The P2Y1 receptor is essential for ADP-induced shape change and aggregation in mouse platelets

Adenosine diphosphate (ADP) is an important platelet agonist, causing the shape change and aggregation required for physiological hemostasis. We have recently demonstrated that the P2Y1 receptor plays an important role in ADP-induced shape change and aggregation in human platelets. The role of the P2Y1 receptor in these physiological responses can be conclusively delineated with gene-knockout approaches in transgenic mice. However, before proceeding to the P2Y1 gene-knockout mice generation, it is important to demonstrate that the P2Y1 receptor plays an essential role in ADP-induced shape change and aggregation in mouse platelets. We examined platelets pooled from twenty 129J mice, a strain used in the generation of knockout mice. Immunofluorescence experiments using P2Y1 specific antiserum detected the presence of the P2Y1 receptor on mouse platelets. ARL 66096, a potent P2T AC receptor antagonist, caused a dose-dependent inhibition of both ADP-induced aggregation and ADP-induced inhibition of adenylyl cyclase, without affecting shape change or calcium mobilization. On the other hand, adenosine-2'-phosphate-5'-phosphate (A2P5P), a P2Y1 receptorselective antagonist, caused a dose-dependent inhibition of ADP-induced aggregation and shape change, as well as inhibiting the mobilization of calcium from intracellular stores. A2P5P had no effect on the inhibition of adenylyl cyclase by ADP. These findings clearly demonstrate the existence of two distinct ADP receptors, the P2Y1 and P2T AC , in mouse platelets with similar function as in human platelets.     

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.     

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-beta,gamma-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.     

Both the ADP receptors P2Y1 and P2Y12, 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 - 10.25 micro mol/l) blocked ADP-induced PSC (by 100%). The median IC(50) was 3.16 micro mol/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.     

An intact PDZ motif is essential for correct P2Y12 purinoceptor traffic in human platelets

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.     

Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation

ADP is an important platelet agonist causing shape change and aggregation required for physiological hemostasis. We recently demonstrated the existence of two distinct G protein-coupled ADP receptors on platelets, one coupled to phospholipase C, P2Y1, and the other to inhibition of adenylyl cyclase, P2T_AC. In this study, using specific antagonists for these two receptors, we demonstrated that concomitant intracellular signaling from both the P2T_AC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Inhibition of signaling through either receptor, by specific antagonists, is sufficient to block ADP-induced platelet aggregation. Furthermore, signaling through the P2T_AC receptor could be replaced by activation of α_2A-adrenergic receptors. On the other hand, activation of serotonin receptors supplements signaling through the P2Y1 receptor. Moreover, this mechanism of ADP-induced platelet aggregation could be mimicked by coactivation of two non-ADP receptors coupled to G_i and G_q, neither of which can cause platelet aggregation by itself. We propose that platelet aggregation results from concomitant signaling from both the G_i and G_q, a mechanism by which G protein-coupled receptors elicit a physiological response.     

Effects of P2Y 1 and P2Y 12 receptor antagonists on platelet aggregation induced by different agonists in human whole blood

ADP plays a major role in the amplification of platelet aggregation induced by other platelet agonists. ADP initiates platelet activation via the P2Y 1 receptor and amplifies platelet activation via the P2Y 12 receptor. Using the selective P2Y 1 receptor antagonist A2P5P and the selective P2Y 12 receptor antagonist AR-C69931MX, we assessed the relative contributions of P2Y 1 receptor and P2Y 12 receptor activation to platelet aggregation in hirudin-anticoagulated whole blood induced by PAF, 5HT, epinephrine, TRAP, streptokinase, U46619 and collagen. The effects of aspirin were assessed concurrently. A2P5P and AR-C69931MX variably inhibited aggregation induced by most of the agonists studied, whereas aspirin only inhibited aggregation induced by streptokinase and collagen. In some experiments, A2P5P and AR-C69931MX yielded additive inhibition of aggregation. All three antagonists interacted synergistically to inhibit collagen-induced aggregation. These studies demonstrate that P2Y 1 receptor activation plays a significant role in amplifying aggregation induced by agonists other than ADP, in addition to the established roles of P2Y 12 receptor activation and thromboxane A 2 synthesis.     

Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation, deaggregation and shape change responses mediated by the purinergic P2Y1 receptor in vitro

Platelet shape change (SC), aggregation and deaggregation responses are integral components of hemostasis that are elicited and modulated in vivo by the simultaneous activation of several membrane receptors. Selective activation of the purinergic P2Y1 receptor in vivo elicits a sustained SC and a small, transient aggregation response that is reversed rapidly by a robust deaggregation response (Platelets 2003; 14: 89). Using a kinetics-based turbidimetric approach to study the modulation of these concurrent components of human platelet responses, we demonstrate that these P2Y1 receptor-related responses and a number of their kinetic and steady-state characteristics are differentially elicited and modulated. P2Y1 receptor agonist concentrations that elicited aggregation (pEC50 for ADP, 2-MeSADP; 5.88, 6.69) were 10-fold greater than those that elicited SC (7.33, 7.67). The magnitude of the aggregation response was agonist concentration-dependent, saturable and was associated with an agonist concentration-dependent deceleration of the deaggregation response. Gi-coupled receptor (alpha 2A-adrenoceptor, EP3 and P2Y12 receptors) agonists also enhanced aggregation through deceleration of the deaggregation response, and an inhibitor of PI3K activity (wortmannin) inhibited aggregation through acceleration of the deaggregation response. Neither treatment affected the extent or the kinetics of the SC response. The aggregation but not the SC response was rapidly desensitized by P2Y1 receptor activation by ADP. The affinity of the presence of a single P2Y1 receptor subtype. The differential characteristics and modulation of the SC and aggregation responses by a single receptor support the idea that different signaling pathways activated at different occupancy states of the same receptor underlie the two responses. P2Y1 receptor-mediated platelet aggregation and SC responses provide a convenient model for studying the phenomenon of agonist-directed signaling by differential occupancy of the same membrane receptor.     

Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation,deaggregation and shape change responses mediated by the purinergic P2Y1 receptor in vitro

Platelet shape change (SC), aggregation and deaggregation responses are integral components of hemostasis that are elicited and modulated in vivo by the simultaneous activation of several membrane receptors. Selective activation of the purinergic P2Y₁ receptor in vitro elicits a sustained SC and a small, transient aggregation response that is reversed rapidly by a robust deaggregation response (Platelets 2003; 14: 89). Using a kinetics-based turbidimetric approach to study the modulation of these concurrent components of human platelet responses, we demonstrate that these P2Y₁ receptor-related responses and a number of their kinetic and steady-state characteristics are differentially elicited and modulated. P2Y₁ receptor agonist concentrations that elicited aggregation (pEC₅₀ for ADP, 2-MeSADP; 5.88, 6.69) were 10-fold greater than those that elicited SC (7.33, 7.67). The magnitude of the aggregation response was agonist concentration-dependent, saturable and was associated with an agonist concentration-dependent deceleration of the deaggregation response. G_i-coupled receptor (α_2A-adrenoceptor, EP3 and P2Y₁₂ receptors) agonists also enhanced aggregation through deceleration of the deaggregation response, and an inhibitor of PI3K activity (wortmannin) inhibited aggregation through acceleration of the deaggregation response. Neither treatment affected the extent or the kinetics of the SC response. The aggregation but not the SC response was rapidly desensitized by P2Y₁ receptor activation by ADP. The affinity of the selective P2Y₁ receptor antagonist, A3P5P, was independent of the response measured and is consistent with the presence of a single P2Y₁ receptor subtype. The differential characteristics and modulation of the SC and aggregation responses by a single receptor support the idea that different signaling pathways activated at different occupancy states of the same receptor underlie the two responses. P2Y₁ receptor-mediated platelet aggregation and SC responses provide a convenient model for studying the phenomenon of agonist-directed signaling by differential occupancy of the same membrane receptor.     

