Figure 3.
Endogenous HAGP accumulates in the absence of AADACL1 activity but is not a substrate of AADACL1. (A) Human platelets were pretreated with 10 μM JW480 or 0.5% DMSO for 10 minutes at 37°C prior to treatment with buffer or 10 μg/mL collagen for 5 minutes. Total lipids were extracted, and ∼10% of the reconstituted lipids were subjected to normal phase LC coupled with high-resolution normal phase liquid chromatography–electrospray ionization tandem MS (NPLC-ESI/MS/MS). HAGP was identified by comparison with a synthetic standard ([M-H]− at m/z 437.2). HAGP levels were normalized to phosphatidylinositol for relative quantitation and expressed as a percentage of HAGP from control platelets treated only with DMSO (*P = .01 for JW480-treated vs DMSO-treated collagen-stimulated samples; n = 3). (B) Human platelets were pretreated with 30 µM HAG or DMSO for the indicated time at 37°C prior to stimulation with 1 μg/mL collagen for 3 minutes. Total lipids were extracted, and ∼2.5% of the reconstituted lipids were subjected to high-resolution MS as in panel A (**P = .001 for HAG-treated 15 minutes plus collagen vs DMSO-treated with no HAG plus collagen; ***P < .0001 for HAG plus collagen 30 minutes vs DMSO-treated with no HAG plus collagen; n = 4). (C) Deacetylation of purified HAGP was tested using AADACL1-expressing microsomes and detected by reverse phase LC-MS. Microsomes from AADACL1-transfected HEK cells (0.1 mg total protein) were incubated with either 20 μM purified HAGP or HAG for 30 minutes at 37°C and extracted with methanol before centrifugation and LC-MS analysis. Integrated peak areas were used for relative quantitation, and HAGP deacetylation (% lipid hydrolysis) was calculated as product/(product + substrate) × 100 (**P < .001 for AADACL1 vs control for HAG; n = 3). (D) Washed human platelets (300 × 109/L) were treated with 10 μM R59022 or 0.5% DMSO for 10 minutes before addition of the indicated concentrations of HAG for an additional 30 minutes. Aggregation (D) and secretion (E) were induced simultaneously with 1 to 2 μg/mL collagen for 4 minutes (*P < .04, **P < .002, and ***P < .001 for R59022 vs control; n = 5). (F) Washed human platelets (3 × 109/L) were incubated with 30 µM HAG or DMSO vehicle control (0.5%) for the indicated times at 37°C. Aggregation was initiated with 0.75 μg/mL collagen, and platelet aggregation was observed for 4 minutes postagonist stimulation (n = 4). (G) Platelets were treated with the indicated concentrations of HAG, and aggregation was observed for 4 minutes postaddition of HAG without collagen (n = 3). (H) Platelets loaded with Fluo-4 were pretreated with either 0.25% DMSO (negative control) or 30 µM HAG for the indicated times prior to stimulation with convulxin (200 ng/mL) in the presence of 1 mM extracellular calcium (n = 5). CVX, convulxin.

Endogenous HAGP accumulates in the absence of AADACL1 activity but is not a substrate of AADACL1. (A) Human platelets were pretreated with 10 μM JW480 or 0.5% DMSO for 10 minutes at 37°C prior to treatment with buffer or 10 μg/mL collagen for 5 minutes. Total lipids were extracted, and ∼10% of the reconstituted lipids were subjected to normal phase LC coupled with high-resolution normal phase liquid chromatography–electrospray ionization tandem MS (NPLC-ESI/MS/MS). HAGP was identified by comparison with a synthetic standard ([M-H]− at m/z 437.2). HAGP levels were normalized to phosphatidylinositol for relative quantitation and expressed as a percentage of HAGP from control platelets treated only with DMSO (*P = .01 for JW480-treated vs DMSO-treated collagen-stimulated samples; n = 3). (B) Human platelets were pretreated with 30 µM HAG or DMSO for the indicated time at 37°C prior to stimulation with 1 μg/mL collagen for 3 minutes. Total lipids were extracted, and ∼2.5% of the reconstituted lipids were subjected to high-resolution MS as in panel A (**P = .001 for HAG-treated 15 minutes plus collagen vs DMSO-treated with no HAG plus collagen; ***P < .0001 for HAG plus collagen 30 minutes vs DMSO-treated with no HAG plus collagen; n = 4). (C) Deacetylation of purified HAGP was tested using AADACL1-expressing microsomes and detected by reverse phase LC-MS. Microsomes from AADACL1-transfected HEK cells (0.1 mg total protein) were incubated with either 20 μM purified HAGP or HAG for 30 minutes at 37°C and extracted with methanol before centrifugation and LC-MS analysis. Integrated peak areas were used for relative quantitation, and HAGP deacetylation (% lipid hydrolysis) was calculated as product/(product + substrate) × 100 (**P < .001 for AADACL1 vs control for HAG; n = 3). (D) Washed human platelets (300 × 109/L) were treated with 10 μM R59022 or 0.5% DMSO for 10 minutes before addition of the indicated concentrations of HAG for an additional 30 minutes. Aggregation (D) and secretion (E) were induced simultaneously with 1 to 2 μg/mL collagen for 4 minutes (*P < .04, **P < .002, and ***P < .001 for R59022 vs control; n = 5). (F) Washed human platelets (3 × 109/L) were incubated with 30 µM HAG or DMSO vehicle control (0.5%) for the indicated times at 37°C. Aggregation was initiated with 0.75 μg/mL collagen, and platelet aggregation was observed for 4 minutes postagonist stimulation (n = 4). (G) Platelets were treated with the indicated concentrations of HAG, and aggregation was observed for 4 minutes postaddition of HAG without collagen (n = 3). (H) Platelets loaded with Fluo-4 were pretreated with either 0.25% DMSO (negative control) or 30 µM HAG for the indicated times prior to stimulation with convulxin (200 ng/mL) in the presence of 1 mM extracellular calcium (n = 5). CVX, convulxin.

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