Phencyclidine binds to blood platelets with high affinity and specifically inhibits their activation by adrenaline
Graham JamiesonA.K. AgrawalNicholas J. GrecoThomas E. TennerG D JonesKenner C. RiceArthur E. JacobsonJames G. WhiteNarendra N. Tandon
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Abstract:
The ion channel probe phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] selectively inhibited aggregation, secretion and ultrastructural changes in platelets induced by adrenaline, but did not affect activation induced by other common platelet agonists such as alpha-thrombin, ADP, collagen or ionophore A23187. [3H]PCP bound to platelets with high affinity (Kd 134 +/- 33 nM; 3600 +/- 1020 sites/platelet), as did the thienyl analogue [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine). PCP binding to platelets was increased 3-4-fold in N-methylglucamine buffer in the absence of Na+ ions. Binding was unaffected by haloperidol and was only weakly inhibited (EC50 10-20 microM), without significant stereoselectivity by the two sets of stereoselective ligands, dexoxadrol/levoxadrol and (+)MK801/(-)MK801. Binding of PCP was not competed for by adrenaline or yohimbine. Only the high-affinity binding of [3H]PCP to platelets was blocked by prior treatment of the platelets with the covalent affinity probe Metaphit, and these platelets no longer aggregated in response to adrenaline although they responded normally to alpha-thrombin, ADP and collagen. These results suggest that platelets contain high-affinity receptors for PCP that can modulate adrenaline-induced platelet activation.Keywords:
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Piperidine
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To learn more about the binding conformation of phencyclidine (PCP) and to arrive at analogues of higher affinity, which may serve as noncompetitive N-methyl-D-aspartate receptor antagonists, eight optically pure PCP analogues were designed with the aid of computer. These compounds represent conformationally constrained versions of PCP in which the motion of the phenyl ring is frozen, thus allowing a determination of the orientation of the phenyl ring relevant to binding. The analogues were synthesized by a Diels-Alder strategy and tested in a radioligand binding assay to evaluate their affinity for the PCP binding site of the N-methyl-D-aspartate receptor complex. One of the analogues was found to bind with nanomolar affinity (IC50 = 19 nM) and to be 73-fold more potent in binding than its enantiomer. These results, which further elucidate the structural determinants of high affinity binding, should aid both in the design of higher affinity molecular probes of the PCP binding site and in the discovery of potential neuroprotective agents.
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The properties of the sigma opioid receptor of rat brain cortex have been characterized using the prototypic ligand (+)-[3H] SKF 10,047. Binding to this receptor was rapid, and equilibrium was obtained within 30 min at 37 degrees C. Specific binding was linear with protein concentration up to 500 micrograms/2 ml and was dependent upon protein integrity. Denaturation by boiling destroyed over 95% of the specific binding. A high-affinity binding site with a KD of 150 +/- 40 nM and a maximum binding of 2.91 +/- 0.84 pmol/mg of protein was determined from a Scatchard plot of the binding data. The addition of salt, either NaCl or CaCl2, to the buffers markedly decreased binding, with CaCl2 being more potent than NaCl. A broad pH optimum for specific binding was observed; maximum binding was at pH 9.0. The affinity of a number of ligands for the sigma site and the phencyclidine receptor were compared. The binding (IC50) of 13 ligands to the sigma site showed a correlation of 0.86 (P less than .01) with binding to the phencyclidine site. The data demonstrate that the biochemical properties of the sigma and phencyclidine receptors are similar and support the view that these receptors are one and the same site.
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Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing γ′ chains, which are fibrinogen γ chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on γ′ chains (Ka, 4.9 × 106−1; n, 1.05 per γ′ chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 × 106−1; n, 1.69 per molecule). The amino-terminal β15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 μ, respectively). The presence of the high affinity γ′ chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on γ′ chains is a constitutive feature of fibrin as well as fibrinogen. Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing γ′ chains, which are fibrinogen γ chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on γ′ chains (Ka, 4.9 × 106−1; n, 1.05 per γ′ chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 × 106−1; n, 1.69 per molecule). The amino-terminal β15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 μ, respectively). The presence of the high affinity γ′ chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on γ′ chains is a constitutive feature of fibrin as well as fibrinogen.
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Two high affinity phencyclidine (PCP) binding sites, labeled by ({sup 3}H)1-(1-(2-thienyl)cyclohexyl)piperidine (({sup 3}H)TCP), have been identified in guinea pig brain, with one site coupled to the N-methyl-D-aspartate (NMDA) receptor (site 1) and the other site associated with the dopamine reuptake carrier complex (site 2). In this study, PCP enhanced the dissociation of ({sup 3}H)TCP from PCP site 1 and site 2, while (+){minus}MK801 only enhanced dissociation of ({sup 3}H)TCP from PCP site 1. Although additional studies will be required to determine the exact mechanism(s) of these effects, these data demonstrate that the interactions of PCP with both site 1 and site 2 are more complex than previously appreciated.
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Abstract The appearance of new drugs within the crime laboratory necessitates the development and improvement of analytical schemes for their detection. One class of particular interest is the phencyclidine-related drugs. The drugs studied are 1-(1-phenylcyclohexyl) piperidine (PCP), commonly known as phencyclidine, “Angel's Dust,” or DOA [1]; the two homologs 1-(1-phenylcyclohexyl) pyrrolidine (PHP) and 1-(1-phenylcyclopentyl) piperidine (PPP); and an analog 1-(2[thienyl]cyclohexyl) piperidine (TCP).
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