ADP-induced Platelet Shape Change Research Articles

The sGC stimulator riociguat inhibits platelet function in washed platelets but not in whole blood.

Stimulation of soluble guanylyl cyclase (sGC) is a valuable therapeutic strategy for the treatment of several cardiovascular diseases. The sGC stimulator riociguat has been approved for the treatment of two forms of pulmonary hypertension. Platelets contain large amounts of sGC and play a key role in the regulation of haemostasis. Therefore, we investigated the effects of riociguat on platelet function. The effect of riociguat treatment on human platelet activation and aggregation was investigated. The sGC-specific effects of riociguat were determined by comparing wild-type and platelet-specific sGC-knockout mice. Riociguat induced cGMP synthesis and subsequent PKG activation in human platelets, suggesting that the inhibitory effects are mediated by cGMP signalling. This finding was confirmed when sGC-knockout platelets were not inhibited by riociguat. In washed human platelets, 100 nM riociguat reduced ADP-induced GPIIb/IIIa activation, while a 10-fold higher concentration was required to reduce convulxin-stimulated GPIIb/IIIa activation. Riociguat inhibited ADP-induced platelet shape change and aggregation, while ATP-induced shape change remained unaffected. However, in PRP and whole blood, 50-100 μM riociguat was required to inhibit platelet activation and aggregation. Riociguat in combination with iloprost significantly inhibited platelet aggregation, even in whole blood. Riociguat inhibits platelet activation in whole blood only at concentrations above 50 μM, while the plasma concentrations in riociguat-treated patients are 150 to 500 nM. This finding indicates that riociguat treatment does not affect platelet function in patients. Nevertheless, the possibility that riociguat acts synergistically with iloprost to inhibit platelet activation should be considered.

Nucleoside triphosphates inhibit ADP, collagen, and epinephrine-induced platelet aggregation: Role of P2Y1 and P2Y12 receptors

Platelets express two ADP receptors namely P2Y1 and P2Y12 that regulate ADP and other agonists-induced platelet aggregation. P2Y1 receptor activation causes platelet shape change while P2Y12 receptor activation induces platelet aggregation. Previously, anti-aggregatory effects of ATP on ADP-induced and pro-aggregatory effects on epinephrine-induced platelet aggregation have been reported. However, the effects of other nucleoside triphosphates on platelet aggregation have never been described. The aim of the present study was to characterise the effects of nucleoside triphosphates (ATP, UTP, GTP, and CTP) on agonist-induced platelet aggregation. The experiments were performed on platelet rich plasma freshly isolated from blood donated by healthy human volunteers. All the nucleoside triphosphates tested inhibited ADP- and collagen-induced platelet aggregation in a concentration-dependent manner with a rank order of potency, 2MeSATP >ATP ≥ α,β,methyleneATP>UTP >>CTP ≥ GTP. The IC50 values against ADP (10 μM)-induced platelet aggregation were 0.039 ± 0.013, 18 ± 7, 25 ± 6, 32 ± 9, 360 ± 130, and 400 ± 160 μM, respectively. Low concentrations of ATP induced platelet shape change which was due to contaminating ADP. However, higher concentrations antagonised ADP and MRS2365-induced platelet shape change. The ATP analogue α,β,methyleneATP and CTP but not UTP and GTP also antagonised ADP-induced platelet shape change. Similarly, low ATP concentrations potentiated epinephrine-induced platelet aggregation that was abolished by P2Y1 antagonist MRS2500 suggesting P2Y1 receptor activation due to contaminating ADP. Higher ATP concentrations, α,β,methyleneATP, UTP, CTP, and GTP antagonised epinephrine-induced platelet aggregation. Thus, the data demonstrate nucleoside triphosphates in general act as P2Y12 receptor antagonists and antagonise ADP-, collagen-, and epinephrine-induced platelet aggregation.

