Distinct Role of Pyk2 in Mediating Thromboxane Generation Downstream of Both G12/13 and Integrin αIIbβ3 in Platelets*

Background: Pyk2 is abundantly expressed in platelets. Results: Pyk2 regulates thromboxane A2 generation induced by both 2-methylthio-ADP and AYPGKF. Conclusion: Pyk2 is an important functional tyrosine kinase that is activated by both G12/13 and integrin αIIbβ3 in platelets. Significance: Understanding the mechanism of activation of Pyk2 enhances our understanding of the platelet inside-out and outside-in signaling events. Proline-rich tyrosine kinase 2 (Pyk2) is activated by various agonists in platelets. We evaluated the signaling mechanism and the functional role of Pyk2 in platelets by using pharmacological inhibitors and Pyk2-deficient platelets. We found that platelet aggregation and secretion in response to 2-methylthio-ADP (2-MeSADP) and AYPGKF were diminished in the presence of Pyk2 inhibitors or in Pyk2-deficient platelets, suggesting that Pyk2 plays a positive regulatory role in platelet functional responses. It has been shown that ADP-, but not thrombin-induced thromboxane (TxA2) generation depends on integrin signaling. Unlike ADP, thrombin activates G12/13 pathways, and G12/13 pathways can substitute for integrin signaling for TxA2 generation. We found that Pyk2 was activated downstream of both G12/13 and integrin-mediated pathways, and both 2-MeSADP- and AYPGKF-induced TxA2 generation was significantly diminished in Pyk2-deficient platelets. In addition, TxA2 generation induced by co-stimulation of Gi and Gz pathways, which is dependent on integrin signaling, was inhibited by blocking Pyk2. Furthermore, inhibition of 2-MeSADP-induced TxA2 generation by fibrinogen receptor antagonist was not rescued by co-stimulation of G12/13 pathways in the presence of Pyk2 inhibitor. We conclude that Pyk2 is a common signaling effector downstream of both G12/13 and integrin αIIbβ3 signaling, which contributes to thromboxane generation.

activation of platelets. ADP induces platelet activation by signaling through G q -coupled P2Y 1 and G i -coupled P2Y 12 receptors. Unlike ADP, a number of receptors can couple to G 12/13 including thrombin and TxA 2 , and platelet shape change induced by these agonists is mediated by both calcium-dependent and -independent mechanisms that occurs through G q and G 12/13 pathways, respectively (2). G 12/13 has been shown to regulate the Rho-dependent response (3), and the RhoA-p160 ROCK pathway plays an important role in G 12/13 -mediated platelet shape change (4). It has also been shown that G 12/13 pathways regulate dense granule secretion through RhoA-p160 ROCK pathways (5) and contribute to the tyrosine phosphorylation of PKC␦ on the Tyr-311 residue (6) that regulates thromboxane generation (7). Recent studies have shown that G 12/13 pathways could be activated directly through integrin outside-in signaling events (8).
The non-receptor, proline-rich protein tyrosine kinase Pyk2 is a member of focal adhesion protein tyrosine kinase (FAK) family. Both Pyk2 and FAK have a molecular mass between 110 and 125 kDa and are closely related in their overall structure. These two kinases lack SH2 and SH3 domains but, in their C-terminal region, possess two proline-rich domains (9). Pyk2 contains an N-terminal FERM domain, a central kinase domain, three proline-rich motifs, and a C-terminal focal adhesion targeting domain. Tyr-402 phosphorylation serves as a binding site for Src with subsequent phosphorylation of activation loop residues Tyr-579, Tyr-580, and Tyr-881 in the focal adhesion targeting domain, which promotes binding of the adapter protein Grb2 (10). Recently, Pyk2 knock-out mice have been generated and the role of Pyk2 in macrophages (11) and osteoclasts (12) have been studied.
