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.

Tissue factor at the crossroad of coagulation and cell signaling

“…Phosphatidyl ethanolamine (PE) is modified by the 12/15 lipoxygenase Alox15 expressed by eosinophils and Alox15 knock‐out mice display reduced thrombosis. This and other studies provide evidence that immune cell‐derived enzymatically oxidized HETE phospholipids play broader roles in prothrombotic and hemostatic reactions .…”

Enzymatically oxidized phospholipids assume center stage as essential regulators of innate immunity and cell death

“…Several studies support an essential role for eoxPLs in promoting PS-dependent coagulation in vitro and in vivo (46,49,50). Specifically, HETE-PE or HETE-PCs enhance the ability of PS to interact with multiple clotting factors, increasing the rates of thrombin generation and ultimately promoting hemostasis (Fig.…”

“…To test the involvement of lipids in disease, Alox-deficient mice are a suitable model; however, these enzymes generate many oxylipins, making it challenging to ascribe functions to specific lipids. To that end, we have administered HETE-PLs to mice, either locally into the tail or systemically through the tail vein and found them to be well tolerated, with acute effects on hemostasis and thrombosis in line with their proposed procoagulant actions (46,49).…”

Platelet lipidome: Dismantling the “Trojan horse” in the bloodstream

“…They exhibit less thrombin generation and increased bleeding risk. 12‐HETE‐PLs restore thrombin generation in postoperative plasma in vitro exemplifying its clinical benefits …”

“…12-HETE-PLs restore thrombin generation in postoperative plasma in vitro exemplifying its clinical benefits. 72 DHA-derived 12-LOX metabolite 14-HDOHE can also be esterified to PE to produce diacyl and plasmalogen-PEs. 73 Additionally, COX-1 generated PGE2 and PGD2 are promptly PE-esterified following activation with thrombin, collagen, or ionophore, in a calcium-dependent manner.…”