Research ArticleBiochemistry

Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis

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Science Signaling  28 Nov 2017:
Vol. 10, Issue 507, eaan2787
DOI: 10.1126/scisignal.aan2787
  • Fig. 1 HETE-PEs and HETE-PCs dose-dependently enhance TF-dependent thrombin generation through a PS-dependent mechanism.

    Thrombin generation was initiated by the addition of liposomes to pooled platelet-poor plasma (PPP) with a thrombinoscope, as described in Materials and Methods. (A) Hydroxyeicosatetraenoic acid–phosphatidylethanolamines (HETE-PEs) enhance tissue factor (TF)–dependent thrombin generation in plasma. Liposomes contained 10 pM recombinant TF with 65% 1,2-di-stearoyl-phosphatidylcholine (DSPC), 5% 1-stearoyl-2-arachidonoyl-phosphatidylserine (SAPS), and 30% SAPE, with 0 to 10% SAPE replaced with 0 to 10% of the indicated HETE-PE species. (B). HETE-PCs enhance TF-dependent thrombin generation in plasma. Liposomes contained 10 pM TF, 55% DSPC, 5% SAPS, and 30% SAPE, with 0 to 10% SAPC replaced with <10% of the indicated HETE-PC species. Data in (A) and (B) are representative traces of experiments that were repeated at least three times. (C) 15-HETE-PE and 15-HETE-PC enhance PS-dependent thrombin generation. Pooled PPP was activated with liposomes as described earlier, where SAPE was replaced with 15-HETE-PE (left graphs) or 15-HETE-PC (right graphs), with or without SAPS replacing PC, as indicated. Representative traces and maximum thrombin at varying PS concentrations are shown. Data are means ± SEM of three runs. (D) HETE-PEs and HETE-PCs promote the activities of coagulation factors in a full reconstitution system. Thrombin generation was initiated by the addition of liposomes to purified factors II, V, VII, VIIII, IX, and X at physiological concentrations (see Materials and Methods). Liposomes contained (left) 65% DSPC, 5% SAPS, and 30% SAPE, with 10% SAPE (control) or 10% HETE-PE, or (middle) 55% DSPC, 5% SAPS, and 30% SAPE, with 10% SAPC (control) or 10% HETE-PC. Right: Summary data presented as fold change for the maximum thrombin generation rate observed (slope of lines in left and middle graphs) for each isomer. Data are means ± SEM of three runs. **P < 0.01 by single-factor analysis of variance (ANOVA) and post hoc Tukey tests.

  • Fig. 2 MD simulation suggests the association of the HETE-PL hydroxyl group with the polar environment, serine, and Ca2+, whereas HETE-PL membranes bind more Ca2+.

    (A to E) Molecular dynamics (MD) simulation shows the HETE hydroxyl group altering membrane behavior. MD simulations were undertaken for 300 ns using 5% SAPS, 5% SAPC, 30% SAPE, 55% dioleoylphosphatidylcholine (DOPC), and 5% 12-HETE-PC, as described in Materials and Methods. (A to D) Side views showing hydrophobic region (yellow), charged phosphate groups (cyan), and PC head groups (gray). (E) Top view looking down on the membrane showing areas of positive charge (blue), negative charge (brown), PS head groups (pink), and HETE hydroxyl groups (red). (F) Binding of calcium to membranes is increased by 15-HETE-PE. Liposomes consisting of 65% DSPC, 30% SAPE, and 5% SAPS, with up to 10% SAPE replaced by 15-HETE PE, were tested for calcium binding by measuring Fluo-FF fluorescence (see Materials and Methods). *P < 0.05, when compared to no added 15-HETE-PE. Data are means ± SEM of three experiments and were analyzed with the Mann-Whitney U test.

  • Fig. 3 HETE-PL liposomes prevent tail bleeding and increase TAT concentrations in vivo, whereas platelets from mice genetically lacking p12-LOX generate few eoxPLs.

