Evaluation of the Binding to Fixed Platelets of Agonists and Antagonists of ADP-Induced Aggregation

1989 ◽  
Vol 62 (04) ◽  
pp. 1103-1106 ◽  
Author(s):  
Ashok K Agarwal ◽  
Narendra N Tandon ◽  
Nicholas J Greco ◽  
Noel J Cusack ◽  
G A Jamieson
Keyword(s):  
Binding Sites ◽  
Binding Assay ◽  
Relative Magnitude ◽  
Affinity Binding ◽  
Inhibitory Potency ◽  
Competitive Binding Assay ◽  
High Affinity ◽  
High Affinity Site ◽  
Purine Ring ◽  
Induced Aggregation

SummarySteady state binding of eleven different ADP analogues to formaldehyde-fixed platelets has been determined in a competitive binding assay using 3H-ADP. The compounds tested were the inactive analogues L-ADP and L-ATP; the agonists 2-chloroadenosine 5’-diphosphate, adenosine 5’-O-(2-thiodiphosphate)and the diastereoisomeric pair Sp-adenosine 5’-(1-thiodiphosphate) (Sp-ADP-α-S) and Rp-adenosine 5’-(1-thiodiphosphate) (Rp-ADP-α-S); and the antagonists adenosine 5’-O-thiomonophosphate, 2-chloroadenosine 5’-O-thiomonophosphate, 2-chloroadenosine 5’-triphoshate, and the diastereoisomeric pair 5’-(1-thiotriphosphate) (Sp-ATP-α-S) and Rp-adenosine 5’-(1-thiotriphosphate) (Rp-ATP-α-S). All compounds tested competed at the high affinity binding sites for ADP previously identified (Blood 1988; 71: 110-6) but in some cases competition could not be demonstrated at the low affinity sites because of the high nucleotide concentrations required. As a group, C2-substituted analogues bound less strongly (Ki >2 μM) than did the analogues without substituents in the purine ring (Ki <0.7 μM). With the pair of diastereoisomeric agonists Sp-ADP-α-S and Rp-ADP-α-S the Ki values at the high affinity site (210 nM and 560 nM) were of the same relative magnitude and in the same direction as their reported potencies as agonists (Ki 4 μM and 20 μM). With the diastereoisomeric antagonists Sp-ATP-α-S and Rp-ATP-α-S a similar relationship was seen between affinity (17 nM and 156 nM) and inhibitory potency (Ki 4 μM and 20 μM). These results may help to differentiate possible mechanisms in the interaction of ADP with its receptors.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Abstract Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

scholarly journals Platelet membrane alterations induced by the local anesthetic dibucaine

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 463-471
Author(s):  
EI Peerschke
Keyword(s):  
Monoclonal Antibody ◽  
Binding Sites ◽  
Periodic Acid ◽  
Platelet Membrane ◽  
Actin Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Induced Aggregation

Tertiary amine local anesthetics modify a variety of platelet membrane- related functions. The present study explored dibucaine (DB)-induced inhibition of platelet cohesion by examining structural and functional alterations of the human platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) and platelet Ca2+ homeostasis. Complete inhibition of ADP-induced aggregation was achieved five minutes after platelet exposure to 0.10 to 0.25 mmol/L of DB when fibrinogen binding was reduced by 50%. At higher concentrations of DB (approximately 1 mmol/L), ADP-induced fibrinogen binding was completely blocked. Scatchard analysis revealed loss of high-affinity binding sites in addition to reduction in Bmax. In contrast, chymotrypsin-treated platelets sustained 50% inhibition of fibrinogen binding when incubated with 0.4 to 0.5 mmol/L DB, and kinetic analysis showed that the high- affinity platelet-fibrinogen interactions were reduced but not absent. Fibrinogen binding to chymotrypsin-treated platelets could not be completely inhibited even at high DB concentrations (1 mmol/L). The inhibition of fibrinogen binding to chymotrypsin-treated platelets correlated with changes in binding of a monoclonal antibody (10E5) specific for an epitope on the GPIIb-IIIa complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and radioelectroimmunoassay of DB-treated platelets, however, showed no evidence of a reduction or degradation of GP IIb or IIIa. Platelet incubation with DB (five minutes, 0.1 to 1.0 mmol/L) was also accompanied by: increased platelet membrane-associated Ca2+ involving low-affinity binding sites [Kd = 5 X 10(-5) mol/L-]; increased 45Ca2+ uptake which correlated with degradation of actin-binding protein (ABP) and digestion of GPIb as visualized on periodic-acid Schiff (PAS)- stained SDS gels and as inferred from decreased binding of a monoclonal antibody (6D1) directed against this glycoprotein; and enhanced Ca2+ exchange. Thus, exposure of platelets to DB results in membrane-related alterations that may contribute to inhibition of platelet cohesion: Decreased fibrinogen receptor exposure by traditional agonists and diminished accessibility of the GPIIb-IIIa complex to extracellular ligands correlate with DB-induced inhibition of platelet aggregation; and increased calcium uptake and exchange across the platelet membrane likely leads to activation of the calcium-dependent protease(s) which was previously shown to correlate with DB-induced inhibition of ristocetin-induced platelet agglutination.

