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 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.
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 – 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.
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.
2. Deoxygenation
3. Red Blood Cell Nitrite Reduction
4. Western Blotting of VASP
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) (Figure 2A-B). Increased hemolysis is not associated with stirring because stirring without deoxygenation does not induce hemolysis (Figure 2C).
We found that the presence of RBCs in lysed samples decreases P-VASPSer239 and VASP detected by Western blotting (Figure 3). Therefore, we first separate platelets and RBCs before running Western blots to detect P-VASPSer239 in platelets.
To show that there is enough time to separate RBCs out of platelets without affecting VASP phosphorylation, we used a short-lived NO donor. PROLI NONOate (NO donor with half-life of 1.8 s at 37 C pH 7.4), rapidly increased P-VASPSer239 in platelets (within 10 s of incubation). P-VASPSer239 induced by PROLI NONOate is detected for 10 min after the incubation with PRP (Figure 4).
Nitrite increases P-VASPSer239 in platelets in the presence of deoxygenated RBCs (pO2 25 mmHg). Nitrite reductase activity of deoxygenated RBCs depends on hematocrit and the maximum effect is observed at 20% hematocrit (Figure 5).
Figure 1: Deoxygenation chamber. Mixture of PRP and RBCs is added into a plastic bottle closed with a rubber septum. The closed bottle is connected to helium (He) line using a needle. Oxygen is flushed out by a small syringe needle (26 G) serving as a gas outlet.
Figure 2: Partial oxygen pressure (pO2) and cell-free hemoglobin after deoxygenation. (A) PRP and RBCs samples were deoxygenated for 3, 5, 10, 15, 30 and 50 min. pO2 of samples were measured by blood gas analyzer (n = 3). Error bars are SEM. Representative pictures of supernatant of PRP+RBCs samples stirred with (B) or without helium (C). Please click here to view a larger version of this figure.
Figure 3: Representative VASP Western blot bands in samples of PRP/RBCs prepared at various hematocrit. RBCs were added into PRP to 1, 5, 10, 15, 20, 40% hematocrit. Mixtures of PRP + RBCs were centrifuged at 500 x g for 5 min and lysed with lysis buffer.
Figure 4: Representative Western blot bands of P-VASPSer239 and VASP in PRP after exposure to NO donor PROLI NONOate. PROLI NONOate (10 M) was added into PRP and incubated at 37 °C for 10 and 30 s and 1, 2, 5, 10, 20 and 40 min.
Figure 5: VASPSer239 phosphorylation depends on pO2 and hematocrit levels. (A) PRP + RBCs (20% hematocrit) were deoxygenated to two different pO2 levels, 40 and 25 mmHg. P-VASPSer239 and VASP were measured after the incubation of deoxygenated samples with 10 M nitrite at 37 C for 10 min. (B) Nitrite (10 M) was added into deoxygenated PRP + RBCs at 0, 10, 20 and 40% hematocrit (pO2 = 25 mmHg). Fold increased (P-VASPSer239) was calculated relative to control (PRP+RBCs without nitrite) for each hematocrit value. Error bars = SEM. Please click here to view a larger version of this figure.
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 span, up to 2 h. To achieve high reproducibility, all experiments should be done only with fresh platelets and within 2 h after blood collection. The presented data were obtained with platelets in PRP, which is considered more physiological than washed platelets. Although the life span and purity increase for washed platelets, repeated centrifugation during preparation of washed platelets can lead to their spontaneous activation and this may result in inconsistent quality of platelet preparations. PRP is preferred over washed platelets since the procedure is faster and further handling could activate platelets. However, in specific cases, when the interference of plasma blood clotting factors could interfere with experimental setup, washing platelets represents solution to avoid these problems.
Oxygen contamination in deoxygenated samples is the most important critical step for this assay. RBCs can be pooled and deoxygenated before being added to the PRP. However, transferring deoxygenated RBCs increases the risk of oxygen contamination. In addition, deoxygenation of concentrated RBCs requires more time than deoxygenation of the full preparation at the desired hematocrit, and longer exposure to He gas leads to increased levels of cell-free hemoglobin. Although cell-free hemoglobin can be washed out of deoxygenated RBCs, the phosphate buffer saline or other buffers used for washing must be carefully deoxygenated to prevent oxygen contamination during the centrifugation. To shorten the preparation process and avoid unnecessary oxygen contamination, each PRP+RBCs sample was prepared at the desired hematocrit first and then deoxygenated before the addition of nitrite.
