Dilthey, Beverly G.

Gross, Richard W.

Guan, Shaoping

Jenkins, Christopher M.

Liu, Xinping

Sims, Harold F.

English

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 1  Supporting Information  12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout  Xinping Liu1, Harold F. Sims1, Christopher M. Jenkins1, Shaoping Guan1, Beverly G. Dilthey1, and Richard W. Gross*1,2,3 From the 1Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO 63110; 2Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO 63110; 3Department of Chemistry, Washington University, Saint Louis, MO 63130 Running Title: Eicosanoid-lysophospholipids are attenuated by iPLA2γ knockout *To whom correspondence should be addressed: Richard W. Gross, M.D., Ph.D. Division of Bioorganic Chemistry and Molecular Pharmacology, Washington University School of Medicine, 660 S. Euclid Ave., Campus Box 8020, St. Louis, MO 63110; rgross@wustl.edu; Tel.: 314-362-2690; Fax: 314-362-1402.    2     Supplementary Fig. 1. Proposed fragmentation pathways upon collision induced dissociation (CID) leading to the observed accurate mass product ions for 12-hydroperoxyl-eicosatetrataenoic-lysophosphatidylcholine (12-HpETE-LPC). The product ion m/z 418.1980 is derived from the unique fragmentation occurring at the labile hydroperoxide bond. Exact masses are calculated theoretical masses; numbers in parentheses indicate the measured mass error in parts per million (ppm) relative to the calculated theoretical mass.    3   Supplementary Fig. 2. Proposed fragmentation pathways upon collision induced dissociation (CID) leading to the observed accurate mass product ions for 12-hydroperoxyl-eicosatetrataenoic-lysophosphatidylethanolamine (12-HpETE-LPE). The product ion m/z 376.1520 is derived from the unique fragmentation occurring at the labile hydroperoxide bond. Exact masses are calculated theoretical masses; numbers in parentheses indicate the measured mass error in parts per million (ppm) relative to the calculated theoretical mass.    4    Supplementary Fig. 3. Production of 1-AA-LPC and 1-AA-LPE in murine platelets from wild-type (WT) and iPLA2γ knockout (KO) mice stimulated with calcium ionophore A23187. Washed platelets were incubated in the presence or absence of calcium ionophore A23187 at 37°C for 15 min followed by extraction of lipids using a modified Bligh and Dyer procedure and analysis by HPLC-MS/MS. A, Representative of LC/MS traces for 1-AA-LPC in the absence (Control) or presence (A23187) of calcium ionophore A23187 in platelets from WT or KO mice; B, Representative of LC/MS traces for 1-AA-LPE in the absence (Control) or presence (A23187) of calcium ionophore A23187 in platelets from WT or KO mice; C, content of 1-AA-LPC in the absence (Control) or presence (A23187) of calcium ionophore A23187 in platelets from WT or KO mice; D, content of 1-AA-LPE in the absence (Control) or presence (A23187) of calcium ionophore A23187 in platelets from WT or KO mice. Data are expressed as mean values ± SEM of six independent replicates. Statistical analyses were performed using an unpaired two-tailed Student t test. Statistically significant p values are indicated above the bracketed data sets (**** p<0.0001).   5   Supplementary Fig. 4. pH dependent kinetics of sn-2 to sn-1 acyl migration for 2-AA-LPC and 2-AA-LPE to 1-AA-LPC and 1-AA-LPE (respectively).  For each reaction, 2-AA-LPC or 2-AA-LPE was added to 1.0 ml of the selected medium or buffer (EBM-2 culture medium, Tyrode’s buffer, 0.05 M Citrate-sodium citrate buffer (pH 4.6 and pH 6.6) or 0.05 M Tris-HCl buffer (pH7.2 and pH7.8) to give a final concentration of 10 μM, and the resulting mixture was incubated at 37°C for the indicated times. The quantities of 1- or 2-AA-lysophospholipids were then determined for the indicated time points by LC-MS.  Plots of remaining 2-AA-LPC (A) or 2-AA-LPE (B) are shown as a percent of total 1- and 2-AA-LPC or 1- and 2-AA-LPE versus time, respectively. Each curve represents the mean ± SEM from three replicate experiments.    6  Supplementary Table 1. Measured apparent Km and Vmax values for 12-HpETE-lysophospholipid products formed upon oxidation of 2-AA-LPC and 2-AA-LPE with human platelet-type 12-LOX (h12LOX) or mouse platelet-type 12-LOX (m12-LOX). Data are expressed as mean values ± SEM of three replicates. Enzyme Substrate Product Km  (µM) Vmax (nmol s-1mg-1) m12-LOX 2-AA-LPC 12-HpETE-LPC 14 (±4) 48 (±6) m12-LOX 2-AA-LPE 12-HpETE-LPE 13 (±2) 33 (±5) h12-LOX 2-AA-LPC 12-HpETE-LPC 12 (± 3) 50 (±8) h12-LOX 2-AA-LPE 12-HpETE-LPE 9 (±1) 27 (±6)   

12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

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12-LOX catalyzes the oxidation of 2-arachidonoyl-lysolipids in platelets generating eicosanoid-lysolipids that are attenuated by iPLA2γ knockout

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