Figure 1.
Dose response curve of Ca2+ mobilization in the presence of extracellular Ca2+ in mouse platelets.
Fura 2-loaded wild type (black circle), PAR3−/− (gray circle), and PAR3+/− (white square) platelets were activated with the indicated concentrations of: (A) thrombin, (0.001–100 nM, (B) AYPGKF (0–2 mM), (C) convulxin (0.01–100 nM), or 20 µM of ADP for 10 min at 37°C 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 (± SD) of three independent experiments (* p<0.05).
Figure 2.
PAR4 expression on mouse platelets.
Flow cytometric analysis of PAR4 expression in wild type (WT) (black line), PAR3−/− (gray line), and PAR4−/− (shaded) mice platelets using anti-PAR4-FITC antibodies.
Figure 3.
Effect of 2MeSAMP on PAR4 enhancing intracellular Ca2+ mobilization in mouse platelets.
Fura 2-loaded wild type (black) and PAR3−/− (gray) platelets were incubated at 37°C for 5 min in the absence or the presence of 100 µM 2MeSAMP. After treatment, platelets were activated 100 nM thrombin (A,) or 2 mM AYPGKF (B) for 10 min at 37°C 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 (± SD) of three independent experiments (* p<0.05).
Figure 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 (1–100 nM) or (C) AYPGKF (0.03–2 mM) was determined by western blotting with phospho-(Ser) PKC substrate antibody. The membranes were re-probed for α-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 (± SD) (* p<0.05).
Figure 5.
Dose response curve of Ca2+ mobilization in the absence of extracellular Ca2+ in mouse platelets.
Fura 2-loaded wild type (black line) and PAR3−/− (gray line) platelets were resuspended in Ca2+-free medium (0.1 mM EGTA was added at the time of experiment). Representative tracings are shown from platelets activated with the indicated concentrations of: (A) thrombin (1–100 nM), (C) AYPGKF (0.15–2 mM), or (E) 3 µM thapsigargin (TG). Quantitation of the change in peak Ca2+ mobilization in platelets stimulated with: (B) thrombin, (D) AYPGKF, or (F) thapsigargin. The results are the mean (± SD) of three independent experiments (* p<0.05).
Figure 6.
RhoA activity measured by G-LISA Kit in mouse platelets.
The level of activated RhoA-GTP is measured by absorbance at 490 nm in response to increasing concentrations of thrombin (1–100 nM). The results are from three independent experiments (* p<0.05, ns: not significant).
Figure 7.
Western blot analysis of Akt phosphorylation in mouse platelets.
(A) The level of Akt phosphorylation at Ser473 in response to increasing concentrations of thrombin (1–100 nM) was determined by western blotting with phospho-Akt (Ser473) antibody. The membrane was re-probed for total Akt to demonstrate protein loading. The blots shown are from a representative of three independent experiments. (B) Quantitation of Akt phosphorylation at (Ser 473) in response to thrombin is represented at the mean (± SD, n = 3) (* p<0.05).
Figure 8.
Bioluminescence resonance energy transfer (BRET) analysis of PAR3 homodimers, PAR4 homodimers, and PAR3-PAR4 heterodimers.
The HEK293 cells were transfected with: (A) PAR4-Luc (1 µg) and PAR3-GFP (0–2.5 µg), (B) PAR3-Luc (1 µg) and PAR3-GFP (0–2.5 µg), or (C) PAR4-Luc (1 µg) and PAR4-GFP (0–2.5 µg). As a control experiment, the HEK293 cells were transfected with: (D) PAR3-Luc (1 µg) and rho-GFP (0–0.12 µg), or (E) PAR4-Luc (1 µg) and rho-GFP (0–0.12 µg). Forty-eight hours post-transfection, the cells were analyzed for GFP expression, Luc expression, and BRET. The curves were plotted as the ratio of GFP to Luc and all points from 3–6 independent experiments were analyzed by global fit to a hyperbolic or linear curve. The surface expression of PAR3 and PAR4 in the HEK293 cells was determined by flow cytometry. (G) V5-PAR4-GFP and V5-PAR3-GFP were detected with a V5 tag antibody conjugated to Alexa Fluor 647. (H) HA-PAR4-LUC and HA-PAR3-LUC were detected with a HA tag antibody conjugated to Alexa Fluor 647. The results are the mean (± SD) of two independent experiments. The number of PAR4 and PAR3 molecules on the HEK293 cells surface is calculated from the V5 or HA antibody standard curve using quantitative flow cytometry (F).