ADP ribose is an endogenous ligand for the purinergic P2Y1 receptor

The mechanism by which extracellular ADP ribose (ADPr) increases intracellular free Ca(2+) concentration ([Ca(2+)](i)) remains unknown. We measured [Ca(2+)](i) changes in fura-2 loaded rat insulinoma INS-1E cells, and in primary β-cells from rat and human. A phosphonate analogue of ADPr (PADPr) and 8-Bromo-ADPr (8Br-ADPr) were synthesized. ADPr increased [Ca(2+)](i) in the form of a peak followed by a plateau dependent on extracellular Ca(2+). NAD(+), cADPr, PADPr, 8Br-ADPr or breakdown products of ADPr did not increase [Ca(2+)](i). The ADPr-induced [Ca(2+)](i) increase was not affected by inhibitors of TRPM2, but was abolished by thapsigargin and inhibited when phospholipase C and IP(3) receptors were inhibited. MRS 2179 and MRS 2279, specific inhibitors of the purinergic receptor P2Y1, completely blocked the ADPr-induced [Ca(2+)](i) increase. ADPr increased [Ca(2+)](i) in transfected human astrocytoma cells (1321N1) that express human P2Y1 receptors, but not in untransfected astrocytoma cells. We conclude that ADPr is a specific agonist of P2Y1 receptors.     

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.     

Reciprocal cross-talk between P2Y1 and P2Y12 receptors at the level of calcium signaling in human platelets

Adenosine diphosphate (ADP), an important platelet agonist, acts through 2 G-protein-coupled receptors (GPCRs), P2Y(1) and P2Y(12), which signal through Gq and Gi, respectively. There is increasing evidence for cross-talk between signaling pathways downstream of GPCRs and here we demonstrate cross-talk between these 2 ADP receptors in human platelets. We show that P2Y(12) contributes to platelet signaling by potentiating the P2Y(1)-induced calcium response. This potentiation is mediated by 2 mechanisms: inhibition of adenylate cyclase and activation of phosphatidylinositol 3 (PI 3)-kinase. Furthermore, the Src family kinase inhibitor PP1 selectively potentiates the contribution to the calcium response by P2Y(12), although inhibition of adenylate cyclase by P2Y(12) is unaffected. Using PP1 in combination with the inhibitor of PI 3-kinase LY294002, we show that Src negatively regulates the PI 3-kinase-mediated component of the P2Y(12) calcium response. Finally, we were able to show that Src kinase is activated through P2Y(1) but not P2Y(12). Taken together, we present evidence for a complex signaling interplay between P2Y(1) and P2Y(12), where P2Y(12) is able to positively regulate P2Y(1) action and P2Y(1) negatively regulates this action of P2Y(12). It is likely that this interplay between receptors plays an important role in maintaining the delicate balance between platelet activation and inhibition during normal hemostasis.     

P2Y1 receptor signaling is controlled by interaction with the PDZ scaffold NHERF-2

P2Y(1) purinergic receptors (P2Y(1)Rs) mediate rises in intracellular Ca(2+) in response to ATP, but the duration and characteristics of this Ca(2+) response are known to vary markedly in distinct cell types. We screened the P2Y(1)R carboxyl terminus against a recently created proteomic array of PDZ (PSD-95/Drosophila Discs large/ZO-1 homology) domains and identified a previously unrecognized, specific interaction with the second PDZ domain of the scaffold NHERF-2 (Na(+)/H(+) exchanger regulatory factor type 2). Furthermore, we found that P2Y(1)R and NHERF-2 associate in cells, allowing NHERF-2-mediated tethering of P2Y(1)R to key downstream effectors such as phospholipase Cbeta. Finally, we found that coexpression of P2Y(1)R with NHERF-2 in glial cells prolongs P2Y(1)R-mediated Ca(2+) signaling, whereas disruption of the P2Y(1)R-NHERF-2 interaction by point mutations attenuates the duration of P2Y(1)R-mediated Ca(2+) responses. These findings reveal that NHERF-2 is a key regulator of the cellular activity of P2Y(1)R and may therefore determine cell-specific differences in P2Y(1)R-mediated signaling.     

Uridine adenosine tetraphosphate is a novel neurogenic P2Y1 receptor activator in the gut

