title: In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow Cytometry.
abstract: Microvesicles (MVs) are submicron vesicles with sizes of 0.1-1.0 μm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxidized low-density lipoprotein (oxLDL), a platelet ligand, induces platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study, we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5 μm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5 μm limit. MV size distribution was analyzed in plasma from 11 healthy volunteers (four females and seven males). MVs were identified as <1 μm and positive for lactadherin binding and cell-specific markers. Platelet-rich plasma (PRP) was incubated without and with oxLDL or LDL (as control) to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41(+) and CD41(+)CD36(+) MVs increased by six to eightfold in PRP, when left at room temperature, and the presence of cell-specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large interindividual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that procoagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great interindividual variability in size distribution of platelet-derived MVs and thereby stress the importance for generation of standardized protocols for MV quantification by flow cytometry.
authors.affiliation: The Doctoral School in Medicine, Faculty of Medicine, Aalborg University , Aalborg , Denmark ; Department of Clinical Biochemistry, Aalborg University Hospital , Aalborg , Denmark.
authors.forename: Tine Bo
authors.initials: TB
authors.lastname: Nielsen
authors.affiliation: Department of Clinical Biochemistry, Aalborg University Hospital , Aalborg , Denmark.
authors.forename: Morten Hjuler
authors.initials: MH
authors.lastname: Nielsen
authors.affiliation: Department of Clinical Biochemistry, Aalborg University Hospital , Aalborg , Denmark ; Department of Clinical Medicine, Faculty of Medicine, Aalborg University , Aalborg , Denmark.
authors.forename: Aase
authors.initials: A
authors.lastname: Handberg
keywords: CD36; extracellular vesicles; flow cytometry; lactadherin; oxidized low-density lipoprotein; platelets