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Published on: November 29, 2024
In vitro Incubation of Platelets with oxLDL Does Not Induce Microvesicle Release When Measured by Sensitive Flow
Tine Bo Nielsen1, Morten Hjuler Nielsen2, Aase Handberg3
1The Doctoral School in Medicine, Faculty of Medicine, Aalborg University , Aalborg , Denmark ; Department of Clinical Biochemistry, Aalborg University Hospital , Aalborg , Denmark.
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.
Insights
Oxidized low-density lipoprotein (oxLDL) binding to platelets does not increase microvesicle (MV) release, despite inducing platelet activation. Significant interindividual variation exists in platelet-derived MV size distribution, highlighting the need for standardized flow cytometry protocols.
Area of Science:
- Cardiovascular Biology
- Hematology
- Cell Biology
Background:
- Microvesicles (MVs) are released from cells, implicated in various diseases.
- Platelets are major MV secretors; oxidized low-density lipoprotein (oxLDL) activates platelets via CD36.
- The role of oxLDL-induced platelet activation in MV release is unclear.
Purpose of the Study:
- To investigate the effect of oxLDL on in vitro platelet microvesicle (MV) release.
- To compare MV quantification using different size gating strategies in flow cytometry.
- To analyze MV size distribution and interindividual variability in healthy volunteers.
Main Methods:
- Platelet-rich plasma (PRP) from healthy volunteers was incubated with oxLDL or LDL (control).
- MV release and size distribution were analyzed using flow cytometry with size-calibrated beads.
- Platelet activation markers and MV-specific markers (lactadherin, CD41, CD36) were assessed.
Main Results:
- Platelet activation markers increased, but total MV count remained unaffected after oxLDL incubation.
- No significant increase in MV release was observed with oxLDL compared to LDL control.
- A substantial interindividual variation (73%) in the size distribution of platelet-derived MVs was found.
Conclusions:
- Platelet activation induced by oxLDL binding to CD36 may not involve microvesicle (MV) release.
- Significant interindividual variability in platelet-derived MV size distribution necessitates standardized quantification protocols.
- Flow cytometry gating strategies impact MV size analysis, emphasizing the need for precise methodology.
