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Platelet metabolism is of interest, particularly as it relates to the role of platelet hyper- and hypoactivity in bleeding and thrombotic disorders. Isolating platelets from plasma is necessary for some metabolic assays; presented here is a method for isolating of intracellular metabolites from washed platelets.
Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%–40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.
Platelets are small (2–4 µm diameter), anucleate cells that play an important role in hemostasis, the tightly regulated process of clot formation1. While vital for vascular integrity, platelets are also implicated in adverse health events. Platelets are involved in deep vein thrombosis (DVT) and arterial thrombosis (AT), which are clots that occlude blood vessels, leading to diminished blood supply locally, or, if pieces of the clot break off (embolize), they can block blood supply to the lungs, heart, or brain2,3,4,
The study received Institutional Review Board approval from the University of Colorado Anschutz Medical Campus. Written consent was obtained from all study participants. Participants reported they did not consume alcohol for the previous 48 h or non-steroidal anti-inflammatory drugs (NSAIDs) for the previous ten days. This project is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under award number R61HL141794.
1. Blood collection.......
Representative results in Figure 2 represent 6 different blood donors, including 3 males and 3 females. The platelet yield relative to whole blood is shown in Figure 2A. Final platelet recovery was an average of 52% ± 3% (standard deviation, n=6). Final platelet count compared to white blood cell contamination was measured using an automated hematology analyzer. White blood cell counts were less than 0.1% of total cells (Figure 2B
Platelets are very sensitive to their environment, including shear stress and presence of agonists38,39. This makes platelets challenging to handle and isolate, making the use of inhibitors and wide bore pipettes crucial40. Proper storage and preparation of PGI2 is vital, as failing to prepare PGI2 in basic PBS will result in rapid degradation of PGI241. To minimize the risk of shear.......
The authors would like to acknowledge Dr. Emily Janus-Bell and Clarisse Mouriaux from the lab of Dr. Pierre Mangin and Katrina Bark from Dr. Jorge DiPaola’s lab for their guidance and advice.
....Name | Company | Catalog Number | Comments |
0.22 µM filter Spin-X tubes | Millapore-Sigma | CLS8160 | Reagent prep |
19 G x 3/4" needle | McKesson Corporation | 448406 | Phlebotomy |
21 G 1.5 inch needle with luer lock | Amazon | B0C39PJD23 | Reagent prep |
96 well plate, half area | Greiner Bio-One | 675101 | Flow cytometry |
ACD-A vaccutainers | Fisher Scientific | 364606 | Phlebotomy |
Adapter | McKesson Corporation | 609 | Phlebotomy |
Alcohol swab | VWR | 15648-916 | Phlebotomy |
Apyrase from potatoes | Sigma | A6410-100UN | Reagent prep |
CD42a Monoclonal Antibody | Thermo Fisher Scientific | 48-0428-42 | Flow cytometry |
Chilled microcentrifuge | ThermoFisher Scientific | 75002441 | Quenching |
D-Glucose | Sigma | G7021 | Reagent prep |
FITC Anti-Fibrinogen antibody | Abcam | 4217 | Flow cytometry |
Flow cytometer | Beckman Coulter | 82922828 | Flow cytometry |
Gauze | VWR | 76049-110 | Phlebotomy |
Glycerol | Sigma Aldrich | G5516 | Reagent prep |
HEPES | Sigma Aldrich | H4034 | Reagent prep |
Human alpha-thrombin | Prolytix | HCT-0020 | Flow cytometry |
KCl | Sigma Aldrich | P9333 | Reagent prep |
KH2PO4 | Sigma Aldrich | P5655 | Reagent prep |
MgCl2 | Sigma | M8266 | Reagent prep |
Microcentrifuge tubes | VWR | 87003-292 | General |
Na2HPO4 | Sigma | S3264 | Reagent prep |
NaCl | Sigma Aldrich | S7653 | Reagent prep |
NaHCO3 | Sigma Aldrich | S5761 | Reagent prep |
Narrow bore transfer pipette | VWR | 16001-176 | Platelet washing |
Paraformaldehyde solution, 4% in PBS | Santa Cruz Biotechnology | sc-281692 | Flow cytometry |
PECy5 Mouse Anti-Human CD62P | BD Pharmingen | 551142 | Flow cytometry |
Plate cover | Thermo Fisher Scientific | AB0626 | Flow cytometry |
Polypropylene 15 mL conical tubes | VWR | 89039-658 | Reagent prep |
Polypropylene 50 mL conical tubes | VWR | 352070 | Platelet washing |
Prostaglandin I2 (sodium salt) | Cayman Chemical | 18220 | Reagent prep |
SKC Inc. C-Chip Disposable Hemocytometers | Fisher Scientific | 22-600-100 | Cell counting |
Syringe | BD Pharmingen | 14-823-41 | Reagent prep |
Tourniquet | VWR | 76235-371 | Phlebotomy |
Vacutainer needle holder | BD | 364815 | Phlebotomy |
Vortexer | VWR | 102091-234 | Reagent prep |
Water bath | Thermo Fisher Scientific | TSGP02 | Platelet washing |
Wide bore transfer pipette | VWR | 76285-362 | Platelet washing |
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