Selective serotonin reuptake inhibitors reduce P2Y12 receptor-mediated amplification of platelet aggregation

Selective serotonin reuptake inhibitors (SSRIs) have been reported to reduce platelet aggregation induced by ADP. ADP induces platelet aggregation through two purinergic receptors P2Y1 and P2Y12. We characterized the inhibitory properties of SSRIs on ADP-induced platelet aggregation and investigated the effects of SSRIs on the signaling pathways downstream of P2Y1 and P2Y12 receptors. Specific antagonists were used to evaluate which purinergic receptor-mediated aggregation was influenced by SSRIs. The primary phase of ADP-induced aggregation was not inhibited by citalopram. Citalopram failed to influence ADP-induced platelet shape change, intracellular calcium mobilization and the early phosphorylation of PKCα. Differently, citalopram inhibited the secondary phase of ADP-induced platelet aggregation in a concentration-dependent manner. Other SSRIs, including fluoxetine and sertraline, exhibited the same anti-platelet effects. Under P2Y1 blockade, citalopram inhibited platelet aggregation and integrin αIIbβ3 activation in response to ADP, indicating that citalopram inhibited P2Y12-mediated aggregation. Citalopram concentration-dependently inhibited the phosphorylation of Akt, GSK3β, p38 MAPK and Syk induced by ADP, but showed no effect on the decrease of cAMP and VASP phosphorylation. With integrin αIIbβ3 blockade, however, the phosphorylation of Akt triggered by ADP was unaltered by the addition of citalopram. Taken together, under the stimulation of ADP, SSRIs inhibit the amplification of platelet aggregation secondary to the activation of P2Y12 receptor, and subsequently reduce the activation of the downstream molecules of the outside-in signaling pathways.

CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways.

The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.

Evidence that the purinergic receptor P2Y12 potentiates platelet shape change by a Rho kinase-dependent mechanism

ADP activates human platelets through two G-protein coupled receptors, P2Y1 and P2Y12, to induce a range of functional responses. Here we have addressed the role and mechanism of P2Y12 in modulating ADP-induced platelet shape change. Although the response depended upon activation of P2Y1, it was potentiated by P2Y12 as the P2Y12-selective antagonists AR-C69931MX and 2MeSAMP partially inhibited shape change in the later phase of the response. This was paralleled by inhibition of pseudopod formation, platelet spheration, actin polymerisation and myosin light chain phosphorylation. P2Y12 is known to couple to activation of PI3 kinase and inhibition of adenylate cyclase, but we showed that neither of these signalling events couples to regulation of shape change by this receptor. However, by assessment of phosphorylation of its major substrate myosin light chain phosphatase, we provide direct evidence for activation of Rho kinase by ADP, and that although P2Y1 is required for activation of Rho kinase, P2Y12 is able to potentiate its activity. We conclude that P2Y12 plays a potentiatory role in ADP-induced shape change through regulation of the Rho kinase pathway, potentiating both myosin phosphorylation and actin polymerisation, and this forms part of an important signalling pathway additional to its well-established Gi-coupled pathways.

C60 - Éruptions eczématiformes sous inhibiteurs calciques. Données issues de la base nationale française de pharmacovigilance

Selective serotonin reuptake inhibitors (SSRIs) have been reported to reduce platelet aggregation induced by ADP. ADP induces platelet aggregation through two purinergic receptors P2Y1 and P2Y12. We characterized the inhibitory properties of SSRIs on ADP-induced platelet aggregation and investigated the effects of SSRIs on the signaling pathways downstream of P2Y1 and P2Y12 receptors. Specific antagonists were used to evaluate which purinergic receptor-mediated aggregation was influenced by SSRIs. The primary phase of ADP-induced aggregation was not inhibited by citalopram. Citalopram failed to influence ADP-induced platelet shape change, intracellular calcium mobilization and the early phosphorylation of PKCα. Differently, citalopram inhibited the secondary phase of ADP-induced platelet aggregation in a concentration-dependent manner. Other SSRIs, including fluoxetine and sertraline, exhibited the same anti-platelet effects. Under P2Y1 blockade, citalopram inhibited platelet aggregation and integrin αIIbβ3 activation in response to ADP, indicating that citalopram inhibited P2Y12-mediated aggregation. Citalopram concentration-dependently inhibited the phosphorylation of Akt, GSK3β, p38 MAPK and Syk induced by ADP, but showed no effect on the decrease of cAMP and VASP phosphorylation. With integrin αIIbβ3 blockade, however, the phosphorylation of Akt triggered by ADP was unaltered by the addition of citalopram. Taken together, under the stimulation of ADP, SSRIs inhibit the amplification of platelet aggregation secondary to the activation of P2Y12 receptor, and subsequently reduce the activation of the downstream molecules of the outside-in signaling pathways.

