In the graphical abstract for the article above, the word ''interferons'' was inadvertently misspelled as ''interterons.'' The graphical abstract has been corrected and is now available online.

1 In the present study we have classi®ed the receptor(s) mediating increases in intracellular calcium concentration ([Ca 2+ ] i ) in human washed platelets and compared the pharmacological pro®le obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y 1 -receptor. 2 The P2Y 1 -receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar a nities (pK B of 5.8 and 6.0, respectively). 3 The selective P2Y ADP antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an a nity (pK A ) of 4.9. This value is consistent with its known P2Y 1 -receptor activity. In platelets, AR-C66096 at a concentration (0.1 mM) approximately 100 fold greater than its known P2Y ADP receptor a nity, had no e ect on ADP-induced increases in [Ca 2+ ] i . 4 The ability of adenine nucleotide analogues to elevate [Ca 2+ ] i in the Jurkat cells was also determined. The rank order of agonist potency (p[A] 50 ) was: 2-MeSADP (8.3)42-ClATP (7.8)4ADP (7.5)=2-MeSATP (7.4)4ATPgS (6.5)4ATP (6.2), with ATP appearing to be a partial agonist. 5 The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPgS and ATP were lower in platelets. 6 The operational model of agonism was used to test whether the agonist concentration-e ect pro®les obtained in these two cell types could be explained on the basis of di erences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y 1 -receptor system. 7 The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an a nity value for ATP similar to that obtained previously at P2Y 1 -receptors. 8 In summary, the results of this study indicate that human washed platelets contain P2Y 1 -receptors which mediate increases in [Ca 2+ ] i and that this receptor population is pharmacologically distinct from P2Y ADP -receptors.

1. In the present study we have investigated the roles of P2Y(1) and P(2T) receptor subtypes in adenosine 5'-diphosphate (ADP)-induced aggregation of human platelets in heparinized platelet rich plasma. 2. The response to ADP can be characterized as the initial rate or the maximum or final extent of aggregation. The response profile is determined by the concentration of ADP used, being transient at lower and sustained at higher concentrations. 3. The P2Y(1) receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) competitively antagonized the initial rate of aggregation (pK(B) 5. 47) and transformed the response profile to a slowly developing but sustained response. Both maximum and final extents were also inhibited by A3P5P although not in a competitive manner (Schild slope <1). 4. The P(2T) receptor antagonist, AR-C67085, competitively antagonized the final extent of aggregation (pK(B) 8.54), transforming the response profile to one of rapid, transient aggregation. Its effect on maximum extent (the most widely used index of aggregation) was complex, and further supported the involvement of both receptor subtypes in the aggregation response. 5. ADP-induced aggregation is a complex phenomenon, the nature of which is determined by the relative occupancy of the two receptor subtypes. While P2Y(1) receptor activation causes a rapid and transient aggregation, the extent of sustained aggregation is determined by the level of P(2T) receptor occupancy. Hence, detailed analysis of the aggregation response is essential to correctly define the purinergic pharmacology of the platelet and interpretation of results is critically dependent on the response index chosen.

1 This paper describes the effects of GRI17289 (1-[[3-bromo-2-[2-(lH-tetrazol-5-yl)phenyl]-5-benzofuranyl]methyl]-2-butyl-4-chloro-lH-imidazole-5-carboxylic acid) at angiotensin receptors and binding sites in rabbit aorta, rat liver and bovine cerebellum preparations in vitro. 2 In rabbit isolated aortic strips, GRI 17289 (0.3, 1 and 3 nM) caused a concentration-related, insurmountable suppression of the concentration-response curve to angiotensin II (All). When the contact time was increased, a greater degree of antagonism of All was observed, suggesting that GRi17289 is slow to reach equilibrium. A pKB of 9.8±0.1 was calculated for GRI17289 after 3h incubation.GR1 17289 (1 tiM) did not affect contractile responses to phenylephrine or 5-hydroxytryptamine (5-HT) in the rabbit aorta. 3 GRI 17289 (1 nM) alone caused a marked suppression and a slight rightward displacement of the All concentration-response curve. Co-incubation with the competitive, surmountable AT, receptor antagonist, losartan (10 nM, 100 nm and 1 ,LM), resulted in a concentration-related upward and rightward displacement of the concentration-response curve to subsequently administered All. In separate experiments in which preparations were pre-incubated with GR1 17289 (1 nM), subsequent addition of losartan (1 JLM) for 2, 15 or 45 min caused a further, but similar, rightward displacement of the concentration-response curve to subsequently administered All with a time-dependent increase in the maximum response. 4 Suppression of All-induced contractile responses, caused by superfusion with GRI17289 (0.3, 1 or 3 nM) was not reversed by continuously washing the tissues for 3 h; in fact, the potency of GRI 17289 was slightly enhanced after this period.5 In rat liver membranes, GRI17289 was a potent competitor with [3H]-AII for AT, binding sites (pKi = 8.7 ± 0.1) but in bovine cerebellum membranes, it was a very weak competitor for AT2 binding sites (pKi<6). Pre-incubation of rat liver membranes with GRI17289 had little effect on its affinity (pKi = 9.1 ± 0.21), but increasing the concentration of bovine serum albumen in the assay buffer from 0.001% to 0.1% w/v decreased affinity (pKi= 7.5 ± 0.1). 6In saturation binding experiments in rat liver membranes, GRI 17289 (12 nM) increased the Kd of[3H]-AII from 0.28 ± 0.06 nM to 0.37 ± 0.02 nM, and decreased Bm. from 10.0 ± 0.1 to 5.6 ± 0.3 fmol mg' tissue. In other experiments, GR1 17289 (1 jIM) did not alter the rate of dissociation of[3H]-AII from AT1 binding sites, following addition of excess unlabelled All.7 In rabbit aorta vascular smooth muscle membranes, GR1 17289 competed with ['25I]-Sar'1le8 All for binding to AT, binding sites. In the presence of 0.1% w/v bovine serum albumen, a pIC50 of 7.6 ± 0.1 was calculated. Under the same conditions, but with rat liver membranes, a pIC50 of 7.8 ± 0.1 was determined.8 Taken together, these results show that GRI17289 is a potent, specific, selective and insurmountable antagonist at angiotensin AT, receptors. Its profile in the rabbit aorta is consistent with ...