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Updated: Feb 24, 2026
Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets
Published on: November 29, 2024
Microvesicles in stored human whole blood increase thrombin generation via phosphatidylserine
Lindsey J Wattley1, Ryan Chae, Rebecca H Schuster
1From the Department of Surgery (L.J.W., R.C., R.H.S., M.D.G., T.A.P.), University of Cincinnati, Cincinnati, Ohio.
Introduction:
Microvesicles are submicron blebs shed from aging cells during storage. Some microvesicles have been shown to be procoagulant; however, the influence of microvesicles on coagulability of stored whole blood has not been elucidated. Additionally, the mechanism of microvesicle influence on coagulation in human blood is unknown. We hypothesized that microvesicles produced during storage express phosphatidylserine on their outer leaflet, which causes an increase in thrombin generation.
Methods:
Whole blood was collected into citrate phosphate double dextrose from 14 healthy donors (6 male, 8 female) aged 20 to 30 years and stored for 21 days. Baseline and end of storage hemostasis was measured with rotational thromboelastometry (Non-Activated ROTEM [NATEM] and Extrinsically Activated ROTEM [EXTEM]). Thrombin generation was measured after stimulation with tissue factor (TF) and phospholipids as well as TF alone. Microvesicles were measured using flow cytometry on day 1 and day 21 of storage. Microvesicle effects were determined by adding microvesicles to platelet-poor plasma (PPP) with and without lactadherin and assaying for changes in thrombin generation.
Results:
Whole blood did not demonstrate differences in NATEM after storage. However, on EXTEM analysis, whole blood demonstrated decreased coagulability. When PPP was stimulated with TF and phospholipids, or TF alone, thrombin generation increased with storage duration. Microvesicle concentrations also increased with storage duration. When microvesicles were added to PPP and then stimulated with TF and phospholipids or TF alone, thrombin generation was increased, and this effect was mitigated by blocking phosphatidylserine on microvesicles with lactadherin.
Conclusion:
Whole blood storage results in increased thrombin generation potential over time. This has previously been viewed as beneficial but could be detrimental when unchecked. These data suggest that changes in thrombin generation are due in part to phosphatidylserine expression on microvesicles produced during storage. These data highlight the need for further studies investigating the use of stored whole blood.
Insights
Stored whole blood shows increased thrombin generation potential due to microvesicles expressing phosphatidylserine. This finding is crucial for understanding blood storage and potential clinical applications.
Area of Science:
- Hematology
- Biochemistry
- Cell Biology
Background:
- Microvesicles, shed from aging cells, are implicated in blood coagulation.
- The precise mechanism by which microvesicles influence coagulation in stored human blood remains unclear.
- This study investigates the role of microvesicles in the coagulability of stored whole blood.
Purpose of the Study:
- To elucidate the influence of microvesicles on the coagulability of stored whole blood.
- To determine the mechanism behind microvesicle-mediated changes in coagulation.
- To test the hypothesis that microvesicles produced during storage express phosphatidylserine, increasing thrombin generation.
Main Methods:
- Whole blood from healthy donors was stored for 21 days.
- Hemostasis was assessed using rotational thromboelastometry (ROTEM).
- Thrombin generation, microvesicle concentration, and microvesicle effects on platelet-poor plasma were measured via flow cytometry and tissue factor assays.
Main Results:
- Stored whole blood showed decreased coagulability on EXTEM analysis.
- Thrombin generation and microvesicle concentration increased with storage duration.
- Microvesicles significantly increased thrombin generation in platelet-poor plasma, an effect reduced by blocking phosphatidylserine.
Conclusions:
- Whole blood storage leads to increased thrombin generation potential.
- Phosphatidylserine expression on microvesicles contributes to altered thrombin generation during storage.
- Further research is needed to investigate the clinical implications of these findings for stored whole blood utilization.
