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J. Biol. Chem., Vol. 277, Issue 48, 46035-46042, November 29, 2002

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Signaling through G_i Family Members in Platelets

REDUNDANCY AND SPECIFICITY IN THE REGULATION OF ADENYLYL CYCLASE AND OTHER EFFECTORS^*

Jing Yang, Jie Wu, Hong Jiang, Richard Mortensen^§, Sandra Austin^¶, David R. Manning, Donna Woulfe, and Lawrence F. Brass

From the Departments of Medicine and Pharmacology and the Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the ^§ Department of Physiology and Medicine (Endocrine), University of Michigan, Ann Arbor, Michigan 48109

Received for publication, August 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Platelet responses at sites of vascular injury are regulated by intracellular cAMP levels, which rise rapidly when prostacyclin (PGI₂) is released from endothelial cells. Platelet agonists such as ADP and epinephrine suppress PGI₂-stimulated cAMP formation by activating receptors coupled to G_i family members, four of which are present in platelets. To address questions about the specificity of receptor:G protein coupling, the regulation of cAMP formation in vivo and the contribution of G_i-mediated pathways that do not involve adenylyl cyclase, we studied platelets from mice that lacked the  subunits of one or more of the three most abundantly expressed G_i family members and compared the results with platelets from mice that lacked the PGI₂ receptor, IP. As reported previously, loss of G_i2 or G_z inhibited aggregation in response to ADP and epinephrine, respectively, producing defects that could not be reversed by adding an adenylyl cyclase inhibitor. Platelets that lacked both G_i2 and G_z showed impaired responses to both agonists, but the impairment was no greater than in the individual knockouts. Loss of G_i3 had no effect either alone or in combination with G_z. Loss of either G_z or G_i2 impaired the ability of ADP and epinephrine to inhibit PGI₂-stimulated adenylyl cyclase activity and caused a 40%-50% rise in basal cAMP levels, whereas loss of G_i3 did not. Conversely, deletion of IP abolished responses to PGI₂ and caused cAMP levels to fall by 30%, effects that did not translate into enhanced responsiveness to agonists ex vivo. From these results we conclude that 1) cAMP levels in circulating platelets reflect ongoing signaling through G_i2, G_z, and IP, but not G_i3; 2) platelet epinephrine (_2A-adrenergic) and ADP (P2Y12) receptors display strong preferences among G_i family members with little evidence of redundancy; and 3) these receptor preferences do not extend to G_i3. Finally, the failure of ADP and epinephrine to inhibit basal, as opposed to PGI₂-stimulated, cAMP formation highlights the need during platelet activation for G_i signaling pathways that involve effectors other than adenylyl cyclase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Platelet activation at sites of vascular injury typically begins with the exposure of collagen within the subendothelial connective tissue matrix and then continues as additional platelets are recruited into the growing platelet mass by soluble agonists such as thrombin, ADP, epinephrine, and thromboxane A₂. To balance the ability of platelets to be rapidly activated when needed, a number of regulators exist to prevent unwarranted platelet activation. Two such regulators are PGI₂¹ and NO, both of which are released from activated endothelial cells. PGI₂ suppresses intracellular signaling by stimulating adenylyl cyclase and causing an increase in intracellular cAMP (1-3). NO causes an increase in cGMP that, among other effects, inhibits cAMP phosphodiesterase (4). Previous investigators have shown that even a modest increase in cAMP levels (far less than the maximum that can be produced by incubating platelets with PGI₂ or PGE₁) will inhibit platelet responses to thrombin and ADP in vitro (5-7). Experiments performed in vivo suggest the same conclusion, because drugs that stimulate PGI₂ receptors, inhibit cAMP phosphodiesterases, or block ADP receptors can be effective anti-platelet agents (8, 9). Conversely, deletion of the gene that encodes the platelet PGI₂ receptor (IP) enhances thrombosis after arterial injury in mice (10), presumably because the inability to respond to PGI₂ enhances platelet responsiveness, although that point has not been fully addressed.