Enteric purinergic motor neurotransmission, acting through P2Y1 receptors (P2Y1R), mediates inhibitory neural control of the intestines. Recent studies have shown that NAD⁺ and ADP ribose better meet criteria for enteric inhibitory neurotransmitters in colon than ATP or ADP. Here we report that human and murine colon muscles also release uridine adenosine tetraphosphate (Up4A) spontaneously and upon stimulation of enteric neurons. Release of Up4A was reduced by tetrodotoxin, suggesting that at least a portion of Up4A is of neural origin. Up4A caused relaxation (human and murine colons) and hyperpolarization (murine colon) that was blocked by the P2Y1R antagonist, MRS 2500, and by apamin, an inhibitor of Ca²⁺-activated small-conductance K⁺ (SK) channels. Up4A responses were greatly reduced or absent in colons of P2ry1^−/− mice. Up4A induced P2Y1R–SK-channel–mediated hyperpolarization in isolated PDGFRα⁺ cells, which are postjunctional targets for purinergic neurotransmission. Up4A caused MRS 2500-sensitive Ca²⁺ transients in human 1321N1 astrocytoma cells expressing human P2Y1R. Up4A was more potent than ATP, ADP, NAD⁺, or ADP ribose in colonic muscles. In murine distal colon Up4A elicited transient P2Y1R-mediated relaxation followed by a suramin-sensitive contraction. HPLC analysis of Up4A degradation suggests that exogenous Up4A first forms UMP and ATP in the human colon and UDP and ADP in the murine colon. Adenosine then is generated by extracellular catabolism of ATP and ADP. However, the relaxation and hyperpolarization responses to Up4A are not mediated by its metabolites. This study shows that Up4A is a potent native agonist for P2Y1R and SK-channel activation in human and mouse colon.Uridine adenosine tetraphosphate (Up4A) is, to the authors’ knowledge, the first dinucleotide isolated from living organisms that contains both purine and pyrimidine moieties. Up4A is a recently-identified, nonpeptide, endothelium-derived vasoconstrictor (1, 2). Up4A is likely associated with blood pressure regulation, because its levels in plasma are elevated in hypertensive subjects (3) and it causes vasoconstriction in deoxycorticosterone acetate-salt hypertensive rats (4) and type 2 diabetic rats (5). Up4A also contracts rat and human airways (6) and rat gastric smooth muscles (7). Pharmacological studies suggest that Up4A causes vasoconstriction via activation of P2X1, P2Y2, and P2Y4 receptors (1) and endothelium-dependent vasodilatation via activation of P2Y1 and P2Y2 receptors (8). In porcine coronary artery Up4A causes vasodilatation via adenosine (P1) receptors (9). Furthermore, Up4A causes vascular smooth muscle cell proliferation and migration (10), stimulates monocyte and lymphocyte oxidative burst activities (11), is a potent proinflammatory agent in the vascular wall (12), and may contribute to the proinflammatory status in patients with chronic kidney disease (11). Plasma of healthy human subjects contains ∼50 nmol/L Up4A, which is sufficient to elicit vascular effects (1). The role of Up4A in the gastrointestinal (GI) tract is unknown.Enteric neural regulation of GI motility includes motor neurotransmission mediated by inhibitory neurons releasing purines that act via P2Y1 receptors (P2Y1Rs) (13–17) and apamin-sensitive small-conductance Ca²⁺-activated K⁺ (SK) channels (13, 14, 18, 19). ATP (20), NAD⁺ (14, 15, 21), and adenosine 5′-diphosphate ribose (ADPR) (17) activate P2Y1R and SK channels and might be inhibitory neurotransmitters in the colon (22). Because Up4A appears to stimulate P2Y1Rs in endothelium, and P2Y1Rs are important for purinergic signaling in the colon, we investigated whether Up4A is released in colonic muscle, whether Up4A affects membrane potentials and contractions of colonic muscles, whether Up4A is an agonist for P2Y1R, whether cells expressing PDGF receptor α (PDGFRα) are targets of Up4A, and how Up4A is metabolized in colons of humans and mice.     

The P2Y1 receptor, necessary but not sufficient to support full ADP-induced platelet aggregation, is not the target of the drug clopidogrel