Pyk2 is abundantly expressed in both megakaryocytes and platelets, in addition to brain tissues and epithelial cells (13,14). Treatment of platelets with several agonists induced the tyrosine phosphorylation of Pyk2 through integrin-dependent and integrin-independent mechanisms (15). They have also shown that Pyk2 tyrosine phosphorylation is regulated by calcium and is mediated through PKC pathways. However, it has been also reported that PKC activation, but not calcium mobilization, is involved in Pyk2 phosphorylation in thrombin-activated platelets (16). Stimulation of human platelets with von Willebrand factor also induces the rapid phosphorylation of Pyk2, which is not affected by either calcium chelation or PKC inhibition (17). In addition, Pyk2 phosphorylation has been shown to be mostly dependent on integrin ␣IIb␤3 and PKC in human platelets (18). Moreover, PI 3-kinase activity has been shown to be involved in Pyk2 tyrosine phosphorylation in low-dose thrombin-stimulated platelets (19). In contrast, it has been shown that the Pyk2 activity is not affected by blocking PI 3-kinase (20). Thus, the signaling mechanism of Pyk2 activation in platelets is complex and controversial and the functional role of Pyk2 in platelet activation has not been fully understood.
TxA 2 is generated from its precursor arachidonic acid through cycloxygenase pathways, acts as a positive feedback mediator, and amplifies the initial platelet responses and stabilizes the hemostatic plug. Previous study has shown that ADPinduced TxA 2 generation depends on outside-in signaling (21). Interestingly, it has been shown that PAR-mediated TxA 2 generation occurs independently of integrin ␣IIb␤3-mediated outside-in signaling, and G 12/13 pathways can rescue the inhibitory effect of fibrinogen receptor antagonist to induce TxA 2 generation (21). These studies have raised the possibility that G 12/13 pathways substitute for integrin-mediated signaling by activating similar effector molecule, but the common signaling molecule downstream of integrins and G 12/13 pathways responsible for this event has not been identified.
In the present study, we demonstrate that Pyk2 is activated downstream of both G 12/13 and integrin pathways, and both 2-MeSADP-and AYPGKF-induced TxA 2 generation is inhibited in Pyk2-deficient platelets. We show that G 12/13 pathways fail to substitute for the integrin-mediated outside-in signaling for TxA 2 generation when Pyk2 is blocked. Moreover, integrindependent TxA 2 generation induced by combined G i and G z stimulation is abolished by blocking Pyk2. Therefore, we conclude that Pyk2 is a common signaling effector downstream of both G 12/13 and integrin ␣IIb␤3 signaling pathways, which plays a crucial role in thromboxane generation in platelets.
Preparation of Human and Mouse Platelets-Human blood was obtained from a pool of healthy volunteers in a one-sixth volume of acid/citrate/dextrose. Platelet-rich plasma was prepared by centrifugation at 230 ϫ g for 20 min at room temperature (RT). Acetylsalicylic acid was added to platelet-rich plasma to a final concentration of 1 mM, and the preparation was incubated for 45 min at 37°C followed by centrifugation at 980 ϫ g for 10 min at RT. In the experiments with TxB 2 measurements, the treatment of platelet-rich plasma with acetylsalicylic acid was omitted.
Mouse blood was collected from anesthetized mice into syringes containing 1/10th blood volume of 3.8% sodium citrate as anticoagulant. Red blood cells were removed by centrifugation at 100 ϫ g for 10 min at RT. Platelet-rich plasma was recovered, and platelets were pelleted at 400 ϫ g for 10 min. The platelet pellet was resuspended in Tyrode's buffer (pH 7.4) containing 0.05 units/ml of apyrase to a density of 2 ϫ 10 8 cells/ml.
Platelet Aggregation and Secretion-Platelet aggregation was measured using a lumi-aggregometer (Chrono-Log, Havertown, PA) at 37°C under stirring conditions. A 0.5-ml sample of washed platelets was stimulated with different agonists, and change in light transmission was measured.
Platelet secretion was determined by measuring the release of ATP by adding luciferin-luciferase reagent. Platelet ATP release and aggregation were performed in a lumi-aggregometer at 37°C simultaneously.
Western Blotting-Platelets were stimulated with agonists for the appropriate time, and phosphorylation events were measured as previously described (22). For outside-in signaling, washed human platelets were plated on fibrinogen-coated coverslips for 45 min at 37°C in a CO 2 incubator, and adherent cells were harvested for immunoblot analysis as described previously (23). In some experiments, platelets were stimulated in the presence of SC57101 (10 M) to eliminate outside-in signaling.