The P2 Receptors and Congenital Platelet Function Defects

Platelets possess three P2 receptors: two (P2Y (1) and P2Y (12)) are receptors for adenosine diphosphate (ADP), and one (P2X1) is a receptor for adenosine triphosphate (ATP). The P2Y (1) receptor, which is coupled to Gq and phospholipase C-beta, is responsible for mobilization of ionized calcium from internal stores and mediates the ADP-induced platelet shape change and initial wave of rapidly reversible aggregation. The other ADP receptor, P2Y (12), is negatively coupled to adenylyl cyclase through Gi and mediates a progressive and sustained ADP-induced aggregation not preceded by shape change. In addition, this receptor plays an important role in the potentiation of platelet secretion induced by several platelet agonists. The combined action of P2Y (1) and P2Y (12) is necessary for the full platelet aggregation response to ADP. Four patients with severe deficiency of P2Y (12) have been described so far. Sequence analysis of the P2Y (12) locus of three of these patients revealed homozygous mutations that produced a frame shift mutation and premature truncation of the protein. The fourth patient had an allele with a frame shift mutation and a normal allele, which could be silenced by an additional, as yet unknown, mutation. More recently, we described a patient with a congenital bleeding disorder and a dysfunctional P2Y (12). The patient is a compound heterozygote, in whom one allele contained a G to A transition resulting in an Arg (256) to Gln codon substitution (R256Q) and the other allele contained a C to T transition resulting in an Arg (265) to Trp codon substitution (R265W). The two substitutions are located in TM6 and EL3 of the receptor. Stable Chinese hamster ovaries (CHO) cell lines were established expressing either wild-type P2Y (12) and P2Y (12)(R256Q) or P2Y (12)(R265W). Neither mutation blocked the ability of the P2Y (12) receptor to translocate to the CHO cell surface. ADP at all tested concentrations (0.1 to 10 muM) greatly inhibited the forskolin-induced increase of cyclic adenosine monophosphate (cAMP) in CHO cells transfected with wild-type P2Y (12), whereas CHO cells transfected with either mutant protein were only partially inhibited by ADP. Thus, the molecular basis for the patient's dysfunctional platelet phenotype is explained by missense mutations and the expression of a dysfunctional P2Y (12) receptor. The localization of both mutations in TM6 and EL3 identifies this region of P2Y (12) as a structurally and functionally critical region of the receptor.

Impaired Platelet Function in a P2Y12-Deficient Patient Caused by a Mutation in the Translation Initiation Codon.

Recent studies have demonstrated that ADP plays a critical role in platelet activation. Platelets possess at least two major G protein-coupled ADP receptors that are largely responsible for platelet responses to ADP: P2Y1 and P2Y12. P2Y12 is the therapeutic target of efficacious antithrombotic agents, such as ticlopidine and clopidogrel, and its congenital deficiency results in a bleeding disorder. In this study, we have identified a patient with the congenital P2Y12 deficiency and analyzed the role of P2Y12 in platelet function. The proband (OSP-1) is a 67-year-old Japanese female with a lifelong history of easy bruising. In response to high concentrations of agonists OSP-1's platelets showed a specifically impaired aggregation to ADP stimulation. The impaired platelet aggregation was neither improved even at 100 μM ADP stimulation nor reduced by adding 1 μM AR-C69931MX, a specific P2Y12 antagonist. ADP induced platelet shape change normally and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets, further suggesting the defect in P2Y12 dependent signal. 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. HEK293 cells transfected with wild-type P2Y12 construct expressed a 60 kD-protein, while cells transfected with mutant P2Y12 construct failed to express any related protein. These data confirmed that the mutation was responsible for the deficiency in P2Y12 and denied the possibility that the substitution might induce an alternative translation starting at downstream ATGs leading to an expression of shorter form of P2Y12.Platelet spreading onto immobilized fibrinogen was impaired in OSP-1 platelets as well as in control platelets in the presence of 1 μM AR-C69931MX. Under static conditions, activation of integrin αIIbβ3 measured by PAC1-binding in OSP-1 platelets was only 5 to 12 % of controls even at high concentrations of PAR1-TRAP, PAR4-TRAP or U46619. These results suggest that ADP released by Gq-coupled receptors and P2Y12-dependent signal are critical for sustained activation of integrin αIIbβ3. Real time observations of thrombogenesis on a type I collagen-coated surface under a high shear rate (2000 s−1) revealed that thrombi were unstable. Most of the aggregates of OSP-1 platelets were unable to resist against high shear stress and loosely-attached aggregates came off into the blood stream. Thrombus height and volume measured at the plateau phase was 50% of control. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signals are critical for platelet spreading, stable integrin αIIbβ3 activation and, as a consequence, for stabilization of thrombus. These results provide a novel insight for the role of P2Y12 and endogenous ADP released into a limited space between thrombus-forming platelets.

Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y1 Receptor Are Crucial for Gq Coupling.

ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y1 and P2Y12 receptors. The Gq-activating P2Y1 receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y1 receptor in Gq activation. Human P2Y1 receptors, either wild type (P2Y1-WT) or a mutant in which the C-terminus was truncated (P2Y1-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y1-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y1-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y1 receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y1-WT- and P2Y1-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y1-ΔR340-L373 and P2Y1-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y1 receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y1 receptor are essential for Gq coupling and subsequent platelet activation.

Platelet Receptors for Adenine Nucleotides and Thromboxane A2

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.

Both the ADP receptors P2Y 1 and P2Y 12 , play a role in controlling shape change in human platelets

Two types of ADP receptors, P2Y 1 and P2Y 12 are involved in platelet aggregation. The P2X 1 receptor is also present but its role, in terms of platelet function, is not yet defined. The aim of this study was to establish if the ADP receptors, P2Y 1, P2Y 12 and P2X 1 play a role in controlling platelet shape change (PSC) in human platelets. PSC is an early phase of platelet activation that precedes aggregation. Using a high-resolution channelyzer, PSC was assessed by measuring the median platelet volume (MPV). The P2Y 1 receptor antagonist MRS 2179 (1.06 m 10.25 µmol/l) blocked ADP-induced PSC (by 100%). The median IC 50 was 3.16 µmol/l. MRS 2179 also significantly ( P = 0.01) inhibited PSC induced by the combination of ADP + serotonin (5HT). The P2Y 12 receptor antagonist AR-C69931MX significantly inhibited (at 10s, P = 0.009; 15 s, P = 0.001 and 30 s, P = 0.015) ADP-induced PSC. The P2X 1 receptor antagonist TNP-ATP had no significant effect on ADP- or ADP + 5HT-induced PSC. We conclude that the IC 50 of a P2Y 1 -blocker can be derived because of the high-resolution and reproducibility of the channelyzer technique. In addition to the P2Y 1 purinoceptor, the P2Y 12 receptor appears to be involved in ADP-induced PSC since this process was significantly inhibited by AR-C69931MX. The channelyzer technique may be more reliable than optical aggregometry to assess PSC.

Dynamic regulation of microtubule coils in ADP-induced platelet shape change by p160ROCK (Rho-kinase)

Platelet shape change is an extremely rapid process mediated by both the calcium-sensitive and p160ROCK pathways. The present study examines how different features of shape change studied by scanning electron microscopy clearly correlate to changes in the pattern of light absorbance measured in an aggregometer. Platelets change shape from the initial 'disc' form by producing: membrane 'blebs', sphere formation (cell-rounding), filopodia extension, and surface membrane folding. The presentation of these features was dramatically slower in the absence of intracellular calcium mobilization. In the presence of the p160ROCK-inhibitor, Y-27632, shape change was initially normal but platelets rapidly transformed back to smooth discs with extended filopodia. The reappearance of the disc shape is reflected by an increase in the amplitude of oscillations in the aggregometer shape change tracing. The kinetics of actin/cytoskeleton association correlated with filopodia formation but not with disc to sphere transformation. Changes in the level of tubulin polymerization correlated with changes from disc to sphere morphology. These experiments are consistent with a role for a RhoA/Rho kinase-regulated pathway in the maintenance of a spherical platelet shape after agonist-dependent activation. Continued disruption of the cytoskeletal microtubule ring, appears to be a Rho kinase-dependent event involved in the transformation of discoid platelets into spheres.