To quantify P-VASPSer239 by Western blot, RBCs must be separated out of the platelets. VASP is expressed in RBCs13, but the levels are nonsignificant when compared to hemoglobin and much lower than in platelets. Since total protein concentration is used for loading controls in the Western blots, the presence of RBCs in lysate samples interferes with P-VASP and VASP measurement by Western blotting. Due to a slow rate of dephosphorylation by phosphatase enzymes in platelets14, P-VASP is sustained for 10 min (Figure 4) and therefore it is possible to separate RBCs by centrifugation before collecting platelet cell lysates.
The EC50 for VASP phosphorylation is almost an order of magnitude lower than the EC50 for the peak cGMP increase, 0.5 nM and 9 nM, respectively3, which makes VASP phosphorylation one of the most sensitive methods to detect the presence of low amounts of NO. However, the VASP phosphorylation curve in response to NO is bell-shaped; a phosphorylation decrease is observed after peaking at 3-30 nM of NO3. This nonlinear response to increasing NO concentrations makes VASP unsuitable for rigorous NO quantification.
Since platelets in PRP have a short life span, the assay must be done in fresh platelets within 2 h after drawing blood. The inconsistency of pO2 levels after the deoxygenation can be found since the deoxygenation of samples is dependent on the flow rate of helium gas and rate of swirling the bottle. Therefore, the time for deoxygenation needs to be determined by trial. For the measurement of P-VASP by Western blots, the antibody choice is critical, and one needs to use a direct NO donor as a positive control to check the specificity. In addition to NO, P-VASPSer239 in platelets can be phosphorylated by the inhibition of phosphodiesterease enzymes and the activation of protein kinase A pathway.
The measurement of P-VASPSer239 has some advantages over cGMP assay. cGMP in platelets has a very short half-life (less than 10 s) and the measurement of cGMP requires the addition of phosphodiesterase inhibitor3. Also, the cGMP measurement uses a rather expensive ELISA technique. VASP can be simply measured by an in-house Western blot protocol and therefore is more readily available.
Increased P-VASPSer239 in platelets was successfully used as a sensor for NO generation by inhaled nitrite in vivo6. Therefore, we showed that it is possible to use P-VASPSer239 in platelets as a marker of NO generation in vitro and in vivo in the blood and possibly in various other situations. The unusually low EC50 makes this method an excellent candidate for detecting the presence of very low NO concentrations in blood. Therefore, this protocol provides a possibility to study NO generation in blood at various physiological stimuli, such as during blood coagulation cascade, increased sheer stress or increased inflammation.
The authors have nothing to disclose.
This work was funded by NIH intramural grant to Dr Alan N. Schechter.
Tri-sodium citrate | Supply by NIH blood bank | ||
Citric acid | Supply by NIH blood bank | ||
Glucose | Sigma | G7528-250G | |
NaCl; sodium chloride | Sigma | S-7653 1kg | |
NaH2PO4; sodium phosphate monobasic, monohydrate | Mallinckrodt Chemical | 7892-04 | |
KCl; potassium chloride | Mallinckrodt Chemical | 6858 | |
NaHCO3; sodium bicarbonate | Mallinckrodt Chemical | 7412-12 | |
HEPES; N-[2-Hydroxyethyl]piperazine-N'-[-ethanesulfonic acid] | Sigma | H3375-500g | |
MgCl2 (1 M); magnesium chloride | Quality Biology | 351-033-721 | |
CaCl2; calcium chloride | Sigma | C5080-500G | |
Nalgene Narrow-mouth HDPE Economy bottles | Nalgene | 2089-0001 | |
Red septum stopper NO.29 | Fisherbrand | FB57877 | |
NaNO2–; sodium nitrite | Sigma | S2252-500G | |
TRIZMA Base; Tris[hydroxymethyl]aminomethane | Sigma | T8524-250G | |
NP-40; 4-Nonylphenyl-polyethylene glycol | Sigma | 74385-1L | |
Protease inhibitor cocktail set III | Calbiochem | 539134 | |
Phospho-VASP (Ser239) antibody | Cell signaling technology | 3114 | |
VASP antibody | Cell signaling technology | 3112 | |
GAPDH (14C10) Rabbit mAb | Cell signaling technology | 2118 | |
2-mercaptoethanol | Sigma | M-6250-10ml | |
Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson Immuno Research Laboratories | 111-035-003 | |
Clarity Western ECL Substrate | BIO-RAD | 1705060-200ml | |
CO-oximeter (ABL 90 flex) | Radiometer |