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.

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-activating factor (PAF-acether) induces high- and low-affinity binding of fibrinogen to human platelets via independent mechanisms

1986 ◽  
Vol 240 (2) ◽  
pp. 403-412 ◽  
Author(s):  
E Kloprogge ◽  
J W Akkerman
Keyword(s):  
Platelet Activation ◽  
Binding Sites ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Affinity Binding ◽  
Binding Studies ◽  
High Affinity Binding ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
A Minor

When human platelets are incubated with 500 nM-PAF-acether (platelet-activating factor. 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) under equilibrium conditions (60 min, 22 degrees C, non-stirred suspensions), two classes of fibrinogen binding sites are exposed: one class with a high affinity [Kd (7.2 +/- 2.1) X 10(-8) M, 2367 +/- 485 sites/platelet, n = 9] and one class with a low affinity [Kd (5.9 +/- 2.4) X 10(-7) M, 26972 +/- 8267 sites/platelet]. Preincubation with inhibitors of cyclo-oxygenase (acetylsalicylic acid, indomethacin) or thromboxane synthetase (UK 38.485) completely abolishes high-affinity binding, leaving low-affinity binding unchanged. In contrast, ADP scavengers (phosphocreatine/creatine kinase or phosphoenol pyruvate/pyruvate kinase) completely prevent low-affinity binding, leaving high-affinity binding unaltered. Initial binding studies (2-10 min incubation) confirm these findings with a major part of the binding being sensitive to ADP scavengers, a minor part sensitive to indomethacin and complete blockade with both inhibitors. Increasing the temperature to 37 degrees C decreases the number of low affinity-binding sites 6-fold without changing high-affinity binding. Aggregation, measured as the rate of single platelet disappearance, then depends on high-affinity binding at 10 nM-fibrinogen or less, whereas at 100 nM-fibrinogen or more low-affinity binding becomes predominant. These findings point at considerable platelet activation during binding experiments. However, arachidonate metabolism [(3H]arachidonate mobilization and thromboxane synthesis) and secretion [(14C]serotonin and beta-thromboglobulin) are about 10% or less of the amounts found under optimal conditions (5 units of thrombin/ml 37 degrees C, stirring). We conclude that PAF-acether induces little platelet activation under binding conditions. The amounts of thromboxane A2 and secreted ADP, however, are sufficient for initiating high- and low-affinity fibrinogen binding via mutually independent mechanisms.

scholarly journals A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 570-577 ◽  
Author(s):  
CG Ruan ◽  
XP Du ◽  
XD Xi ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Scatchard Analysis ◽  
Type I ◽  
Glycoprotein Ib ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

Abstract A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

Characterization of intestinal smooth muscle responses and binding sites for endothelin

1992 ◽  
Vol 70 (3) ◽  
pp. 377-384 ◽  
Author(s):  
Gordon T. Bolger ◽  
Francine Liard ◽  
Michel Garneau ◽  
Jorge Jaramillo
Keyword(s):  
Smooth Muscle ◽  
Binding Sites ◽  
Contractile Activity ◽  
Benzodiazepine Receptor ◽  
Intestinal Smooth Muscle ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Receptor Antagonists ◽  
High Affinity Binding ◽  
High Affinity

The contractile activity of and binding sites for endothelin-1 (ET-1) were investigated in isolated guinea-pig ileal longitudinal smooth muscle (GPILM). ET-1 produced concentration-dependent contractions of GPILM that either slowly subsided in the continued presence of ET-1 or rapidly subsided following washing of the tissue. The ED50 value for ET-1 contractions was 4.2 ± 1.3 × 10−9 M. The removal of extracellular calcium or pretreatment with nifedipine produced a complete inhibition of the contractions to ET-1. The IC50 value of nifedipine for inhibition of ET-1 mediated contractions was 3.0 ± 0.8 × 10−8 M. ET-1 produced a marked prolonged homologous desensitization of its contractile response but did not affect the responses mediated by carbachol, histamine, serotonin, substance P, and PLA2. High-affinity binding sites for 125I-labelled ET-1 were identified on microsomal membranes prepared from GPILM with Kd and Bmax values obtained by Scatchard analysis of 3.5 ± 0.6 × 10−10 M and 2138 ± 159 fmol/mg protein, respectively. The binding of 125I-labelled ET-1 to GPILM microsomes was characterized by a rapid association (kob value of 0.077 min−1 at a radioligand concentration of 0.45 nM and an extremely slow dissociation (kl value of 0.011 min−1; t1/2 value of 793 min). The binding was unaffected by the calcium channel antagonists nifedipine, verapamil, and diltiazem (10−6 M); the receptor antagonists phenoxybenzamine, atropine, and naloxone (10−6 M) and propranolol; and the peripheral benzodiazepine receptor antagonists Ro 5-4864 and PK 11195 and psychotomimetic drug phencyclidine (10−5 M). Incubation of GPILM with ET-1 (2 × 10−8 M) for 10 min followed by washing of the tissue for 1 h resulted in a significant (p < 0.05 unpaired Student's t-test) reduction (33%) of 125I-labelled ET-1 binding that partially recovered following 2 h of washing the tissue. These results demonstrate that ET-1 is an intestinal smooth muscle spasmogen that produces its pharmacologic effects by a mechanism(s) that is not shared by other major intestinal neurotransmitters. Furthermore, intestinal smooth muscle contains specific high-affinity binding sites that likely mediate the contractile responses to ET-1.Key words: intestine, smooth muscle, endothelin, calcium channels, contraction.

