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The intracellular signaling pathways by which G protein–coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the Gi pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet Gαi subtype, Gαi2, has been disrupted. In intact platelets from Gαi2-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin αIIbβ3 (GPIIb-IIIa) were strongly reduced in platelets from Gαi2-deficient mice. In addition, Gαi2-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase–coupled platelet ADP receptor (P2Y12) on wild-type platelets with a selective antagonist. These observations suggest that Gαi2 is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the Gαi2–linked ADP receptor.
Platelets play a critical role in hemostasis and thrombosis, and drugs inhibiting platelet aggregation are effective antithrombotics. Platelet aggregation requires induction of an active conformation of the integrin αIIbβ3 (GPIIb-IIIa), capable of binding soluble fibrinogen and thus cross-linking the platelets. The signaling pathways leading to integrin activation (inside-out signaling) are not well understood (1). Most platelet agonists (e.g., thrombin, ADP, thromboxane A2, epinephrine, and serotonin) bind to G protein–coupled receptors on the platelet surface. This results in the activation of heterotrimeric G proteins consisting of various α and βγ subunits that initiate multiple signaling pathways by interacting with downstream effectors. Platelets express Gαs, Gαq, Gα12/13, Gαz, Gαi2, and Gαi3 (2–5). Whereas Gαs appears to mediate prostacyclin-dependent platelet inhibition by stimulating adenylyl cyclase, Gαq is known to activate phospholipase C, resulting in release of intracellular calcium and activation of protein kinase C. Gα12/13 might be involved in events leading to platelet shape change, and Gαz is capable of inhibition of adenylyl cyclase. Similarly, it has been shown in other cells that Gαi2 and Gαi3 can mediate repression of cAMP formation whereas the released βγ subunits may activate phospholipase Cβ or other signaling molecules (4, 5). The requirement of intracellular calcium mobilization and the Gq pathway for platelet aggregation has been well established. Platelets from mice in which the gene for Gαq has been disrupted are defective in calcium mobilization and aggregation by all agonists (6). Of the inhibitory G proteins in platelets, only Gαz has been investigated in vivo. Platelets from Gαz-knockout mice appear abnormal only for epinephrine-mediated inhibition of adenylyl cyclase and the potentiating effect of epinephrine on aggregation induced by other agonists (7).
Recently, however, the potential relevance of the Gi pathway for platelet activation has been implied from pharmacological studies. For example, ADP-dependent aggregation requires not only activation of a receptor mediating calcium mobilization (P2Y1), but also concomitant stimulation of an ADP receptor coupled to the inhibition of adenylyl cyclase (P2Y12, previously called P2TAC, P2YADP, P2YAC, P2Ycyc) (8–14). Furthermore, after blockade of the P2Y12 receptor, ADP-dependent aggregation could be rescued by epinephrine, which lowers cAMP levels (10, 15). Therefore, in addition to the Gq pathway, it appears that full platelet aggregation requires activation of receptors that are capable of inhibiting adenylyl cyclase and are likely to couple to a G protein of the Gi family. Indeed, in isolated human platelet membranes, stimulation of ADP receptors or the PAR1 thrombin receptor resulted in activation of Gαi2 (3). However, this does not mean that adenylyl cyclase inhibition or a Gi protein is part of the signaling cascade leading to integrin activation and platelet aggregation. In fact, lowering cAMP levels using an adenylyl cyclase inhibitor did not induce platelet aggregation, even with concomitant activation of the Gq pathway (16–18). Thus, it was assumed previously that repression of the cAMP pathway does not lead to aggregation, although low cAMP levels are required to permit aggregation.