    (A) 12-HETE-PE/TF intradermal administration prevents tail bleeding in adult mice. Eleven-week-old male C57BL/6J mice were injected with liposomes containing TF with or without 12-HETE-PE or 12-HETE-PC immediately proximal to a tail cut, and bleeding time and blood loss were recorded as described in Materials and Methods. Data are from 10 to 16 mice per group. **P ≤ 0.01, *P ≤ 0.05 by Mann-Whitney U test. (B) 12-HETE-PE increases thrombin-antithrombin (TAT) complexes in vivo. Control or HETE-PE–containing liposomes were injected intravenously into wild-type (WT) mice, and plasma was obtained after 1 hour. TAT concentrations were measured by enzyme-linked immunosorbent assay. Data are from six mice per group. *P ≤ 0.05. (C) Thrombin stimulates the production of enzymatically oxidized phospholipid (eoxPL) species from washed murine platelets in a p12-lipoxygenase (LOX)–dependent manner. Washed platelets from WT or ALOX12/−/− mice were activated for 30 min with thrombin (0.2 U/ml), and then the lipids were extracted and analyzed to detect eoxPL species. Data are means of three experiments. Data in the heatmap are normalized to basal amounts of the indicated eoxPLs in WT mouse platelets. (D) Time course for the generation of 12-HETE-PE and 12-HETE-PCs. Platelets from WT or ALOX12 −/− mice (2 × 108/ml) were activated with human thrombin (0.2 U/ml) for 0 to 30 min at 37°C. The lipids were then extracted and quantified. HETE-PE represents the sum of 16:0p/12-HETE-PE, 18:1p/12-HETE-PE, 18:0/12-HETE-PE, and 18:0a/HETE PE. HETE-PC represents the sum of 16:0a/12-HETE-PC and 18:0a/12-HETE-PC. Data are means ± SEM of eight mice per group. ns, not significant.

  • Fig. 4 Mice lacking either 12/15-LOX or p12-LOX have impaired venous coagulation, which is restored by the administration of 12-HETE-PE liposomes.

    (A and B) ALOX12-deficient mice show reduced thrombus formation in vivo after challenge. Venous thrombosis was induced in WT and ALOX12−/− mice. (A) Thrombus weights were measured. Data are means ± SEM of seven to nine mice per group. *P < 0.05 by Mann-Whitney U test. (B) A representative thrombus is shown for each genotype. (C) ALOX12-deficient mice have impaired hemostasis in vivo. Male WT C57BL/6J or ALOX12−/− mice (8 to 11 weeks old) were administered a tail cut, and bleeding time and blood loss were recorded. Liposomes containing 19 ng of 12-HETE-PE or control liposomes were administered 10 ml of phosphate-buffered saline into the tail tissue just ahead of the cut and immediately before the cut was made. Data are means ± SEM of 6 to 12 mice per group. (D) ALOX15-deficient mice have impaired hemostasis in vivo. Experiments were performed, and data were collected as described in (C) except that WT C57BL/6J mice were compared to ALOX15-deficient mice. Data are means ± SEM of 12 to 19 mice per group. Data in (C) and (D) were analyzed by Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.005.

  • Fig. 5 HETE-PEs are increased in leukocytes and platelets from APS patients.

    (A and B) 15- and 5-HETE-PEs are increased in leukocytes from antiphospholipid syndrome (APS) patients. Total leukocytes were isolated from healthy control (HC) and APS patients, left unstimulated (US) or stimulated at 4 × 106 cells/ml with 10 μM A23187 for 30 min at 37°C, and then the lipids were analyzed for HETE-PEs. HETE-PEs were quantified as individual molecular species and then combined. Data are means ± SEM of 34 (HC) or 17 (APS) subjects. (C) 12-HETE-PEs are increased in platelets from APS patients. Washed platelets from HC and APS patients were isolated and left unstimulated or stimulated with thrombin (0.2 U/ml) for 30 min at 37°C before the amounts of 12-HETE-PE were determined. Data are means ± SEM of 18 (HC) or 12 (APS) subjects. (D) Platelets from APS patients that spontaneously aggregated contain substantial amounts of 12-HETE-PEs. During the final washing step, a number of APS platelet isolates spontaneously aggregated (n = 7 patients). Lipids were extracted and analyzed for 12-HETE-PEs. (E) Urinary 11-thromboxane B2 (TXB2) is increased in APS patients. Urine from HC and APS patients was analyzed by gas chromatography–mass spectrometry (MS) to determine the amounts of TXB2. Data are means ± SEM of 32 (HC) or 9 (APS) subjects. ng/mg creatinine (Cr). (F and G) 15- and 5-HETE-PEs are basally increased in concentration in platelets from APS patients. Platelets were prepared as described in (C) and then were analyzed to determine the amounts of the indicated HETE-PEs. Data are means ± SEM of 18 (HC) or 12 (APS) subjects. (H) 12-HETE-PEs are basally increased in abundance in leukocytes from APS patients. Total leukocytes were isolated and left unstimulated or stimulated at 4 × 106 cells/ml with 10 μM A23187 for 30 min at 37°C and then analyzed for 12-HETE-PEs. Data are means ± SEM of 34 (HC) or 18 (APS) subjects. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05 by Mann-Whitney U test.

