Abstract
Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs.
Highlights
Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL)
Annexin A5 is commonly used in chromogenic factor Xa (FXa) assays, where the activity measured is based on the EVs exposing PS that are bound to the microplate
We thoroughly validated a modified and easy to use PPL assay for the measurement of procoagulant phospholipids in test specimens and compared its performance with the STA-Procoag-PPL assay
Summary
Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs. Procoagulant phospholipid (PPL) activity has regained interest in recent years, mainly due to the increased understanding of the role of extracellular vesicles (EVs) in thrombosis and h aemostasis[1,2]. Connor and colleagues demonstrated that the amount of annexin A5 positive EVs, measured by flow cytometry, showed a significant and inverse correlation with clotting time[4] These findings suggest that EVs play a significant role in coagulation, apparently due to exposure of phospholipids, and phosphatidylserine (PS) in particular, on their surface. We aimed to develop a modified PPL-dependent clotting assay, capable of measuring the PPL activity in human plasma and cell supernatants of in vitro experiments, by removing PPL from plasma by sequential centrifugation, including final ultracentrifugation. The performance of the modified assay was validated against the commercially available Stago STA-Procoag-PPL assay
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
