Molecular Mechanism of Thromboxane A2-induced Platelet Aggregation

Thromboxane A2 is a positive feedback lipid mediator produced following platelet activation. The Gq-coupled thromboxane A2 receptor subtype, TPα, and Gi-coupled TPβ subtype have been shown in human platelets. ADP-induced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T AC , coupled to Gq and Gi, respectively. We investigated whether the stable thromboxane A2 mimetic, (15S)-hydroxy-9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through Gq and Gi, through co-activation of TPα and TPβ receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP- or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T AC receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an α2A-adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the Gi pathway by selective activation of either the P2T AC receptor or the α2A-adrenergic receptor. We conclude that whereas thromboxane A2 causes intracellular calcium mobilization and shape change independently, thromboxane A2-induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate Gi-coupled receptors.

Upon exposure to activating agonists (e.g. thrombin, ADP, and collagen), platelets liberate arachidonic acid stored as phospholipid in the platelet plasma membrane that is con-verted into thromboxane A 2 by sequential oxygenation of arachidonic acid by cycloxygenase and thromboxane A 2 synthase (1). The released thromboxane A 2 acts as a positive feedback mediator in the activation and recruitment of more platelets to the primary hemostatic plug (2). Thromboxane A 2 exerts its actions via specific G protein-coupled receptors and has been described as either a potent platelet agonist (2,3) or as a weak agonist with an important role in amplifying the response of platelets to more potent agonists (4).
Pharmacological studies indicate the presence of two potential thromboxane A 2 receptor (TP receptor) 1 subtypes on human platelets (5,6). The TP receptor gene has been cloned and encodes two subtypes of the TP receptor that result from alternative splicing of the primary transcript (7). The subtypes share the identical first 293 amino acids but possess different carboxyl-terminal domains. A complete cDNA of the 343 amino acid TP␣ isoform was isolated from both a placental cDNA library and human megakaryocytic leukemia cells (8,9) and a chronic myelogenous leukemia cell line (10). A cDNA for the 407-amino acid TP␤ subtype was cloned from a vascular endothelial library (11,12). Both the TP␣ and TP␤ subtypes mediate the stimulation of phospholipase C and an increase in intracellular concentrations of inositol 1,4,5-triphosphate and diacylglycerol. The formation of inositol 1,4,5-triphosphate induces an increase in the cytosolic concentration of Ca 2ϩ , whereas the release of diacylglycerol activates PKC (13)(14)(15)(16). In transfected cell lines the two subtypes were shown to oppositely regulate levels of cAMP. The TP␣ receptor stimulated cAMP formation in contrast to the TP␤ receptor that inhibited the level of intracellular cAMP (15). Pertussis toxin was shown to block TP␤ receptor-mediated inhibition of adenylyl cyclase; however, its effect on phospholipase C activation was not determined (15). By using isoform-specific antibodies Habib et al. (17) only detected the presence of the TP␣ receptor in human platelets. Hirata et al. (15) have shown the presence of mRNA encoding both TP␣ and TP␤ subtypes in platelets using reverse transcriptase-polymerase chain reaction.
ADP-induced platelet aggregation results from concomitant signaling through the P2Y1 and P2T AC receptors that couple to G q and G i , respectively (18 -21). Thrombin has been shown to activate both G q -and G i -signaling cascades (22,23). Contrary to previous studies, we have demonstrated that epinephrine and serotonin activating only G i or G q pathways, respectively, are not true platelet-aggregating agents (18). Offermanns et al. (24) have provided evidence showing that U46619 couples to G q . Since thromboxane A 2 couples to two TP receptor subtypes and TP␤ has been shown to inhibit adenylyl cyclase, we investigated whether U46619 (a stable thromboxane A 2 analog) also causes platelet aggregation by co-activation of TP␣ and TP␤ receptor subtypes coupled to G q and G i , respectively.
We report here that U46619 causes intracellular calcium mobilization and shape change in human platelets independently of secretion. However, TxA 2 -induced platelet aggregation depends upon secretion of other platelet agonists capable of coupling to G i pathways. In the absence of G i signaling by other agonists, U46619 cannot cause inhibition of adenylyl cyclase or platelet aggregation. We provide evidence for the involvement of the P2T AC and ␣ 2A -adrenergic receptors as well as other G i -coupled receptors in U46619-induced platelet aggregation.
