Inhibition of localized thrombosis in P2Y1-deficient mice and rodents treated with MRS2179, a P2Y1 receptor antagonist

Summary.  Previous studies in experimental models revealed a role for the P2Y₁ platelet ADP receptor in systemic vascular thromboembolism models. In the present work, we used models of localized arterial and venous thrombosis to assess the role of the P2Y₁ receptor in these processes. Arterial thrombosis was induced in one mesenteric arteriole of a mouse using FeCl₃, while venous thrombosis was studied in a Wessler model adapted to rats. P2Y₁-deficient mice and mice treated with the P2Y₁ antagonist MRS2179 displayed significantly less arterial thrombosis than their respective controls. Combination of P2Y₁ deficiency with P2Y₁₂ inhibition led to a significant additive effect. Venous thrombosis was slightly but significantly inhibited in MRS2179-treated rats. These results demonstrate a role for the P2Y₁ receptor in both arterial and venous thrombosis, further establishing this receptor as a potential target for antithrombotic drugs.     

Critical role of ADP interaction with P2Y12 receptor in the maintenance of αIIbβ3 activation: association with Rap1B activation

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.     

Inhibition of the platelet P2Y12 receptor for adenosine diphosphate potentiates the antiplatelet effect of prostacyclin

BACKGROUND: Activation of two receptors for adenosine diphosphate (ADP), P2Y(1) and P2Y(12), is necessary for ADP-induced platelet aggregation (PA). It is generally believed that the antithrombotic effects of drugs inhibiting P2Y(12), such as clopidogrel, are uniquely mediated by inhibition of P2Y(12)-dependent PA. However, as P2Y(12) is negatively coupled to adenylyl cyclase (AC), its inhibition may also exert antithrombotic effects through the potentiation of prostacyclin (PGI(2)), which inhibit PA by stimulating AC. OBJECTIVES: To test whether inhibition of P2Y(12) potentiates the antiplatelet effects of PGI(2). METHODS: We measured the effects of PGI(2) (0.01-10 microm) on PA of washed human platelets induced by thrombin (0.5 U mL(-1)) in the presence or absence of ARC69931MX (anti-P2Y(12)) or MRS2500 (anti-P2Y(1)). Results: PGI(2) inhibited PA in the presence of anti-P2Y(12), but not in the presence of anti-P2Y(1) or in the absence of inhibitors. In contrast, dibutyryl-cyclicAMP inhibited PA both in the presence and absence of anti-P2Y(1) or anti-P2Y(12). PGI(2) increased platelet cyclicAMP levels only in the absence of thrombin or in the presence of thrombin plus anti-P2Y(12). CONCLUSIONS: PGI(2) did not inhibit PA induced by thrombin, because its effect on AC was prevented by released ADP interacting with P2Y(12). Anti-P2Y(12) drugs, by rescuing AC activity, potentiate the antiplatelet effect of PGI(2), which may contribute to their antithrombotic effect.     

A differential role of the platelet ADP receptors P2Y1 and P2Y12 in Rac activation

The dynamics of the actin cytoskeleton, largely controlled by the Rho family of small GTPases (Rho, Rac and Cdc42), is critical for the regulation of platelet responses such as shape change, adhesion, spreading and aggregation. Here, we investigated the role of adenosine diphosphate (ADP), a major co-activator of platelets, on the activation of Rac. ADP rapidly activated Rac in a dose-dependent manner and independently of GPIIb/IIIa and phosphoinositide 3-kinase. ADP alone, used as a primary agonist, activated Rac and its effector PAK via its P2Y1 receptor, through a G(q)-dependent pathway and independently of P2Y12. The P2Y12 receptor appeared unable to activate the GTPase per se as also observed for the adenosine triphosphate receptor P2X1. Conversely, secreted ADP strongly potentiated Rac activation induced by FcgammaRIIa clustering or TRAP via its P2Y12 receptor, the target of antithrombotic thienopyridines. Stimulation of the alpha(2A)-adrenergic receptor/G(z) pathway by epinephrine was able to replace the P2Y12/G(i)-mediated pathway to amplify Rac activation by FcgammaRIIa or by the thrombin receptor PAR-1. This co-activation appeared necessary to reach a full stimulation of Rac as well as PAK activation and actin polymerization and was blocked by a G-protein betagamma subunits scavenger peptide.     

