Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies
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
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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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 2

HETE-PLs enhance thrombin generation in the absence of FIX or FXI, or in the absence of tissue factor pathway inhibitor (TFPI) and FVIII combined.

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HETE-PLs enhance thrombin generation in the absence of FIX or FXI, or in...
(A and B) Thrombin generation was initiated by addition of liposomes to plasma deficient in FIX, as described in Methods. Liposomes were generated as described in Methods, and 10% 1-stearoyl-2-arachidonyl-PE (SAPE) was replaced with 10% 15-HETE-PE where indicated. (C and D) Thrombin generation was initiated by addition of liposomes to plasma deficient in FXI, as described in Methods. Liposomes were generated as described in Methods, and 10% SAPE was replaced with 10% 15-HETE-PE where indicated. (A–D) n = 3, mean ± SEM, *P < 0.05. A representative trace is shown for each. (E–H) Thrombin generation was initiated by direct addition of liposomes to plasma as described in Methods, at 10% HETE-PE, and measured in the absence (E and G) or presence (F and H) of 100 nmol/l anti-TFPI antibody preincubated for 15 minutes, in either normal (E and F) of FVIII-deficient plasma (G and H). (I–L) Thrombin generation was initiated by direct addition of liposomes to plasma as described in Methods, at 10% HETE-PC, and measured in the absence (I and K) or presence (J and L) of 80 nmol/l anti-TFPI antibody preincubated for 15 minutes, in either normal (I and J) of FVIII-deficient plasma (K and L). Data is shown as Tukey box plots, with 1-way ANOVA with Tukey post hoc multicomparison test. *P < 0.05.