It remains to be clarified which of the inhibitory G proteins (Gi2, Gi3, Gz) mediate inhibition of adenylyl cyclase in intact platelets, and more importantly, whether a Gi protein is involved in the pathway leading to integrin activation and aggregation. In the present study we have investigated the role of the predominant platelet Gαi protein, Gαi2, in thrombin, epinephrine, and ADP-dependent platelet activation by using platelets from mice in which the gene for the α subunit Gαi2 has been disrupted. Gαi2–/– mouse strains have been established, but their platelet function has not been reported (19, 20). Because released ADP appears to be a crucial cofactor of other platelet agonists as well (21), we have used the selective ADP receptor antagonist 2-methylthioadenosine 5′-monophosphate (2MeSAMP) to determine the contribution of released ADP activating a Gαi2-coupled ADP receptor in thrombin-induced platelet signaling.
Mice genetically deficient in Gαi2 were produced by homologous recombination in embryonic stem cells and blastocyst-mediated transgenesis as described previously (20). Mice used for these experiments were generally sex-matched littermates of heterozygous breeding pairs of a mixed 129/Svter and C57BL/6J genetic background. For some experiments, platelets were pooled from littermates and age- and sex-matched offspring of a large set of heterozygous mating pairs. In these cases mutant and control mice were selected randomly to avoid introducing systematic differences in potential modifier loci derived from the two genetic backgrounds used. All procedures conformed to institutional guidelines and to the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, Maryland, USA).
Following cardiac puncture 0.7 ml of mouse blood was drawn into 0.56 ml of 0.9% NaCl and 0.14 ml of TSC (3.8% trisodium citrate, pH 7) plus 100 nM prostaglandin E1 (PGE1; Sigma Chemical Co., St. Louis, Missouri, USA) for platelet-rich plasma (PRP), or 0.14 ml ACD (85 mM sodium citrate, 111 mM glucose, 71.4 mM citric acid) plus 100 nM PGE1 for washed platelets (WPs) (22). Diluted PRP was obtained after centrifugation of blood from individual mice at 80 g for 10 minutes in an Eppendorf microcentrifuge. For WPs, after removal of the PRP, the lower phase was diluted with 1 vol of 0.9% NaCl/10% ACD and centrifuged again. This step was repeated, the three PRP samples pooled, centrifuged at 730 g for 15 minutes, and the resulting platelet pellet resuspended in CGS (13 mM sodium citrate, 30 mM glucose, 120 mM NaCl) containing 1 U/ml apyrase (grade VII; Sigma Chemical Co.). After incubation for 15 minutes at 37°C, platelets were collected by centrifugation and resuspended at 2 × 108/ml in HEPES/Tyrode’s buffer/BSA (10 mM HEPES, 138 mM NaCl, 5.5 mM glucose, 2.9 mM KCl, 12 mM NaHCO3 , pH 7.4, 0.1% BSA) at room temperature.
Washed mouse platelets were lysed in hypotonic buffer (10 mM HEPES, 5 mM EDTA, pH 7.4), including a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, Indiana, USA), by sonication for three times for 3 seconds on ice. After centrifugation at 600 g for 10 minutes at 4°C, crude platelet membranes were collected from the supernatant by centrifugation at 20,000 g for 30 minutes at 4°C. Protein samples (30 μg) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with PBS-T (PBS, 0.05 % Tween 20) containing 2.5% nonfat dry milk, followed by overnight incubation at 4°C with anti–G protein α and β subunit Ab’s (Gαi2 mouse mAb, Chemicon International, Temecula, California, USA; Gαq , Gαi1 , and Gαi3 rabbit antisera, Calbiochem-Novabiochem Corp., La Jolla, California, USA; Gαz and Gβ1-4 rabbit antisera, Santa Cruz Biotechnology Inc., Santa Cruz, California, USA). After treatment with horseradish peroxidase–conjugated goat anti-mouse or mouse anti-rabbit Ig (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA), the blots were developed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, New Jersey, USA).