Measurement of Thromboxane A 2 Generation-Washed platelets without aspirin treatment were prepared at a concentration of 2 ϫ 10 8 platelets/ml. Stimulations were performed for 3.5 min and the reaction was stopped by snap freezing. Levels of TxB 2 were determined in duplicate using a Correlate-EIA thromboxane B 2 enzyme immunoassay kit (Assay Designs, Inc., Ann Arbor, MI), according to the manufacturer's instructions.
Statistical Analysis-All statistical tests were carried out using Prism software (version 3.0). Data are presented as mean Ϯ S.E. Statistical significance was determined by Student's t test and analysis of variance. p Ͻ 0.05 was considered statistically significant.

Time-and Concentration-dependent Phosphorylation of
Pyk2 in Platelets-It has been shown that treatment of platelets with various agonists including thrombin induces phosphorylation of Pyk2 in platelets. To determine the kinetics of Pyk2 phosphorylation, Tyr-402 and Tyr-881 phosphorylation in response to PAR4-activating peptide AYPGKF were monitored over a time range of 0.5-2 min. Fig. 1A shows a time-dependent increase in Pyk2 phosphorylation in which a rapid increase in Pyk2 phosphorylation in response to AYPGKF was detectable as early as 30 s after stimulation. We also exposed platelets to different concentrations of AYPGKF, and Tyr-402 phosphorylation was measured at 2 min after the addition of agonist. Fig.  1B shows a concentration-dependent increase in Pyk2 phosphorylation. An increase in Tyr 402 phosphorylation was detectable at concentrations above 100 M AYPGKF, and higher concentrations induced further phosphorylation that peaked at concentrations above 500 M AYPGKF. A similar pattern of time-and concentration-dependent phosphorylation of Tyr-402 in response to 2-MeSADP, SFLLRN, and thrombin was also detected (data not shown).
Characterization of the Activation of Pyk2 Downstream of G 12/13 and Integrin ␣IIb␤3-dependent Outside-in Pathways in Platelets-Previous studies have shown that Pyk2 is regulated by various cell type-dependent mechanisms. To investigate the signaling mechanism of Pyk2 activation in platelets, we first evaluated the role of integrin ␣IIb␤3 in Pyk2 activation. As shown in Fig. 2A, Pyk2 was phosphorylated downstream of ADP receptors, which was completely blocked by fibrinogen receptor antagonist SC-57101. Similarly, 2-MeSADP failed to induce Pyk2 phosphorylation under non-stirring conditions (data not shown). ADP stimulates both G q and G i pathways to induce fibrinogen receptor activation. Thus, these results suggest that Pyk2 phosphorylation by ADP occurs in an integrindependent manner, and either G q or G i pathways cannot directly activate Pyk2. Interestingly, AYPGKF-induced Pyk2 phosphorylation was significantly but not completely inhibited in the presence of SC-57101 (Fig. 2B), indicating that AYPGKFinduced Pyk2 phosphorylation occurs through both integrindependent and -independent pathways. Unlike ADP, it has been shown that PAR agonists can stimulate G 12/13 pathways. Thus, it has raised the possibility that G 12/13 pathways can induce Pyk2 phosphorylation in the presence of SC57101 through the integrin-independent pathways.
To confirm the contribution of G 12/13 and integrin ␣IIb␤3mediated outside-in signaling to Pyk2 phosphorylation, we have investigated whether selective activation of G 12/13 pathways and integrin ␣IIb␤3-mediated outside-in signaling can activate Pyk2. We have previously shown that YM-254890 selectively inhibits G q signaling in platelets, in which AYPGKF caused G 12/13 -induced platelet shape change in the presence of YM-254890 that was further abolished by the addition of Rho kinase inhibitor Y-27632 (22). Selective activation of G 12/13 pathways by AYPGKF in the presence of G q selective inhibitor YM254890 resulted in Pyk2 phosphorylation (Fig. 3A). It appears that G 12/13 -mediated Pyk2 phosphorylation plateaus faster in the absence of integrin-mediated signaling because Pyk2 phosphorylation at later time points is mainly mediated by integrin-mediated signaling. Akt phosphorylation was measured to verify the selective activation of G 12/13 pathways because we have shown that G 12/13 pathways alone cannot induce Akt phosphorylation (22). Selective activation of G q pathways (2-MeSADP ϩ AR-C69931MX) or G i pathways (2-MeSADP ϩ MRS2179) failed to induce Pyk2 phosphorylation (Fig. 3, B and C), confirming that G q and G i alone cannot cause Pyk2 activation. We have measured Akt phosphorylation to verify the selective blockade of G q , G i , or integrin pathways because we and others have shown that ADP-induced Akt phosphorylation is only dependent on G i pathways (24,25). In addition, platelet adhesion to immobilized fibrinogen resulted in an increase in the phosphorylation of Pyk2 (Fig. 3D), confirming the role of integrin ␣IIb␤3-mediated signaling in Pyk2 phosphorylation. Platelet adhesion to fibrinogen also caused an increase in the phosphorylation of Src Tyr-416, which has been identified as a prominent signaling complex downstream of integrin ␣IIb␤3 (23). Thus, these results confirm that Pyk2 is  activated by both G 12/13 pathways and integrin ␣IIb␤3-mediated outside-in signaling in platelets.
