Targeted metabolomics identifies a metabolic switch to mitochondrial respiration in antiphospholipid syndrome that drives platelet procoagulant activity Free

Introduction Antiphospholipid syndrome (APS), characterized by persistent circulating antiphospholipid antibodies (aPL) is associated with increased risk of thrombosis. Platelets have been shown to be activated or their responses to agonist were potentiated by aPL as indicated by integrin activation, P-selectin expression and platelet aggregation. Surprisingly, studies in APS have so far not assessed platelet procoagulant activity. Although we and others have demonstrated a skewing of energy metabolism towards glycolysis and pentose phosphate pathway (PPP) in agonist-stimulated platelets, a definitive metabolic phenotyping is yet to be undertaken for procoagulant platelets, which are an activated platelet subset with low integrin activity and high phosphatidylserine (PS) exposure providing a surface for assembly of tenase and prothrombinase complexes. We explored the changes in energy metabolism that might underlie the procoagulant platelet phenotype in APS using a targeted metabolomics approach.

Methods Venous blood was drawn from 9 patients with APS and 6 healthy controls. Washed human platelets were prepared by differential centrifugation. We performed metabolomic profiling (MetwareBio Inc.) of the isolated platelets through a targeted approach looking at 69 metabolites related to pathways of energy metabolism including glycolysis, PPP, TCA cycle, amino acid catabolism, as well as nucleotides such as ATP and NADPH. The differential levels analysis, pathway analysis, and PCA were performed using Metaboanalyst. Another fraction of washed platelets either remained unstimulated or were treated with thrombin, labelled with Alexa-Fluor488-anti-C4d antibody, PE-anti-CD62P antibody, PE-Annexin V, Mitotracker Red, Rhod2-AM for 30 min in the dark, and then analyzed by flow cytometry. In another set of experiments (n=3-6), washed platelets from healthy donors were supplemented with calcium and incubated with sera (10%) from APS patients or healthy donors for 1 or 2h in the presence of PE-Annexin V, APC-anti-CD62P and Alexa-Fluor488-anti-C5b-9 antibodies for simultaneous analysis of procoagulant platelets and complement binding. Platelets were then fixed and analyzed by flow cytometry. On certain occasions platelets were pre-incubated with antimycin (complex III inhibitor) (2 µg/ml) or oligomycin (ATP synthase inhibitor) (10 µg/ml) before serum treatment.

Results We found that unstimulated platelets from 6/9 APS patients exhibited higher PS exposure and P-selectin expression than healthy individuals suggesting induction of procoagulant activity. While platelet PS exposure in APS was associated with increased mitochondrial calcium it was not accompanied by mitochondrial depolarization and was strongly correlated with platelet surface complement C4d deposition. Targeted metabolomics revealed a significant downregulation (<0.5 fold) of 16 metabolites including pyruvate (fold-change=0.102, p=0.05), xylulose-5-phosphate (FC=0.369, p=0.067), ribulose-5-phosphate (FC=0.387, p=0.08) in platelets from APS patients with procoagulant platelets (n=6) compared to those from healthy controls (n=6). Pathway analysis revealed PPP (Holm p=0.1, impact score=0.55) glycolysis (Hp=0.1, IS=0.53) and TCA cycle (Hp=0.1, IS=0.28), to be the principal pathways that were altered in APS compared to controls. Metabolic profile of APS patients without procoagulant platelets (n=3) were intermediate of those with procoagulant platelets and healthy controls. Incubating healthy donor platelets with sera from APS patients reproduces procoagulant platelet phenotype with complement deposition characteristic of APS patients. Platelet procoagulant activity induced by APS serum was significantly inhibited by pre-incubating platelets with either antimycin or oligomycin that force glucose metabolism through glycolysis and PPP away from mitochondrial metabolism.

Conclusion We unravel a metabolic switch towards mitochondrial metabolism from glycolysis/PPP in platelets from APS patients using a targeted metabolomics approach. We also establish its relevance to thrombotic APS by showing that forcing metabolic flux in platelets back towards glycolysis/PPP using mitochondrial inhibitors abolishes APS-associated procoagulant activity. These findings improve our understanding of metabolic changes underlying platelet procoagulant activity in APS and potentially also in other contexts.