The Effect of Clopidogrel (Plavix) on Platelet Function in Patients with Peripheral Vascular Disease ‐ Comparison with Aspirin

Aims: To establish if clopidogrel (Plavix) is a more effective platelet inhibitor than aspirin in patients with peripheral vascular disease (PVD). Introduction:PVD is a common condition that is associated with a considerable increase in the risk of vascular events, like myocardial infarction (MI) and stroke. Aspirin resistance has been reported in PVD patients. These patients tend to have hyperactive platelets. Aspirin is an inhibitor of the synthesis of thromboxane A2 (TXA2), a promoter of platelet aggregation. In contrast, clopidogrel acts at the level of the platelet ADP receptor. ADP is released by activated platelets to further induce aggregation on other platelets. The rationale behind combination therapy is that aspirin and clopidogrel act by different mechanisms. Methods: Twenty PVD patients (14 men; 6 women: mean age 70 years) took part in this study. Citrated venous blood was collected at baseline (visit 1, n = 20), 10 days after clopidogrel (75 mg/day) or aspirin (75 mg/day) (visit 2, n = 10 for each drug), and then 10 days after combination therapy with aspirin plus clopidogrel, (visit 3, n = 20). Platelet aggregation was measured in whole blood using a MD18 Coulter counter, after adding ADP (5–10 μmol/l) or 5HT (serotonin, 5.0 μmol/l). Spontaneous platelet aggregation (SPA) was followed for up to 15 min. Platelet shape change (PSC) was assessed after adding agonists (5-HT, 0.5 μmol/l; ADP, 0.4-0.8 μmol/l -in platelet rich plasma) using a high-resolution (0.07 fl) channelyzer (ZM34 coupled to a Coulter counter). Median platelet volume (MPV) was measured at various time points. Results: There was a significant decrease in platelet aggregation, induced with ADP (measured as% free platelet count, FPC), after taking clopidogrel for 10 days, P = 0.0001. In contrast taking aspirin for 10 days showed no significant increase in FPC after adding ADP. The combination of clopidogrel plus aspirin significantly decreased SPA (P = 0.001) and also ADP-induced aggregation (P = <0.0001). SPA was not significantly altered by either drug used alone. The effect of either drug or both combined on 5HT-induced platelet aggregation was not significant. ADP-induced PSC (using% increase in MPV) was more significantly inhibited by clopidogrel (P = 0.004) than by aspirin (P = 0.01). This was also true for 5HT-induced PSC (clopidogrel P = 0.01, aspirin P = 0.03). However, with combination therapy the PSC effect was more marked (ADP, P = 0.0002, 5HT, P = 0.0006) than with either drug alone. Conclusions:In patients with PVD, clopidogrel is a more potent inhibitor of ADP-induced platelet activation than aspirin. There appears to be a beneficial combined effect of clopidogrel plus aspirin on platelet activation in PVD.

A general approach toward the synthesis of C-nucleoside pyrazolo[1,5-a]-1,3,5-triazines and their 3',5'-bisphosphate C-nucleotide analogues as the first reported in vivo stable P2Y(1)-receptor antagonists.