Effect of Pyk2 Inhibition on Human Platelet Aggregation and Secretion-To determine the functional role of Pyk2 in platelets, we first examined the effect of the Pyk2 selective inhibitor AG17 on 2-MeSADP-induced aggregation and dense granule secretion. As shown in Fig. 4A, 2-MeSADP-induced secondary aggregation and dense granule secretion in washed non-aspirin-treated human platelets were inhibited in the presence of AG17. Thromboxane production in response to ADP in nonaspirin-treated platelets results in dense granule secretion and subsequent secondary aggregation. Thus, it raised the possibility that Pyk2 plays a role in ADP-induced platelet responses through the regulation of TxA2 generation. To rule out the secondary effects of TxA2 on 2-MeSADP-induced aggregation, we evaluated the effect of AG17 on aspirin-treated platelets. There was no significant difference in 2-MeSADP-induced platelet aggregation in the presence and absence of AG17 (Fig.  4B), indicating the role of Pyk2 in regulating TxA 2 generation.
We also observed that platelet aggregation and dense granule release induced by AYPGKF were inhibited in the presence of the Pyk2 inhibitor AG17 (Fig. 4C). A highly selective TATmediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2 (amino acid residues 680 -1009), has been recently developed to block the activation of Pyk2 (26). Consistent with the result in Fig. 4C, AYPGKFinduced platelet aggregation and secretion were inhibited in the presence of TAT-Pyk2-CT, whereas TAT-GFP control had no effect (Fig. 4D). We also compared the effect of Pyk2 inhibition and aspirin treatment in response to AYPGKF, and aspirin treatment had a similar inhibitory effect on AYPGKF-induced platelet aggregation and secretion compared with AG17, confirming that Pyk2 inhibits platelet aggregation through the regulation of TxA 2 generation. These agonist-induced platelet aggregation and secretions were diminished upon blockade of Pyk2 over a wide range of agonist concentrations. Platelet response was more significantly diminished at lower concentrations of agonist, and differences became minor at higher doses of agonist. These results show that inhibition of Pyk2 in platelets was found to be defective in their function ex vivo, strongly indicating that Pyk2 plays an important role in regulation of platelet function.
Role of Pyk2 in AYPGKF-induced Platelet Aggregation, Secretion, and TxA 2 Generation in Pyk2-deficient Platelets-As pharmacological inhibitors are known to have off-target and broad-spectrum effects, we have examined platelets from Pyk2 Ϫ/Ϫ mice to ascertain the role of Pyk2 in platelet function. Consistent with the results obtained with Pyk2 inhibitors, platelet aggregation (Fig. 5A) and ATP secretion (Fig. 5B) were diminished in Pyk2-deficient platelets compared with the wild type platelets. In addition, the Pyk2 inhibitor AG17 did not show an additional inhibitory effect on AYPGKF-induced platelet aggregation in Pyk2-deficient platelets, indicating that the effects of AG17 are likely mediated by Pyk2. Similarly, the concentration-response curves for AYPGKF-induced TxA 2 generation were shifted to the right in Pyk2-deficient platelets as the level of TxB 2 generation in Pyk2-deficient platelets in response to AYPGKF was significantly decreased compared with WT platelets (Fig. 5C), suggesting that Pyk2 positively regulates TxA 2 generation. Because TxA 2 has a short half-life and is rapidly converted to stable product TxB 2 , TxB 2 was measured as TxA 2 by ELISA. It has been shown that PAR agonists cause TxA 2 generation independently of integrin signaling, and we have observed that Pyk2 is activated by both G 12/13 pathways and integrin signaling (Fig. 3). Thus, our results have raised the possibility that Pyk2 is the common signaling molecule downstream of both G 12/13 pathways and integrin signaling, which plays an essential role in AYPGKF-induced TxA 2 generation.
