SummarySimultaneous measurements of [Ca2+]i and light transmission were used to examine the relationship between P2X1 receptor activation and functional platelet responses. The P2X1 agonist α,β-MeATP evoked a transient [Ca2+]i increase and a reversible decrease in light transmission; both responses required external Ca2+ and the nucleotidase apyrase. The transmission response was due to shape change only, verified by scanning electron microscopy and insensitivity to Reopro, a GPIIbIIIa antagonist. α,β-MeATP stimulated smaller shape changes than ADP, however P2X1 responses had a lifespan of <2 h following resuspension in saline and may be considerably larger in vivo. A peak [Ca2+]i increase of >50 nM was required for detectable shape change. Overlap of concentration-response relationships for α,β-MeATP-evoked [Ca2+]i and shape change suggests that other second messengers are not involved. Therefore, the physiological P2X1 agonist ATP can contribute to platelet activation, in contrast to its previously described inhibitory action at metabotropic platelet purinoceptors.

Road, Leicester LE1 9HN 1 ADP, an important agonist in thrombosis and haemostasis, has been reported to activate platelets via three receptors, P2X 1 , P2Y 1 and P2T AC . Given the low potency of ADP at P2X 1 receptors and recognized contamination of commercial samples of adenosine nucleotides, we have re-examined the activation of P2X 1 receptors by ADP following HPLC and enzymatic puri®cation. 2 Native P2X 1 receptor currents in megakaryocytes were activated by a,b-meATP (10 mM) and commercial samples of ADP (10 mM), but not by puri®ed ADP (10 ± 100 mM). 3 Puri®ed ADP (up to 1 mM) was also inactive at recombinant human P2X 1 receptors expressed in Xenopus oocytes. Puri®cation did not modify the ability of ADP to activate P2Y receptors coupled to Ca 2+ mobilization in rat megakaryocytes. 4 In human platelets, P2X 1 and P2Y receptor-mediated [Ca 2+ ] i responses were distinguished by their di erent kinetics at 138C. In 1 mM Ca 2+ saline, a,b-meATP (10 mM) and commercial ADP (40 mM) activated a rapid [Ca 2+ ] i increase (lag time 40.5 s) through the activation of P2X 1 receptors. Hexokinase treatment of ADP shifted the lag time by &2 s, indicating loss of the P2X 1 receptor-mediated response. 5 A revised scheme is proposed for physiological activation of P2 receptors in human platelets. ATP stimulates P2X 1 receptors, whereas ADP is a selective agonist at metabotropic (P2Y 1 and P2T AC ) receptors.

1 We have examined the role of ATP-dependent P2X 1 receptors in megakaryocytes (MKs) and platelets using receptor-de®cient mice and selective agonists. 2 a,b-meATP-and ATP-evoked ionotropic inward currents were absent in whole-cell recordings from MKs of P2X 1 7/7 mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X 1 receptors. 3 P2X 1 receptor de®ciency had no e ect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. 4 P2Y receptor-stimulated [Ca 2+ ] i responses were una ected in MKs from P2X 1 7/7 mice, however the inward cation current associated with Ca 2+ release was reduced by *50%, suggesting an interaction between the membrane conductances activated by P2X 1 and P2Y receptors. 5 Interaction between P2X 1 and P2Y receptors in human platelets was also examined using [Ca 2+ ] i recordings from cell suspensions. a,b-meATP (10 mM) evoked a rapid transient P2X 1 receptormediated increase in [Ca 2+ ] i , whereas ADP-(10 mM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co-application of a,b-meATP and ADP resulted in marked acceleration and ampli®cation of the peak [Ca 2+ ] i response. 6 We conclude that ionotropic P2X 1 receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.

Defining an appropriate and efficient assessment of drug‐induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc‐prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14‐based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B‐based “double‐negative” nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high‐dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double‐negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile establishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development.

SummaryG-protein-coupled P2Y1 and P2Y12 receptors play key roles in platelet activation, however the importance of ionotropic P2X1 receptors remains unclear. Platelet P2X1 responses are highly labile in vitro, but were greatly enhanced by increasing [Ca2+]o in the range 1–10 mM. The P2X1 agonist α,β-MeATP stimulated a shape change which saturated at peak [Ca2+]i of ≥ 400 nM, without evidence for aggregation. The maximal P2X1-evoked transmission decrease was 82% of that obtained via P2Y1 receptors. α., β-MeATP caused a disc to sphere transformation in virtually all platelets, but lacked the long processes produced by ADP. Following block of P2Y1 receptors with A3P5PS, co-stimulation with α., β-MeATP and ADP failed to induce aggregation despite the generation of peak [Ca2+]i responses similar to those stimulated via P2Y1 receptors. Therefore early, transient Ca2+ influx via P2X1 receptors can contribute to platelet activation by stimulating a significant morphological change, but does not readily synergise with P2Y12 receptors to support aggregation.

Purine nucleotides acting through P2 receptors play key roles in platelet signaling. The P2X 1 receptor is an adenosine triphosphate (ATP)-gated ion channel that mediates a rapid calcium influx signal, but can also synergize with subsequent adenosine diphosphate (ADP)-evoked P2Y 1 receptormediated responses and thus may contribute to platelet activation during hemostasis. Recent studies have shown that P2X 1 receptors contribute to the formation of platelet thrombi, particularly under conditions of high shear stress. Based on intracellular Ca 2؉ measurements a previous report has suggested that a splice variant of the P2X 1 receptor, P2X 1del , is expressed in platelets and, in contrast to the full-length P2X 1 WT receptor, is activated by ADP. In the present study we show that the P2X 1del receptor fails to form functional ion channels and is below the limit of detection in human platelets. Furthermore, ADP does not contribute to the rapid ionotropic P2X receptor-mediated response in platelets. These results support the notion that ATP is the principal physiologic agonist at P2X 1 receptors and that it plays a role in the activation of platelets. IntroductionPurine nucleotides acting through P2 receptors play key roles in platelet signaling and hemostasis. P2 receptors can be subdivided into P2X ionotropic and P2Y metabotropic receptors, and transgenic mice models have now firmly established roles of P2X 1 , P2Y 1 , and P2Y 12 receptors in platelet function. 1-6 P2X 1 receptors for adenosine triphosphate (ATP) mediate rapid transient increases in calcium concentration that can synergize with the larger, more sustained response through the adenosine diphosphate (ADP)-dependent P2Y 1 receptor, leading to speeding and amplification of the calcium increase. 4 In addition, P2X 1 receptor-deficient mice have recently been shown to have a decreased aggregation response and to exhibit protection against thromboembolism. 7 A major problem that has complicated the understanding and characterization of P2 receptors is the use of agonists to define P2 receptors because impurity or interconversion of the agonist has led to the pharmacologic misclassification of receptors. For example uridine diphosphate (UDP) appears as a weak agonist at the uridine triphosphate (UTP)-sensitive P2Y 4 receptor; however, when contaminating UTP is removed with hexokinase, agonist activity is essentially abolished. 8 The same problem has also influenced the study of P2 receptors in platelets and characterization of the specificity of action of different purines at individual receptors. For the P2X 1 receptor we have shown that the apparent activity of ADP at the receptor can be accounted for by ATP contamination of commercially available ADP. 9,10 This has important implications for the physiologic activation of platelet purinoceptors because ATP acts as a competitive antagonist rather than an agonist at platelet P2Y 1 receptors as a result of their low level of expression. 11,12 Thus, in platelets ATP and ADP are selective stimuli at P2X 1 an...

No abstract