In our effort to identify potent purinergic P2Y(1) receptor antagonists as potent platelet aggregation inhibitors with enhanced metabolic stability, we developed an efficient route for the large-scale preparation of 2'-deoxy-C-nucleosides of pyrazolo[1,5-a]-1,3,5-triazine. The key strategic elements of this novel synthetic approach involved the following: (i) the use of a novel activating group, the N-methyl-N-phenylamino group, which was easily generated in high yield by treatment of the pyrazolo[1,5-a]-1,3,5-triazin-4-one (5) with phosphorus oxychloride and dimethylaniline under high pressure, (ii) a regio- and stereospecific palladium-mediated coupling reaction of the readily available unprotected glycal 1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol (4b) and the 8-iodo derivative (16), and (iii) the stereoselective reduction of the ketone group of the furanosyl ring followed by the subsequent displacement of the N-methyl-N-phenylamino group upon treatment with methylamine. The beta configuration at the anomeric C-1' position of the glycal moieties was perfectly retained throughout this conversion. This procedure afforded 8-(2'-deoxy-beta-D-ribofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (21) and 8-(2'-deoxy-beta-D-xylofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (24) with an overall yield of 50% and 39%, respectively. Finally, the conversion of nucleosides 21 and 24 to the pyrazolotriazine C-nucleotides 3',5'-bisphosphate 2 and 3',5'-cyclophosphate 26 is also described herein and represents the first reported nucleotide derivatives within the pyrazolo[1,5-a]-1,3,5-triazine series. Preliminary biological testing has shown that compound 2 strongly inhibits ADP-induced human platelet aggregation and shape change and possesses significant efficacies 30 min after injection in rat, highlighting a strong P2Y(1)-receptor antagonist activity in vitro combined with a prolonged duration of action in vivo.

Novel antagonists acting at the P2Y(1) purinergic receptor: synthesis and conformational analysis using potentiometric and nuclear magnetic resonance titration techniques.

The human P2Y(1) receptor is widely distributed in many tissues and has a classical structure of a G protein-coupled receptor. Activated by adenosine-5'-diphosphate (ADP), this receptor is essential for platelet aggregation. In the present paper, we describe the synthesis of novel P2Y(1) antagonists that could be of interest at least as tools to define the physiological roles of the P2Y(1) receptor, at best as new antithrombotic agents. Thus, we prepared the 2,N(6)-dimethyl-2'-deoxyadenosine-3',5'-bisphosphate derivative, 1e. The biological activity was demonstrated by the ability of compound 1e to inhibit ADP-induced platelet aggregation, shape change, and intracellular calcium rise. This compound was a full antagonist at the P2Y(1) receptor with a pA(2) value of 7.11 +/- 0.11 and was found to be 4-fold more potent than the reference N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (1a, pA(2) = 6.55 +/- 0.05), revealing the potency-enhancing effects of the 2-methyl group. The better activity of 1e as compared to 1a was analyzed using both potentiometric and nuclear magnetic resonance titration techniques, which highlighted specific conformational features of this compound. These results clearly indicate the preference for both compounds for an anti conformation at the N-glycosyl linkage. Furthermore, the percentage of S conformer of 1e is close to that of 1a, which is nearly 70% at pH = 2.8 and increases dramatically when pH increases. From the macroprotonation constants, it can be noted that compound 1e is significantly more basic than 1a. This is indeed expected for the N1 adenine nitrogen due to the electron-donating character of the methyl moiety. By considering the microconstants of the phosphate groups, the higher basicity of P3 and P5 for 1e may be due to the decrease in the local dielectric constant induced by the substitution of the hydrogen atom by a more lipophilic methyl group. Thus, it may be suggested that the gain in activity of 1e when compared to the reference compound 1a would result from its gain in basicity rather than steric and conformational modifications. The synthesis of the first selective radioligand acting at the P2Y(1) receptor ([(33)P]-N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate, 17) is also reported and will be used in the future for efficient screening needed for drug optimization.

Effects of P2Y 1 and P2Y 12 receptor antagonists on ADP-induced shape change of equine platelets: comparison with human platelets

Platelet activation by adenosine 5' -diphosphate (ADP) is via both P2Y 1 and P2Y 12 receptors and leads to shape change and aggregation. The effects on ADP-induced platelet shape change of two P2Y 1 antagonists, adenosine 3'-phosphate, 5'-phosphosulfate (A3P5PS) and 2-deoxy- N 6 -methyladenosine 3', 5'-diphosphate (MRS-2179) and a P2Y 12 antagonist 2-propylthio- D - g , n -dichloromethylene-adenosine 5'-triphosphate (AR-C67085MX) were determined by turbidimetric aggregometry and scanning electron microscopy (SEM) on equine and human platelets. The platelet aggregation was inhibited during aggregometry by 4-[4-[4(aminoiminomethyl)phenyl]-1-piperazinyl]-1-piperidin acid hydrochloride trihydrate (GR 144053F), an inhibitor of fibrinogen binding. From aggregation profiles, concentration-response curves and SEM we conclude that the shape change of equine platelets was susceptible to inhibition by the P2Y 1 antagonists A3P5PS and MRS-2179, but less so than human platelets. The P2Y 12 antagonist AR-C67085 did not influence significantly the shape change of either equine or human platelets.