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.

Diphenyl Ether Herbicides, a Tool to Elucidate the Mechanism of Photophosphorylation

1990 ◽  
Vol 45 (5) ◽  
pp. 552-557 ◽  
Author(s):  
Bernhard Huchzermeyer ◽  
Andreas Löhr
Keyword(s):  
Binding Sites ◽  
Diphenyl Ether ◽  
Coupling Factor ◽  
Conformational State ◽  
Nucleotide Exchange ◽  
Inhibitory Potency ◽  
High Affinity ◽  
High Affinity Site ◽  
Chloroplast Coupling Factor ◽  
Regulatory Functions

At least two different classes of ADP binding sites on chloroplast coupling factor are described in the literature. High-affinity sites are assumed to entail regulatory functions while low-affinity sites are involved in catalysis. Diphenyl ether herbicides, acting as energy transfer inhibitors, interfere with nucleotide exchange on both categories of ADP binding sites. Their inhibitory potency varies depending on their substitution. We found that each diphenyl ether assayed revealed identical I50 values for inhibition of nucleotide interaction with both classes of binding sites. We show here that diphenyl ether inhibition of energytransfer is primary based upon the interference with ADP binding to high-affinity binding sites. Thereby the control of proton permeability through the coupling factor complex is affected. Moreover, we found that the three β-subunits are not absolutely fixed in one conformational state: After covalently blocking the high-affinity site by an azido-label. a new high affinity state appeared on another β-subunit.

scholarly journals Two Binding Sites for [3H]PBR28 in Human Brain: Implications for TSPO PET Imaging of Neuroinflammation

2010 ◽  
Vol 30 (9) ◽  
pp. 1608-1618 ◽  
Author(s):  
David R Owen ◽  
Owain W Howell ◽  
Sac-Pham Tang ◽  
Lisa A Wells ◽  
Idriss Bennacef ◽  
...  
Keyword(s):  
Binding Sites ◽  
Marked Reduction ◽  
Specific Binding ◽  
Translocator Protein ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Binding Characteristics ◽  
High Affinity ◽  
High Affinity Site ◽  
Site Binding

[11C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [11C]PK11195, another TSPO radioligand. We measured the specific binding signals with [3H]PK11195 and [3H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [3H]PBR28, although all samples showed [3H]PK11195 binding. There was a marked reduction in the affinity of [3H]PBR28 for TSPO in samples with no visible [3H]PBR28 autoradiographic signal ( K i=188±15.6 nmol/L), relative to those showing normal signal ( K i=3.4±0.5 nmol/L, P<0.001). Of this latter group, [3H]PBR28 bound with a two-site fit in 40% of cases, with affinities ( K i) of 4.0±2.4 nmol/L (high-affinity site) and 313±77 nmol/L (low-affinity site). There was no difference in Kd or Bmax for [3H]PK11195 in samples showing no [3H]PBR28 autoradiographic signal relative to those showing normal [3H]PBR28 autoradiographic signal. [3H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [11C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.

High Affinity Binding Sites for Activated Protein C and Protein C on Cultured Human Umbilical Vein Endothelial Cells

1994 ◽  
Vol 72 (03) ◽  
pp. 465-474 ◽  
Author(s):  
Neelesh Bangalore ◽  
William N Drohan ◽  
Carolyn L Orthner
Keyword(s):  
Binding Sites ◽  
Protein C ◽  
Umbilical Vein ◽  
Activated Protein C ◽  
Affinity Binding ◽  
Cell Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Gla Domain ◽  
Umbilical Vein Endothelial Cells

SummaryActivated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4° C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal γ-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for ∼30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4°C compared to 37 °C were consistent with association of ∼25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37° C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombotic system to the vascular endothelium.

Sign in / Sign up

Export Citation Format

Share Document