Observations such as these contribute to the current view that maintenance of the intracellular cAMP concentration within narrowly defined limits is essential for normal platelet function in vivo. As in other types of cells, cAMP formation in platelets is regulated bimodally by activation of G protein-coupled receptors that stimulate or inhibit adenylyl cyclase. PGI₂ increases cAMP formation via G_s-coupled IP receptors, whereas ADP and epinephrine inhibit cAMP formation via receptors coupled to G_i family members. Four G_i family members are expressed in platelets: G_i1, G_i2, G_i3, and G_z. Of these, G_i2, G_z, and G_i3 are the most abundant (11, 12). Previous studies with receptor antagonists and with genetically engineered mice show that ADP inhibits adenylyl cyclase via P2Y12 purinergic receptors primarily coupled to G_i2 (13), whereas epinephrine inhibits adenylyl cyclase via _2A-adrenergic receptors primarily coupled to G_z (14). However, a number of fundamental questions remain unanswered. How is the basal cAMP concentration established in circulating platelets? Will relatively small chronic changes in the cAMP concentration produce the same global changes in platelet responsiveness that are seen after acute changes?

How critical is the ability of G_i-coupled agonists to inhibit adenylyl cyclase when platelets are not exposed to PGI₂? How much redundancy and selectivity exist among the G_i family members in platelets, three of which (G_i1, G_i2, and G_i3) have  subunits that are nearly identical? If selectivity exists, how much of it occurs at the level of receptor:G protein coupling and how much occurs at the level of G protein:effector interactions? To what extent is the well established requirement for G_i-mediated signaling during platelet activation caused by the need to suppress cAMP formation, and to what extent is it caused by the activation of other downstream effectors such as phosphatidylinositol 3-kinase ?

To answer these questions we examined the regulation of cAMP formation and agonist responsiveness in platelets from mice that lack the  subunit of one or more G_i family members or the platelet PGI₂ receptor, IP. The results provide further evidence for functional differences among the G_i family members in platelets and show that maintenance of a normal basal cAMP concentration reflects ongoing signaling through G_i2, G_z, and IP but not G_i3. The results also show that a decrease or increase in the basal cAMP concentration does not by itself make platelets more or less sensitive to agonists, and that unless PGI₂ is present, platelet agonists have little or no effect on platelet cAMP levels. Finally, the results emphasize the importance of G_i-coupled effectors other than adenylyl cyclase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- U46619 was obtained from Calbiochem. Collagen was obtained from Chrono-log (Havertown, PA). PGI₂, PGE₁, and SQ22536 were obtained from Sigma. The PAR4 agonist peptide, AYPGQV, was synthesized by core facilities at the University of Pennsylvania School of Medicine.

Mice-- G_z(/) (14), G_i2(/) (13, 15, 16), G_i3(/) (15, 16) and IP(/) (17, 18) mice were generated as described previously. Double knockout mice were produced by breeding single knock-outs with heterozygous mice.

Platelet Aggregation-- Blood was collected from the inferior vena cava of anesthetized mice (100 mg/kg pentobarbital) by using a heparinized syringe (15 units/ml blood). Samples from four mice were pooled and diluted with 3 ml of HEPES/Tyrode buffer (129 mM NaCl, 8.9 mM NaHCO₃, 2.8 mM KCl, 0.8 mM KH₂PO₄, 10 mM HEPES, 0.8 mM MgCl₂, 5.6 mM dextrose, pH 7.4). Red cells were removed by centrifugation, and the final platelet count was adjusted to 2-3 × 10⁸/ml by using HEPES/Tyrode buffer. Aggregation was measured in a lumi-aggregometer (Chrono-log).

cAMP Assays-- Washed platelets were incubated as noted in each experiment in the absence of a phosphodiesterase inhibitor (unless otherwise indicated). The reaction was then stopped by adding ice-cold, 10% trichloroacetic acid. cAMP was measured by radioimmunoassay (PerkinElmer Life Sciences).