The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor.

P2Y receptor activation in many cell types leads to phospholipase C activation and accumulation of inositol phosphates, while in blood platelets, C6-2B glioma cells, and in B10 microvascular endothelial cells a P2Y receptor subtype, which couples to inhibition of adenylyl cyclase, historically termed P2Y(AC), (P2T(AC) or P(2T) in platelets) has been identified. Recently, this receptor has been cloned and designated P2Y(12) in keeping with current P2 receptor nomenclature. Three selective P(2T) receptor antagonists, with a range of affinities, inhibited ADP-induced aggregation of washed human or rat platelets, in a concentration-dependent manner, with a rank order of antagonist potency (pIC(50), human: rat) of AR-C78511 (8.5 : 9.1)>AR-C69581 (6.2 : 6.0)>AR-C70300 (5.4 : 5.1). However, these compounds had no effect on ADP-induced platelet shape change. All three antagonists had no significant effect on the ADP-induced inositol phosphate formation in 1321N1 astrocytoma cells stably expressing the P2Y(1) receptor, when used at concentrations that inhibit platelet aggregation. These antagonists also blocked ADP-induced inhibition of adenylyl cyclase in rat platelets and C6-2B cells with identical rank orders of potency and overlapping concentration - response curves. RT - PCR and nucleotide sequence analyses revealed that the C6-2B cells express the P2Y(12) mRNA. These data demonstrate that the P2Y(AC) receptor in C6-2B cells is pharmacologically identical to the P2T(AC) receptor in rat platelets.

Inhibition of platelet function by administration of MRS2179, a P2Y 1 receptor antagonist

The effects of a potent P2Y 1 receptor antagonist, N 6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179) on adenosine-5′-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca 2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [ 33P]MRS2179 showed that washed human platelets displayed 134±8 binding sites per platelet with an affinity ( K d) of 109±18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y 1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y 1 receptor with a view to antithrombotic therapy.

ADP-induced platelet shape change: an investigation of the signalling pathways involved and their dependence on the method of platelet preparation

Shape change is an important early event in platelet activation. In this study we show that the Ca2+ chelator BAPTA and the Rho-kinase inhibitor Y-27632 inhibit ADP-induced myosin light chain (MLC) phosphorylation and platelet shape change through distinct pathways and with distinct kinetics. Ca2+ is largely responsible for the initial onset of shape change, whilst Rho-kinase plays a major role in the maintenance of the response. The relative contribution of these two pathways to each stage of the response was dependent on the method of platelet preparation, but in all cases shape change was shown to be downstream of the P2Y1 receptor. Similar observations were made in murine platelets. The shape change response was modulated via changes in cAMP levels, possibly via the P2T AC receptor, but not by tyrosine phosphorylation. We conclude that ADP-induced A shape change occurs via the P2Y1 receptor, which can be differentially coupled to Rho-kinase and Ca2+-linked pathways dependent on the method of platelet preparation.

Platelet Shape Change Is Mediated by both Calcium-dependent and -independent Signaling Pathways: ROLE OF p160 Rho-ASSOCIATED COILED-COIL-CONTAINING PROTEIN KINASE IN PLATELET SHAPE CHANGE

Platelets undergo shape change upon activation with agonists. During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia. When agonist-induced cytosolic Ca(2+) increases were prevented using the cytosolic Ca(2+) chelator, 5, 5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5, 5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic Ca(2+), agonist-stimulated myosin light chain phosphorylation was significantly inhibited. The myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, thrombin, or U46619-induced Ca(2+)-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and myosin light chain phosphorylation. Our results indicate that Ca(2+)/calmodulin-stimulated myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to myosin light chain phosphorylation and platelet shape change, through Ca(2+)-sensitive and Ca(2+)-insensitive pathways, respectively.