Role of Pyk2 in 2-MeSADP-induced Secretion and TxA2 Generation in Pyk2-deficient Platelets-It has been shown that ADP-induced TxA 2 generation and subsequent secretion is dependent on integrin activation. To confirm the role of Pyk2 downstream of integrins, we next measured TxA 2 generation and ATP secretion in response to 2-MeSADP in Pyk2-deficient  JUNE 21, 2013 • VOLUME 288 • NUMBER 25
Effect of Pyk2 on Regulation of TxA2 Generation Downstream of Integrin-dependent Outside-in and G 12/13 Pathways-We have previously shown that the combined stimulation of G i and G z pathways causes platelet aggregation and thromboxane generation, which is dependent on integrin-mediated outside-in signaling (27). To verify the role of Pyk2 in outside-in signaling, we first tested the effect of Pyk2 inhibition on platelet aggregation under these conditions. As shown in Fig. 7A, a combination of G i stimulation (2-MeSADP plus P2Y 1 antagonist MRS2179) with G z pathways (epinephrine) caused robust aggregation and dense granule secretion in non-aspirin-treated human plate- lets. However, pre-treating the platelets with AG17 caused an inhibition in secondary platelet aggregation and dense granule secretion. Because secondary platelet aggregation and dense granule secretion in this condition is mediated by TxA 2 generation, which is dependent on outside-in signaling, we then measured the effect of Pyk2 inhibition on TxA 2 generation induced by co-stimulation of G i and G z pathways. Selective stimulation of G i (2-MeSADP plus MRS2179) or G z pathways (epinephrine) alone failed to cause TxA 2 generation (Fig. 7B). However, co-stimulation of G i and G z pathways caused a sig- nificant increase in TxA 2 generation as previously described (27). When platelets were pre-treated with SC57101 or AG17, co-stimulation of G i and G z pathways failed to induce TxA 2 generation, indicating that Pyk2 is necessary for thromboxane generation mediated by outside-in signaling.
The study from our group has also shown that inhibition of ADP-induced thromboxane generation by a fibrinogen receptor antagonist was rescued by co-stimulation of G 12/13 pathways by YFLLRNP (21), suggesting that G 12/13 pathways can substitute integrin-mediated signaling by probably activating common signaling effectors downstream of both integrins and G 12/13 pathways. To confirm the role of Pyk2 downstream of G 12/13 pathways in TxA 2 generation, we co-stimulated platelets with 2-MeSADP and YFLLRNP in the presence of the fibrinogen receptor antagonist SC57101 and then compared the effect of Pyk2 inhibitor AG17 on TxA 2 generation. As shown in Fig.  7C, 2-MeSADP-induced TxA 2 generation was completely blocked in the presence of SC57101 or AG17, confirming the role of Pyk2 in ADP-induced TxA 2 generation, which depends on integrin-mediated signaling. Selective activation of G 12/13 pathways with YFLLRNP failed to cause TxA 2 generation, but co-stimulation of platelets with 2-MeSADP and YFLLRNP in the presence of SC57101 caused a significant increase in TxA 2 generation, indicating that G 12/13 pathways substitute for integrin signaling for TxA 2 generation. However, TxA 2 generation under these conditions was completely blocked by AG17, confirming that G 12/13 pathways rescue integrin-mediated outside-in signaling for TxA2 generation through Pyk2.