Isolation of Platelets-Human blood was collected from a pool of informed healthy volunteers all of whom are students or staff at Temple University School of Medicine. The donated blood was collected into a one-sixth volume of ACD (2.5 g of sodium citrate, 1.5 g of citric acid, and 2.0 g of glucose in 100 ml of deionized H 2 O). Platelet-rich plasma (PRP) was isolated by centrifugation of citrated blood at 180 ϫ g for 15 min at room temperature. PRP was incubated with 1 mM acetylsalicylic acid (aspirin treated) for 1 h at 37°C followed by centrifugation at 1000 ϫ g for 10 min at room temperature. The platelet pellet was resuspended in HEPES-buffered Tyrode's solution (138 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 3.0 mM NaH 2 PO 4 , 5 mM glucose, 10 mM HEPES, adjusted to pH 7.4) supplemented with 0.2% bovine serum albumin, and 0.05 units/ml apyrase. The platelet count was adjusted to 2 ϫ 10 8 cells/ml. All experiments were repeated at least three times using platelets from different donors.
Analysis of Platelet Aggregation and Shape Change-Agonist-induced platelet aggregation was determined by measuring the transmission of light through a 0.5-ml sample of aspirin-treated washed platelets (2 ϫ 10 8 cells/ml) with stirring (900 rpm) in a lumi-aggregometer at 37°C (Chrono-Log, Havertown, PA). The recorder output speed was set to 0.2 mm/s. The base line was set using 0.5 ml of HEPES-buffered Tyrode's solution as a blank. Aggregation of washed platelets required the addition of fibrinogen (1 mg/ml) prior to the addition of an agonist. Platelet shape change was observed by the addition of 1 M SC-57101 before agonist stimulation. SC-57101 is a known inhibitor of platelet aggregation through blocking fibrinogen binding to its receptor (25). All experiments were performed in the presence of 2 mM CaCl 2 which was added first before either fibrinogen or SC-57101. All experiments were repeated at least three times using platelets from different donors.
Measurement of Platelet Secretion-Platelet secretion was determined by measuring the release of [ 14 C]5-HT and expressed as the percentage of the total [ 14 C]5-HT content. The activation of labeled [ 14 C]5-HT platelets was performed in the lumi-aggregometer at 37°C with stirring (900 rpm) and was stopped after 2 min with the addition of formaldehyde/EDTA according to the method of Costa and Murphy (26). Imipramine was added to the HEPES-buffered Tyrode's solution at a final concentration of 1 M in order to prevent re-uptake of secreted [ 14 C]5-HT. Samples were collected and centrifuged at 5000 ϫ g for 1 min, and the radioactivity of the supernatant was measured using an LKB (Amersham Pharmacia Biotech) liquid scintillation counter.
Measurement of Cytoplasmic Concentrations of Ionized Ca 2ϩ -Plate-let-rich plasma was incubated at 37°C with 3 M Fura PE-3 acetoxymethyl ester and 1 mM acetylsalicylic acid for 45 min followed by 15 min at room temperature. The platelet-rich plasma was centrifuged at 1000 ϫ g for 10 min at room temperature. The platelet pellet was resuspended in HEPES-buffered Tyrode's solution supplemented with 0.2% bovine serum albumin, and 0.05 units/ml apyrase. The platelet count was adjusted to 2 ϫ 10 8 cells/ml. Aliquots (1.0 ml) of the platelet suspension were stirred (900 rpm) in a water-jacketed cuvette maintained at 37°C during activation. Fluorescence was constantly measured using a Perkin-Elmer LS-5 spectrofluorimeter with settings of 340 Tyrode's solution including 1 M imipramine to prevent re-uptake of secreted 5-HT. A, platelet aggregation was measured in the presence of extracellular fibrinogen (1 mg/ml) and 2 mM CaCl 2 as described under "Experimental Procedures." Aggregation was performed in a cuvette maintained at 37°C with stirring. The ordinate represents the observed changes in light absorbance (optical density) due to light scattering by the platelets. These tracings are representative of results observed on three separate occasions from three different donors. B, Ro 31-8220 or dimethyl sulfoxide (control) was added to a 0.5-ml volume of platelets and incubated for 5 min at 37°C with stirring before the addition of U46619. Each data point is the mean Ϯ S.E. of three measurements. The experiment was repeated three times using platelets from different donors.
