Lycerol (DAG) and inositol-1,4,5-triphosphate (IP3) to activate PKC and Ca2+ store depletion, respectively [28]. In platelets, the major Ca2+ entry pathway is mediated by Ca2+ channels known as store-operated calcium entry (SOCE). The SOCE channels are activated by depletion of intracellular Ca2+ stores induced by IP3 generated downstream of Gq [29]. In this study, we have shown that platelets from PAR32/2 mice have 1.6fold increase in the maximum intracellular Ca2+ mobilization (Figure 1), an increase in phosphorylation level of PKC substrates (Figure 4), and a 2-fold increase in Ca2+ release from the stores (Figure 5) in ML-281 site response to thrombin (30?00 nM) or AYPGKF. Our results from Ca2+ store depletion are consistent with previous data that show an increase in IP3 formation in COS7 cells transfected with PAR4 compared to COS7 transfected with both receptorsPAR3 and PAR4 form constitutive homodimers and heterodimersTo 1326631 address the mechanism of how Indolactam V chemical information down-regulation of mouse PAR3 affects mouse PAR4 signaling, we investigated the possibility that PAR3 and PAR4 physically interact using bioluminescent resonance energy transfer (BRET) [21]. Initial studies examined the PAR3-PAR4 heterodimer (Figure 8A). PAR3 and PAR4 formed heterodimers as indicated by a hyperbolic BRET signal in response to an increase in the PAR3-GFP: PAR4Luc ratio. We next determined that PAR3 and PAR4 also formed homodimers (Figure 8 B and C) and PAR3 or PAR4 were unable to form heterodimers with rhodopsin (Rho) (Figure 8 D and E). These data demonstrate that PAR3 specifically interact withPAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 4. Western blot analysis of protein kinase C (PKC) substrate phosphorylation in mouse platelets. The level of PKC substrate phosphorylation on serine residues in response to increasing concentrations of: (A) thrombin (1?00 nM) or (C) AYPGKF (0.03? mM) was determined by western blotting with phospho-(Ser) 15755315 PKC substrate antibody. The membranes were re-probed for a-actinin to demonstrate protein loading. The blots shown are from a representative of three independent experiments. Quantitation of PKC substrate phosphorylation in 
response to (B) thrombin or (D) AYPGKF is represented at the mean (6 SD) (* p,0.05). doi:10.1371/journal.pone.0055740.g(PAR4 and PAR3) in response to thrombin (10?00 nM) [6]. It has been shown that PAR1, but not PAR4, negatively regulates intracellular Ca2+ mobilization and procoagulant phosphatidylserine (PS) exposure in a PKC-dependent mechanism in human platelets [30]. Our data show that PAR3 negatively regulates Ca2+ mobilization and PKC activation in response to high thrombin concentration or PAR4 agonist peptide, perhaps by a physical interaction with PAR4 in mouse platelets. Further, platelets from PAR3+/2 had an intermediate increase in Ca2+ mobilization (Figure 1A and B). These data support that PAR3 is directly influencing signaling from PAR4. In platelets, PAR4 also interacts with the P2Y12 receptor in response to thrombin [23]. Therefore, it is also possible that PAR4 and P2Y12 heterodimers are increased in the absence of PAR3, 
which influences PAR4 mediated increase in the maximum Ca2+ mobilization. However, our results show that blocking ADP signaling with 2MeSAMP does not affect the Ca2+ mobilization in response to thrombin (30 and 100 nM) or AYPGKF (1.5 and 2 mM) in PAR32/2 platelets. These dataconfirm that PAR3 is affecting the Ca2+ signaling downstream of PAR4 independently of P2Y12. PAR subtypes.Lycerol (DAG) and inositol-1,4,5-triphosphate (IP3) to activate PKC and Ca2+ store depletion, respectively [28]. In platelets, the major Ca2+ entry pathway is mediated by Ca2+ channels known as store-operated calcium entry (SOCE). The SOCE channels are activated by depletion of intracellular Ca2+ stores induced by IP3 generated downstream of Gq [29]. In this study, we have shown that platelets from PAR32/2 mice have 1.6fold increase in the maximum intracellular Ca2+ mobilization (Figure 1), an increase in phosphorylation level of PKC substrates (Figure 4), and a 2-fold increase in Ca2+ release from the stores (Figure 5) in response to thrombin (30?00 nM) or AYPGKF. Our results from Ca2+ store depletion are consistent with previous data that show an increase in IP3 formation in COS7 cells transfected with PAR4 compared to COS7 transfected with both receptorsPAR3 and PAR4 form constitutive homodimers and heterodimersTo 1326631 address the mechanism of how down-regulation of mouse PAR3 affects mouse PAR4 signaling, we investigated the possibility that PAR3 and PAR4 physically interact using bioluminescent resonance energy transfer (BRET) [21]. Initial studies examined the PAR3-PAR4 heterodimer (Figure 8A). PAR3 and PAR4 formed heterodimers as indicated by a hyperbolic BRET signal in response to an increase in the PAR3-GFP: PAR4Luc ratio. We next determined that PAR3 and PAR4 also formed homodimers (Figure 8 B and C) and PAR3 or PAR4 were unable to form heterodimers with rhodopsin (Rho) (Figure 8 D and E). These data demonstrate that PAR3 specifically interact withPAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 4. Western blot analysis of protein kinase C (PKC) substrate phosphorylation in mouse platelets. The level of PKC substrate phosphorylation on serine residues in response to increasing concentrations of: (A) thrombin (1?00 nM) or (C) AYPGKF (0.03? mM) was determined by western blotting with phospho-(Ser) 15755315 PKC substrate antibody. The membranes were re-probed for a-actinin to demonstrate protein loading. The blots shown are from a representative of three independent experiments. Quantitation of PKC substrate phosphorylation in response to (B) thrombin or (D) AYPGKF is represented at the mean (6 SD) (* p,0.05). doi:10.1371/journal.pone.0055740.g(PAR4 and PAR3) in response to thrombin (10?00 nM) [6]. It has been shown that PAR1, but not PAR4, negatively regulates intracellular Ca2+ mobilization and procoagulant phosphatidylserine (PS) exposure in a PKC-dependent mechanism in human platelets [30]. Our data show that PAR3 negatively regulates Ca2+ mobilization and PKC activation in response to high thrombin concentration or PAR4 agonist peptide, perhaps by a physical interaction with PAR4 in mouse platelets. Further, platelets from PAR3+/2 had an intermediate increase in Ca2+ mobilization (Figure 1A and B). These data support that PAR3 is directly influencing signaling from PAR4. In platelets, PAR4 also interacts with the P2Y12 receptor in response to thrombin [23]. Therefore, it is also possible that PAR4 and P2Y12 heterodimers are increased in the absence of PAR3, which influences PAR4 mediated increase in the maximum Ca2+ mobilization. However, our results show that blocking ADP signaling with 2MeSAMP does not affect the Ca2+ mobilization in response to thrombin (30 and 100 nM) or AYPGKF (1.5 and 2 mM) in PAR32/2 platelets. These dataconfirm that PAR3 is affecting the Ca2+ signaling downstream of PAR4 independently of P2Y12. PAR subtypes.
