Ellular free Ca2+ using equation reported by Grynkiewicz et al. [20].Materials and Methods Reagents and AntibodiesHuman a-thrombin (specific activity of 5380 NIH units/mg) was purchased from Haematological Technologies (Essex Junction, VT). PAR4 activating peptide (AYPGKF-NH2) was synthesized at PolyPeptide Laboratories (San Diego, CA). Convulxin was purchased from Enzo Life Sciences Inc. (Farmingdale, NY). ADP was purchased from Chrono-log Corporation (Havertown, PA). Fura-2AM and all cell culture reagents were purchased from Invitrogen. Prostaglandin I2 was purchased from Calbiochem. Heparin, thapsigargin, and 2-Methylthioadenosine 59-monophosphate triethylammonium salt hydrate (2MeSAMP) were purchased from Sigma Chemical Co. The anti-phospho-(Ser) PKC substrates, anti-PKC, anti-phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). The anti-a-actinin antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The 117793 anti-PAR4-FITC antibody was purchased from Alamone Labs Ltd. (Jerusalem, Israel). The anti-HA tag Alexa Fluor 647 (6E2) antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA). The anti-V5 tag Alexa Fluor 647 antibody was purchased from AbD Serotec. (Raleigh, NC).Measurement of PAR4 expression in mouse plateletsMouse platelet surface expression of PAR4 was determined by flow cytometry using a Beckman Coulter LSRII (Case Comprehensive Cancer Center Flow Core). Washed mouse platelets were adjusted to a final concentration of 406106 platelets/mL in PD1-PDL1 inhibitor 1 cost HEPES-Tyrode buffer (pH 7.4). Twenty-five microliter aliquots were incubated with 25 mg/mL of anti-PAR4-FITC at 4uC for 20 min. Platelets were then diluted (1:8) and 10,000 events were acquired on a Beckman Coulter LSRII. Flow cytometry data were analyzed with Flowjo software.Western blottingWashed platelets were adjusted to a final concentration of 36108 platelets/mL. Fifty microliter aliquots were activated with thrombin(1, 10, 30, or 100 nM) or AYPGKF (0.03, 0.5, 1.5, or 2 mM) for 3 min at 37uC. The reaction was stopped by adding 66 Laemmli reducing buffer and samples were resolved by SDSPAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with primary rabbit antibody to phospho-(Ser) PKC substrates, PKC, or phospho-Akt (Ser473). To demonstrate protein loading, the membrane was reprobed with a rabbit antibody to a-actinin. Detection was performed with HRP-conjugated anti-rabbit secondary antibody and an enhanced chemiluminescence system (Pierce Chemical). The optical densities of proteins in the blot were quantified using Image J (1.45) software.AnimalsPAR3 knockout (PAR32/2) and PAR3 heterozygous (PAR3+/2) mice have been described and were obtained from Mutant Mouse Regional Resource Center (MMRRC, Chapel Hill, NC) [5]. All animal studies were approved by the Institutional Animal 
Care and Use Committee at Case Western Reserve University School of Medicine.Platelet preparationMice were anesthetized with intraperitoneal injection of pentobarbital (62 mg/kg). Blood was collected from mice by heparinized capillary puncture of the retro-orbital venous sinus and immediately combined with (1/5) volume of acid citrate dextrose (ACD) as an anticoagulant. The whole blood was centrifuged at 23006 g for 20 sec at room temperature (RT) to isolate platelet-rich plasma (PRP). The platelets were pelleted and washed once at 22006g for 3 min at RT in HEPES-Tyrode buffer pH 7.4 (10.Ellular free Ca2+ using equation reported by Grynkiewicz et al. [20].Materials and Methods Reagents and AntibodiesHuman a-thrombin (specific activity of 5380 NIH units/mg) was purchased from Haematological Technologies (Essex Junction, VT). PAR4 activating peptide (AYPGKF-NH2) was synthesized at PolyPeptide Laboratories (San Diego, CA). Convulxin was purchased from Enzo Life Sciences Inc. (Farmingdale, NY). ADP was purchased from Chrono-log Corporation (Havertown, PA). Fura-2AM and all cell culture reagents were purchased from Invitrogen. Prostaglandin I2 was purchased from Calbiochem. Heparin, thapsigargin, and 2-Methylthioadenosine 59-monophosphate triethylammonium salt hydrate (2MeSAMP) were purchased from Sigma Chemical Co. The anti-phospho-(Ser) PKC substrates, anti-PKC, anti-phospho-Akt (Ser473) antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). The anti-a-actinin antibody was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The anti-PAR4-FITC antibody was purchased from Alamone Labs 
Ltd. (Jerusalem, Israel). The anti-HA tag Alexa Fluor 647 (6E2) antibody was purchased from Cell Signaling Technology Inc. (Danvers, MA). The anti-V5 tag Alexa Fluor 647 antibody was purchased from AbD Serotec. (Raleigh, NC).Measurement of PAR4 expression in mouse plateletsMouse platelet surface expression of PAR4 was determined by flow cytometry using a Beckman Coulter LSRII (Case Comprehensive Cancer Center Flow Core). Washed mouse platelets were adjusted to a final concentration of 406106 platelets/mL in HEPES-Tyrode buffer (pH 7.4). Twenty-five microliter aliquots were incubated with 25 mg/mL of anti-PAR4-FITC at 4uC for 20 min. Platelets were then diluted (1:8) and 10,000 events were acquired on a Beckman Coulter LSRII. Flow cytometry data were analyzed with Flowjo software.Western blottingWashed platelets were adjusted to a final concentration of 36108 platelets/mL. Fifty microliter aliquots were activated with thrombin(1, 10, 30, or 100 nM) or AYPGKF (0.03, 0.5, 1.5, or 2 mM) for 3 min at 37uC. The reaction was stopped by adding 66 Laemmli reducing buffer and samples were resolved by SDSPAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with primary rabbit antibody to phospho-(Ser) PKC substrates, PKC, or phospho-Akt (Ser473). To demonstrate protein loading, the membrane was reprobed with a rabbit antibody to a-actinin. Detection was performed with HRP-conjugated anti-rabbit secondary antibody and an enhanced chemiluminescence system (Pierce Chemical). The optical densities of proteins in the blot were quantified using Image J (1.45) software.AnimalsPAR3 knockout (PAR32/2) and PAR3 heterozygous (PAR3+/2) mice have been described and were obtained from Mutant Mouse Regional Resource Center (MMRRC, Chapel Hill, NC) [5]. All animal studies were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University School of Medicine.Platelet preparationMice were anesthetized with intraperitoneal injection of pentobarbital (62 mg/kg). Blood was collected from mice by heparinized capillary puncture of the retro-orbital venous sinus and immediately combined with (1/5) volume of acid citrate dextrose (ACD) as an anticoagulant. The whole blood was centrifuged at 23006 g for 20 sec at room temperature (RT) to isolate platelet-rich plasma (PRP). The platelets were pelleted and washed once at 22006g for 3 min at RT in HEPES-Tyrode buffer pH 7.4 (10.
