Molecular Basis for ADP-induced Platelet Activation

Acting through cell surface receptors, ADP activates platelets resulting in shape change, aggregation, thromboxane A2 production, and release of granule contents. ADP also causes a number of intracellular events including inhibition of adenylyl cyclase, mobilization of calcium from intracellular stores, and rapid calcium influx in platelets. However, the receptors that transduce these events remain unidentified and their molecular mechanisms of action have not been elucidated. The receptor responsible for the actions of ADP on platelets has been designated the P2T receptor. In this study we have used ARL 66096, a potent antagonist of ADP-induced platelet aggregation, and a P2X ionotropic receptor agonist, α,β-methylene adenosine 5′-triphosphate, to distinguish the ADP-induced intracellular events. ARL 66096 blocked ADP-induced inhibition of adenylyl cyclase, but did not affect ADP-mediated intracellular calcium increases or shape change. Both ADP and 2-methylthio-ADP caused a 3-fold increase in the level of inositol 1,4,5-trisphosphate over control levels which peaked in a similar fashion to the Ca2+ transient. The increase in inositol 1,3,4-trisphosphate was of similar magnitude to that of inositol 1,4,5-trisphosphate. α,β-Methylene adenosine 5′-triphosphate did not cause an increase in either of the inositol trisphosphates. These results clearly demonstrate the presence of two distinct platelet ADP receptors in addition to the P2X receptor: one coupled to adenylyl cyclase and the other coupled to mobilization of calcium from intracellular stores through inositol trisphosphates.

ADP was the first low molecular weight agent recognized to cause platelet aggregation (1,2). It is stored in the dense granules of human platelets and is an important platelet agonist as evidenced by the fact that patients with defective ADP storage have bleeding tendencies (3,4). Activation of platelets by ADP follows a defined sequence. The first event, shape change, occurs when discoid shaped resting cells are rapidly converted to spiculated spheres. Shape change is followed by platelet aggregation and granule secretion which releases more ADP as well as many other substances (5). Acting extracellularly, ADP causes a number of intracellular events including rapid calcium influx (6,7), mobilization of intracellular calcium stores (8), and inhibition of adenylyl cyclase (9). An increase in intracellular Ca 2ϩ may be due to an increase in inositol trisphosphate (10 -13) but this finding remains controversial (14,15). In addition, arachidonic acid, liberated from platelet membranes due to activation of phospholipase A 2 , is converted to thromboxane A 2 , itself a powerful platelet agonist. Despite this knowledge the exact identity of platelet ADP receptors responsible for functional responses of ADP are not fully defined and the mechanism by which aggregation occurs is still under investigation.
The idea that ADP's effects on platelets are receptor-mediated was indicated by the finding that ATP shows true competitive inhibition of ADP-induced aggregation and adenylyl cyclase inhibition (16). Several other nucleoside triphosphates are also competitive antagonists of ADP and have similar activity ratios for inhibition of aggregation and for inhibition of ADP's effect on adenylyl cyclase (17). Evidence that an ADP receptor couples to G-proteins is based on the fact that ADP stimulates the binding of GTP␥S 1 to platelet membranes in humans and rats (18). The receptors activated by extracellular nucleotides have been classified as P2 receptors and are divided into two subclasses: P2X intrinsic ion channels and P2Y metabotropic receptors coupled to heterotrimeric G-proteins (19). These receptor subtypes are numbered in the order of cloning and to date eight subtypes of P2X receptors and six subtypes of P2Y receptors have been cloned (20). Because of its pharmacologic profile, the identity of platelet ADP receptor(s) could not be assigned to any cloned member of the P2Y receptor family and hence has been designated P2T (21,22). Several molecules have been proposed to be the platelet ADP receptor, including aggregin, a 100-kDa protein that covalently binds to 5Ј-p-fluorosulfonyl benzoyladenosine (23), and platelet glycoprotein IIb, a component of the fibrinogen receptor on platelets (24), but none has been conclusively demonstrated to be a platelet ADP receptor.
Using ␣,␤-MeATP as the agonist, MacKenzie et al. (7) demonstrated an ADP receptor in platelets, suggested to be the P2X1 receptor, that could cause a rapid calcium influx but not internal Ca 2ϩ mobilization. However, it is unclear whether ␣,␤-MeATP can activate any physiological platelet responses (7). Léon et al. (25) detected mRNA for the P2Y1 receptor, a metabotropic receptor, in platelets. They expressed this receptor in Jurkat cells and demonstrated that ATP is an antagonist at this receptor and postulated that the P2Y1 receptor is the platelet ADP receptor coupled to adenylyl cylase inhibition (25,26).
