MM HEPES, 12 mM NaHCO3, 130 mM NaCl, 5 mM KCl, 0.4 mM Na2HPO4, 1 mM MgCl2, 5 mM glucose, 0.33 w/v human serum albumin) containing 0.5 mM prostaglandin I2 (PGI2) and 10 U/mL Heparin. Washed platelets were counted on a Hemavet 950FS (Drew Scientific Inc, Waterbury, CT, USA) and the final platelet count adjusted with HEPESTyrode’s buffer.ELISA-based RhoA 25033180 activation assayRhoA GTPase activity was determined in freshly prepared mouse platelets lysates (adjusted to a final concentration of 36108 platelets/mL) by using the absorbance based G-LISA RhoA activation assay kit (Cytoskeleton, Inc.). Fifty microliter aliquots were activated with thrombin (1, 10, 30, or 100 nM) for 3 min at 37uC. Activated mouse platelets were then lysed using an equal volume of supplied cell lysis buffer, and the lysates were immediately used for the Rho G-LISA assay. All subsequent incubation and detection followed the instructions provided by the manufacturer.PAR3 Regulates PAR4 Signaling in Mouse PlateletsBioluminescence resonance energy transfer (BRET) assay and cell surface expression of mouse PAR3 and mouse PAR4 measurementThe cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library. An amino terminal HA (YPYDVPDYA) or V5 (GKPIPMPLLGLDST) epitope tag was added to PAR3 or PAR4 by PCR, and the cDNAs were ligated into pLUC-N2 (HA-tagged) or pGFP2-N2 (V5 tagged). The mouse rhodopsin in pGFP2-N2 (V5 tagged) has been described [21] . The BRET vectors were purchased from PerkinElmer. All constructs were verified by DNA sequencing. For BRET assays, HEK293 cells (American Type Culture Collection) were transfected with Lipofectamine 2000 according to manufacturer’s instructions. BRET assays were performed as previously described [21]. Cell surface expression of mouse PAR3 and mouse PAR4 was determined by flow Cy5 NHS Ester price cytometry using BD LSRFortessa (Center for Aids Research, Immune Function Core, Case Western Reserve University). HEK293 cells (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 CTX-0294885 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.MM HEPES, 12 mM NaHCO3, 130 mM NaCl, 5 mM KCl, 0.4 mM Na2HPO4, 1 mM MgCl2, 5 mM glucose, 0.33 w/v human serum albumin) containing 0.5 mM prostaglandin I2 (PGI2) and 10 U/mL Heparin. Washed platelets were counted on a Hemavet 950FS (Drew Scientific Inc, Waterbury, CT, USA) and the final platelet count adjusted with HEPESTyrode’s buffer.ELISA-based RhoA 25033180 activation assayRhoA GTPase activity was determined in freshly prepared mouse platelets lysates (adjusted to a final concentration of 36108 platelets/mL) by using the absorbance based G-LISA RhoA activation assay kit (Cytoskeleton, Inc.). Fifty microliter aliquots were activated with thrombin (1, 10, 30, or 100 nM) for 3 min at 37uC. Activated mouse platelets were then lysed using an equal volume of supplied cell lysis buffer, and the lysates were immediately used for the Rho G-LISA assay. All subsequent incubation and detection followed the instructions provided by the manufacturer.PAR3 Regulates PAR4 Signaling in Mouse PlateletsBioluminescence resonance energy transfer (BRET) assay and cell surface expression of mouse PAR3 and mouse PAR4 measurementThe cDNA for mouse PAR3 was purchased from Origene Technologies Inc. (Rockville, MD) and the cDNA for mouse PAR4 was isolated from a BaF3 cDNA library. An amino terminal HA (YPYDVPDYA) or V5 (GKPIPMPLLGLDST) epitope tag was added to PAR3 or PAR4 by PCR, and the cDNAs were ligated into pLUC-N2 (HA-tagged) or pGFP2-N2 (V5 tagged). The mouse rhodopsin in pGFP2-N2 (V5 tagged) has been described [21] . The BRET vectors were purchased from PerkinElmer. All constructs were verified by DNA sequencing. For BRET assays, HEK293 cells (American Type Culture Collection) were transfected with Lipofectamine 2000 according to manufacturer’s instructions. BRET assays were performed as previously described [21]. Cell surface expression of mouse PAR3 and mouse PAR4 was determined by flow cytometry using BD LSRFortessa (Center for Aids Research, Immune Function Core, Case Western Reserve University). HEK293 cells (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.