(excitation) and 510 nm (emission). Fura PE-3 fluorescence signals were calibrated as described previously (27). F min was determined by the addition of 2 mM EGTA, 20 mM Tris base, and 40 M digitonin. F max was determined by addition of a saturating concentration of CaCl 2 to the lysed cells. All experiments were performed in the presence of 2 mM CaCl 2 and repeated at least three times using platelets from different donors. Calibration curves for experiments that included Ro 31-8220 were performed in the presence of Ro 31-8220 due to its slight quenching of the fluorescent signal.
Measurement of cAMP-PRP was incubated with 2 Ci/ml [ 3 H]ade-nine and aspirin (1 mM) for 1 h at 37°C. Platelets were isolated from PRP by centrifugation as described above and resuspended in HEPESbuffered Tyrode's solution. Reactions were stopped with 1 M HCl, and 4,000 dpm of [ 14 C]cAMP was added as the recovery standard. The level of cAMP was determined as described previously (28) and measured as a fraction of total [ 3 H]adenine nucleotides. Results are normalized to the level of forskolin (20 M)-stimulated cAMP and expressed as a percentage.

RESULTS
Effect of Ro 31-8220, a Protein Kinase C Inhibitor, on U46619-induced Platelet Responses-Platelets respond to increasing concentrations of ADP by first undergoing shape change and then, at a higher concentrations, aggregation (29). This is because ADP-induced platelet shape change results from activation of the high affinity P2Y1 receptor (19), and higher concentrations of ADP are needed for co-stimulation of both the high affinity P2Y1 receptor and a low affinity P2T AC receptor to induce aggregation (19). In order to determine if concomitant higher affinity G q -coupled signaling and lower affinity G i -coupled signaling also occurs in response to U46619 and to determine whether aggregation requires lower concentration of U46619 than secretion, we exposed platelets to different concentrations of the agonist. Similar to the response observed for ADP, the platelets first responded to lower concentrations of U46619 (100 nM) by changing shape. Aggregation occurred at significantly higher concentrations (300 nM) (Fig. 1A). Secretion did not occur at concentrations of U46619 below 300 nM (Fig. 1B); furthermore, the onset of aggregation appears to correlate with the initiation of secretion. PKC has been shown to play an important role in the induction of platelet secretion, and secretion can be blocked using the cell-permeable inhibitor of PKC, Ro 31-8220 (30 -32). We investigated the role of secretion in platelet aggregation in response to ADP, thrombin, and U46619. Secretion in response to U46619 is totally abolished by 10 M Ro 31-8220 (Fig. 1B). In the presence of Ro 31-8220, U46619 caused shape change but did not induce aggregation (Fig. 2). Platelet aggregation induced by thrombin was slightly slowed down indicating the participation of secreted agonists, whereas aggregation in response to ADP was unaffected (Fig. 2).
Effect of Ro 31-8220 on U46619-induced G q -coupled Platelet Responses-ADP-mediated G q -coupled signaling has been shown to be required for both platelet shape change and aggregation (19,28). Stimulation of the TP receptor with 30 -100 nM U46619 leads to platelet shape change resembling selective stimulation of the G q -coupled P2Y1 receptor. In order to assess the possible effects of secretion on G q -mediated signaling, both platelet shape change and intracellular Ca 2ϩ mobilization were measured in the presence and absence of Ro 31-8220, a protein kinase C (PKC) inhibitor. Platelet shape change in response to U46619 was not affected by Ro 31-8220 (Fig. 3A) indicating that these signaling pathways are not dependent upon either secretion or PKC activity. Furthermore, the U46619-induced increase in cytosolic Ca 2ϩ was unaffected by the presence of Ro 31-8220 (Fig. 3B) indicating that G q -coupled signaling initiated by TP receptor stimulation is independent of released granule contents.
Effect of Receptor-selective Antagonists on U46619-induced G q -coupled Platelet Responses-Platelet secretion releases ADP and serotonin at the site of injury in order to activate and recruit more platelets into the forming primary hemostatic plug (2). By using receptor-selective antagonists, we investigated the contribution of these agonists to U46619-induced G q -coupled responses. The compound A3P5P is an antagonist of the G q -coupled P2Y1 receptor (33). Cyproheptadine is an antagonist at the 5-HT 2A receptor (34 -37). Aggregation was not affected by the presence of either compound (data not shown). U46619-induced platelet shape change was not affected by the presence of A3P5P (Fig. 4A) or cyproheptadine (not shown) indicating the lack of any contribution by the P2Y1 or serotonin receptors to this event. The possible contribution of both the P2Y1 and 5-HT 2A receptors in the mobilization of intracellular Ca 2ϩ was investigated. Intracellular Ca 2 ϩ mobilization in response to U46619 was not affected by A3P5P and/or cyproheptadine (Fig. 4B).
