Abstract
Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.
Highlights
IntroductionTissue factor (TF) has been established as a pivotal element in activation of the clotting cascade. It is well described that TF expressed subendothelially in blood vessels comes into contact with blood upon tissue injury and, in combination with activated FVII, initiates the coagulation process. TF is produced in monocytes and may be expressed in platelets and activated endothelial cells. In plasma, TF circulates both in its full-length form incorporated in the membranes of circulating cell–derived extracellular vesicles (EVs) and in an alternatively spliced soluble form. The role of blood-borne TF in the coagulation process is not clear, but TF-bearing EVs (TFþ EVs) have been suggested to play a role in hemostasis and thrombus formation, and TF activity and TFþ EVs quantitated by flow cytometry (FC) have been shown to be increased in various thrombogenic conditions.1 consensus on the nomenclature of different types of EVs has not been achieved, smaller sized EVs, typically with a diameter of less than 150 nm, may be termed exosomes, whereas larger EVs are often referred to as microvesicles (MVs). While these two types of EVs are formed and released from cells by different mechanisms, indications exist that both types possess the ability to carry TF in their phospholipid bilayer membrane
While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins
Flow Cytometry flow cytometry (FC) results on the fasting and postprandial samples are shown in ►Fig. 1 and ►Fig. 2
Summary
Tissue factor (TF) has been established as a pivotal element in activation of the clotting cascade. It is well described that TF expressed subendothelially in blood vessels comes into contact with blood upon tissue injury and, in combination with activated FVII, initiates the coagulation process. TF is produced in monocytes and may be expressed in platelets and activated endothelial cells. In plasma, TF circulates both in its full-length form incorporated in the membranes of circulating cell–derived extracellular vesicles (EVs) and in an alternatively spliced soluble form. The role of blood-borne TF in the coagulation process is not clear, but TF-bearing EVs (TFþ EVs) have been suggested to play a role in hemostasis and thrombus formation, and TF activity and TFþ EVs quantitated by flow cytometry (FC) have been shown to be increased in various thrombogenic conditions.1 consensus on the nomenclature of different types of EVs has not been achieved, smaller sized EVs, typically with a diameter of less than 150 nm, may be termed exosomes, whereas larger EVs are often referred to as microvesicles (MVs). While these two types of EVs are formed and released from cells by different mechanisms, indications exist that both types possess the ability to carry TF in their phospholipid bilayer membrane.. It is well described that TF expressed subendothelially in blood vessels comes into contact with blood upon tissue injury and, in combination with activated FVII, initiates the coagulation process.. Consensus on the nomenclature of different types of EVs has not been achieved, smaller sized EVs, typically with a diameter of less than 150 nm, may be termed exosomes, whereas larger EVs are often referred to as microvesicles (MVs).. Consensus on the nomenclature of different types of EVs has not been achieved, smaller sized EVs, typically with a diameter of less than 150 nm, may be termed exosomes, whereas larger EVs are often referred to as microvesicles (MVs).9 While these two types of EVs are formed and released from cells by different mechanisms, indications exist that both types possess the ability to carry TF in their phospholipid bilayer membrane.
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