  • Fig. 6 Circulating IgG against HETE-PEs is increased in abundance in APS, and β2BP1 binding to membranes is enhanced by HETE-PEs.

    (A to C) Immunoglobulin G (IgG) against HETE-PEs is statistically significantly increased in abundance in plasma from APS patients. The amounts of IgG antibodies against HETE-PEs in HC and APS patient plasma were determined by diluting plasma 1:12 and testing binding to the indicated antigens. SAPE was used as an unoxidized lipid for comparison. All samples were analyzed in triplicate. Data are means ± SEM of 18 (HC) or 9 (APS) subjects. *P < 0.05, **P 0.01 by Mann-Whitney U test. (D) IgG titers are comparable in plasma from HC and APS patients. Total IgG amounts in plasma from HC and APS patients were determined. Data are means ± SEM of 34 (HC) and 10 (APS) subjects. (E) HETE-PEs enhance the binding of β2-glycoprotein 1 (β2GP1) to lipid membranes. Binding of β2GP1 to liposomes in the presence of cardiolipin, 15-HETE-PE, 5-HETE-PE, or 12-HETE-PE was determined as described in Materials and Methods. (F) HETE-PEs enhance the cardiolipin-dependent binding of β2GP1 to lipids. Binding of β2GP1 to liposomes in the presence of cardiolipin with or without 15-HETE-PE, 5-HETE-PE, or 12-HETE-PE was determined. In (D) and (E), data are means ± SEM of three experiments, each performed in triplicate. Statistical significance was determined by one-way ANOVA and Tukey-Kramer test. *P < 0.05, **P < 0.01, ***P < 0.001. RLU, relative light units.

  • Fig. 7 Lipidomic profiling of 47 eoxPL defines their enzymatic origin and regulatory networks.

    (A and B) Washed platelets were isolated from HC or APS patients and were left unstimulated or were stimulated with thrombin (0.2 U/ml, 30 min) before lipids were extracted and analyzed by liquid chromatography-MS/MS for 47 eoxPL species, as described in Materials and Methods. Data are from 16 (HC) and 10 (APS) subjects. (A) p12-LOX– and cyclooxygenase 1 (COX-1)–derived lipids cluster into distinct families based on Sn2 fatty acid composition. A heatmap of the effect of thrombin on the abundances of the indicated lipids is shown. In the heatmap, an increase in abundance is denoted in red, whereas a decrease in abundance is denoted in blue. Deeper tones indicate greater differences, with comparisons for individual lipids made across samples. (B) Plotting correlation between individual lipids illustrates additional relatedness between families of ions. Correlations between lipids across the whole cohort were plotted in a grid in order of decreasing correlation with 18:0a/12-HETE-PE (see fig. S4 and Materials and Methods). Red, p12-LOX; blue, COX-1; green, polyoxygenated PL; black, unknown origin. Lipids marked with red arrows indicating a relationship are listed. All lipid abundances were normalized to the mean of those of the control unstimulated values.

  • Fig. 8 Cytoscape network confirms the enzymatic origin of lipids and identifies an additional group regulated independently of known pathways; p12-LOX–derived eoxPLs are statistically significantly increased in APS patient platelets.