Western Blots-- Platelet lysates in hypotonic buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, and 1 mM dithiothreitol) plus a protease inhibitor mixture (Sigma) were centrifuged at 660 × g for 10 min at 4 °C to remove cell debris and then frozen and thawed three times. Membranes were pelleted at 10,000 × g for 30 min at 4 °C and resuspended in radioimmune precipitation assay buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in phosphate-buffered saline). Protein samples (20 µg) were loaded on 12% SDS-PAGE gels, separated, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat dry milk in TBS for 1 h at room temperature and then incubated with the appropriate antisera (1:200 dilution) in TBS with 1% gelatin for 2 h at room temperature. Afterward, the polyvinylidene difluoride membranes were washed three times with ×0.2% Tween 20 in TBS for 10 min and then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin (1:2000 × dilution, Amersham Biosciences) for 1 h at room temperature. The blot was then washed three times with Tris-buffered saline/Tween and analyzed by using an ECL Western blot detection kit (Amersham Biosciences). ERK (extracellular signal-regulated kinase)-1 was detected by using a rabbit polyclonal antiserum purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Regulation of Basal cAMP Levels and Effects on Agonist Responses-- Basal cAMP concentration is determined by the balance between cAMP formation and hydrolysis and potentially sets the tone for platelet responsiveness to agonists. Therefore, as a first step we measured the basal cAMP concentration in platelets isolated from mice that lacked G_i2, G_z, G_i3, or the PGI₂ receptor (IP) and compared them with their wild type littermates. "Basal" was defined as the intracellular cAMP concentration in the absence of either a phosphodiesterase inhibitor or an adenylyl cyclase activator. The basal cAMP concentration in the wild type mice was 4.7 ± 0.7 pmol/10⁸ platelets (mean ± S.E., n = 16), which is very similar to values reported previously for human platelets (6, 7, 19). Deletion of the genes that encode G_i2 or G_z caused a 40%-50% increase in the basal cAMP concentration, whereas loss of IP caused the basal cAMP concentration to fall by 30% (Fig. 1). Loss of G_i3 had no effect (not shown). These results suggest that the set point for the basal cAMP concentration in platelets is determined in part by exposure to endothelium-derived PGI₂ and in part by signaling through G_i family members. This fact is presumably the result of ongoing exposure of platelets to agonists having receptors that are coupled to G_i2 or G_z but not to G_i3.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Basal cAMP levels in platelets that lack Gi2, Gz, or the PGI2 receptor (IP). The cAMP concentration was determined in freshly isolated platelets that had been exposed to neither PGI2 nor a phosphodiesterase inhibitor. All comparisons were made between wild type (WT) and knock-out mice that were produced by crossing heterozygotes. The results are expressed relative to the wild type and are shown as mean ± S.E. A, platelets from Gi2(/) mice (n = 4). B, platelets from Gz(/) mice (n = 3). C, platelets from IP(/) mice (n = 4). The p values shown were derived by paired Student's t test analysis of the actual cAMP concentrations.

