Preparation of Washed Human Platelets for Quantitative Metabolic Flux Studies

I studied the impact of acute estrogen exposure on platelet central carbon metabolism and resting and thrombin activated platelets. We are attempting to determine if acute estrogen exposure on platelets contributes to the increase in thrombotic risk imparted by oral contraceptive use. Women's health research is largely underfunded.

This project was part of a larger call from the NIH to address gaps in knowledge about women's health. Despite contraceptives being available for more than 50 years, we still don't know the exact mechanism that results in higher thrombotic risk. Biological variability.

Traditional cell culturing techniques don't apply to platelets, meaning platelets come from donors with different genotypes. Large sample sizes are needed, emphasizing the need for a repeatable platelet isolation procedure. Existing platelet washing protocols are written for experts in the field of platelet biology.

There are a number of unique challenges associated with working on platelets that my protocol highlights for the novice platelet researcher. To begin, preheat the water bath to 37 degrees Celsius and place the modified Tyrode's buffer containing glucose in it. Combine the whole blood collected from all vacutainers into a 50-milliliter polypropylene conical tube.

Using a bevel-cut pipette tip, take an appropriate sample volume to conduct a cell count. Carefully aspirate with a narrow bore transfer pipette to remove any bubbles generated. Then let the blood rest in a 37 degree Celsius water bath for 25 minutes.

To prevent activation during centrifugation, add prostaglandin I2 and apyrase to the whole blood and invert the tube once to mix the sample. Next centrifuge the whole blood at 250G for 15 minutes without a break at 37 degrees Celsius. Collect the PRP, which appears as the top yellow layer above the buffy coat and red blood layer into a new tilted 50-milliliter conical tube along the wall.

Allow the PRP to rest for 10 minutes at 37 degrees Celsius. After incubation, mix the PRP with 500 nanomolar prostaglandin I2 and 0.02 units per milliliter of apyrase. Centrifuge the mixture at 1, 000G for 10 minutes at 37 degrees Celsius with no acceleration and no break.

Now using a plastic wide bore transfer pipette, aspirate the supernatant for the bulk volume and then use a one milliliter pipette with an uncut tip to aspirate the remaining liquid near the pellet. Then add prostaglandin I2 and apyrase-containing Tyrode's buffer to the pellet, down the side of the conical tube. Using a bevel-cut tip and a one milliliter pipette tip, gently resuspend the pellet several times.

Once the pellet is resuspended, use a wide bore transfer pipette to transfer it to a new conical tube, leaving any red cells or visibly clumped cells behind. Then take a sample for flow cytometry using a bevel-cut pipette tip. Incubate the rest of the platelets for one hour at 37 degrees Celsius to enable inhibitors to wear off.

After incubation, gently mix the sample with a wide bore pipette and aliquot the samples for flow cytometry. The average platelet recovery from whole blood was around 52%with white blood cell contamination remaining under 0.1%after the wash process. P-selectin exposure was low throughout the wash process, but increased significantly after thrombin treatment.

Bound fibrinogen levels initially spiked after the first 1, 000G spin, but dropped below 5%after the second spin, increasing again significantly post thrombin treatment. Platelets were successfully gated for size, singlets, and CD42a positivity before measuring activation markers. Thrombin treatment led to a significant increase in P-selectin and fibrinogen expression compared to control.

To begin, obtain the washed platelets from human blood samples. After pre-cooling the microcentrifuge to zero degrees Celsius, collect the platelet suspension in partially frozen normal saline in a 1:6 ratio. Centrifuge the mixture at 16, 000G for 10 minutes at zero degrees Celsius.

Aspirate the supernatant into a separate microcentrifuge tube. Next, add 0.5 milliliters of prechilled methanol:water at minus 20 degrees Celsius to the quenched pellet and vortex vigorously for one minute. Freeze the sample in liquid nitrogen and thaw at zero degrees Celsius.

Centrifuge the suspension again at 16, 000G for 10 minutes at four degrees Celsius, and collect the supernatant in a new microcentrifuge tube. After drying the supernatant overnight, resuspend the dry extract in 150 microliters of LC/MS grade water and mix for 15 minutes at four degrees Celsius. Briefly vortex and transfer the extract to a 0.22-micrometer microcentrifuge tube to remove cell debris.

Centrifuge the sample for five minutes at 16, 000G and four degrees Celsius. Post-centrifugation, pipette an additional 50 microliters of water onto the filter and centrifuge again for five minutes at 16, 000G and four degrees Celsius. Finally, collect the filtrate for analysis.

Lactate production was positive in all donor samples while glucose and acetate were consistently consumed, with thrombin enhancing these fluxes.