Alox15 via H2O2 mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation

Figures

• 

  Figure 1. 12/15-LOX is required for TXA2-induced platelet activation and hemostasis (A–C) Eight-weeks-old WT and 12/15-LOX−/− mice were subjected to measurement of body weight (A), tail bleeding time (B), and whole blood clotting time (C) (n = 10). (D) Platelet-rich plasma (PRP) from WT and 12/15-LOX−/− mice were incubated with and without F2-TXA2 (1 μM) for the indicated periods at RT and photographed. The percentage of clot retraction and extruded serum volume were calculated as described in the methods (n = 3). (E) Wahed platelets were plated onto fibrinogen-coated coverslips and after 1 h stained with phalloidin and DAPI and observed under a Zeiss inverted microscope (Axiovision Observer.z1; 40×/NA 0.6). The pictures were captured by a Zeiss AxioCam MRm camera using the microscope operating and image analysis software ZEN 2.6. (F) Washed platelets from WT mice were labeled with calcein acetoxymethyl ester (10 μM) for 30 min and placed onto fibrinogen-coated wells in a 96-well plate. Platelets were then incubated with and without F2-TXA2 at the indicated concentrations for 30 min, washed with PBS and the bound platelets were lysed with lysis buffer and the fluorescence intensity was…

• 

  Figure 2. SQ29548 attenuates TXA2-induced platelet activation and hemostasis (A–D) Washed platelets from WT mice with the indicated treatments were subjected to adhesion (A), and spreading (B), while PRP was assayed for platelet aggregation (C) and clot retraction (D) as described in Figure 1 (n = 3). (E and F) Tail bleeding time (E) and whole blood clotting time (F) were measured using WT mice that received vehicle or SQ29548 by immunoprecipitation (ip) injections (5 mg/kg body weight) (n = 10). (G–J) Washed platelets (G and I) or PRP (H and J) from WT and 12/15-LOX−/− mice with the indicated treatments were subjected adhesion assay (G and I) (n = 3) or aggregation assay (H and J) (n = 3). All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗p < 0.01 versus control; #p < 0.01 versus F2-TXA2. Scale bars, 10 μm in (B).

• 

  Figure 3. 12/15-LOX is required for TXA2-induced TP receptor membrane trafficking (A and B) Platelets from WT and 12/15-LOX−/− mice were treated with and without F2-TXA2 for indicated periods, and either mRNA or protein extracts were prepared and analyzed by qRT-PCR (A) or western blotting (B) for TP receptor and β-actin levels using their specific primers (Table 1) or antibodies (n = 3). (C) Platelets from WT and 12/15-LOX−/− mice were treated with and without F2-TXA2 for 30 min, labeled with sulfo-NHS-SS-Biotine, washed with PBS and the membrane and cytoplasmic proteins were isolated as described in STAR Methods and analyzed by western blotting for the indicated proteins using their specific antibodies (n = 3). (D) WT mouse platelets were treated with and without collagen type I (10 μg/mL) in the presence and absence of aspirin (10 μM) for 30 min, labeled with sulfo-NHS-SS-Biotine, washed with PBS and the membrane and cytoplasmic proteins were isolated and analyzed by western blotting for the indicated proteins using their specific antibodies. (E) Platelets from 12/15-LOX−/− mice were treated with and without F2-TXA2 in the presence or absence of 12(S)-HETE (0.5 μM) for 30 min and…