New P2Y12 blockers

Summary.  A number of new antiplatelet agents currently in development are anticipated to improve clinical outcomes and safety benefits in patients with acute coronary syndrome (ACS). This article reviews the pharmacology and clinical development of three of these agents: prasugrel, cangrelor, and ticagrelor. Prasugrel, a third-generation, oral thienopyridine, has been shown to be superior to clopidogrel, the current gold standard, in preventing ischemic events in patients with ACS undergoing percutaneous coronary intervention (PCI), although the bleeding rate was higher. Cangrelor, a chemical analog of adenosine triphosphate, is a potent direct platelet P2Y₁₂ antagonist. In development as an intravenous agent, cangrelor is currently being evaluated in two phase III studies in patients requiring PCI. Ticagrelor is the first of a new class of orally available antiplatelet agents antagonizing the effects of ADP mediated by P2Y₁₂; it is currently being studied in a phase III trial in patients with ACS.     

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .     

Transcriptional down-regulation of the platelet ADP receptor P2Y12 and clusterin in patients with systemic lupus erythematosus

Cardiovascular complications are common in systemic lupus erythematosus (SLE) and myocardial infarctions are the leading cause of increased mortality. The ADP receptor P2Y(12) plays a central role in platelet activation and the P2Y(12) blocker clopidogrel reduces the incidence of cardiovascular events. Clusterin, a complement inhibitory protein suggested to be involved in the pathogenesis of SLE, has been found recently in a microarray study to be expressed at very high levels in platelets. Using a new protocol for mRNA quantification in platelets we set out to study if gene expression is altered in SLE patients compared with a healthy control group. Quantitative assay based on real-time PCR was used to measure mRNA expression, Western blot for P2 receptor protein expression and PFA-100 for platelet aggregation. The P2Y(12) receptor expression was decreased in SLE compared to the controls (P < 0.05), while expression of P2Y(1) and P2X(1) were unaltered. These findings were consistent at the protein level. The clusterin mRNA expression was very high. However, SLE patients had significantly lower levels than controls (P < 0.05). Platelet aggregation was similar in both groups. It may be suggested that a decreased level of P2Y(12) receptors could represent a protective response in SLE against thrombotic complications. Lowered clusterin levels could be involved in the pathogenesis of SLE due to decreased protective effects. These findings could help to achieve a better understanding of the platelet function in SLE and serve as a guide for further research and drug use.     

P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.     

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP-induced receptor signaling and platelet aggregation

Summary. Background: P2Y₁₂ plays an important role in regulating platelet aggregation and function. This receptor is the primary target of thienopyridine antiplatelet agents, the active metabolites of which bind irreversibly to the receptor, and of newer agents that can directly and reversibly modulate receptor activity. Objective: To characterize the receptor biology of the first reversibly binding oral P2Y₁₂ antagonist, ticagrelor (AZD6140), a member of the new cyclopentyltriazolopyrimidine (CPTP) class currently in phase III development. Methods: Ticagrelor displayed apparent non‐competitive or insurmountable antagonism of ADP‐induced aggregation in human washed platelets. This was investigated using competition binding against [³H]ADP, [³³P]2MeS‐ADP and the investigational CPTP compound [¹²⁵I]AZ11931285 at recombinant human P2Y₁₂. Functional receptor inhibition studies were performed using a GTPγS‐binding assay, and further binding studies were performed using membranes prepared from washed human platelets. Results: Radioligand‐binding studies demonstrated that ticagrelor binds potently and reversibly to human P2Y₁₂ with K_on and K_off of (1.1 ± 0.2) × 10^?4 nm ^?1 s^?1 and (8.7 ± 1.4) × 10^?4 s^?1, respectively. Ticagrelor does not displace [³H]ADP from the receptor (K_i > 10 μm ) but binds competitively with [³³P]2MeS‐ADP (K_i = 4.3 ± 1.3 nm ) and [¹²⁵I]AZ11931285 (K_i = 0.33 ± 0.04 nm ), and shows apparent non‐competitive inhibition of ADP‐induced signaling but competitive inhibition of 2MeS‐ADP‐induced signaling. Binding studies on membranes prepared from human washed platelets demonstrated similar non‐competitive binding for ADP and ticagrelor. Conclusions: These data indicate that P2Y₁₂ is targeted by ticagrelor via a mechanism that is non‐competitive with ADP, suggesting the existence of an independent receptor‐binding site for CPTPs.     