In this report, we provide evidence that there are two separate receptors for ADP on the platelet surface; one coupled to inhibition of adenylyl cyclase, designated P2T AC , and a second coupled to mobilization of intracellular calcium stores through inositol phosphate production, designated P2T PLC , in addition to the intrinsic ion channel P2X, coupled to rapid calcium influx previously demonstrated by MacKenzie et al. (7). ATP is an antagonist at both the receptor coupled to inhibition of adenylyl cyclase and that coupled to increases in inositol phosphates. (Type V) 4 , Fura-2-labeled Platelets-Human blood was collected from informed healthy volunteers in acid/citrate/dextrose. Platelet-rich plasma was obtained by centrifugation at 180 ϫ g for 15 min at ambient temperature and was recentrifuged (800 ϫ g for 15 min, ambient temperature). The platelet pellet was resuspended in 0.5 volumes of autologous plasma and incubated with 32 PO 4 (0.25 mCi/ml) at 37°C. After 15 min, fura-2 (3 M) and aspirin (1 mM) were added and the incubation at 37°C continued for another 45 min. Platelets were isolated from the incubation medium by gel filtration as described previously (27). The final buffer consisted of 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 3.0 mM NaH 2 PO 4 , 5 mM glucose, 10 mM HEPES (pH 7.4), 0.2% bovine serum albumin, and 20 g/ml apyrase. The platelet count was adjusted to 2 ϫ 10 8 cells/ml. All experiments were carried in buffers with no added Ca 2ϩ unless otherwise noted.

Materials-Apyrase
Measurement of Ca 2ϩ with Fura-2 and Platelet Activation-Platelets (1.0 ml) were activated in a Perkin-Elmer LS-5 spectrofluorimeter in a water jacketed cuvette maintained at 37°C with stirring. Fura-2 fluorescence was monitored continuously using settings of 340 nm (excitation) and 510 nm (emission). Fura-2 fluorescence signals were calibrated as described by Pollock et al. (28). Reactions were stopped by the addition of 1:10 volume of ice-cold 6.6 N HClO 4 and kept on ice until further processing.
Measurement of Inositol Trisphosphates-After addition of HClO 4 , 0.02 Ci each of [ 3 H]Ins(1,4,5)P 3 and [ 3 H]Ins(1,3,4)P 3 were added to each sample and the samples were centrifuged. Supernatants were adjusted to pH 7.5-8.0 by addition of 2 M K 2 CO 3 and the precipitate of KClO 4 was removed by centrifugation. The resulting supernatant was treated at 37°C overnight with inorganic pyrophosphatase (10 units/ ml) in the presence of 5 mM MgCl 2 , 10 mM Tris-HCl (pH 8.0). Each sample was treated with 100 mg of charcoal (Darco, G-60) for 5 min on ice to remove nucleotides. Charcoal was removed by centrifugation (3 min at 5000 ϫ g) followed by filtration through Rainin microfilterfuge tubes. Separation of inositol phosphates was accomplished using a Rainin Rabbit HP HPLC and a Whatman Partisil 10 SAX column. The column was eluted at a flow rate of 1.5 ml/min with a 45-min gradient of water as initial buffer and 1.5 M ammonium formate adjusted to pH 3.7 with H 3 PO 4 , as final buffer. Fractions were collected and counted in a LKB liquid scintillation counter with windows set to separate 3 H and 32 P. The [ 3 H]Ins(1,4,5)P 3 and [ 3 H]Ins(1,3,4)P 3 standards were used to indicate the positions of their 32 P counterparts and to correct for recovery.
Platelet Aggregation-For aggregation experiments, aspirated platelets were isolated from plasma by centrifugation at 800 ϫ g for 15 min and resuspended in the same buffer that was used for gel filtration. Platelet shape change and aggregation (0.5-ml samples) were measured in a Chronolog lumi-aggregometer with stirring at 37°C. Fibrinogen (400 g/ml) was added to all samples. The base line was set using the platelet suspension diluted 1:1 with platelet suspension buffer to increase the gain of the aggregometer output.
Measurement of Cyclic AMP Formation in Intact Platelets-Plateletrich plasma was incubated with 2 Ci/ml [ 3 H]adenine and aspirin (1 mM) for 1 h at 37°C (29). Platelets were isolated from plasma by centrifugation at 800 ϫ g for 15 min and resuspended in the same buffer as was used for gel filtration. Reactions were stopped with 1 M HCl and 4000 disintegrations/min of [ 14 C]cAMP as recovery standard. Cyclic AMP was determined by the method of Salomon (30) and expressed as percentage of total [ 3 H]adenine nucleotides. correlation between the concentration dependence of Ca 2ϩ mobilization and IP 3 metabolism for both agonists. The EC 50 for ADP was about 1 M, which is comparable with published results for induction of shape change and Ca 2ϩ mobilization (21). The EC 50 for 2-MeSADP was about 4-fold lower which is in agreement with the published activity of 2-MeSADP for induction of platelet Ca 2ϩ mobilization (21).