On the basis of studies in which the cAMP concentration was manipulated in vitro by incubation of platelets with PGE₁ (6, 7), in theory even the small increase in basal cAMP levels caused by deletion of G_i2 or G_z could be sufficient to inhibit platelet responses to agonists. If so, this loss of responsiveness should extend to agonists whose receptors are not directly coupled to the missing G protein as well as those that are. Defects in platelet aggregation for mice that lack either G_i2 or G_z have been described previously (13, 14). In the absence of G_z, epinephrine is unable to potentiate the effects of other platelet agonists except when added at supraphysiologic concentrations (14). Responses to other agonists are normal. In the absence of G_i2, platelet responses to ADP are reduced, whereas responses to other agonists are normal except to the extent that they require reinforcement by secreted ADP (Ref. 13 and Fig. 2A). Notably, deletion of the genes that encode G_i2 or G_z appears to have no effect on the level of expression of other G_i family members or G in platelets (Refs. 13 and 14 and Fig. 2B). In general, we found that the decrease in ADP-induced aggregation and suppression of cAMP formation (Fig. 2) caused by loss of G_i2 was less striking than the loss of epinephrine responses in G_z(/) platelets (14). In contrast, loss of G_i3 had no apparent effect on platelet activation by ADP, epinephrine, or AYPGQV, a peptide agonist for the murine PAR4 thrombin receptor (Fig. 3). Western blotting confirmed that G_i3 was no longer expressed in the G_i3(/) platelets and that no compensatory changes occurred in the levels of expression of other G proteins (not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Analysis of platelets from Gi2(/) mice. A, aggregation. The results shown are representative of those from at least three experiments. The arrowheads in this and subsequent aggregation figures show the approximate point of agonist addition. B, immunoblots of total platelet lysates. The results shown are representative of those obtained in two studies. Western blots performed with additional anti-G antibodies (not shown) gave results identical to those described in Ref. 13. C, inhibition of PGI2-stimulated cAMP formation. The final PGI2 concentration was 20 µM. The results shown are the mean ± S.E. from four experiments. WT, wild type.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Analysis of platelets from Gi3(/) mice. A, platelet aggregation in response to (top) 10 µM ADP or (middle) 0.2 µM ADP plus 1 µM epinephrine or (bottom) 200 µM AYPGQF, a PAR4 agonist peptide. The results shown are representative of three experiments. B, inhibition of PGI2-stimulated cAMP formation. The final PGI2 concentration was 20 µM. The results shown are the mean from two experiments. WT, wild type.

Redundancy and Specificity-- Although loss of G_z impaired responses to epinephrine and loss of G_i2 impaired responses to ADP, neither deletion abolished aggregation. To determine whether the remaining responses in each case are mediated by the other G_i family members, double knockouts were produced by crossing the respective single knockouts. Platelets from mice that lacked both G_i2 and G_z showed a pattern of aggregation abnormalities that was approximately the sum of those seen in the absence of G_i2 and G_z individually (Fig. 4A). That is, aggregation of the double knockout platelets was diminished in response to both ADP and epinephrine. However, just as in the G_z(/) platelets, the impaired ability of epinephrine to potentiate responses to other agonists in the G_i2(/)/G_z(/) platelets can be partially overcome by increasing the epinephrine concentration. As illustrated in the experiment shown in Fig. 4A, this increase is achieved by adding epinephrine to platelets along with a suboptimal concentration of the thromboxane A₂ receptor agonist, U46619. The response to these agonists could mean that one of the two remaining G_i family members (G_i1 and G_i3) is able to substitute for G_z when G_i2 is absent. Mice that lacked G_i1 were not available. G_i3(/)/G_z(/) mice were produced by crossing mice carrying the individual knock-outs. The mice themselves were viable and had no evident defects. In general, platelet aggregation by the G_i3(/)/ G_z(/) platelets was indistinguishable from G_z(/) platelets. Reduced potentiation by epinephrine was observed, but responses to other agonists were normal (Fig. 4B and data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Aggregation responses in platelets that lack two Gi family members. A, platelets from Gi2(/)/Gz(/) mice. B, platelets from Gi3(/)/Gz(/) mice. The results shown are representative of those obtained in three experiments. A, ADP; E, epinephrine; U, U46619; WT, wild type; KO, knock-out.

