Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies
David A. Slatter, … , Peter W. Collins, Valerie B. O’Donnell
David A. Slatter, … , Peter W. Collins, Valerie B. O’Donnell
Published March 22, 2018
Citation Information: JCI Insight. 2018;3(6):e98459. https://doi.org/10.1172/jci.insight.98459.
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Research Article Hematology

Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies

Abstract

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell–derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid–phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

Authors

David A. Slatter, Charles L. Percy, Keith Allen-Redpath, Joshua M. Gajsiewicz, Nick J. Brooks, Aled Clayton, Victoria J. Tyrrell, Marcela Rosas, Sarah N. Lauder, Andrew Watson, Maria Dul, Yoel Garcia-Diaz, Maceler Aldrovandi, Meike Heurich, Judith Hall, James H. Morrissey, Sebastien Lacroix-Desmazes, Sandrine Delignat, P. Vincent Jenkins, Peter W. Collins, Valerie B. O’Donnell

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Figure 5

HETE-PLs containing membranes directly bind Gla domain proteins, and specific HETE-PL positional isomers optimally support coagulation.

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HETE-PLs containing membranes directly bind Gla domain proteins, and spe...
(A–C) Liposomes were analyzed using DLS for ζ potential, electrophoretic mobility, and polydispersity. Liposomes (65% DSPC, 5% DSPC, 10%–0% SAPE, 0%–10% 15-HETE-PE) were analyzed using a Zetasizer Nano, as described in Methods (n = 3–5 separate liposome preparations, each analyzed as 4 replicates) and compared for differences against PC liposomes, using 1-way ANOVA with multicolumn comparison using Tukey. **P < 0.01. (D) Liposomes (65% DSPC, 5% DSPC, 30%–0% SAPE, 0%–0% 15-HETE-PE) were analyzed by nanoparticle tracking analysis to determine the mean diameter (n = 4, mean ± SEM). (E) X-ray diffraction was carried out at beamline I22 (Diamond Light Source). Samples in glass capillary tubes were held at 37°C ± 0.1°C, and images were integrated as described in Methods (n = 3, mean ± SEM). (F–H) SPR analysis of protein binding to nanodiscs demonstrates increased binding in the presence of HETE-PE. Control discs were composed of 15% SAPS, 30% SAPE, and 55% DSPC. Nanodiscs containing HETE-PE were composed of 15% SAPS, 20% SAPE, 55% DSPC, and 10% HETE-PE. (F) Representative SPR-derived binding isotherms for FX binding to HETE-PE and control nanodiscs. Discs used were of the same composition as described for A. (G) Representative SPR-derived binding isotherms for prothrombin binding to HETE-PE containing and control nanodiscs. Discs used were of the same composition as described A. (H) Molecules of protein bound per leaflet of nanodiscs in the presence or absence of HETE-PE. Summary data for experiments, n = 4 (FX), 2 (FII). (I–K) Positional isomer of HETE-PL influences potency of thrombin stimulation. Thrombin generation was initiated by addition of tissue factor containing liposomes to plasma as described in Methods. (I) liposomes contained 65% DSPC, 5% SAPS, and 30% SAPE, with 10% SAPE replaced with 10% HETE-PE. (J) Liposomes contained 55% DSPC, 5% SAPS, and 30% SAPE, with 10% SAPC replaced with 10% HETE-PC. (K) Summary data for maximum thrombin generation (n = 3, mean ± SEM). ***P < 0.005, **P < 0.01, *P < 0.05, as determined by 1-way ANOVA and post hoc Tukey tests.