After pretreatment with 0.5 mM IBMX (3-isobutyl-1-methylxanthine; Research Biochemicals International, Natick, Massachusetts, USA) for 5 minutes at 37°C, washed mouse platelets (108/ml in HEPES/Tyrode’s buffer/BSA) were incubated in triplicate with antagonists, agonists, and 50 μM forskolin (Sigma Chemical Co.) for 5 minutes at 37°C (13). Levels of cAMP were determined in ethanol extracts using a radioimmunoassay (Amersham Pharmacia Biotech).
Aggregation was determined in an aggregometer (Chrono-Log Corp., Havertown, Pennsylvania, USA) with stirring (1, 200 rpm) at 37°C using washed mouse platelets (2 × 108/ml in HEPES/Tyrode’s buffer/BSA, 1 mM MgCl2 , 1 mM CaCl2) after a recovery period of 1 hour. Human fibrinogen (0.4 mg/ml, Grade L; American Diagnostics, Inc. Greenwich, Connecticut, USA) was added for ADP-dependent aggregation (ADP from Roche Molecular Biochemicals and human α-thrombin from Haematologic Technologies, Essex Junction, Vermont, USA). The P2Y12 antagonist 2MeSAMP was obtained from Research Biochemicals International. For platelet shape-change measurements 5 mM EDTA was added to washed platelets (5 × 107/ml) after a 2-hour recovery period to prevent aggregation, and the decrease in light transmittance upon agonist addition was traced in an aggregometer with an amplified recorder scale setting.
Diluted mouse PRP was prepared as described above and incubated at 5 × 107 platelets/ml with antagonists, agonists, and 60 μg/ml FITC-conjugated fibrinogen (human fibrinogen from Enzyme Research Laboratories, South Bend, Indiana, USA, labeled with FITC celite from Molecular Probes Inc., Eugene, Oregon, USA) for 30 minutes at room temperature. To thrombin-containing samples, 2.5 mM of the peptide GlyProArgPro (Bachem Bioscience Inc., King of Prussia, Pennsylvania, USA) was added to prevent thrombin-mediated fibrin generation. The samples were then transferred to 4°C and fixed with 1% p-formaldehyde for 30 minutes. After dilution with 0.5 ml 1% p-formaldehyde/PBS the fluorescence of platelet-bound FITC-fibrinogen was analyzed with a FACSort flow cytometer (Becton-Dickinson Immunocytometry Systems, San Jose, California, USA) (22). A FITC-conjugated Ab raised against mouse integrin β3 subunit (anti-mouse CD61; PharMingen, San Diego, California, USA) was used as a positive control.
Mouse platelets were prepared using the washed platelet procedure until the CGS step described above, except that PGE1 (100 nM) was not added to the blood, but to the pooled diluted PRP. Platelets in CGS were incubated with 1 U/ml apyrase and 4 μM Fura-2AM/0.008% Pluronic F-127 (Molecular Probes Inc.) for 30 minutes at 37°C, collected by centrifugation, and resuspended at 2 × 108/ml in HEPES/Tyrode’s buffer/BSA buffer containing 0.1 U/ml apyrase and 1 mM MgCl2. After 1.5–2 hours, peak intracellular calcium ion concentrations in response to ADP and thrombin were determined in a SLM-Aminco AB2 spectrofluorometer (SLM-Aminco, Urbana, Illinois, USA) using the ratio method as described (13).