• 

  Figure 4. Palmitoylation is required for TP receptor membrane transport (A) Alignment of mouse TP receptor (ENSMUSG00000034881) protein sequence. Palmitoylation sites were shown in bold red color text. Palmitoylation sites were identified using http://cppdd.cn/seqpalm software. (B) Platelets from WT and 12/15-LOX−/− mice were treated with and without F2-TXA2 for 30 min and subjected to resin-assisted capture of S-acylated proteins (Acyl-RAC) assay as described in STAR Methods. (C) All the conditions were the same as in (B) except that platelets from WT mice were preincubated with and without 2-bromo palmitate (2-BP, 10 μM) and treated with and without F2-TXA2 for 30 min. (D) Platelets from WT mice that were pre-incubated with vehicle or 2-BP (10 μM, 30 min) were treated with without F2-TXA2 for 30 min and labeled with sulfo-NHS-SS-Biotin (250 μg/mL; 10 min at RT). Proteins from membrane and soluble fractions were isolated as described in Figure 3C, and analyzed by western blotting for the indicated proteins using their specific antibodies (n = 3). (E–H) All the conditions were the same as in (D), except that the platelets were subjected to adhesion (E), and spreading (G), while PRP was…

• 

  Figure 5. A role for PKCθ in TXA2-induced 12/15-LOX-mediated TP receptor palmitoylation (A) Platelets from WT and 12/15-LOX−/− mice were treated with and without F2-TXA2 for the indicated periods, and protein extracts were prepared and analyzed by western blotting for the indicated phospho and total PKCs using their specific antibodies (n = 3). (B) Platelets from WT mice were treated with and without F2-TXA2 in the presence and absence of PKCα/β or PKCθ inhibitors, Go6976 (1 μM) or C27 (5 μM), respectively, for 30 min, and subjected to Acyl-RAC assay as described in STAR Methods. (C) Platelets from WT mice were treated with and without F2-TXA2 in the presence and absence of C27 for 30 min and labeled with sulfo-NHS-SS-Biotin. Proteins from membrane and soluble fractions were isolated as described in Figure 3C, and analyzed by western blotting for the indicated proteins using their specific antibodies (n = 3). (D–G) All the conditions were same as in (C) except that the platelets were subjected to adhesion (D), and spreading (F), while the PRP was used for platelet aggregation (E) and clot retraction (G) (n = 3). (H and I) Tail bleeding time (H) and whole blood clotting times (I) were…

• 

  Figure 6. A requirement for ROS in TXA2-induced 12/15-LOX-mediated PKCθ activation and TP receptor palmitoylation (A) Platelets from WT mice were treated with and without F2-TXA2 at the indicated concentrations for 30 min and H2O2 production was measured using Amplex Red Hydrogen Peroxide/Peroxidase Assay kit (n = 3). (B) Platelets from WT and 12/15-LOX−/− mice were treated with and without F2-TXA2 for 30 min and H2O2 production was measured (n = 4). (C) Platelets from WT mice were treated with and without F2-TXA2 in the presence and absence of allopurinol (50 μM) for 30 min and H2O2 production was measured (n = 4). (D) Platelets from WT and 12/15-LOX−/− mice were treated with and without U46619 for 30 min and H2O2 production was measured (n = 3). (E) Platelets from WT mice were treated with and without U46619 in the presence or absence of allopurinol for 30 min and H2O2 production was measured (n = 4). (F and G) All the conditions were the same as in (D and E) except that platelets were treated with and without collagen type I. (H) Platelets from WT mice were treated with and without F2-TXA2 in the presence and absence of allopurinol for 30 min and protein extracts were prepared and…

• 

  Figure 7. H2O2 but not uric acid plays a role in platelet activation and hemostasis (A) WT mice platelets were incubated with and without H2O2 (200 μM) or uric acid (100 μM) for 30 min, and protein extracts were prepared and analyzed by western blotting for the levels of phospho and total PKCθ using their specific antibodies (n = 3). (B) Platelets from WT mice were treated with and without H2O2 or uric acid for 30 min and subjected to Acyl-RAC assay as described in Figure 4B. (C) Platelets from WT mice were treated with and without H2O2 or uric acid for 30 min, labeled with sulfo-NHS-SS-Biotin and membrane and soluble fractions were isolated and analyzed by western blotting for TP receptor, Na/K ATPase and MEK1 levels using their specific antibodies (n = 3). (D–G) All the conditions were the same as in (A) except that the platelets were subjected to adhesion (D) and spreading (F) and the PRP was used for platelet aggregation (E) and clot retraction (G) (n = 3). All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗p < 0.01 versus vehicle; #p < 0.01 versus H2O2. Scale bars: 10 μm in (F). HA, hydroxylamine; UA, uric acid; M, membrane fraction; S, soluble fraction.