P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

Gi-dependent and -independent mechanisms downstream of the P2Y12 ADP-receptor

Summary.  The P2Y₁₂ ADP receptor is one of the major regulators of platelet activation and the target of antithrombotic thienopyridines (ticlopidine and clopidogrel). It has been recently cloned but the signaling pathways triggered by this receptor are still poorly documented. Here, we show that stimulation of the human P2Y₁₂ receptor stably expressed in Chinese hamster ovary cells activates two major intracellular signaling mechanisms leading either to cell proliferation or to actin cytoskeleton reorganization. Both effects were blocked by the active metabolite of clopidogrel, a specific antagonist of P2Y₁₂. The P2Y₁₂-mediated stimulation of proliferation required the pertussis toxin-sensitive activation of PI3-kinase/Akt upstream of MAP-kinases. A partial contribution of a transactivation mechanism, through the tyrosine kinase receptor platelet-derived growth factor (PDGF)-R-β, was also observed. Conversely, the P2Y₁₂-mediated reorganization of the actin cytoskeleton was Gi-independent, requiring activation of RhoA and Rho-kinase. Our results provide new insights into the molecular basis of P2Y₁₂-mediated intracellular signaling. These data may prove to be useful for a better understanding of the physiological role of P2Y₁₂, particularly in platelets and glial cells which express this important therapeutic target.     

Adenosine reduces airway excitatory non-cholinergic (e-NC) contraction through both A1 and A2 adenosine receptor activation in the guinea pig

Summary— The influence of adenosine and selective A₁ and A₂ agonists and antagonists was investigated on the cholinergic and the excitatory non-cholinergic (e-NC) contractions induced by electrical field stimulation in the guinea-pig bronchi. Adenosine (10 nM-1 mM) induced a concentration-dependent inhibition of the e-NC contraction (EC₅₀ = 90 ± 14 μM), whereas the cholinergic peak was only slightly affected. Preincubation of the tissue with the adenosine uptake blocker dipyridamole (10 μM) significantly shifted the concentration-inhibition curve to adenosine to the left (EC₅₀ = 10 ± 1 μM), suggesting an interaction with extracellular adenosine receptors of A₁ and/or A₂ subtype. To characterize the receptor type involved in this effect, selective adenosine derivatives were studied. The agonist to both A₁ and A₂ adenosine receptors, 5′-N-ethylcarboxamidoadenosine (NECA) was more potent than the selective A₁ agonist, (-)-R-6-phenylisopropyladenosine (R-PIA), in inhibiting the e-NC contraction (EC₅₀ = 0.10 ± 0.04 and 0.60 ±0.12 μM, respectively, with a maximal inhibition of 70 and 45%, respectively). The concentration-response curve to NECA was shifted to the right by the A₂ receptor selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) (10 μM) (EC₅₀ = 1.4 ± 0.5 μ) as well as by the specific A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (10 μM) (EC₅₀ = 0.7 ± 0.3 μM). The inhibitory effect induced by the association of both antagonists, DPCPX and DMPX, was considerably potentiated (EC₅₀ > 22 ± 2.5 μM). The effect of R-PIA was also shifted to the right by DPCPX (EC₅₀ = 8.2 ± 1.6 μM) but was not modified by DMPX. The contractile response to exogenous substance P was unaffected by NECA pretreatment (0.3 μM). Altogether, these results suggest that adenosine-induced inhibition of e-NC contraction of guinea-pig bronchi is mediated through activation of both A₁ and A₂ adenosine receptors linked to inhibition of the release of neuropeptides from C-fibre nerve endings.     

