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 6

Altered generation and action of HETE-PLs in a clinical setting of hemostatic failure associated with cardiopulmonary bypass surgery.

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Altered generation and action of HETE-PLs in a clinical setting of hemos...
(A–E) Platelets after CPB generate less HETE-PE and externalize less HETE-PE and PS than those isolated before CPB. Platelets isolated from peripheral blood of patients, before (shaded) and after (unshaded) CPB were measured for HETE-PE, HETE-PC, and externalized PE, PS, and HETE-PE basally and following thrombin or collagen activation, as described in Methods (n = 12). (F–H) Stimulation of thrombin generation in preoperative plasma by 12-HETE-PL was significantly reduced in patients who received hemostatic treatment after CPB. Plasma from patients before CPB was added to 50 pmol/l TF liposomes with/without 10% HETE-PL, and thrombin was measured as described in Methods. ETP (F), peak thrombin (G), and velocity index (H) were determined and compared with responses to liposomes that contained native PL, to give fold changes for each. (I) Reduced thrombin generation seen in post-CPB plasma was restored using HETE-PL. Thrombin generation was compared in plasma before or after CPB using liposomes containing HETE-PL, as indicated (10%). For A–I, horizontal black lines represent median, boxes represent the interquartile range, bars represent values falling within 1.5 times the interquartile range, and asterisks/circles represent outliers (values 1.5–3 times lower or higher than the interquartile range). n = 87 for panels F–I. Data were analyzed using Wilcoxon rank test for paired variables, Mann Whitney U test for unpaired variables and Friedman’s 2-way analysis of variance with post hoc testing to compare repeated measures of related variables, **P < 0.01, *P < 0.05.