Mice with a disruption of the Gαi2 gene did not display any obvious abnormalities of the hemostatic system, but the effects on platelet function have not been investigated (19, 20). We have found no significant difference in the platelet counts between wild-type and Gαi2 –/– mice (599 ± 73 × 103 /μl, n = 5, versus 621 ± 67 × 103/μl, n = 5). The expression of Gαi2 and other Gα subtypes was analyzed in crude platelet membranes using Western blot analysis. As expected, no Gαi2 protein could be detected in membranes from Gαi2–/– mice, and the expression of Gαi2 was reduced in platelet membranes from heterozygous mice (Figure (Figure1).1). By comparison, no difference was seen in the abundance of Gαq. To determine whether the absence of Gαi2 resulted in upregulation of other G proteins capable of inhibiting adenylyl cyclase, as reported for Gαi3 in SV40-transformed embryonic fibroblasts from Gαi2–/– mice (19), the expression levels for Gαi1 , Gαi3 , and Gαz were compared in platelet membranes prepared from wild-type and Gαi2–/– mice. No significant difference in the abundance of Gαi3 and Gαz proteins could be detected (Figure (Figure1).1). There was also no change in the level of Gβ protein that could reflect different expression of βγ subunits in Gαi2–/– platelets (data not shown). In confirmation of a previous report on human platelets (3), we were unable to detect expression of Gαi1 in wild-type mouse platelet membranes (data not shown). It thus appears that the lack of Gαi2 does not lead to an increased expression in platelets of other G protein subunits capable of mediating the inhibition of adenylyl cyclase.
The G protein subunit Gαi2 is known to be capable of mediating inhibition of stimulated adenylyl cyclase in many cell types and is also the most abundant Gαi form in platelets. To determine the role of Gαi2 in the regulation of platelet adenylyl cyclase in intact mouse platelets, we have compared the ability of ADP, thrombin, and epinephrine to inhibit forskolin-stimulated cAMP accumulation in washed platelets from wild-type and Gαi2–/– mice. ADP inhibited cAMP formation in platelets from wild-type mice resulting in 60% and 76% inhibition at 1 and 10 μM ADP, respectively (Figure (Figure2a).2a). In platelets from Gαi2–/– mice, however, ADP-dependent repression of cAMP levels was impaired with only 30% and 52% inhibition observed at 1 and 10 μM ADP, respectively. This suggests that Gαi2 is important for ADP-dependent inhibition of mouse platelet adenylyl cyclase in vivo. Interestingly, this defect can be detected even in the presence of other inhibitory G proteins, such as Gαi3 and Gαz , which may partially substitute for Gαi2 in Gαi2–/– platelets.
Thrombin repressed forskolin-stimulated cAMP accumulation in wild-type mouse platelets, reaching 65% and 62% inhibition at 10 and 100 nM thrombin, respectively. This inhibition was impaired in platelets prepared from Gαi2–/– mice with 38% and 46% inhibition at 10 and 100 nM thrombin (Figure (Figure2b).2b). To determine whether this defect is based on a direct coupling of Gαi2 to mouse thrombin receptors or to thrombin-dependent release of ADP, which then activates the Gαi2 -coupled platelet ADP receptor P2Y12 , the cAMP experiment with wild-type platelets was performed in the presence of 2MeSAMP. It has been reported previously that 2MeSAMP is a selective antagonist of the human platelet ADP receptor (P2Y12), mediating the inhibition of adenylyl cyclase (13, 14). We have confirmed the properties of this antagonist on mouse ADP receptors (data not shown). Interestingly, addition of 5 μM 2MeSAMP prevented the inhibition of cAMP accumulation even at the relatively high thrombin concentration of 100 nM (Figure (Figure2c).2c). These results indicate that thrombin-dependent inhibition of platelet adenylyl cyclase is not mediated by thrombin receptors, but by ADP released upon stimulation of platelets activating the Gαi2 -coupled ADP receptor (P2Y12) in wild-type mice. Therefore, thrombin-dependent inhibition of adenylyl cyclase is defective in Gαi2 –/– mice.
By contrast, no significant impairment of epinephrine-mediated inhibition of adenylyl cyclase could be detected (Figure (Figure2d).2d). Epinephrine inhibited forskolin-stimulated cAMP formation in wild-type platelets by 21%, 53%, and 80% and in Gαi2–/– platelets by 9%, 55%, and 86% at 0.01, 0.1, and 1 μM epinephrine, respectively. This inhibition suggests either that Gαi2 does not mediate epinephrine-dependent inhibition of platelet adenylyl cyclase or that other inhibitory G proteins, e.g., Gαi3 or Gαz , can functionally replace Gαi2 in Gαi2–/– platelets. The former interpretation is consistent with the suggestion based on studies with Gαz –/– platelets that the effects of epinephrine appear to be predominantly mediated by Gαz (7).
