Ls (16105) in 6-well plates were transfected with 1.5 mg of HA-PAR4-LUC or V5-PAR4-GFP and 1 mg of HA-PAR3-LUC or V5-PAR3-GFP. Transfected cells were removed from plates 48 h post-transfection by rinsing with PBS. Surface detection of HA- Calciferol web tagged PAR3 or PAR4 was detected with an HA tag antibody conjugated to Alexa Fluor 647 (Cell Signaling Technology Inc) with 1:50 dilution. Surface detection of V5tagged PAR3 or PAR4 was detected with a V5 tag antibody conjugated to Alexa Fluor 647 (AbD Serotec) with 1:5 dilution. HEK293 labeled cells (1.256105) were then diluted (1:8) and 10,000 events were acquired on a BD LSRFortessa. Cell surface expression of HA- or V5-tagged PAR4 or PAR3 was determined by quantitative flow cytometry and performed essentially as described [21].treatment, Benzocaine site platelets from PAR32/2 and PAR3+/2 mice had a 2.6fold or 1.9-fold increase in the maximum Ca2+ mobilization, respectively, compared to wild type platelets in response to AYPGKF (Figure 1B). The EC50 for AYPGKF-induced Ca2+ mobilization is not statistically significant between wild type and PAR32/2 platelets (392 mM vs. 614 mM, respectively, p = 0.45). Next, we investigated whether the increase in the maximum Ca2+ mobilization in the PAR32/2 mice was specific to PAR4 stimulation. Wild type and PAR32/2 platelets were stimulated with convulxin, the specific GPVI agonist, or with a high concentration of ADP [22], the specific P2Y12 and P2Y1 agonist. There was no significant difference in the maximum Ca2+ mobilization or the EC50 in response to convulxin (Figure 1C) or in the maximum Ca2+ mobilization in response to 20 mM ADP (Figure 1D) between wild type and PAR32/2 platelets. These data indicate that the increase in the maximum Ca2+ mobilization was specific to PAR activation, but independent of the PAR4 agonist. These data suggest that PAR3 influences PAR4 at the level of the receptor. To verify that the increase in the maximal Ca2+ mobilization was not due to an increase in surface expression of PAR4 in PAR32/2 22948146 platelets, PAR4 expression was measured by flow cytometry. Platelets from wild type and PAR32/2 mice had the same level of PAR4 expression (Figure 2).P2Y12 inhibition does not influence PAR4 enhanced Ca2+ mobilization in PAR32/2 mouse plateletsPAR4 and P2Y12 physically interact in human platelets after thrombin or AYPGKF stimulation and the association is reduced by P2Y12 inhibitor 2MeSAMP [23]. To determine if the increase in the maximum Ca2+ mobilization was caused by crosstalk between PAR4 and P2Y12 in the absence of PAR3, wild type and PAR32/2 platelets were stimulated with thrombin or AYPGKF in the presence of 2MeSAMP (P2Y12 antagonist). There was no significant difference in the maximum Ca2+ mobilization between wild type and PAR32/2 platelets activated with 30 nM thrombin (p = 0.64, data not shown) or 100 nM thrombin (p = 0.99, Figure 3A). Similarly, there was no significant difference in maximum Ca2+ mobilization when platelets were stimulated with 1.5 mM AYPGKF (p = 0.10, data not shown) or 2 mM AYPGKF (p = 0.06, Figure 3B). These data indicate that the increase in the maximum Ca2+ mobilization was independent of the PAR4-P2Y12 interaction after thrombin or AYPGKF stimulation.Data analysisDifferences between means were determined by unpaired Student’s t test and by one way ANOVA test and were considered significant when p,0.05.Results Intracellular Ca2+ mobilization is increased in PAR32/2 mouse plateletsWe first determined if the absence of PAR3 aff.Ls (16105) in 6-well plates were transfected with 1.5 mg of HA-PAR4-LUC or V5-PAR4-GFP and 1 mg of HA-PAR3-LUC or V5-PAR3-GFP. Transfected cells were removed from plates 48 h post-transfection by rinsing with PBS. Surface detection of HA- tagged PAR3 or PAR4 was detected with an HA tag antibody conjugated to Alexa Fluor 647 (Cell Signaling Technology Inc) with 1:50 dilution. Surface detection of V5tagged PAR3 or PAR4 was detected with a V5 tag antibody conjugated to Alexa Fluor 647 (AbD Serotec) with 1:5 dilution. HEK293 labeled cells (1.256105) were then diluted (1:8) and 10,000 events were acquired on a BD LSRFortessa. Cell surface expression of HA- or V5-tagged PAR4 or PAR3 was determined by quantitative flow cytometry and performed essentially as described [21].treatment, platelets from PAR32/2 and PAR3+/2 mice had a 2.6fold or 1.9-fold increase in the maximum Ca2+ mobilization, respectively, compared to wild type platelets in response to AYPGKF (Figure 1B). The EC50 for AYPGKF-induced Ca2+ mobilization is not statistically significant between wild type and PAR32/2 platelets (392 mM vs. 614 mM, respectively, p = 0.45). Next, we investigated whether the increase in the maximum Ca2+ mobilization in the PAR32/2 mice was specific to PAR4 stimulation. Wild type and PAR32/2 platelets were stimulated with convulxin, the specific GPVI agonist, or with a high concentration of ADP [22], the specific P2Y12 and P2Y1 agonist. There was no significant difference in the maximum Ca2+ mobilization or the EC50 in response to convulxin (Figure 1C) or in the maximum Ca2+ mobilization in response to 20 mM ADP (Figure 1D) between wild type and PAR32/2 platelets. These data indicate that the increase in the maximum Ca2+ mobilization was specific to PAR activation, but independent of the PAR4 agonist. These data suggest that PAR3 influences PAR4 at the level of the receptor. To verify that the increase in the maximal Ca2+ mobilization was not due to an increase in surface expression of PAR4 in PAR32/2 22948146 platelets, PAR4 expression was measured by flow cytometry. Platelets from wild type and PAR32/2 mice had the same level of PAR4 expression (Figure 2).P2Y12 inhibition does not influence PAR4 enhanced Ca2+ mobilization in PAR32/2 mouse plateletsPAR4 and P2Y12 physically interact in human platelets after thrombin or AYPGKF stimulation and the association is reduced by P2Y12 inhibitor 2MeSAMP [23]. To determine if the increase in the maximum Ca2+ mobilization was caused by crosstalk between PAR4 and P2Y12 in the absence of PAR3, wild type and PAR32/2 platelets were stimulated with thrombin or AYPGKF in the presence of 2MeSAMP (P2Y12 antagonist). There was no significant difference in the maximum Ca2+ mobilization between wild type and PAR32/2 platelets activated with 30 nM thrombin (p = 0.64, data not shown) or 100 nM thrombin (p = 0.99, Figure 3A). Similarly, there was no significant difference in maximum Ca2+ mobilization when platelets were stimulated with 1.5 mM AYPGKF (p = 0.10, data not shown) or 2 mM AYPGKF (p = 0.06, Figure 3B). These data indicate that the increase in the maximum Ca2+ mobilization was independent of the PAR4-P2Y12 interaction after thrombin or AYPGKF stimulation.Data analysisDifferences between means were determined by unpaired Student’s t test and by one way ANOVA test and were considered significant when p,0.05.Results Intracellular Ca2+ mobilization is increased in PAR32/2 mouse plateletsWe first determined if the absence of PAR3 aff.