• 

  Figure 8. 12/15-LOX via COP9 signalosome protects XO from proteasomal degradation (A and B) Platelets from WT and 12/15-LOX−/− mice were treated with and without F2-TXA2 for 30 min, and mRNA or protein extracts were prepared and analyzed by qRT-PCR (A) or western blotting (B) for XO and β-actin mRNA or protein levels, respectively, using their specific primers (Table 1) or antibodies (n = 3). (C) Platelets from WT mice were incubated with and without F2-TXA2 in the presence and absence of rapamycin (100 nM) for 30 min, and protein extracts were analyzed by western blotting for XO and β-actin levels using their specific antibodies (n = 3). (D) All the conditions were same as in (B) except that the protein extracts were immunoprecipitated with anti-XO antibody and the immunocomplexes were analyzed by western blotting using anti-ubiquitin antibody. The blot was sequentially reprobed for CSN5, cullin 2, and XO. The input protein was analyzed by western blotting for CSN5, cullin 2, and β-actin levels. All data are presented as mean ± SD and analyzed by paired Student’s t test. ∗p < 0.01 versus control; #p < 0.01 versus F2-TXA2.

• 

  Figure 9. 15-LOX1 mediates TP receptor palmitoylation and its membrane trafficking leading to platelet activation in humans (A) Platelets were isolated from the venous blood of the healthy human volunteers, treated with and without F2-TXA2 for 30 min, RNA was isolated and analyzed by qRT-PCR for ALOX12 and ALOX15 mRNA levels using their specific primers (Table 1). β-actin was used as an endogenous control (n = 3). (B) Human platelets were treated with and without F2-TXA2 for the indicated periods, and protein extracts were prepared and analyzed by western blotting for 12-LOX, 15-LOX1 and β-actin levels using their specific antibodies (n = 3). (C) Human platelets were treated with and without F2-TXA2 in the presence and absence of ML351 (10 μM) for 30 min and protein extracts were prepared and analyzed by western blotting for TP receptor and β-actin levels using their specific antibodies (n = 3). (D) Alignment of human TP receptor (ENSG00000006638) protein sequence with a potential palmitoylation site shown in bold red color text. Palmitoylation sites were identified using http://cppdd.cn/seqpalm software. (E) Human platelets were treated with and without F2-TXA2 in the presence and absence…

• 

  Figure 10. ROS plays a role in F2-TXA2-induced platelet activation in humans (A) Human platelets were treated with and without F2-TXA2 in the presence and absence of ML351 for 30 min and protein extracts were prepared and analyzed by western blotting for XO and β-actin levels using their specific antibodies (n = 3). (B) Human platelets were treated with and without F2-TXA2 in the presence and absence of ML351 for 30 min and H2O2 production was measured (n = 4). (C) All the conditions were same as in (A) except that the protein extracts were immunoprecipitated with anti-XO antibody and the immunocomplexes were analyzed by western blotting using anti-ubiquitin antibody. The blot was sequentially reprobed for CSN5, cullin 2, and XO. The input protein was analyzed by western blotting for CSN5, cullin 2, and β-actin levels. (D) Human platelets were treated with and without F2-TXA2 in the presence and absence of allopurinol (50 μM) for 30 min and H2O2 production was measured (n = 4). (E) Human platelets were treated with and without F2-TXA2 in the presence and absence of allopurinol for 30 min and subjected to Acyl-RAC assay as described in Figure 4B. (F) Human platelets were treated with and…

• 

  Figure 11. Schematic diagram depicting the potential signaling events mediated by 12/15-LOX/15-LOX1 in TXA2-induced TP receptor palmitoylation and its membrane trafficking promoting platelet activation and hemostasis