To determine the role of Gαi2 in platelet aggregation, we compared ADP- and thrombin-induced aggregation of washed platelets from wild-type and Gαi2–/– mice. ADP induced half-maximal aggregation of wild-type platelets at about 0.3 μM and full aggregation at 10 μM (Figure (Figure3a).3a). In Gαi2–/– platelets, the final extent of aggregation was reduced by 35–67% and the slopes of aggregation tracings were diminished as well. Even at the very high ADP concentration of 100 μM, aggregation was still impaired by 21–38% (data not shown). These results support the conclusion that Gαi2 is not only important for the inhibition of platelet adenylyl cyclase but also for platelet aggregation. ADP-dependent aggregation was also investigated in platelets from heterozygous mice. No defect was detected, which is consistent with the assumption that the amount of Gαi2 in platelets from heterozygous mice is sufficient to saturate the small number of ADP receptors (data not shown).
Thrombin-induced aggregation was also impaired in platelets from Gαi2–/– mice, but only at threshold concentrations of thrombin that induce slightly reversible aggregation (Figure (Figure3b).3b). With platelets from Gαi2–/– mice, maximum aggregation was reduced to about 50% when compared with wild-type platelets, and aggregation was almost completely reversible within 5 minutes. At a fivefold higher concentration of thrombin, which induces irreversible aggregation in wild-type platelets, no effect of Gαi2 gene disruption was observed. Since aggregation induced by low concentrations of thrombin has been shown previously to be dependent on released ADP (21), the P2Y12 ADP receptor antagonist 2MeSAMP was included in the experiment. Blockade of the P2Y12 receptor on wild-type platelets by 10 μM 2MeSAMP mimicked essentially the observations with thrombin-induced platelet aggregation of platelets from Gαi2–/– mice (Figure (Figure3c).3c). Apparently, Gαi2 does not couple to mouse platelet thrombin receptors in the pathway leading to aggregation, or Gαi3 together with Gq, and possibly other G proteins can functionally replace Gαi2.
Platelet aggregation requires activation of the platelet integrin αIIbβ3 (GPIIb-IIIa) enabling the binding of soluble fibrinogen with high affinity and avidity. Integrin activation was determined as an ADP or thrombin-induced increase in binding of FITC-conjugated fibrinogen to platelets using flow cytometry. FITC-fibrinogen binding to wild-type platelets was induced 4.1-fold, 13-fold, and 18-fold by 1, 10, and 100 μM ADP, respectively (Figure (Figure4a).4a). By comparison, in Gαi2–/– platelets, induction of FITC-fibrinogen binding was strongly reduced even at high ADP concentrations, resulting in an induction factor of only 1.4, 1.8, and 2.5. Apparently, Gαi2 is required for ADP-mediated inside-out integrin signaling with little substitution by other G proteins still present in Gαi2–/– platelets. Thrombin-induced FITC-fibrinogen binding is also reduced in Gαi2–/– platelets, however, to a lesser degree (Figure (Figure4b).4b). FITC-fibrinogen binding was induced 15- and 23-fold by 10 and 100 nM thrombin in wild-type platelets and 7- and 20-fold in Gαi2–/– platelets, respectively. In wild-type mouse platelets, blockade of the P2Y12 ADP receptor linked to inhibition of adenylyl cyclase with 100 μM 2MeSAMP also resulted in a reduction of thrombin-mediated FITC-fibrinogen binding (Figure (Figure4c).4c). These data support the hypothesis that Gαi2 is involved in inside-out integrin signaling by thrombin predominantly through released ADP.
