Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

This protocol addresses the potential use of platelets as highly sensitive nitric oxide sensor both in vitro and in vivo within the blood. Increased Phosphorylated Vasodilator-Stimulated Phosphoprotein or P-VASP serine 239 in platelets nitric oxide has some advantages, including an ability to detect the presence of nitric oxide in nanomolar lineages. Within two hours of collecting 30 to 50 milliliters of whole blood from a healthy donor, add five milliliter aliquots of the blood into the appropriate number of 15 milliliter conical tubes for centrifugation.

Use a plastic transfer pipette to carefully collect the platelet-rich plasma from the upper portion of the supernatants. Pellet the plasma-rich platelets with a second centrifugation and gently wash the pellets in five milliliters of CGS buffer. Centrifuge at 400 g for 10 minutes at room temperature.

At the end of the centrifugation, resuspend the pellets in three milliliters of modified Tyrode buffer. Use a hemocytometer for counting and adjust the density of the platelet suspensions to three times 10 to the eighth cells per milliliter in fresh Tyrode buffer. Then incubate the platelet suspensions for one hour at 37 degrees Celsius.

Meanwhile, collect the red blood cells from the plate-free plasma layers of the whole blood samples by centrifugation followed by four washes in five milliliters of PBS per wash. After the last wash, discard the supernatants and mix the red blood cells with one milliliter of the plasma-rich platelets at the appropriate experimental hematocrit in polypropylene bottles closed with rubber stoppers. Insert a needle connected to a helium gas tank into each septa and insert 26 gauge needles to make gas outlets.

Then slowly swirl the bottles at room temperature and use an oximeter to measure the partial oxygen pressure for monitoring of the deoxygenation process. The rate of oxygen decrease is dependent on the helium flow rate and the stirring speed. Therefore, the time of the oxygenation should be optimized before beginning an experiment as this is highly dependent on the geometry of the experimental setup.

For red blood cell nitrite reduction, use a microsyringe to inject nitrite through the septum into the deoxygenated samples of PRP and RBCs so that a 10 micromolar final concentration is achieved. Incubate the samples for at least 10 minutes at 37 degrees Celsius and transfer one milliliter of each sample into a microcentrifuge tube for centrifugation. Transfer 300 microliters of platelet suspension from the upper portion of the supernatants into new microcentrifuge tubes and add acid citrate dextrose to each tube at a one to nine ratio.

Centrifuge the platelets again and resuspend the pellets in 80 microliters of ice cold lysis buffer containing protease inhibitor cocktail three to each tube. Next, load 15 micrograms of protein from each sample to two separate 10%SDS-PAGE gels. Run the gels at 120 volts for 1.3 hours.

At the end of the run, transfer the gels to individual nitrous cellulose membranes and block any nonspecific binding with an appropriate volume of 5%nonfat dry milk. Incubate the membranes with the appropriate primary antibodies overnight at four degrees Celsius followed by labeling with an appropriate secondary horse radish peroxidase antibody for 45 minutes at room temperature. Then expose the membranes with an imager to quantify the band density with an appropriate image analysis program.

Healthy venous blood samples have partial oxygen pressure values between 50 to 80 millimeters of mercury. Deoxgenation by helium rapidly decreases the partial pressure of oxygen to 25 millimeters of mercury within 10 minutes. Further decreasing of partial oxygen pressure can be achieved with an increased deoxygenation time.

Increased deoxygenation leads to increased levels of hemolysis as indicated by increasingly red color plasma. This is an effect of deoxygenation and is not caused by stirring itself as indicated by the lack of hemolysis in samples stirred without helium. Both Vasodilator-Stimulated Phosphoprotein or VASP and Phosphorylated-VASP expression levels in platelets decreased with increasing hematocrit in deoxygenated samples as detected by western blot.

Treatment with a short-lived nitric oxide donor increases phosphorylated vasodilator-stimulated phosphoprotein expression in platelets within 10 seconds of treatment. Further, levels of Phosphorylated-VASP or P-VASP remained stable up to 10 minutes after incubation of the NO donor with platelet-rich plasma indicating that there is sufficient time for separating RBCs from platelets without affecting existing P-VASP levels. Nitrite also increases platelet's P-VASP levels in the presence of deoxygenated RBCs.

Here the ratio of P-VASP to total VASP depends on the level of hematocrit. VASP is highly expressed in platelet and it is rapidly phosphorylated in the presence of nitric oxide. Therefore, this protocol provides a method for studying the induction of nitric oxide by various physiological stimuli.

With this method, we have successfully used increased P-VASP in platelet as a marker of nitric oxide stimulation by inhaled nitrite in vivo. Don't forget that working with platelet with a biological specimen should always be performed using the appropriate personal protection equipment.