Name: platelet-based detection of nitric oxide in blood by measuring vasp phosphorylation
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation at JoVE.com

This work was funded by NIH intramural grant to Dr Alan N. Schechter.

NOTE: Blood samples were obtained from NIH blood bank (IRB approved protocol: 99-CC-0168).
1. Blood Sample Preparation
NOTE: To avoid platelet activation, draw blood slowly and mix gently with citrate by inverting the tube several times.
<ol>
	<li>Platelet-rich plasma (PRP) preparation
	<ol>
		<li>Draw 30&#8211;50 mL of blood using a 20 G or larger diameter needle and add into a tube conta...

<ol>
	<li>Huang, K. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+between+nitrite+and+deoxyhemoglobin.+Reassessment+of+reaction+kinetics+and+stoichiometry.">The reaction between nitrite and deoxyhemoglobin. Reassessment of reaction kinetics and stoichiometry.</a> Journal of Biological Chemistry. 280 (35), 31126-31131 (2005).</li><li>Cosby, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrite+reduction+to+nitric+oxide+by+deoxyhemoglobin+vasodilates+the+human+circulation.">Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.</a> Nature Medicine. 9 (12), 1498-1505 (2003).</li><li>Mo, E., Amin, H., Bianco, I. H., Garthwaite, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+a+cellular+nitric+oxide/cGMP/phosphodiesterase-5+pathway.">Kinetics of a cellular nitric oxide/cGMP/phosphodiesterase-5 pathway.</a> Journal of Biological Chemistry. 279 (5), 26149-26158 (2004).</li><li>Park, J. W., Piknova, B., Nghiem, K., Lozier, J. N., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+effect+of+nitrite+on+coagulation+processes+demonstrated+by+thromboelastography.">Inhibitory effect of nitrite on coagulation processes demonstrated by thromboelastography.</a> Nitric oxide. 40, 45-51 (2014).</li><li>Wajih, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+red+blood+cell+S-nitrosation+in+nitrite+bioactivation+and+its+modulation+by+leucine+and+glycose.">The role of red blood cell S-nitrosation in nitrite bioactivation and its modulation by leucine and glycose.</a> Redox Biology. 8, 415-421 (2016).</li><li>Akrawinthawong, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+flow+cytometric+analysis+of+the+inhibition+of+platelet+reactivity+due+to+nitrite+reduction+by+deoxygenated+erythrocytes.">A flow cytometric analysis of the inhibition of platelet reactivity due to nitrite reduction by deoxygenated erythrocytes.</a> PLoS One. 9 (3), e92435 (2014).</li><li>Friebe, A., Koesling, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+nitric+oxide-sensitive+guanylyl+cyclase.">Regulation of nitric oxide-sensitive guanylyl cyclase.</a> Circulation Research. 93 (2), 96-105 (2003).</li><li>Srihirun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+inhibition+by+nitrite+is+dependent+on+erythrocytes+and+deoxygenation.">Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.</a> PLoS One. 7 (1), e30380 (2012).</li><li>Smolenski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+regulation+of+vasodilator-stimulated+phosphoprotein+serine+239+phosphorylation+in+vitro+and+in+intact+cells+using+a+phosphospecific+monoclonal+antibody.">Analysis and regulation of vasodilator-stimulated phosphoprotein serine 239 phosphorylation in vitro and in intact cells using a phosphospecific monoclonal antibody.</a> Journal of Biological Chemistry. 273 (32), 20029-20035 (1998).</li><li>Burkhart, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+comprehensive+and+quantitative+analysis+of+human+platelet+protein+composition+allows+the+comparative+analysis+of+structural+and+functional+pathways.">The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways.</a> Blood. 120 (15), e73-e82 (2012).</li><li>Parakaw, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+inhibition+and+increased+phosphorylated+vasodilator-stimulated+phosphoprotein+following+sodium+nitrite+inhalation.">Platelet inhibition and increased phosphorylated vasodilator-stimulated phosphoprotein following sodium nitrite inhalation.</a> Nitric oxide. 66, 10-16 (2017).</li><li>Srihirun, S., Piknova, B., Sibmooh, N., Schechter, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+vasodilator-stimulated+phosphoprotein+(P-VASPSer239)+in+platelets+is+increased+by+nitrite+and+partially+deoxygenated+erythrocytes.">Phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets is increased by nitrite and partially deoxygenated erythrocytes.</a> PLoS One. 13 (3), e0193747 (2018).</li><li>Mal Cortese-Krott, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+soluble+guanylate+cyclase+in+RBCs:+preserved+activity+in+patients+with+coronary+artery+disease.">Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease.</a> Redox Biology. 14, 328-337 (2018).</li><li>Abel, K., Mieskes, G., Walter, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+the+focal+adhesion+protein+VASP+in+vitro+and+in+intact+human+platelets.">Dephosphorylation of the focal adhesion protein VASP in vitro and in intact human platelets.</a> FEBS letter. 370 (3), 184-188 (1995).</li></ol>

Since platelets are easily activated, gentle handling of platelet-containing samples is required. Fast pipetting and vigorous shaking should be avoided. Platelet inhibitors such as prostacyclin (PGI2) can be used to prevent platelet activation; however, this may affect some signaling pathways inside the platelets. For the preparation of platelet pellets, we add ACD to the platelet suspensions and use low speed centrifugation.
Freshly prepared platelets in PRP have a limited life spa...

