Purification and Activation of Platelets from Murine Whole Blood

Following vascular injury, platelets are recruited and activated at the injury site to form a platelet plug and initiate tissue repair.

To purify platelets from murine whole blood and activate them in vitro, take a suitable density gradient medium. Carefully layer the blood sample on top of the medium. Centrifuge to separate the platelets from the plasma and the red and white blood cell layers.

Transfer the platelets to two new tubes containing a suitable staining buffer. Add the synthetic GPRP tetrapeptide containing the glycine-proline-arginine-proline amino acid sequence to one tube, followed by thrombin.

Thrombin cleaves specific extracellular domains of the platelet-surface thrombin receptors, initiating a downstream signaling cascade and activating the platelets. The activated platelets' granules release CD62P protein, which rapidly translocates to the membrane surface.

The GPRP tetrapeptide blocks fibrinogen — a glycoprotein that binds to the membrane-spanning integrin CD41/CD61 on activated platelets to initiate platelet aggregation — thereby maintaining the activated platelets in suspension.

Add fluorescently-labeled anti-CD41 and anti-CD62P antibodies to the platelet-containing tubes.

The anti-CD41 antibodies bind to the CD41 ubiquitously expressed on resting and activated platelets. However, anti-CD62P specifically binds to the CD62P translocated to the membrane surface of activated platelets.

Analyze the cells using flow cytometry. Resting platelets appear as CD41+ cells, whereas activated platelets are characterized as CD41+ CD62P+ cells.

To purify the platelets, use a wide-bore pipette tip to layer 200 microliters of freshly-harvested whole blood, slowly down the side of a 1.5-milliliter tube onto 600 microliters of iohexol gradient medium without mixing.

After centrifugation in a swinging bucket rotor to isolate the platelets, use a new wide-bore pipette tip to collect most of the platelet-rich layer, and a small fraction of the platelet-poor layer without aspirating the white blood cell or red blood cell layer. Add the platelet sample to a new tube, and add 1 milliliter of PBS to the platelets.

Mix the platelets by inversion, before performing another centrifugation. Then, resuspend the pellet in 200 microliters of PBS with mild pipetting. For platelet activation, transfer 1 to 2 times 10 to the 6th platelets to a new tube containing 100 microliters of staining buffer. Add 1 to 2 times 10 to the 6th platelets to a different tube containing 100 microliters of staining buffer supplemented with 0.4 millimolar GPRP peptide.

Add thrombin to the tube with the GPRP peptide to activate the platelets, and add the antibody cocktail of interest to both tubes. As a positive control, add 1 microliter of whole blood in up to 100 microliters of staining buffer in a tube containing 0.4 millimolar GPRP peptide, and add thrombin to activate these platelets.