scholarly journals UTP binding and phosphoinositidase C activation in ampulla from frog semicircular canal

2000 ◽  
Vol 279 (3) ◽  
pp. R803-R812 ◽  
Author(s):  
Marie Teixeira ◽  
Evelyne Ferrary ◽  
Daniel Butlen
Keyword(s):  
Semicircular Canal ◽  
Binding Sites ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
Reactive Blue 2 ◽  
High Affinity ◽  
Pyrimidine Nucleotide ◽  
Protein Dissociation

Pyrimidine nucleotide-sensitive phosphoinositidase C activity (PLC), previously identified in frog semicircular canal ampulla, was pharmacologically characterized. Binding of [3H]UTP and abilities of unlabeled nucleotide analogs to inhibit binding and to stimulate PLC in myo-[3H]inositol-loaded ampullas were determined. Specific [3H]UTP binding was competitively inhibited by UTP [apparent dissociation binding constant = 0.8 μM; Hill coefficient = 0.7]. Scatchard analysis revealed a minor class of high-affinity binding sites [45 fmol UTP bound/μg protein; dissociation constant ( K D1) = 0.4 μM] and a major class of moderate-affinity binding sites (365 fmol UTP bound/μg protein; K D2 = 10 μM). The stereospecificity pattern for UTP analog recognition was UMP > UDP ≥ ADP = UTP = dTTP > adenosine 5′- O-(3-thiotriphosphate) = ATP = CTP = 2′-and 3′- O-4-(benzoylbenzoyl)-ATP (Bz-ATP) ≥ AMP ≥ 2-methylthio-ATP = α,β-methylene-ATP > uridine = diadenosine tetraphosphate (Ap4A); cAMP and adenosine were inactive. Antagonist recognition pattern was DIDS = pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) = reactive blue 2 > suramin. The rank order of potencies for agonist-induced PLC activation was UDP ≥ UTP ≥ Ap4A ≥ UMP = Bz-ATP; uridine was inactive. UTP-stimulated PLC activity was inhibited by DIDS = reactive blue 2 = PPADS > suramin. These results suggest that the population of [3H]UTP-labeled binding sites is heterogeneous, with a low number of high-affinity UTP receptors whose function(s) need to be determined and a large number of moderate-affinity receptors triggering PLC activation.

scholarly journals Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex

Blood ◽  
1983 ◽  
Vol 61 (1) ◽  
pp. 140-148
Author(s):  
G Di Minno ◽  
P Thiagarajan ◽  
B Perussia ◽  
J Martinez ◽  
S Shapiro ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Arachidonic Acid ◽  
Binding Sites ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Scatchard Analysis ◽  
Single Class ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
High Affinity Site

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

scholarly journals A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 570-577 ◽  
Author(s):  
CG Ruan ◽  
XP Du ◽  
XD Xi ◽  
PA Castaldi ◽  
MC Berndt
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelet ◽  
Platelet Glycoprotein ◽  
Scatchard Analysis ◽  
Type I ◽  
Glycoprotein Ib ◽  
Induced Aggregation ◽  
Bernard Soulier Syndrome

A new monoclonal antibody (MoAb), SZ 2, reactive with the human platelet glycoprotein Ib complex has been produced by the hybridoma technique. SZ 2 immunoprecipitated the components of the glycoprotein Ib complex, glycoprotein Ib and glycoprotein IX, from Triton-X-100- solubilized, periodate-labeled platelets. Western blot analysis indicated that the epitope for SZ 2 was on the alpha-subunit of glycoprotein Ib. Scatchard analysis of SZ 2 binding to formaldehyde- fixed, washed platelets revealed a single class of binding sites with Kd = 6.6 +/- 3.3 X 10(-10) mol/L and 15,200 +/- 4,100 binding sites per platelet (mean +/- SD, n = 10). Intact antibody and its purified (Fab')2 fragments not only inhibited the ristocetin-dependent binding of von Willebrand factor to platelets and ristocetin-induced platelet agglutination but also inhibited platelet aggregation induced by Type I collagen and platelet-activating factor (PAF). SZ 2 inhibited platelet serotonin and beta-thromboglobulin release in response to these stimuli and also platelet thromboxane A2 formation in response to ristocetin and collagen. SZ 2 was without effect on platelet aggregation or release in response to other platelet stimuli such as ADP, thrombin, or arachidonic acid. The inhibition by SZ 2 of collagen- and PAF-induced platelet aggregation is surprising in that Bernard-Soulier syndrome platelets, which lack the glycoprotein Ib complex, respond normally to both these stimuli. SZ 2 was unreactive toward Bernard-Soulier syndrome platelets, as evaluated by fluorescence-associated cell sorting, and had no effect on the collagen- and PAF-induced aggregation of Bernard- Soulier syndrome platelets. The combined results suggest that the inhibition by SZ 2 of collagen- and PAF-induced aggregation of normal platelets is steric and are consistent with the glycoprotein Ib complex and the platelet collagen and PAF receptor(s) being adjacent in the human platelet plasma membrane.

