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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.

SUBMITTER: Slatter DA

PROVIDER: S-EPMC5926910 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies.

Slatter David A DA Percy Charles L CL Allen-Redpath Keith K Gajsiewicz Joshua M JM Brooks Nick J NJ Clayton Aled A Tyrrell Victoria J VJ Rosas Marcela M Lauder Sarah N SN Watson Andrew A Dul Maria M Garcia-Diaz Yoel Y Aldrovandi Maceler M Heurich Meike M Hall Judith J Morrissey James H JH Lacroix-Desmazes Sebastien S Delignat Sandrine S Jenkins P Vincent PV Collins Peter W PW O'Donnell Valerie B VB

JCI insight 20180322 6

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 p ...[more]

PMID: 29563336

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Project description:BackgroundFactor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS.ObjectiveTo investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG).MethodsIn plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers.ResultsHigher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p < 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients.ConclusionIn MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression.

Project description:Phosphoproteomic analysis of platelets activated by pro-thrombotic oxidized phospholipids and thrombin Alejandro Zimman, Bjoern Titz, Evangelia Komisopoulou, Sudipta Biswas, Thomas G. Graeber, and Eugene A. Podrez ABSTRACT Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have now employed mass spectrometry-based tyrosine, serine, and threonine global phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide a new resource to study platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCy2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple novel insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36. Methods Human platelets isolated by gel filtration (2.7 x 108 / mL) were incubated in Tyrode's buffer with 50 uM KODA-PC or PLPC (as control) for 30 min at 37 degrees C. Platelets were then centrifuged at 3,700 g (10 min, 35 degrees C) and lysed in 8 M urea, 50 mM Tris-HCl (pH 7.4), 1 mM Na3VO4, and 1 mM NaF with sonication. Phosphoproteome changes induced by the agonists were assessed based on trypsin digestion of the protein lysate, phosphopeptide enrichment, mass spectrometry analysis, chromatography alignment, and label-free quantitation as described in the Supplemental Materials and Methods of the publication. The same protocol was applied when studying platelet activation by thrombin, where gel-filtered platelets were incubated in Tyrode's buffer with 0.05 U/mL thrombin or in buffer alone (Resting) for 3 min at 37 degrees C.

Project description:Development of inhibitory antibodies to coagulation factor VIII (fVIII) is the primary obstacle to the treatment of hemophilia A in the developed world. This adverse reaction occurs in 20-30% of persons with severe hemophilia A treated with fVIII-replacement products and is characterized by the development of a humoral and neutralizing immune response to fVIII. Patients with inhibitory anti-fVIII antibodies are treated with bypassing agents including recombinant factor VIIa (rfVIIa). However, some patients display poor hemostatic response to bypass therapy and improved treatment options are needed. Recently, we demonstrated that fVIII inhibitors display widely variable kinetics of inhibition that correlate with their respective target epitopes. Thus, it was hypothesized that for antibodies that display slow rates of inhibition, supplementation of rfVIIa with fVIII would result in improved thrombin generation and be predictive of clinical responses to this novel treatment regimen. In order to test this hypothesis, 10 murine monoclonal antibodies (MAbs) with non-overlapping epitopes spanning fVIII, differential inhibition titers, and inhibition kinetics were studied using a thrombin generation assay. Of the 3 MAbs with high inhibitory titers, only the one with fast and complete (classically defined as "type I") kinetics displayed significant inhibition of thrombin generation with no improvement upon supplementation of rfVIIa with fVIII. The other two MAbs that displayed incomplete (classically defined as "type II") inhibition did not suppress the potentiation of thrombin generation by fVIII. All antibodies that did not completely inhibit fVIII activity demonstrated potentiation of thrombin generation by the addition of fVIII as compared to rfVIIa alone. In conclusion, fVIII alone or in combination with rfVIIa corrects the thrombin generation defect produced by the majority of anti-fVIII MAbs better than single agent rfVIIa. Therefore, combined fVIII/rfVIIa therapy may provide better hemostatic control than current therapy in some patients with anti-fVIII inhibitors.

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Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies.

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Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies.

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