ADP mediates platelet shape change through the P2Y1 receptor, which is linked to intracellular calcium mobilization. Antagonists of the P2Y12 ADP receptor coupled to inhibition of adenylyl cyclase do not block shape change (8, 13, 21). Furthermore, no ADP-dependent shape change has been detected in platelets from mice in which the Gαq gene has been disrupted (6, 23). However, signaling pathways independent of Gαq and calcium have been proposed to be involved in ADP-mediated shape change (5, 24). As shown in Figure Figure5,5, platelet shape change upon addition of ADP was not affected in Gαi2–/– mice. This is consistent with Gαq being at least the predominant mediator of this response, although some involvement of Gαi2 , substituted by Gαi3 and Gαz in Gαi2–/– platelets, cannot be excluded.
The Gαq pathway and intracellular calcium mobilization do not appear to be required for thrombin-dependent platelet shape change, but Gα12 and/or Gα13 might be involved (5, 6). The contribution of GI proteins has not been investigated. Here we show that the shape change response in platelets from Gαi2–/– mice was not impaired, even at low concentrations of thrombin (0.1 nM; Figure Figure5),5), suggesting that Gαi2 is not a major component in the signaling pathway for thrombin-dependent platelet shape change.
Intracellular calcium mobilization is a critical event for platelet activation and has been attributed to the Gq pathway. Consequently, in platelets from Gαq –/– mice no intracellular calcium mobilization was detected in response to ADP, thrombin, or other platelet agonists (6). However, the contribution of other G proteins, e.g., βγ subunits released from Gαi2 and capable of activation of phospholipase Cβ, cannot be excluded from those data. Wild-type and Gαi2–/– platelets were analyzed for intracellular calcium mobilization using the fura-2 ratio method. In three independent experiments no impairment of responses to 1 and 10 μM ADP or 1 nM thrombin was found in Gαi2–/– platelets (data not shown). These findings are consistent with the hypothesis that Gαi2 does not play any role in this signaling pathway.
This study provides direct evidence for the importance of Gαi2 for platelet activation resulting in aggregation. Platelets from Gαi2–/– mice have been found to be defective for ADP-dependent inhibition of adenylyl cyclase, integrin activation, and aggregation. Intracellular calcium mobilization and shape change were not affected, which is consistent with previous results with Gαq–/– platelets and selective ADP receptor antagonists, which suggested a predominant role of the Gq pathway for these responses (6, 23). Lack of expression of Gαi2 resulted in only partial signaling defects. It is conceivable that other inhibitory G proteins expressed in the Gαi2–/– platelets, such as Gαi3 and maybe Gαz , can be activated by ADP receptors even though they might not couple efficiently to these receptors in wild-type platelets when Gαi2 is present. This would be consistent with the finding that Gαz–/– platelets display normal cAMP signaling in response to ADP and thrombin-receptor stimulation (7). The fact that any defects could be detected in the presence of a likely excess of other inhibitory G proteins supports the crucial role of Gαi2 in this signaling pathway.
The finding that integrin activation was more affected in the Gαi2–/– platelets than inhibition of adenylyl cyclase points to a pathway between Gαi2 and the integrin that is independent of cAMP and strongly suggests a pivotal role for Gαi2 in inside-out signaling. However, the almost complete loss of ADP-dependent integrin activation did not result in a similar dramatic effect on platelet aggregation, although integrin activation is thought to be a prerequisite of aggregation. There are at least two possible explanations for this apparent discrepancy. First, platelets from β3 integrin heterozygous -/+mice that express only 50% of wild-type level of GPIIb-IIIa are still capable of full aggregation (25). Second, in contrast to measurements of FITC-fibrinogen binding, aggregation assays are performed with an excess of fibrinogen and stirring, facilitating integrin outside-in signaling that enhances aggregation (1). A contribution of released thromboxane A2 to the observed aggregation can be excluded, because addition of a thromboxane A2 receptor antagonist did not affect aggregation induced by ADP or thrombin (H.-M. Jantzen, unpublished observations).