Recently we showed that the P2Y₁ receptor coupled to calcium mobilization is necessary to initiate ADP-induced human platelet aggregation. Since the thienopyridine compound clopidogrel specifically inhibits ADP-induced platelet aggregation, it was of interest to determine whether the P2Y₁ receptor was the target of this drug. Therefore we studied the effects of clopidogrel and of the two specific P2Y₁ antagonists A2P5P and A3P5P on ADP-induced platelet events in rats. Although clopidogrel treatment (50 mg/kg) greatly reduced platelet aggregation in response to ADP as compared to untreated platelets, some residual aggregation was still detectable. In contrast, A2P5P and A3P5P totally abolished ADP-induced shape change and aggregation in platelets from both control and clopidogrel-treated rats. A2P5P and A3P5P (100 μM ) totally inhibited the [Ca²⁺]_i rise induced by ADP (0.1 μM ) in control and clopidogrel-treated platelets, whereas clopidogrel treatment had no effect. Conversely, the inhibition of adenylyl cyclase induced by ADP (5 μM ) was completely blocked by clopidogrel but not modified by A2P5P or A3P5P (100 μM ). A3P5P (1 m M ) reduced the number of [³³P]2MeSADP binding sites on control rat platelets from 907 ± 50 to 611 ± 25 per platelet. After clopidogrel treatment, binding of [³³P]2MeSADP decreased to 505 ± 68 sites per platelet and further decreased to 55 ± 12 sites in the presence of A3P5P (1 m M ). In summary, these results demonstrate that the platelet P2Y₁ receptor responsible for the initiation of aggregation in response to ADP is not the target of clopidogrel. Platelets may express another, as yet unidentified, P2Y receptor, specifically coupled to the inhibition of adenylyl cyclase and necessary to induce full platelet aggregation, which could be the target of this drug.     

Blockade of adenosine diphosphate receptors P2Y(12) and P2Y(1) is required to inhibit platelet aggregation in whole blood under flow.

Using heparinized whole blood and flow conditions, it was shown that adenosine 5'-diphosphate (ADP) receptors P2Y(12) and P2Y(1) are both important in direct shear-induced platelet aggregation and platelet aggregation subsequent to initial adhesion onto von Willebrand factor (vWf)-collagen. In the viscometer, whole blood was subjected to shear rates of 750, 1500, and 3000 s(-1) for 30 seconds at room temperature. The extent of aggregation was determined by flow cytometry. The P2Y(12) antagonist AR-C69 931MX (ARMX) reduced shear-induced aggregation at these rates by 56%, 54%, and 16%, respectively, compared to control samples. Adenosine 3',5'-diphosphate (A3P5P; P2Y(1) antagonist) inhibited shear-induced aggregation by 40%, 30% and 29%, respectively, compared to control samples. Blockade of both ADP receptors at 3000 s(-1) with ARMX plus A3P5P further reduced the platelet aggregation by 41% compared to the addition of ARMX alone (57% compared to control samples). Using a parallel-plate flow chamber, whole blood was perfused over bovine collagen type 1 at a wall shear rate of 3000 s(-1) for 60 seconds. Platelet deposition was quantified with epifluorescence video microscopy and digital image processing. Blockade of P2Y(12) alone or blockade of P2Y(1) alone did not reduce thrombus formation on vWf-collagen. In contrast, blockade of both P2Y(12) and P2Y(1) reduced platelet deposition by 72%. These results indicate that combinations of antagonists of the ADP receptors P2Y(12) and P2Y(1) are effective inhibitors of direct shear-induced platelet aggregation and of platelet aggregation subsequent to initial adhesion under flow conditions. Inhibitors of these pathways are potentially useful as antiarterial thrombotic agents.     

The melanocortin 2 receptor accessory protein exists as a homodimer and is essential for the function of the melanocortin 2 receptor in the mouse y1 cell line

The ACTH receptor [melanocortin 2 receptor (MC2R)] gene produces a functional receptor only when transfected into cells of adrenocortical origin, implying that it may require an adrenal-specific accessory factor. Recently we showed that the MC2R accessory protein (MRAP) is essential for the cell surface expression of the MC2R in such models. Using RNA interference (RNAi) technology, we have further explored the action of MRAP in the functioning of the MC2R in Y1 mouse adrenocortical cells that endogenously express MRAP and MC2R. We created stable cell lines expressing mouse MRAP short hairpin RNA (shRNAs) by transfecting cells with an expression vector containing the MRAP small interfering RNA sequence. The knockdown of MRAP resulted in a reduction in MC2R signaling. The overexpression of a mouse MRAP-Flag construct did not restore the expression of MRAP due to its degradation by the mouse shRNAs. The introduction of human MRAP that is resistant to silencing by mouse MRAP shRNAs resulted in the rescue of the MC2R signaling. MRAP migrates on SDS-PAGE with markedly lower mobility than predicted for a 14.1-kDa protein. Coimmunoprecipitation and mass spectroscopy suggests that MRAP exists as a homodimer that is resistant to dissociation by sodium dodecyl sulfate and reducing agents.     

ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'- p-fluorosulfonylbenzoyladenosine is a weak platelet agonist

Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5'-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.