Ected PAR4 mediated intracellular Ca2+ mobilization in PAR32/2 (-)-Calyculin A platelets in response to thrombin. The EC50 for thrombin-induced Ca2+ mobilization is Rubusoside custom synthesis increased ,10-fold in PAR32/2 platelets compared to wild type platelets (4.1 nM vs 0.6 nM, with a 95 confidence interval of 0.24?.5 nM or 2.3?5 nM, respectively) (Figure 1A). Heterozygous mice (PAR3+/2) had an intermediate value (1.1 nM with a 95 confidence interval of 0.5?.7 nM). These results agree with published data showing that PAR3 is a cofactor for PAR4 activation at low thrombin concentrations [6]. However, at thrombin concentrations above 10 nM, platelets from PAR32/2 mice had a ,1.6-fold increase in the maximum Ca2+ mobilization compared to wild type platelets. Platelets from PAR3+/2 had an intermediate increase in the maximum Ca2+ mobilization (,1.2-fold) (Figure 1A). These data indicated that the absence of PAR3 affects the Ca2+ mobilization in response to high thrombin concentrations (30?00 nM). We next determined if the increase in the maximum Ca2+ mobilization in PAR32/2 platelets was dependent on thrombin’s interaction with PAR4 by using a specific PAR4 activating peptide (AYPGKF). Similar to thrombinProtein Kinase C (PKC) activation is increased in PAR32/2 mouse plateletsIntracellular Ca2+ mobilization and PKC activation are both downstream of Gq. We next determined if PKC activation was also increased in PAR32/2 platelets by measuring the serine phosphorylation of PKC substrates, which reflects the activation of PKC. Serine phosphorylation on PKC substrates in response to 1 nM thrombin was absent in PAR32/2 platelets compared to wild type platelets (Figure 4A and B). These results are expected because PAR3 is required for PAR4 activation at low concentrations of thrombin. Importantly, we show that the level of serine phosphorylation of PKC substrates was increased in response to high concentration of thrombin (30?00 nM) in PAR32/2 compared to wild type mouse platelets. The level of serine phosphorylation on PKC substrates was also increased in response to AYPGKF in PAR32/2 platelets (Figure 4C and D). To show that the increased PKC activity in PAR32/2 platelets was not due to increased expression of PKC, we reprobed the membranes for total PKC. There was no significant difference in the level of PKC between PAR32/2 and wild type platelets. These results arePAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 1. Dose response curve of Ca2+ mobilization in the presence of extracellular Ca2+ in mouse platelets. Fura 2-loaded wild type (black circle), PAR32/2 (gray circle), and PAR3+/2 (white square) platelets were activated with the indicated concentrations of: (A) thrombin, (0.001?100 nM, (B) AYPGKF (0? mM), (C) convulxin (0.01?00 nM), or 20 mM of ADP for 10 min at 37uC in the presence of 2 mM of CaCl2. The difference between the maximum increase and the basal intracellular Ca2+ mobilization was measured. The results are the mean (6 SD) of three independent experiments (* p,0.05). doi:10.1371/journal.pone.0055740.gconsistent with the Ca2+ mobilization data and indicate that PAR3 negatively regulates PAR4-induced Gq-mediated signaling in mouse platelets.Intracellular Ca2+ store depletion is increased in PAR32/2 mouse plateletsWe next examined if the increase in the maximum Ca2+ mobilization was caused by an increase in the depletion of intracellular Ca2+ stores. Platelets from PAR32/2 and wild 1407003 type mice were stimulated with thrombin (1, 10, 30, or 100 nM) in Ca2+-free buf.Ected PAR4 mediated intracellular Ca2+ mobilization in PAR32/2 platelets in response to thrombin. The EC50 for thrombin-induced Ca2+ mobilization is increased ,10-fold in PAR32/2 platelets compared to wild type platelets (4.1 nM vs 0.6 nM, with a 95 confidence interval of 0.24?.5 nM or 2.3?5 nM, respectively) (Figure 1A). Heterozygous mice (PAR3+/2) had an intermediate value (1.1 nM with a 95 confidence interval of 0.5?.7 nM). These results agree with published data showing that PAR3 is a cofactor for PAR4 activation at low thrombin concentrations [6]. However, at thrombin concentrations above 10 nM, platelets from PAR32/2 mice had a ,1.6-fold increase in the maximum Ca2+ mobilization compared to wild type platelets. Platelets from PAR3+/2 had an intermediate increase in the maximum Ca2+ mobilization (,1.2-fold) (Figure 1A). These data indicated that the absence of PAR3 affects the Ca2+ mobilization in response to high thrombin concentrations (30?00 nM). We next determined if the increase in the maximum Ca2+ mobilization in PAR32/2 platelets was dependent on thrombin’s interaction with PAR4 by using a specific PAR4 activating peptide (AYPGKF). Similar to thrombinProtein Kinase C (PKC) activation is increased in PAR32/2 mouse plateletsIntracellular Ca2+ mobilization and PKC activation are both downstream of Gq. We next determined if PKC activation was also increased in PAR32/2 platelets by measuring the serine phosphorylation of PKC substrates, which reflects the activation of PKC. Serine phosphorylation on PKC substrates in response to 1 nM thrombin was absent in PAR32/2 platelets compared to wild type platelets (Figure 4A and B). These results are expected because PAR3 is required for PAR4 activation at low concentrations of thrombin. Importantly, we show that the level of serine phosphorylation of PKC substrates was increased in response to high concentration of thrombin (30?00 nM) in PAR32/2 compared to wild type mouse platelets. The level of serine phosphorylation on PKC substrates was also increased in response to AYPGKF in PAR32/2 platelets (Figure 4C and D). To show that the increased PKC activity in PAR32/2 platelets was not due to increased expression of PKC, we reprobed the membranes for total PKC. There was no significant difference in the level of PKC between PAR32/2 and wild type platelets. These results arePAR3 Regulates PAR4 Signaling in Mouse PlateletsFigure 1. Dose response curve of Ca2+ mobilization in the presence of extracellular Ca2+ in mouse platelets. Fura 2-loaded wild type (black circle), PAR32/2 (gray circle), and PAR3+/2 (white square) platelets were activated with the indicated concentrations of: (A) thrombin, (0.001?100 nM, (B) AYPGKF (0? mM), (C) convulxin (0.01?00 nM), or 20 mM of ADP for 10 min at 37uC in the presence of 2 mM of CaCl2. The difference between the maximum increase and the basal intracellular Ca2+ mobilization was measured. The results are the mean (6 SD) of three independent experiments (* p,0.05). doi:10.1371/journal.pone.0055740.gconsistent with the Ca2+ mobilization data and indicate that PAR3 negatively regulates PAR4-induced Gq-mediated signaling in mouse platelets.Intracellular Ca2+ store depletion is increased in PAR32/2 mouse plateletsWe next examined if the increase in the maximum Ca2+ mobilization was caused by an increase in the depletion of intracellular Ca2+ stores. Platelets from PAR32/2 and wild 1407003 type mice were stimulated with thrombin (1, 10, 30, or 100 nM) in Ca2+-free buf.
