Abstract
Thrombus formation is complex process involving both cellular and molecular (protein) components. Platelets are responsible for maintaining hemostasis and for preventing excessive bleeding. These cells aggregate along with other plasma components and blood cells to form blood clots. Undesirable platelet aggregation may lead to life-threatening conditions such as stroke. Thrombogenicity is the property of a material to induce the formation of a thrombus, which results in partial or complete occlusion of a blood vessel. The tendency to cause platelet aggregation and perturb plasma coagulation can serve as an in vitro measure of a nanomaterial’s likelihood to be thrombogenic in vivo. This chapter describes a procedure for in vitro analyses of platelet aggregation and plasma coagulation time. Platelet-rich plasma (PRP) is obtained from freshly derived human whole blood and incubated with nanoparticles. Then the plasma is examined using a particle count and size analyzer to determine the number of active platelets. The percent aggregation is calculated by comparing the number of active platelets in the nanoparticle-exposed sample to control plasma. To measure the plasma coagulation time, platelet-poor plasma from human whole blood is exposed to nanoparticles in vitro and analyzed in prothrombin (PT), activated partial thromboplastin (APTT), and thrombin time assays.
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Acknowledgments
This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
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Appendix: Instrument Settings, Times, and Volumes
Appendix: Instrument Settings, Times, and Volumes
Assay | Control | Settings | Volumes | Normal coagulation time (s) | ||||
|---|---|---|---|---|---|---|---|---|
Max time (s) | Incubation time (s) | Single/duplicate | Precision (%) | Plasma | Reagent | |||
PT (neoplastine) | CoagControlN+ABN | 60 | 120 | Duplicate | 5 | 100 μL Plasma | Neoplastine reagent: 100 μL (PIP Position 4) | ≤13.4 |
APTT | CoagControlN+ABN | 120 | 180 | Duplicate | 5 | 50 μL Plasma + 50 μL PTT-A Reagent | CaCl2: 50 μL (PIP Position 2) | ≤34.1 |
Thrombine | CoagControlN+ABN | 60 | 60 | Duplicate | 5 | 100 μL Plasma | Thrombine: 100 μL (PIP Position 4) | ≤21 |
Reptilase | System Control N+P | 60 | 120 | Duplicate | 5 | 50 μL Plasma + 50 μL Owren-Koller Reagent | Reptilase: 100 μL (PIP Position 4) | ≤20 |
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Neun, B.W., Dobrovolskaia, M.A. (2011). Method for In Vitro Analysis of Nanoparticle Thrombogenic Properties. In: McNeil, S. (eds) Characterization of Nanoparticles Intended for Drug Delivery. Methods in Molecular Biology, vol 697. Humana Press. https://doi.org/10.1007/978-1-60327-198-1_24
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DOI: https://doi.org/10.1007/978-1-60327-198-1_24
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