Potentiation of platelet activation through the stimulation of P2X1 receptors

Summary.  The platelet P2X₁ purinergic receptor is a ligand-gated ion channel that responds to ATP. The precise role of P2X₁ in platelet function is unknown, though stimulation with the P2X₁ agonist α,β-Me-ATP is known to result in platelet shape change through elevation of calcium levels. The aim of the present study was to examine further the effects of P2X₁ stimulation on platelet activation. Stimulation of P2X₁ with α,β-Me-ATP resulted in shape change and small aggregate formation in heparin-anticoagulated platelet preparations. Given the ability of heparin to potentiate platelet activation, subsequent experiments were performed in hirudin. In these platelet preparations, aggregate formation in response to α,β-Me-ATP alone was less than that observed in heparin; however, α,β-Me-ATP significantly potentiated platelet aggregate formation when added in conjunction with other weak platelet agonists [epinephrine or thrombopoietin (TPO)]. Platelet aggregate formation was confirmed by single platelet loss (microaggregate formation), microscopy, and light transmittance studies. Further, the P2X₁ antagonist MRS-2159 inhibited platelet shape change and aggregation responses induced by α,β-Me-ATP. Overall, this study demonstrates that P2X₁ stimulation can induce/potentiate platelet activation in combination with other platelet agonists. These results are the first demonstration of platelet aggregation mediated through direct P2X₁ stimulation, supporting a role for this receptor in regulating platelet activation.     

ERK2 activation in arteriolar and venular murine thrombosis: platelet receptor GPIb vs. P2X1

The functional significance of extracellular signal-regulated kinase 2 (ERK2) activation was investigated during shear induced human platelet aggregation (SIPA) in vitro and during shear controlled thrombosis in vivo in intestinal arterioles and venules of wild type (WT) and transgenic (TG) mice with platelet-specific overexpression of human P2X(1) (TG). In SIPA, ERK2 was rapidly phosphorylated during GPIb stimulation, its activation contributing to SIPA for 50%, independently of P2X(1) regulation. Thrombotic occlusion of injured arterioles occurred considerably faster in TG (4.3 +/- 2.3 min) than in WT (38 +/- 8 min) arterioles, but occlusion times in TG (19 +/- 12) and WT (48 +/- 4.5 min) venules differed less. Both the alphabeta-meATP triggered desensitization of platelet P2X(1), as well as P2X(1) antagonism by NF279 or NF449 prolonged mean occlusion to about 75 min in WT and 65 min in TG arterioles, but venular occlusion times were less affected. Preventing ERK2 activation by U0126 prolonged occlusion times in TG (41 +/- 10 min) and WT (51 +/- 17) arterioles more than in TG (46 +/- 5 min) and WT (56 +/- 6 min) venules, uncovering a role for ERK2 in shear controlled thrombosis. Antagonism of GPIb by a recombinant murine von Willebrand factor (VWF)-A1 fragment prolonged occlusion times to comparable values, ranging from 55 to 58 min, both in TG and WT arterioles and venules. Further inhibition strategies, combining VWF-A1, U0126 and NF449 in WT and TG mice and resulting in occlusion in various time windows, identified that inhibition by VWF-A1 largely abrogated the ERK2 contribution to thrombosis. In conclusion, P2X(1) and ERK2 both participate in shear stress controlled thrombosis, but ERK2 activation is initiated predominantly via GPIb-VWF interactions.     

Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon

In this study, we have identified a patient (OSP-1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP-1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets. Molecular genetic analysis revealed that OSP-1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild-type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP-1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real-time observations of thrombogenesis under a high shear rate (2000 s(-1)) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP-1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.     

Pharmacological control of the human gastric histamine H2 receptor by famotidine: comparison with H1, H2 and H3 receptor agonists and antagonists