Effect of ADP and 2-MeSADP on Inositol
Effect of ␣,␤-MeATP on Inositol Phosphate Formation and Adenylyl Cyclase Inhibition-␣,␤-MeATP induced a small Ca 2ϩ transient similar to that shown by MacKenzie et al. (7) but only if the suspension buffer contained 1 mM Ca 2ϩ . However, it did not induce a significant increase in Ins(1,4,5)P 3 formation (Table I). In addition, ␣,␤-MeATP did not inhibit forskolin-stimulated adenylate cyclase activity. An increase in external Ca 2ϩ from nominal (approximately 10 M) to 1 mM had no effect on the amount of Ins(1,4,5)P 3 or cyclic AMP formed when platelets were stimulated by ADP (Table I).
Effect of ARL 66906 on Platelet Aggregation and Shape Change-ARL 66096 is an ATP analog that is a highly potent and selective inhibitor of ADP-induced platelet aggregation (31). As shown in Fig. 3, ARL 66096 was an effective inhibitor of ADP-induced platelet aggregation, resulting in complete inhibition of aggregation at 100 -300 nM. However, ARL 66096 failed to block shape change even when a concentration of 1 M was used (Fig. 3).
Effect of ARL 66096 on Ca 2ϩ Mobilization, IP 3 Metabolism, and Adenylyl Cyclase-Since ARL 66096 inhibited ADP-induced platelet aggregation, we expected that it would be equally effective as an inhibitor of ADP-induced Ca 2ϩ mobilization and IP 3 metabolism. However, concentrations known to effectively inhibit aggregation had no effect on either Ca 2ϩ mobilization (Fig. 4) or IP 3 formation (Table II). Since inhibition of adenylyl cyclase is another major intracellular effect of ADP on platelets, we investigated the ability of ARL 66096 to block the ADP-induced inhibition of forskolin-stimulated adenylyl cyclase activity (Fig. 5). ARL 66096 inhibited this response in the same concentration range as it inhibited aggregation. In contrast to ARL 66096, ATP blocked ADP-induced   3. The effect of ARL 66096 on ADP-induced platelet aggregation and shape change. Platelet aggregation was measured as described. The ordinate represents apparent changes in optical density due to light scattering by the platelets. The first arrow indicates the addition of ARL 66096 or solvent; the second arrow indicates the addition of 10 M ADP. This scale was set to give a full response for control ADP-induced aggregation.
Ca 2ϩ mobilization, shape change, and aggregation within the same concentration range (data not shown). DISCUSSION ADP has been known to be a platelet agonist for more than three decades and the ADP-induced physiological responses and several intracellular responses are well characterized. However, the molecular mechanisms of the effect of ADP on platelets remain obscure. It is well accepted that the effects of ADP in platelets are receptor-mediated and this receptor has been designated P2T receptor. However, several observations have remained unexplained. First, 2-MeSADP is at least 100fold more potent than ADP in causing platelet aggregation and adenylyl cyclase inhibition, but only 4-fold more potent in mo-bilization of calcium from intracellular stores (21). Second, the thienopyridine analogs ticlopidine and clopidogrel when used in vivo are potent and specific inhibitors of ADP-induced platelet aggregation and antagonize ADP inhibition of prostaglandin E 1 -activated adenylyl cyclase in intact platelets (21). However, these compounds do not block ADP-induced shape change or intracellular calcium mobilization (32) nor do they affect ADP-induced phosphorylation of myosin light chain and pleckstrin in platelets (33). Third, a congenital defect in platelet function attributed to an ADP receptor defect has been described by Nurden et al. (34). Platelet aggregation was abnormal and ADP-induced inhibition of adenylyl cyclase activity was abolished. However, ADP-induced shape change was unaltered. Finally, several megakaryocytic cell lines and human erythroleukemia cells have been reported to express platelet ADP receptors (35-37) but ADP does not inhibit adenylyl cyclase in these cell lines (38).
Hourani and Hall (22) have proposed a two-receptor model to explain the actions of ADP on platelet function. In this model, rapid calcium influx is mediated by a receptor-operated Ca 2ϩ channel while adenylyl cyclase inhibition and intracellular Ca 2ϩ mobilization are modulated by the P2T receptor through multiple G-proteins. A similar model was advocated by Gachet and co-workers (26), who proposed that the P2Y1 receptor is the P2T receptor mediating inhibition of adenylyl cyclase. They propose that calcium entering through P2X receptor activation could cause mobilization of calcium from intracellular stores by a calcium-induced calcium release mechanism. Severe deficiencies of the latter model are first that the P2X receptor agonist, ␣,␤-MeATP, does not produce either the extent or duration of Ca 2ϩ mobilization that ADP produces (7) and second that ADP can cause intracellular Ca 2ϩ mobilization in the absence of external Ca 2ϩ .