    (A) A Cytoscape network correlation identifies three groups of eoxPLs. Cytoscape 1.2.3 correlation was performed (correlation > 0.8) using data shown in Fig. 7A, with nodes as individual lipids and size determined by the number of links to others. Edge thickness represents the extent of the correlation between individual nodes. Lipids were identified as COX-1– or p12-LOX–derived based on aspirin sensitivity or their absence in ALOX12−/− mice. The plasmalogen-PE group was not associated with any enzymatic system. All lipid abundances were normalized to the mean of the control unstimulated values. (B) p12-LOX–derived lipids are statistically significantly increased in abundance in platelets from patients with APS. Platelet lipids identified as originating from p12-LOX (n = 31) or COX-1 (n = 9) as determined with the Cytoscape network were examined as separate groups for statistically significant differences using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001. A/IS, analyte/internal standard.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/507/eaan2787/DC1

    Fig. S1. Primary data for the generation of eoxPLs by washed wild-type and ALOX12−/− platelets.

    Fig. S2. Analysis of HC and APS demographics for effects on eoxPL generation shows no effect of age, gender, or arterial thrombosis on platelet 12-HETE-PEs.

    Fig. S3. Primary data for the generation of eoxPLs by washed platelets from HC and APS patients.

    Fig. S4. Correlation plots for lipids from all groups show that lipids group according to enzymatic origin.

    Fig. S5. Correlation plots for lipids from either HC or APS patients show that lipids group according to enzymatic origin.

    Table S1. Patient demographics and relevant medical details.

    Table S2. Age and gender demographics of study participants.

    Movie S1. Simulation of the movement of HETE-PC in a lipid biomembrane.

  • Supplementary Materials for:

    Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis

    Sarah N. Lauder, Keith Allen-Redpath, David A. Slatter, Maceler Aldrovandi, Anne O'Connor, Daniel Farewell, Charles L. Percy, Jessica E. Molhoek, Sirpa Rannikko, Victoria J. Tyrrell, Salvatore Ferla, Ginger L. Milne, Alastair W. Poole, Christopher P. Thomas, Samya Obaji, Philip R. Taylor, Simon A. Jones, Phillip G. de Groot, Rolf T. Urbanus, Sohvi Hörkkö, Stefan Uderhardt, Jochen Ackermann, P. Vince Jenkins, Andrea Brancale, Gerhard Krönke, Peter W. Collins,* Valerie B. O'Donnell*

    *Corresponding author. Email: o-donnellvb{at}cardiff.ac.uk (V.B.O.); collinspw{at}cardiff.ac.uk (P.W.C.)

    This PDF file includes:

    • Fig. S1. Primary data for the generation of eoxPLs by washed wild-type and ALOX12−/− platelets.
    • Fig. S2. Analysis of HC and APS demographics for effects on eoxPL generation shows no effect of age, gender, or arterial thrombosis on platelet 12-HETE-PEs.
    • Fig. S3. Primary data for the generation of eoxPLs by washed platelets from HC and APS patients.
    • Fig. S4. Correlation plots for lipids from all groups show that lipids group according to enzymatic origin.
    • Fig. S5. Correlation plots for lipids from either HC or APS patients show that lipids group according to enzymatic origin.
    • Table S1. Patient demographics and relevant medical details.
    • Table S2. Age and gender demographics of study participants.
    • Legend for movie S1

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.49 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Simulation of the movement of HETE-PC in a lipid biomembrane.

    Citation: S. N. Lauder, K. Allen-Redpath, D. A. Slatter, M. Aldrovandi, A. O'Connor, D. Farewell, C. L. Percy, J. E. Molhoek, S. Rannikko, V. J. Tyrrell, S. Ferla, G. L. Milne, A. W. Poole, C. P. Thomas, S. Obaji, P. R. Taylor, S. A. Jones, P. G. de Groot, R. T. Urbanus, S. Hörkkö, S. Uderhardt, J. Ackermann, P. Vince Jenkins, A. Brancale, G. Krönke, P. W. Collins, V. B. O'Donnell, Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis. Sci. Signal. 10, eaan2787 (2017).

    © 2017 American Association for the Advancement of Science

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