No Increase in Agonist Responses in IP(/) Mice-- As already noted, deletion of the gene that encodes platelet PGI₂ receptors caused a 30% decrease in basal cAMP levels (Fig. 1C). It also abolished the increase in cAMP that would otherwise be caused by either PGI₂ or PGE₁ (Fig. 5A). On the basis of these observations and the sensitivity of platelet responses to small changes in cAMP concentrations, it might be predicted that IP(/) platelets would have increased responses to platelet agonists. Using an independently derived strain of IP(/) mice, Murata et al. (10) observed increased thrombosis after carotid artery injury. However, they found no differences between IP(/) and wild type platelets in the rate or extent of aggregation in response to 5 µM ADP (10). To see whether such differences could be elicited, we measured aggregation at suboptimal concentrations of ADP, U46619, and collagen. No differences were observed under any of the conditions tested (Fig. 5B). Therefore, it is apparently not the change in basal cAMP levels that predisposes IP(/) mice to thrombosis.

View larger version (50K): [in this window] [in a new window]   Fig. 5.   Analysis of platelets from IP(/) mice. A, the ability of 20 µM PGI2 or PGE1 to stimulate an increase in the intracellular cAMP concentration was compared in IP(/) platelets and their wild type (WT) littermates. The data are expressed as a fold-increase over basal cAMP levels and plotted as mean ± S.E. from three studies. B, platelet aggregation. The results are representative of those seen in three experiments. A, ADP; C, collagen; U, U46619.

Inhibition of Basal Versus Stimulated cAMP Formation-- We have shown previously that the impaired ability of epinephrine to potentiate platelet aggregation in G_z(/) mice is accompanied by a reduced ability of epinephrine, but not ADP, to inhibit PGI₂-stimulated cAMP formation (14). Conversely, inhibition of PGI₂-stimulated cAMP formation by ADP is reduced in G_i2(/) platelets, whereas inhibition by epinephrine is intact (Ref. 13 and Fig. 2C). Deletion of G_i3 had no effect on this response to either agonist (Fig. 3B). Given these results, we asked whether ADP or epinephrine would decrease the basal cAMP concentration in wild type platelets and whether a direct inhibitor of adenylyl cyclase would restore the defects in platelet aggregation caused by deletion of a G_i family member. Representative results are shown in Fig. 6. In contrast to their ability to inhibit PGI₂-stimulated adenylyl cyclase activity, neither ADP nor epinephrine caused a decrease in the cAMP concentration in wild type platelets in the absence of PGI₂. Conversely, preincubation of G_z(/) platelets with the membrane-permeable adenylyl cyclase inhibitor, SQ22536, at a concentration sufficient to inhibit PGI₂-stimulated cAMP formation to the same extent as ADP or epinephrine in wild type platelets (data not shown) failed to restore a normal response to epinephrine (Fig. 6B). Thus, under basal conditions the ability to inhibit cAMP formation appears to be neither necessary nor sufficient for platelet aggregation to occur.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Basal cAMP and adenylyl cyclase inhibitors. A, the effects of ADP and epinephrine on basal cAMP levels in wild type (WT) mice (mean ± S.E., n = 16). B, direct inhibition of adenylyl cyclase by SQ22536 does not restore normal responses in Gz(/) platelets. The results shown are representative of those seen in two experiments. A, ADP; E, epinephrine.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human platelets express members of the G_s, G_i, G_q, and G₁₂ families of heterotrimeric G proteins. Current models of platelet activation suggest that each family has a defined role: G_s stimulates adenylyl cyclase, G_q activates phospholipase C, and G₁₂ helps to initiate reorganization of the actin cytoskeleton (13, 14, 20, 21). The four G_i family members that are expressed in platelets are something of a conundrum. Three of them have  subunits that are nearly identical at the protein level. The  subunit of the fourth, G_z, is only 60% identical to the others. It is also not a substrate for pertussis toxin and has a slower intrinsic rate of GTP hydrolysis than the others. Even with these differences and similarities, it is not entirely clear what benefit derives from the expression of so many G_i family members. Recent studies on platelets from mice that lacked G_z or G_i2 suggest part of the answer. G_z is the preferred partner for _2A-adrenergic receptors (14), whereas G_i2 is the preferred partner for the P2Y12 ADP receptor (Ref. 13 and the present study). As a result, loss of G_i2 inhibits ADP responses and loss of G_z inhibits epinephrine responses. Issues left unanswered include the role of the other two G_i family members, G_i3 and G_i1, the basis for the residual response to ADP in the G_i2(/) platelets and to epinephrine in the G_z(/) platelets, the role of G_i family members in the maintenance of the basal cAMP concentration, and the contribution of effectors other than adenylyl cyclase to the requirement for G_i-dependent signaling during platelet activation. These and related issues have been addressed in the present study. We propose that several conclusions can be drawn from the results.