Histamine 0.1 microM-0.1 mM increased adenylate cyclase activity five- to ten-fold in human fundic membranes, with a potency Ka = 3 microM. The histamine dose-response curve was mimicked by the H3 receptor agonist (R) alpha-MeHA, but at 100 times lower potency, Ka = 0.3 mM. Histamine-induced adenylate cyclase activation was abolished by H2, H1 and H3 receptor antagonists, according to the following order of potency IC50: famotidine (0.3 microM) greater than triprolidine (0.1 mM) thioperamide (2 mM), respectively. Famotidine has no action on membrane components activating the adenylate cyclase system, including the Gs subunit of the enzyme stimulated by forskolin and cell surface receptors sensitive to isoproterenol (beta 2-type), PGE2 and VIP. The Schild plot was linear for famotidine (P less than 0.01) with a regression coefficient r = 0.678. The slope of the regression line was 0.64 and differs from unity. Accordingly, famotidine showed a slow onset of inhibition and dissociation from the H2 receptor in human cancerous HGT-1 cells. The results demonstrate that famotidine is a potent and selective H2 receptor antagonist with uncompetitive actions in human gastric mucosa. Consequently, famotidine might be a suitable drug with long-lasting actions in the treatment of Zollinger-Ellison syndrome. The results also confirm and extend the previous observations that (R) alpha-MeHA and thioperamide are two selective ligands at histamine H3 receptor sites. In the human gastric mucosa, these drugs are respectively 330 and 6700 times less potent than histamine and famotidine on the adenylate cyclase system. The possible involvement of histamine H3 receptors in the regulation of gastric secretion is proposed.     

5HT2 receptors in cerebral cortex of migraineurs studied using PET and 18F-fluorosetoperone

Since the brain 5HT₂ receptors might be implicated in migraine pathogenesis, we have used positron emission tomography and ¹⁸F-fluorosetoperone, a 5HT₂ specific radioligand, to investigate in vivo the cortical 5HT₂ receptors in migraine subjects. Nine migraineurs who had either migraine with and without aura ( n = 5) or only migraine without aura ( n = 4) were studied between attacks. Twelve unmedicated healthy subjects of similar mean age were used as controls. Brain radioactivity was measured after ¹⁸F-setoperone IV injection for 90 min. A decrease of the regional specific distribution volumes (SDV) of the ligand was observed both in migraineurs and in controls. The age adjusted group means of SDV did not differ between patients and controls for the whole and for the right or left frontal, temporal, parietal and occipital cortex. These results suggest that cortical 5HT₂ receptors may be unaltered between attacks in migraine sufferers.     

Increased platelet activation and thrombosis in transgenic mice expressing constitutively active P2Y12

Summary. Background: In our previous in vitro study, we reported a constitutively active chimeric P2Y₁₂ (cP2Y₁₂) and found that AR‐C78511 is a potent inverse agonist at this receptor. The role of cP2Y₁₂ in platelet activation and thrombosis is not clear. Objectives: To investigate the physiologic implications of cP2Y₁₂ for platelet activation and thrombus formation, and to evaluate the antiplatelet activity of AR‐C78511 as an inverse agonist. Methods and Results:  We generated transgenic mice conditionally and platelet‐specifically expressing cP2Y₁₂. High‐level expression of cP2Y₁₂ in platelets increased platelet reactivity, as shown by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice showed a shortened bleeding time, and more rapid and stable thrombus formation in mesenteric artery injured with FeCl₃. The constitutive activity of cP2Y₁₂ in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR‐C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited a superior antiplatelet effect to that of AR‐C69931MX in transgenic mice. Conclusions:  These findings further emphasize the importance of P2Y₁₂ in platelet activation, hemostasis, and thrombosis, as well as the prothrombotic role of the constitutive activity of P2Y₁₂. Our data also validate the in vivo inverse agonist activity of AR‐C78511, and confirm its superior antiplatelet activity over neutral antagonists.     

In the presence of strong P2Y12 receptor blockade,aspirin provides little additional inhibition of platelet aggregation

Summary. Background: Aspirin and antagonists of platelet ADP P2Y₁₂ receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y₁₂ receptors can inhibit thromboxane A₂ (TXA₂)‐dependent pathways of platelet activation independently of aspirin. Objectives: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y₁₂ receptor blockade. Methods: With the use of platelet‐rich plasma from healthy volunteers, determinations were made in 96‐well plates of platelet aggregation, TXA₂ production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP‐6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1–10 μmol L^?1), aspirin (30 μmol L^?1), PAM + aspirin or vehicle. Results: PAM concentration‐dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 μmol L^?1. In further tests of PAM (3 μmol L^?1), aspirin (30 μmol L^?1) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA₂ and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. Conclusions: P2Y₁₂ receptors are critical to the generation of irreversible aggregation through the TXA₂‐dependent pathway. As a result, strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.