Figure 5
Figure 5. oxLDL inhibits cGMP signaling through a mechanism that requires CD36, Src kinases, and PKC. (A) Human platelets (3 × 108 platelets/mL) were incubated with nLDL (50 µg/mL), oxLDL (50 µg/ml), oxPCCD36 (5 µmol/L), or PAPC (5 µmol/L) prior to the addition of 8-pCPT-cGMP (50 µmol/L) for 2 minutes. Platelets were washed and lysed, and intracellular cGMP concentrations were measured by enzyme immunoassay (n = 4). (B) Human platelets (5 × 108 platelets/mL) were treated with nLDL or oxLDL (50 μg/mL) for 15 minutes, lysed, and PKG1 immunoprecipitated. Immunoprecipitates were incubated with exogenous cGMP and PKG activity measured spectrophotometrically at 450 nm. *P < .05 compared with the absence of oxLDL. (C) In an in vitro kinase assay, constitutively active rPKG (0.5 µg) was incubated with rRhoA (1 µg) for 30 minutes at 37°C. In some cases, rPKG was preincubated for 30 minutes with either xanthine (100 µmol/L)/xanthine oxidase (5 mU/mL) (X/XO) or KO2 (1 mmol/L) before addition to rRhoA. Phosphorylation was terminated by Laemmli buffer, the mixture separated by SDS-PAGE and immunoblotted for phosphoRhoA-Ser188, total RhoA, or PKG1. **P < .01 for rPKG alone compared with the presence of X/XO or KO2. (D) Washed human platelets (5 × 108 platelets/mL) preincubated with apyrase (2 U/mL), indomethacin (10 µmol/L), and EGTA (1 mmol/L) were treated with oxLDL (50 µg/mL) in the presence or absence of the CD36 blocking antibody FA6-152 or IgG (5 µg/mL) prior to the addition of 8-pCPT-cGMP (50 µmol/L) for 2 minutes. Platelets were lysed, separated by SDS-PAGE, and immunoblotted (IB) for phospho–VASP-ser239 and β-tubulin. *P < .05 compared with the absence of oxLDL and oxLDL in the presence and absence of FA6. (E) As in panel D, except platelets were treated with PP2 or PP3. *P < .05 compared with the absence of oxLDL and oxLDL in the presence and absence of PP2). (F) As in panel D, except platelets were treated with Ro31-8220, BIMI, BIMV, U73122, or BAPTA-AM prior to the addition of oxLDL (50 µg/mL) for 15 minutes and 8-pCPT-cGMP (50 µmol/L) for 2 minutes. In all figures, subpanel i shows blots representative of at least 3 experiments using different blood donors, and subpanel ii shows densitometric analysis of the blots. *P < .05 compared with oxLDL alone.

oxLDL inhibits cGMP signaling through a mechanism that requires CD36, Src kinases, and PKC. (A) Human platelets (3 × 108 platelets/mL) were incubated with nLDL (50 µg/mL), oxLDL (50 µg/ml), oxPCCD36 (5 µmol/L), or PAPC (5 µmol/L) prior to the addition of 8-pCPT-cGMP (50 µmol/L) for 2 minutes. Platelets were washed and lysed, and intracellular cGMP concentrations were measured by enzyme immunoassay (n = 4). (B) Human platelets (5 × 108 platelets/mL) were treated with nLDL or oxLDL (50 μg/mL) for 15 minutes, lysed, and PKG1 immunoprecipitated. Immunoprecipitates were incubated with exogenous cGMP and PKG activity measured spectrophotometrically at 450 nm. *P < .05 compared with the absence of oxLDL. (C) In an in vitro kinase assay, constitutively active rPKG (0.5 µg) was incubated with rRhoA (1 µg) for 30 minutes at 37°C. In some cases, rPKG was preincubated for 30 minutes with either xanthine (100 µmol/L)/xanthine oxidase (5 mU/mL) (X/XO) or KO2 (1 mmol/L) before addition to rRhoA. Phosphorylation was terminated by Laemmli buffer, the mixture separated by SDS-PAGE and immunoblotted for phosphoRhoA-Ser188, total RhoA, or PKG1. **P < .01 for rPKG alone compared with the presence of X/XO or KO2. (D) Washed human platelets (5 × 108 platelets/mL) preincubated with apyrase (2 U/mL), indomethacin (10 µmol/L), and EGTA (1 mmol/L) were treated with oxLDL (50 µg/mL) in the presence or absence of the CD36 blocking antibody FA6-152 or IgG (5 µg/mL) prior to the addition of 8-pCPT-cGMP (50 µmol/L) for 2 minutes. Platelets were lysed, separated by SDS-PAGE, and immunoblotted (IB) for phospho–VASP-ser239 and β-tubulin. *P < .05 compared with the absence of oxLDL and oxLDL in the presence and absence of FA6. (E) As in panel D, except platelets were treated with PP2 or PP3. *P < .05 compared with the absence of oxLDL and oxLDL in the presence and absence of PP2). (F) As in panel D, except platelets were treated with Ro31-8220, BIMI, BIMV, U73122, or BAPTA-AM prior to the addition of oxLDL (50 µg/mL) for 15 minutes and 8-pCPT-cGMP (50 µmol/L) for 2 minutes. In all figures, subpanel i shows blots representative of at least 3 experiments using different blood donors, and subpanel ii shows densitometric analysis of the blots. *P < .05 compared with oxLDL alone.

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