The data in the present study support a role of Gαi2 in thrombin-dependent inhibition of adenylyl cyclase, integrin activation, and aggregation, although the effect was less pronounced than with ADP. Interestingly, blockade of the ADP receptor mediating inhibition of adenylyl cyclase (P2Y12) by 2MeSAMP in wild-type platelets had effects on thrombin signaling that were similar to those of the Gαi2 knockout. This supports the hypothesis that Gαi2 does not directly couple to mouse platelet thrombin receptors, but instead ADP released upon thrombin activation of platelets activates Gαi2 -coupled ADP receptors. These results are also consistent with other reports emphasizing the role of released ADP and a Gi-linked ADP receptor in platelet activation by other agonists (21, 26–29). The observation that the P2Y12 antagonist 2MeSAMP interfered with thrombin effects on adenylyl cyclase and fibrinogen binding to a larger extent than did the Gαi2 gene disruption, substantiates the conclusion above that the P2Y12 receptor couples to other proteins of the Gi family in addition to Gαi2. The findings presented here suggesting that mouse thrombin receptors do not directly mediate inhibition of adenylyl cyclase in intact platelets support the observation that the cloned thrombin receptors PAR3 and PAR4 do not couple to adenylate cyclase (30). Unlike mouse platelets, human platelets express PAR1 instead of PAR3, and PAR1 is capable of mediating inhibition of adenylyl cyclase, indicating a different role for Gαi2 in thrombin signaling in human platelets (31).
The signaling cascade between Gi2 and activation of the platelet integrin αIIbβ3 (GPIIb-IIIa) still remains to be elucidated. Gαi2 mediates inhibition of adenylyl cyclase, but direct inhibition of the enzyme, even with concomitant activation of the Gq pathway, is not sufficient for platelet aggregation (16–18). Since high cAMP levels prevent platelet activation, the phosphorylation-dephosphorylation equilibrium of substrates of the cAMP-dependent protein kinase A might be important for inside-out signaling. Alternatively, cAMP and Gαi2 could directly interact with Epac and Rap1GAPII, respectively, which are regulators of Rap1, a small guanine nucleotide-binding protein activated by ADP and thrombin (32–34).
This study demonstrates a critical role for Gαi2 in platelet activation, especially by ADP. However, it is possible that the absence of the βγ subunits normally released from activated Gαi2 and not the lack of Gαi2 per se, is responsible for some of the observed defects. For example, βγ subunits can activate isoforms of phosphoinositide-3-OH kinase (PI 3-kinase). PI 3-kinases and released ADP acting on the adenylyl cyclase–coupled ADP receptor (P2Y12) appear to be important for the stabilization of platelet aggregates (1, 21, 27, 29). Alternatively, released βγ subunits may activate nonreceptor tyrosine kinases and other signaling proteins (1, 35). The challenge for future research will be the identification of the missing links of the signaling cascade between Gαi2 and platelet integrin αIIbβ3 (GPIIb-IIIa). The analysis of platelet function from mice lacking Gαi2 , other signaling proteins, or the Gαi2 -coupled ADP receptor P2Y12 in combination with selective inhibitors will facilitate progress toward this goal.
We thank F. Deguzman and the COR Therapeutics Inc. animal facility, G. Li and E. Thompson for technical assistance. We thank E. Reynolds and C. Homcy for help with initiating the project; D. Oksenberg, D. Law, D. Phillips, C. Homcy, V. Ramakrishnan, and R. Scarborough for advice or critical reading of the manuscript; and R. Wong, D. DeGuzman and D. Stockett for help with the figures. D.S. Milstone was supported by grants from the NIH (HL-5409 and HL-30628). R.M. Mortensen was supported by NIH grant R01 HL-58606.