The preference of platelet ADP (P2Y12) receptors and epinephrine (_2A-adrenergic) receptors for G_i2 versus G_z does not appear to extend to G_i3. Deletion of G_i3 had no detectable effect on platelet responses to a variety of agonists. On the other hand, unless it is caused by the small amount of G_i1 expressed in platelets no single G_i family member appears to account for the residual responses to epinephrine in G_z(/) platelets or the remaining ADP response in G_i2(/) platelets. The double knockout of G_i2 and G_z produced a pattern of impaired agonist responses that was approximately the sum of the individual knockouts, not a more profound defect. The remaining responses to ADP and epinephrine in the G_i2(/)/G_z(/) platelets were nearly the same as in the individual knockouts. Likewise, deletion of both G_i3 and G_z had no greater effect than loss of G_z alone. Evidently a strongly preferred partner exists, but in the absence of the preferred partner other G_i family members can substitute for the missing G protein in a fairly promiscuous, if less efficient, manner. Perhaps because of their greater similarity in G sequence, G_i3 or G_i1 may be able to replace G_i2 to a greater extent than all three of these can replace G_z. This ability might account in part for the observation that P2Y12-mediated ADP responses are affected by deletion of G_i2 to a lesser extent than are _2A-adrenergic receptor-mediated responses to epinephrine in the absence of G_z. Regardless, it now seems clear that the two receptors most commonly associated with G_i-dependent events in platelets (P2Y12 and _2A-adrenergic) have strong preferences for particular G_i family members. It also appears that, at least at one level, platelets express more than one G_i family member to accommodate those preferences.

A second conclusion from these studies concerns the regulation and impact of small changes in the basal cAMP concentration in platelets. Ample evidence exists that small, acute changes in cAMP concentration can have large effects on platelet responses to agonists (5-7). Among other consequences, an increase in cAMP concentration essentially shuts down agonist-induced phosphoinositide hydrolysis (20), impairs resynthesis of phosphatidylinositol-4,5-P₂ (21), and accelerates the uptake of Ca²⁺ into the dense tubular system (22, 23). Known substrates for protein kinase A in platelets include glycoprotein Ib (24, 25), actin-binding protein (26), myosin light chain kinase (27, 28), Rap1B (29), and G₁₃ (30). The cAMP concentration in mouse platelets (4-5 pmol/10⁸ platelets) is very similar to the basal cAMP concentration in human platelets (6, 7, 19). Deletion of G_i2 or G_z caused this value to increase, whereas deletion of the only known PGI₂ receptor in platelets, IP, caused it to fall. The magnitude of the changes was sufficient to affect responses to thrombin when imposed acutely in vitro (7). Nonetheless, in the present study such changes in cAMP levels appeared to have no effect on agonist responses ex vivo or at least no effect that could not be attributed to the loss of a preferred G_i family member when ADP or epinephrine responses were measured. If nothing else, this fact suggests that the basal cAMP concentration in circulating platelets reflects ongoing signaling through PGI₂ receptors and through G_i2 and G_z as well as the balance that is maintained between ongoing cAMP synthesis and hydrolysis. This finding may also suggest that small, chronic changes in basal cAMP concentration have less of an impact than acute changes of similar magnitude, although this particular point was not addressed directly.