DISCUSSION
It has been shown that Pyk2 is abundantly expressed in platelets and is activated by various agonists including thrombin, collagen, or von Willebrand factor in platelets (15). However, the signaling mechanism of Pyk2 activation in platelets is complex and controversial and the functional role of Pyk2 in platelet activation has not been fully understood. Therefore, we have used pharmacological inhibitors of Pyk2 and Pyk2-deficient platelets to identify the signaling pathways of Pyk2 and its role in platelet function. Pyk2 has been shown to be phosphorylated through integrinmediated pathways, intracellular Ca 2ϩ mobilization, and PKC activation in several cells, including human B cells (28), neurons (29), CMK megakaryocytic cells (30,31), and PC12 cells (14,32). However, it has been shown that Pyk2 activation mediated by thrombin is dependent on intracellular calcium but not dependent on PKC, Src, or PI 3-kinase in human endothelium (33). It has also been shown that several platelet agonists induce the tyrosine phosphorylation of Pyk2 through integrin-dependent and integrin-independent mechanisms (15). Thus, there appears to be a cell type-dependent mechanism regulating Pyk2 activation. We have investigated whether selective activation of G 12/13 pathways, G q pathways, G i pathways, and integrin-mediated outside-in signaling can activate Pyk2. We found that 2-MeSADP-induced Pyk2 phosphorylation was induced by integrin-dependent pathways, whereas PAR agonists-induced Pyk2 phosphorylation was dependent on both integrin-dependent and -independent mechanism in platelets. PAR agonists can couple to G 12/13 pathways, and we observed that selective activation of G 12/13 pathways resulted in Pyk2 phosphorylation. We also observed that 2-MeSADP failed to induce Pyk2 phosphorylation in the presence of P2Y 1 or P2Y 12 receptor antagonists indicating that G q or G i pathways alone cannot directly activate Pyk2. Thus it appears that some of inhibitory effects of other signaling molecules on Pyk2 activation in previous studies might not be due to their direct inhibitory effect on Pyk2 but might be related to their inhibitory effect on platelet aggregation and the subsequent outside-in signaling.
Only a few selective inhibitors of Pyk2 have been identified. As pharmacological inhibitors are known to have off-target and broad-spectrum effects, the effect of Pyk2 in platelets has not been completely determined. Salicylate has been shown to inhibit Pyk2 phosphorylation, but it also exhibits an inhibitory effect on c-Src (34). It has recently been shown that the Pyk2 inhibitor AG17 reduces neutrophil adhesion to adherent platelets (35). A highly selective TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT) has been recently developed and used in several studies to selectively inhibit Pyk2 activity (26, 36 -38). We have demonstrated the efficacy of these inhibitors on Pyk2 phosphorylation induced by AYPGKF. Importantly, Pyk2 knock-out mice have been generated and the role of Pyk2 in macrophages (11) and osteoclasts (12) have been studied. We found that platelet aggregation and dense granule release induced by various agonists including 2-MeSADP and AYPGKF were inhibited in the presence of Pyk2 inhibitors AG-17 and TAT-Pyk2-CT in human platelets suggesting that Pyk2 positively regulates platelet function. Consistently, platelet aggregation and secretion induced by 2-MeSADP and AYPGKF were diminished in Pyk2deficient platelets compared with WT platelets.