Vagus nerve modulates secretin binding sites in the rat forestomach

1999 ◽  
Vol 276 (4) ◽  
pp. G1052-G1058 ◽  
Author(s):  
Hyeok Y. Kwon ◽  
Ta-Min Chang ◽  
Kae Y. Lee ◽  
William Y. Chey
Keyword(s):  
Gastric Motility ◽  
Binding Sites ◽  
Intravenous Infusion ◽  
Intraperitoneal Injection ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Secretin Receptor

Secretin is well known for its inhibitory action on gastric motility. It has been reported that secretin in a physiological dose inhibits gastric motility through mediation by the vagal afferent pathway. Secretin also elicited relaxation of carbachol-stimulated rat forestomach muscle strips by binding to its receptors, suggesting a direct action on this peripheral tissue. We hypothesized that vagal input may affect the action of secretin by modulating the level of secretin receptor in the forestomach. Several treatments, including vagal ligation, vagotomy, perivagal application of capsaicin or colchicine, intravenous infusion of tetrodotoxin, and intraperitoneal injection of atropine, were performed to investigate their effects on secretin receptor binding to forestomach membranes. Specific binding of125I-labeled secretin to forestomach membranes was significantly decreased (45%) by vagal ligation, vagotomy (50%), or perivagal colchicine treatment (40%). On the contrary, specific binding of125I-secretin was not affected by perivagal capsaicin treatment, intravenous infusion of tetrodotoxin, or intraperitoneal injection of atropine. By Scatchard analysis of the binding data, the capacity of the high-affinity binding sites in forestomach membranes was found to decrease significantly after vagal ligation compared with membranes from the sham-operated group. However, the affinity at the high-affinity binding sites, the binding parameters of the low-affinity binding sites, and binding specificity were not changed. Vagal ligation but not perivagal capsaicin treatment reduced the inhibitory effect of secretin on bethanechol-stimulated contraction of isolated forestomach muscle strips, causing a right shift in the dose-response curve. These results suggest that vagal input through axonal transport plays a significant role on secretin action by modulating the capacity of secretin binding sites (but not affinity or specificity), at least in rat forestomach.

A Novel Mechanism for Exposure of Fibrinogen Binding Sites on GPIIb-IIIa by a Monoclonal Antibody

1995 ◽  
Vol 73 (01) ◽  
pp. 138-143 ◽  
Author(s):  
Takaaki Hato ◽  
Akito Watanabe ◽  
Shingo Nakatani ◽  
Yoko Minamoto ◽  
Shigeru Fujita
Keyword(s):  
Monoclonal Antibody ◽  
Conformational Changes ◽  
Binding Sites ◽  
Platelet Membrane ◽  
Cross Linking ◽  
Membrane Glycoprotein ◽  
Cross Link ◽  
Specific Domain ◽  
Fibrinogen Binding ◽  
The Difference

SummaryConformational changes in platelet membrane glycoprotein (GP) IIb-IIIa, whose nature is not defined, lead to exposure of fibrinogen binding sites. We have reported previously that F(ab’)2fragments of a monoclonal antibody, PMA4, directed against the GPIIb-IIIa complex- specific domain, induced binding of fibrinogen to platelets without causing intracellular activation, whereas Fab did not. In this study, we examined the mechanism responsible for the difference in the ability of PMA4 F(ab’)2and Fab to expose fibrinogen binding sites. PMA4 Fab had affinity for GPIIb-IIIa similar to that of PMA4 F(ab’)2. Addition of F(ab’)2goat anti-mouse Fab antibody to cross-link PMA4 Fab-bound GPIIb-IIIa molecules induced fibrinogen binding. There was a direct correlation between the number of molecules of PMA4 F(ab’)2and the amount of fibrinogen bound. PMA4 did not recognize ligand-induced binding sites (LIBS). These results suggest that the cross-linking of special sites on the GPIIb-IIIa complex-specific domain by bivalent antibody alters the conformation of GPIIb-IIIa to a state competent to bind soluble fibrinogen and that conformational changes in non-LIBS are involved in the mechanism for exposing fibrinogen binding sites on GPIIb-IIIa.

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