A third conclusion from the present observations regards the different roles that are played by G_i family members during platelet activation. Recent studies, including those by Kunapuli and co-workers (31, 32), have shown that optimal platelet activation requires signaling through G_i-coupled receptors as well as those that couple to G_q and G₁₂ family members. The present study shows that the requirement for G_i-dependent signaling cannot be solely the result of the ability of these proteins to inhibit adenylyl cyclase. First, in the absence of PGI₂ neither of the prototypical G_i-coupled agonists (ADP and epinephrine) had a measurable effect on cAMP levels and second, the defect in G_z(/) platelets could not be reversed by the simple addition of a membrane-permeable inhibitor of adenylyl cyclase, even though that inhibitor was found to suppress PGI₂-stimulated cAMP formation as effectively as ADP or epinephrine. Therefore, although G_i family members can clearly inhibit PGI₂-stimulated cAMP formation in platelets, their ability to regulate other effectors presumably accounts for their essential role in promotion of platelet activation when PGI₂ is absent. This conclusion is consistent with observations on human platelets with the use of receptor antagonists and adenylyl cyclase inhibitors (33). The full range of effectors for G_i family members other than adenylyl cyclase is still being explored, but recent studies show that at least one of those pathways involves phosphatidylinositol 3-kinase  and the Ras family member, Rap1B, possibly via G_i-derived G (34, 35). The particular relevance of this pathway is suggested by the recent demonstration that Rap1B supports _IIb3 activation (36). Similar to the inhibition of adenylyl cyclase, Rap1B activation can be triggered in platelets by ADP (via G_i2) and epinephrine (via G_z) (35). The fact that more than one G_i family member can do so suggests again that the specificity in the roles of the G_i family members in platelets lies at the level of receptor coupling and not at the level of effector interactions.

Finally, the results of the present study also have implications for the role of PGI₂ in the regulation of platelet responsiveness. In theory the tendency toward injury-induced thrombosis in IP(/) mice could be the result of either the observed fall in basal cAMP levels or of a failure to respond to local accumulations of PGI₂. Our inability to detect enhanced sensitivity of IP(/) platelets to low concentrations of agonists calls into question the impact of the decrease in basal cAMP. PGI₂ is synthesized in endothelial cells and released in response to endothelial cell agonists, including thrombin (37, 38). The fact that thrombin is produced locally in response to vascular injury suggests that part of the response to injury is to generate an inhibitor of platelet activation (PGI₂) whose effects on platelet function must then be overcome to permit the formation of a platelet plug. Although this process seems somewhat cumbersome, it is likely that the system exists to place a threshold on platelet responsiveness when the extent of injury does not warrant extensive platelet activation. Only when the impetus for platelet plug formation is sufficiently great does the ability of agonists to inhibit adenylyl cyclase via G_i family members become essential. It also suggests that the increased tendency toward thrombosis in IP(/) mice after arterial injury is not caused by a generalized increase of agonist responsiveness in the circulating platelet population but rather by a loss of the regulatory effects of PGI₂, specifically at the site of injury.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL-45181 and HL-54500 and by an American Heart Association postdoctoral fellowship (awarded to D. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Vascular Biology, Centocor, Inc., Malvern, PA 19355.

^¶ Current address: Clinical Pharmacology, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, Ireland.

To whom correspondence should be addressed: University of Pennsylvania, Rm. 913 BRB-II, 421 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-573-3540; Fax: 215-573-2189; E-mail: Brass@mail.med.upenn.edu.

Published, JBC Papers in Press, September 23, 2002, DOI 10.1074/jbc.M208519200

	ABBREVIATIONS

The abbreviations used are: PGI₂, prostacyclin; IP, prostacyclin receptor; PAR4, proteinase-activated receptor-4; TBS, Tris-buffered saline.

	REFERENCES

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				S. Mukhopadhyay and A. C. Howlett Chemically Distinct Ligands Promote Differential CB1 Cannabinoid Receptor-Gi Protein Interactions Mol. Pharmacol., June 1, 2005; 67(6): 2016 - 2024. [Abstract] [Full Text] [PDF]

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