Effect of Ro 31-8220 or Receptor-selective Antagonists on U46619-induced Inhibition of Platelet Adenylyl Cyclase-Previous studies have shown that U46619 causes a decrease in the intracellular concentration of cAMP in platelets (38,39). In order to determine whether TP receptors can couple to G isignaling pathways, we utilized two approaches. The first was to block secretion using Ro 31-8220. In the absence of Ro 31-8220, U46619 inhibited forskolin-stimulated adenylyl cyclase (Fig. 5). In the presence of Ro 31-8220, U46619 failed to inhibit adenylyl cyclase. The second approach utilized receptorselective antagonists to the P2T AC and ␣ 2A -adrenergic receptors. AR-C66096 is an antagonist at the G i -coupled P2T AC receptor (28), and yohimbine is an antagonist at the G i -coupled ␣ 2 -adrenergic receptor (40,41). Platelet dense granules contain both ADP and epinephrine which cause the inhibition of cAMP following activation at their respective receptors (2). The level of cAMP was measured following stimulation of platelets in the absence and presence of the antagonists AR-C66096 and yohimbine. These antagonists effectively prevented the contribution of G i -coupled signaling by either the P2T AC or the ␣ 2Aadrenergic receptor, respectively. As shown in Fig. 5, U46619induced adenylyl cyclase inhibition was also blocked by these receptor antagonists, suggesting that U46619-induced G i stimulation depends on secreted ADP and epinephrine.  (18) and others (20,21) have provided evidence that concomitant signaling through both G icoupled and G q -coupled receptors is required for platelet aggregation. Since the TP receptor does not couple to G i , independently of secreted ADP and epinephrine (Fig. 5), we utilized receptor-selective antagonists to elucidate the role of these G i -coupled receptors in U46619-induced platelet aggregation. AR-C66096 dramatically inhibited ADP-induced platelet aggregation (18,28). The rate and extent of U46619-induced aggregation were diminished in the presence of AR-C66096 (Fig. 6). In the presence of AR-C66096, yohimbine further inhibited U46619-induced platelet aggregation (Fig. 6). However, yohimbine alone was without any significant effect (not shown). These results indicated that the P2T AC receptor is essential for U46619-induced platelet aggregation.

Effect of Receptor-selective Antagonists on U46619-induced Platelet Aggregation-We
Restoration of U46619-induced Aggregation Blocked by Ro 31-8220 -In order to verify that signaling through a G i -coupled receptor only occurs following U46619-induced secretion, we investigated the effects of selective activation of G i -coupled receptor stimulation in the presence of Ro 31-8220. Control experiments were performed to ensure that platelets respond normally to ADP and thrombin in the presence of Ro 31-8220 or vehicle (not shown). The P2T AC receptor was selectively activated by ADP in the presence of A3P5P. As shown in Fig. 7, selective activation of the P2T AC receptor reversed the effects of secretion blockade on U46619-induced aggregation. AR-C66096 blocked this reversal, providing further evidence that ADP is selectively activating the G i -coupled P2T AC receptor (Fig. 7). Epinephrine also reversed the inhibitory effects of Ro 31-8220 on U46619-induced aggregation. Addition of ADP and epinephrine together potentiated this reversal. Thus platelet aggregation in response to U46619 is mediated by concomitant signaling through the G q -coupled TP receptor and the G i -coupled P2T AC and ␣ 2A receptors. DISCUSSION The molecular mechanisms leading to aggregation following platelet exposure to thromboxane A 2 have yet to be clearly elucidated. Four explanations for the stimulatory action caused by U46619 or other thromboxane A 2 mimetics are possible. First, U46619 may activate G q and G i through the TP␣ and TP␤ receptors, respectively. Second, it is conceivable that U46619 only activates the G q pathway and that secreted ADP activates the G i pathway. Although unlikely, a third explanation is that U46619 activates G i or G o through TP␤ leading to the activation of phospholipase C and the inhibition of cyclase. Following secretion, released ADP would activate the G q pathway. Finally, U46619 may activate an unidentified G proteincoupled pathway that results in secretion of ADP which activates both G q and G i through the P2Y1 and the P2T AC receptors, respectively. We used three complementary approaches to identify the molecular mechanisms of U46619induced platelet activation as follows: 1) determination of the minimum concentration required for platelet aggregation and secretion by U46619, 2) blockade of secretion, and 3) receptor subtype-selective antagonists in order to eliminate the positive feedback from granule contents. Here we report that although thromboxane A 2 causes intracellular calcium mobilization and shape change independently, thromboxane A 2 -induced inhibition of adenylyl cyclase and platelet aggregation depend exclusively on ADP and other released granule contents.