Although phospholipase C activation and inositol phosphate formation have been shown to be important in mobilization of calcium from intracellular stores in many cellular systems, this intracellular effect of ADP-induced platelet activation has been controversial. In this paper, we have confirmed our earlier findings that ADP causes an increase in inositol trisphosphates using high performance liquid chromatography to separate inositol trisphosphate isomers and show that there is a significant, 3-fold increase in Ins(1,4,5)P 3 , which is the isomer that causes release of intracellular stores of Ca 2ϩ (39). Overall the increases in Ins(1,4,5)P 3 correlate well with changes in intracellular Ca 2ϩ . However, Ca 2ϩ levels start to decline at 10 s while IP 3 levels remain relatively high. This suggests that the initial release of Ca 2ϩ may trigger resequestration mechanisms that rapidly lower intracellular Ca 2ϩ levels. Levels of intracellular Ca 2ϩ , even through much lower than the peak concentration, remain above basal for 30 s (Fig. 1), especially in the presence of 1 mM external Ca 2ϩ (7).
We were surprised to find that ARL 66096 had no effect on ADP-induced intracellular Ca 2ϩ mobilization, in the range where it is known to inhibit ADP-induced platelet aggregation (31). The possibility that the effects of ADP on platelets are mediated through at least two different receptors has long been the subject of conjecture (21,22). Using ARL 66096, we can clearly separate two distinct responses of platelets to ADP. ARL 66096 was able to completely inhibit ADP-induced aggregation at a concentration as low as 100 nM while it had no effect platelet shape change at concentrations over 10-fold higher. ARL 66096 also did not inhibit ADP-induced Ca 2ϩ mobilization or IP 3 production in this range.
MacKenzie et al. (7) have shown a receptor of the P2X family, P2X1, on platelets that is coupled to rapid Ca 2ϩ influx. ␣,␤-MeATP, a P2X receptor agonist, while causing a small tran-   Incubations were continued for 2 min after ADP addition after which stopping solution was added. Forskolin-stimulated cyclic AMP level in the absence of ADP was 4.2% of total [ 3 H]adenine nucleotide. The curve represents the hyperbola that best fit the data using the program Kaleidagraph (Synergy Software, Reading PA). sient increase in intracellular Ca 2ϩ of a few seconds duration, failed to cause prolonged mobilization of intracellular calcium, IP 3 formation, or inhibition of adenylyl cyclase (Fig. 3). Thus IP 3 formation and inhibition of adenylyl cyclase are independent of the P2X receptor activation.
These data can best be explained if there are two separate receptors for ADP on platelets apart from the P2X1 receptor on platelets that is coupled to rapid Ca 2ϩ influx, one coupled to inhibition of adenylyl cyclase and the other coupled to phospholipase C-Ca 2ϩ mobilization. Thus, we propose a three-receptor model to explain the effects of ADP on platelet activation. This model explains several of the inconsistencies that two receptors models fail to explain. We propose that 2-Me-SADP has relatively higher affinity at the ADP receptor coupled to adenylyl cyclase inhibition, that thienopyridines selectively inhibit signal transduction through this same receptor, and that the patient described by Nurden et al. (34) has a defect in this receptor but not in the ADP receptor coupled to activation of phospholipase C. Finally, hemopoetic cell lines have been shown to lack the ADP receptor coupled to adenylyl cyclase inhibition but express the ADP receptor coupled to activation of phospholipase C.
Currently the platelet ADP receptor has been designed P2T (21,22). Since this nomenclature does not distinguish between the two physiological receptors that we have defined, we propose a new nomenclature that will functionally distinguish the platelet ADP receptors; a P2T AC receptor coupled to inhibition of adenylyl cyclase, a P2T PLC receptor coupled to activation of phospholipase C, and the P2X1 receptor responsible for rapid Ca 2ϩ influx ( Fig. 6 and Table III). The IUPHAR committee has recommended that the uncloned ADP receptor on platelets should be designated P2Y ADP , as the P2Y receptor at which ADP is an agonist (20). However, since both the P2T PLC and P2T AC receptors respond to ADP and are not cloned at this stage, the name P2Y ADP would not distinguish the two receptors that we have defined. After cloning, these receptors will be designed with standard P2Y numbering. The accompanying paper (40) describes the molecular identity of the P2T PLC receptor and its functional role in platelet activation. The fact that P2Y1 has been shown to couple through a G-protein dependent mechanism to phospholipase C activation, IP 3 formation, and Ca 2ϩ mobilization (25,41,42) is fully consistent with our observations that ADP produces IP 3 through activation of this receptor on platelets.