It has been shown that ADP-induced TxA 2 generation requires outside-in signaling, whereas thrombin-mediated TxA 2 generation occurs independently of outside-in signaling. Platelets from patients with Glanzmann's thrombasthenia or platelets treated with fibrinogen receptor antagonist have been shown to be defective in ADP-, but not thrombin-induced TxA 2 generation and secretion (39,40). Unlike ADP, thrombin can couple to G 12/13 pathways, and previous study has shown that inhibition of ADP-induced TxA 2 generation by integrin ␣IIb␤3 blockade is rescued by co-stimulation of G 12/13 pathways (21). These studies suggest that thrombin substitutes outside-in signaling through G 12/13 pathways thus enables thromboxane to be generated in the absence of outside-in signaling. These observations also suggest the existence of a common signaling effector downstream of both integrins and G 12/13 pathways. If Pyk2 is the common effector molecule downstream of both integrins and G 12/13 pathways contributing to thromboxane generation, we anticipated that integrin-mediated TxA 2 generation by 2-MeSADP and G 12/13 -dependent TxA 2 generation by AYPGKF would be inhibited upon inhibition of Pyk2. Interestingly, we found that 2-MeSADP-and AYPGKF-induced TxA 2 generation was inhibited in Pyk2 Ϫ/Ϫ platelets compared with WT platelets. Our data showed that the extent of inhibition of platelet aggregation caused by Pyk2 inhibition was similar to the one caused by aspirin treatment, confirming the role of Pyk2 in TxA2 generation. In addition, we found that Pyk2 inhibition had no effect on ADP-induced platelet aggregation in aspirin-treated platelets, which is consistent with a previous study (41) showing that there was no difference in ADP-induced platelet aggregation from WT or TxA 2 receptor null mice. Recent study (42) has characterized the platelets from Pyk2 knock-out mice and no significant differences are observed between WT and Pyk2 knock-out mice. In addition, arachidonic acid induces normal platelet aggregation in Pyk2deficient platelets, and Pyk2 is linked to cPLA2 activation (42). In the experimental model adopted from a previous study (21) to confirm the role of Pyk2 downstream of G 12/13 pathways in TxA 2 generation, we found that G 12/13 pathways failed to substitute for integrin-mediated signaling for TxA 2 generation in the presence of the Pyk2 inhibitor confirming the contribution of Pyk2 downstream of G 12/13 pathways. In addition, we have previously shown that combined P2Y 12 receptor and ␣ 2A adrenergic receptor stimulation causes TxA 2 generation with a requirement for outside-in signaling through ␣IIb␤3 (27). Our data showed that the Pyk2 inhibitor and ␣IIb␤3 antagonist completely inhibited TxA 2 generation caused by costimulation of G i and G z signaling, further confirming an essential role of Pyk2 in TxA 2 generation that is activated by outside-in signaling. Combined with our results showing that Pyk2 is activated downstream of both G 12/13 and integrin-mediated outside-in pathways, these results strongly suggest that Pyk2 is the common effector molecule downstream of both G 12/13 and outside-in signaling pathways, which contribute to TxA 2 generation in platelets.
Recently, it has been shown that Pyk2 is activated after integrin ␣2␤1 engagement in platelets and Pyk2 regulates PI 3-kinase ␤ activity after integrin ␣2␤1-mediated adhesion (43). PI 3-kinase ␤ has been shown to play an important role in signaling downstream of ␣IIb␤3 (44), and our work has shown that PI 3-kinase ␤ plays an important role in ADP-induced TxA 2 generation (45). Thus, it is possible that Pyk2 mediates ADP-induced TxA 2 generation by regulating PI 3-kinase ␤ activity in platelets.
We have previously shown that ADP-induced TxA 2 generation requires both P2Y 1 -and P2Y 12 -mediated signaling (46). However, the present study showed that selective activation of either P2Y 1 or P2Y 12 signaling failed to induce Pyk2 activation and identified an important role of Pyk2 downstream of integrins in ADP-induced TxA 2 generation that is dependent on outside-in signaling. ADP fails to induce platelet aggregation when either the P2Y 1 or P2Y 12 receptor is blocked (47). Thus this suggests that the inhibitory effect of either P2Y 1 or P2Y 12 on ADP-induced TxA 2 generation might be due to their inhibitory effect on platelet aggregation, which leads to the blockade of integrin-mediated signaling and Pyk2 activation.
Pyk2 is highly homologous to FAK, which plays a key role in mediating signaling downstream of integrins (48). The expression level of FAK in Pyk2 knock-out platelets has been shown to be normal. A previous study (21) has shown that FAK is activated downstream of integrins and G 12/13 pathways in platelets, and 2-MeSADP-induced TxA 2 generation is inhibited in the presence of the FAK inhibitor TAE-226. However, 2-MeSADPinduced TxA 2 generation was not affected in Pf4-Cre/FAKfloxed mice, indicating that FAK does not contribute to TxA 2 generation induced by outside-in signaling. Because TAE-226 also inhibits Pyk2 at a higher concentration (10), the inhibitory effect of TAE-226 on TxA 2 generation was probably through the inhibition of Pyk2, which is consistent with our results using Pyk2 inhibitors. In conclusion, we have demonstrated that Pyk2 is an important functional tyrosine kinase that is activated by both G 12/13 and integrin ␣IIb␤3-mediated outside-in signaling pathways and plays an important role in regulation of TxA 2 generation in platelets.