Platelets are small disc-shaped cell fragments derived from megakaryocytes that are crucial for blood clotting. The clotting cascade is initiated by various bioactive molecules (such as collagen or ADP), released after the injury of vascular wall. The blood clotting process can be modified, among various effectors by nitric oxide (NO). NO, naturally produced by mammalian cells, is one of the most versatile physiological signals. It acts as a potent vasodilator, neurotransmitter and immune modulator, to name a few of its many functions. In the bloodstream, NO also helps to regulate the extent of blood clotting by inhibiting platelet aggregation. One of the most likely sources of NO in the bloodstream is nitrite, an inorganic ion that has been shown to serve as a precursor of NO. Reacting with red blood cells (RBCs), nitrite is reduced to NO and deoxyHb is oxidized to methemoglobin (metHb)1. NO released from RBCs is vasoactive and causes vasorelaxation2. This nitrite reduction pathway is an alternate NO generation pathway, acting together with and complementing the classical NO generation path by endothelial nitric oxide synthase at hypoxic conditions.
Platelets themselves are not able to reduce nitrite into NO but are very sensitive to its presence. In intact platelets, NO in the nanomolar range increases cGMP (EC50 = 10 nM) and phosphorylation of VASP (EC50 = 0.5 nM)3. Therefore, platelets may serve as an excellent sensor of nitrite reduction by RBCs and NO release into blood. There are several methods that can directly measure the extent of platelet activation - such as aggregometry and thromboelastography (TEG)4,5. However, these methods require expensive specialized instrumentation and rather large amounts of material. It is also possible to monitor events downstream, after NO is released from RBCs, using the changes in platelet surface protein expression &#8211; such as P-selectin6. NO is also known to increase the amount of cGMP in the platelets7. Previously, we used cGMP to monitor NO release into blood after nitrite reduction by deoxygenated RBC8. This proved to be a very sensitive method; however, cGMP is a short-lived molecule and its detection involves extensive labor. Another possibility, described in the presented protocol, uses phosphorylation of the vasodilator-stimulated phospho (VASP)-protein to detect the presence of NO in blood. VASP is a substrate of protein kinase G activation, which is phosphorylated upon the interaction with NO through the sGC/cGMP pathway9. Detectable VASP phosphorylation occurs at very low NO concentrations, which could make platelets a very sensitive detector of NO presence in blood. VASP is highly expressed in platelets, but not in other blood cells, which allows to follow selectively the events involving platelets10.
The main goal of this protocol is to describe the method in detail for the detection of NO release in whole blood using its interaction with platelets by monitoring VASP phosphorylation11,12. The described method allows early detection of low NO concentrations - theoretically in the nanomolar range which makes the present protocol more sensitive than cGMP determination, due to the use of standard Western blot techniques achievable in most laboratory settings.

<table>
	<tbody>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>Supply by NIH blood bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Supply by NIH blood bank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7528-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl; sodium chloride</td>
			<td>Sigma</td>
			<td>S-7653 1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4; sodium phosphate monobasic, monohydrate</td>
			<td>Mallinckrodt Chemical</td>
			<td>7892-04</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl; potassium chloride</td>
			<td>Mallinckrodt Chemical</td>
			<td>6858</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3; sodium bicarbonate</td>
			<td>Mallinckrodt Chemical</td>
			<td>7412-12</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES; N-[2-Hydroxyethyl]piperazine-N&#39;-[-ethanesulfonic acid]</td>
			<td>Sigma</td>
			<td>H3375-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2 (1 M); magnesium chloride</td>
			<td>Quality Biology</td>
			<td>351-033-721</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2; calcium chloride</td>
			<td>Sigma</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Narrow-mouth HDPE Economy bottles</td>
			<td>Nalgene</td>
			<td>2089-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Red septum stopper NO.29</td>
			<td>Fisherbrand</td>
			<td>FB57877</td>
			<td></td>
		</tr>
		<tr>
			<td>NaNO2-; sodium nitrite</td>
			<td>Sigma</td>
			<td>S2252-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIZMA Base; Tris[hydroxymethyl]aminomethane</td>
			<td>Sigma</td>
			<td>T8524-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>NP-40; 4-Nonylphenyl-polyethylene glycol</td>
			<td>Sigma</td>
			<td>74385-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail set III</td>
			<td>Calbiochem</td>
			<td>539134</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-VASP (Ser239) antibody</td>
			<td>Cell signaling technology</td>
			<td>3114</td>
			<td></td>
		</tr>
		<tr>
			<td>VASP antibody</td>
			<td>Cell signaling technology</td>
			<td>3112</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH (14C10) Rabbit mAb</td>
			<td>Cell signaling technology</td>
			<td>2118</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M-6250-10ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson Immuno Research Laboratories</td>
			<td>111-035-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Clarity Western ECL Substrate</td>
			<td>BIO-RAD</td>
			<td>1705060-200ml</td>
			<td></td>
		</tr>
		<tr>
			<td>CO-oximeter (ABL 90 flex)</td>
			<td>Radiometer</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

platelet-based detection of nitric oxide in blood by measuring vasp phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Venous blood samples have pO2 values between 50-80 mmHg. Deoxygenation by helium rapidly decreases pO2 to 25 mmHg within 10 min. Increased deoxygenation time slightly further decreases pO2. However, increased time of deoxygenation also leads to significantly increased levels of cell-free hemoglobin (determined by CO-Oximeter, visually seen on Figure 2 as increasingly red coloration of plasma) (Figu...

Watch this Scientific Journal Video about Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation at JoVE.com

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

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

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.

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