of Fibrinogen

1981 ◽  
Author(s):  
Joan Ross ◽  
Graham D Kemp
Keyword(s):  
Calcium Ions ◽  
Binding Sites ◽  
Calcium Ion ◽  
Specific Binding ◽  
Ion Release ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Scatchard Plots ◽  
Systems Studies ◽  
Flow Dialysis

There is considerable evidence that fibrinogen contains a number of strongly bound calcium ions and these appear to have a significant role in the structure and properties of the molecule. Most of the evidence suggests that there are three such strongly bound calcium ions in fibrinogen and each of the two fragments D contains one of these. It has been suggested that the section of the (A) α chain which is the region of the molecule first attacked by plasnin is involved in binding calcium ions. Should this constitute the third site it follows that this calcium ion must link the two (A) α chains and the site may well be destroyed by minimal plasnin attack. The figure of three calcium ions bound, however, must be open to sane doubt due to the difficulty in evaluating data from Scatchard plots prepared from. a system, such as fibrinogen, which contains a number of identical ligands with more than one binding affinity. Accordingly we have developed methods to prepare fibrinogen in as intact a form as possible, and used such fibrinogen in flow dialysis systems. Studies of calcium ion release during proteolytic degradation of fibrinogen lead us to conclude that there are probably only two high affinity, calcium ion specific binding sites in fibrinogen.

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