Evidence exists for a dissociation of platelet activation responses following stimulation of the TP receptor. First, the EC 50 values of the TP receptor agonists, U46619 (42) and STA 2 (43), for an increase in cytosolic Ca 2ϩ and platelet shape change are lower than the EC 50 values for secretion and aggregation. Our data indicate that platelet aggregation correlates with the occurrence of secretion. We observed that platelet shape change occurs at lower concentrations of U46619 and that aggregation occurs at higher concentrations (Fig. 1A). Furthermore, the same concentration of U46619 that leads to the initiation of aggregation also initiates secretion (Fig. 1B). However, from this evidence it is not clear if platelet aggregation results in part from P2 receptor stimulation.
Substantial evidence exists that PKC activation is required for platelet secretion (31). In platelets activated by U46619 in the presence of Ro 31-8220, it was reported that P47 phosphorylation, fibrinogen binding, and serotonin release were all inhibited (32). In agreement with previous studies, our results show that Ro 31-8220 prevented U46619-induced platelet aggregation (Fig. 2). We observed that Ro 31-8220 inhibited U46619-induced secretion in platelets loaded with [ 14 C]serotonin in the presence of 2 mM Ca 2ϩ (Fig. 1B) and that Ro 31-8220 did not inhibit the increase in cytosolic Ca 2ϩ induced by U46619 (Fig. 3B).
Ro 31-8220 failed to inhibit ADP-or thrombin-induced platelet aggregation (Fig. 3) suggesting that the Ro 31-8220 inhibitable PKC isoforms do not directly contribute to fibrinogen receptor activation. Ro 31-8220 has been shown to block PKC isoforms ␣, ␤, ␥, and ⑀ (44). Hence these PKC isoforms do not contribute to the inside-out signaling leading to fibrinogen receptor activation by either ADP or thrombin.
Considering that secretion and aggregation both occur at the same concentration of U46619 (Fig. 1) and that blocking secretion prevents aggregation (Fig. 2), it is reasonable to suggest that thromboxane A 2 -induced aggregation is dependent upon secretion. The role of ADP in thromboxane A 2 -induced platelet aggregation has been investigated using enzymes that deplete released ADP. This work suggested that the aggregation response is mediated by the secretion of platelet ADP (45)(46)(47)(48)(49). It was concluded that U46619-induced platelet aggregation depends on the release of stored ADP. The use of apyrase could have enhanced the generation of adenosine from AMP. Adenosine binds to the G s -coupled A 2 receptor resulting in an in- crease in the intracellular concentration of cAMP and inhibits platelet activation (50,51). Moreover, these studies did not clearly determine how ADP and the other components of the dense and ␣-granules contribute to TxA 2 -induced platelet aggregation. The use of creatine phosphate/creatine phosphokinase converts ADP to ATP, an antagonist at the platelet ADP receptors (2). ATP can also potentially stimulate adenylyl cyclase activity resulting in inhibition of platelet activation (52,53). Our experiments make use of the receptor subtype-selective antagonists AR-C66096 and yohimbine, which block stimulation of G i signaling.
Evidence exists to support the presence of the TP␣ and TP␤ receptor subtypes in platelets (8,17); these isoforms, when expressed in Chinese hamster ovary cells, have been shown to couple to G s and G i pathways, respectively. However, in the presence of Ro 31-8220, high concentrations of U46619 did not alter the level of cAMP, indicating that TP receptor subtypes do not couple to adenylyl cyclase in platelets. Our observation also is supported by two studies. By using platelet membranes, U46619 was found to have no effect upon levels of cAMP (54). Furthermore, Klages et al. (55) have shown that U46619 does not stimulate G i proteins in mouse platelets. G protein coupling may be affected by levels of heterologous receptor expression; futhermore, high levels of receptor expression can lead to promiscuous coupling to multiple G proteins.
U46619-induced aggregation requires concomitant stimulation of both a G q -coupled receptor and a G i -coupled receptor. Granule contents appear to mediate the stimulation of G icoupled signaling as is evident by the lack of cyclase inhibition when U46619-induced platelet secretion is prevented (Fig. 5). The fact that signaling through the G q -coupled TP receptor is unaffected under such conditions is apparent by both the robust shape change response (Fig. 3A) and the normal level of cytosolic Ca 2ϩ mobilization (Fig. 3B).
