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

Summary

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.

Abstract

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-VASP^Ser239) 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.

Introduction

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 ....

Protocol

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.

1. Platelet-rich plasma (PRP) preparation
   1. Draw 30–50 mL of blood using a 20 G or larger diameter needle and add into a tube conta.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
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