An alternative explanation for the effect of Ro 31-8220 on U46619-induced platelet aggregation is that U46619 causes platelet aggregation involving activation of a PKC isoform through a mechanism different from that of ADP. Hence, Ro 31-8220 would inhibit U46619-induced aggregation by inhibiting this PKC isoform in addition to blocking secretion. This possibility was ruled out using receptor-selective antagonists.
Through the use of receptor-selective antagonists, we were able to identify clearly the contribution of receptors mediating aggregation following U46619-induced secretion. Antagonists at G q -coupled receptors such as cyproheptadine and A3P5P had no effect on aggregation, shape change, or the increase in cytosolic Ca 2ϩ concentration. In contrast, both of the G i -coupled P2T AC and ␣ 2A -adrenergic receptors were found to mediate aggregation and inhibition of adenylyl cyclase, following U46619-induced secretion (Fig. 6). The compound AR-C66096 had the greatest inhibitory effect indicating the large contribution to G i -coupled signaling by the P2T AC receptor. In the absence of AR-C66096, yohimbine failed to affect U46619-induced aggregation, indicating that G i stimulation could be compensated by P2T AC receptor stimulation. The amount of epinephrine found in platelets is extremely small (1.1-3.8 pmol/ 1 ϫ 10 8 platelets) (56); however, the initial concentration of this secreted amount in the microenvironment of the platelet could be much greater. As observed, the secreted epinephrine makes a significant contribution as revealed by the inhibition of aggregation by yohimbine only in the absence of P2T AC receptor stimulation (Fig. 6). This suggests that secretion of the G icoupled receptor stimulating agonists (ADP and epinephrine) are required for full aggregation following activation of G qcoupled signaling by thromboxane A 2 . When U46619-induced secretion was blocked by Ro 31-8220 aggregation was pre- U46619 acts at the G q -coupled TP receptor to cause secretion of granule contents. Activation of G i -coupled receptors is dependent upon secretion. Concomitant stimulation of G q -coupled and G i -coupled receptors leads to platelet aggregation.
vented. Under these conditions the selective activation of either the G i -coupled P2T AC receptor or the ␣ 2A -adrenergic receptor restored aggregation (Fig. 7).
Further evidence for the important role of the P2T AC receptor in mediating the platelet response to TxA 2 is provided by reports of patients with congenital ADP receptor defects (57)(58)(59). In these cases the shape change and cytosolic Ca 2ϩ mobilization responses to ADP are present indicating function of the P2Y1 receptor, whereas ADP-induced aggregation and inhibition of adenylyl cyclase are absent. Such findings suggest that the defect involves the P2T AC receptor. The lack of signaling due to a defective P2T AC receptor affects the response of these platelets to thromboxane A 2 mimetics. In both cases, U46619induced activation of the integrin ␣ IIb ␤ 3 was inhibited (58,59). Inhibition of adenylyl cyclase by epinephrine in platelets from both patients was normal, suggesting that the residual fibrinogen receptor activation could be due to activation of ␣ 2Aadrenergic receptors by secreted epinephrine. On the other hand, we predict that in the case of a hypothetical P2Y1 receptor defect, platelet aggregation in response to U46619 would appear normal as G i stimulation, although the P2T AC receptor and the ␣ 2 -adrenergic receptor would be intact.
Even in the presence of both AR-C66096 and yohimbine we still observed some residual aggregation (Fig. 6). We propose that this residual aggregation results from G i signaling by other components of the granules. This prediction is supported by the fact that secretion blockade completely eliminates U46619-induced platelet aggregation. Based on previous and recent reports describing the mechanism of action by thrombospondin, a major constituent of the ␣ granules, in platelet activation and aggregation (60 -62), we suggest that it too may be mediating TxA 2 mimetic-induced aggregation. A recent study has demonstrated that thrombospondin can stimulate the G i -signaling pathways (60).
In conclusion, as outlined in Fig. 8, our results show that U46619 causes platelet shape change and intracellular Ca 2ϩ mobilization independently of secreted granule contents. However, U46619-induced platelet aggregation depends exclusively on G i stimulation by ADP and other released granule contents. The P2T AC receptor appears to be the predominant stimulator of the G i pathway. These results further support the hypothesis that platelet fibrinogen receptor activation requires concomitant signaling from the G q -and G i -signaling pathways.