Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Podsumowanie

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Streszczenie

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

Wprowadzenie

Platelets are crucial components of blood and their main function-together with coagulation factors-is to stop bleeding after blood vessel injury. Platelets are small (2-3 µm) anuclear fragments derived from megakaryocytes of the bone marrow¹. Platelets circulate in non-activated state, during which they appear as lens-shaped structures. Upon interruption of the endothelium, platelets gather to the site of blood vessel injury to plug the hole, a process called primary hemostasis. Initially, platelets attach to sub-endothelial molecules, such as collagen and von Willebrand factor, that are exposed as a result of the injury-adhesion step². Then, they change shape and secrete chemical messengers-activation step. Finally, they connect to each other by bridging receptors-aggregation step. Primary hemostasis is followed by a secondary process involving activation of the coagulation cascade with fibrin deposition, which stabilizes the initial thrombus².

Acute ischemic events such as myocardial infarction³ often result from thrombi that form because of physical disruption (rupture) of an atherosclerotic plaque. Current anti-platelet drugs are the cornerstone of the treatment of this widespread disease but their clinical benefit is limited by an increased risk for bleeding. The most prescribed drugs in cardiovascular patients, aspirin and anti-P2Y12 compounds, target the thromboxane A2 and the ADP pathways⁴, respectively, which are the major pathways leading to platelet activation. However, innovative research towards new targets that would optimally balance antithrombotic effects and haemorragic risk is still necessary.

From the 1960s⁵ to today, turbidimetric aggregometry has played a crucial role in research, enhancing our knowledge of platelet reactivity and in the monitoring of the potency of anti-thrombotic reagents in humans. Turbidimetry was initially applied to PRP extracted from blood samples. Indeed, blood collection performed in tubes containing citrate allows fast and large production of PRP without having any effect on platelet integrity and function. However, the short-term stability (about 3 h) of PRP and the remaining plasmatic enzymes, such as thrombin, and the low calcium concentration associated with potentially artefactual aggregation profiles are of major inconvenience for the use of PRP. An important step forward has been the development of a method for platelet isolation with additional centrifugation and washing steps⁶. In short, PRP is isolated from whole blood collected on acid-citrate-dextrose (ACD) and platelets are isolated after serial centrifugation steps before being resuspended in an iso-osmotic phosphate buffer (Tyrode's buffer) containing glucose, human serum albumin and divalent cations (Ca²⁺ and Mg²⁺). To avoid changes in platelet reactivity, the pH of Tyrode's buffer is carefully kept at 7.35-7.4. Moreover, undesired activation of platelets is prevented by adding prostacyclin (PGI₂) before some centrifugation steps. Finally, addition of apyrase prevents washed platelets from becoming resistant against the action of ATP/ADP. The resulting platelet suspension lacks coagulant factors and the stability of platelets is increased by at least two-fold as compared to PRP solutions. In addition, the fact that platelets are inactive but intact warrants the reproducibility of turbidimetric measurements and provides the ability to study the action of agonists or antagonists of platelet aggregation in an optimal way.

Using this method, we have shown in a recent study that inhibiting the formation of Panx1 channels by a genetic approach (knock-out mice) or decreasing Panx1 channel activity by pharmacological approaches reduced collagen-induced platelet aggregation⁷. Panx1 forms ATP-release channels, which are ubiquitously expressed in many cell types including human platelets^7,8. In fact, we demonstrated by turbidimetry on human washed platelets that a 7 min preincubation with a panel of more-or-less specific chemical blockers (probenecid, mefloquine and ¹⁰Panx1 peptides) prior to the addition of various agonists, inhibited specifically collagen-induced platelet aggregation while platelet responses to AA and ADP were not affected. We demonstrated that ATP release through Panx1 channels specifically interferes in the GPVI signaling pathway leading to collagen-induced aggregation. Interestingly, multiple FDA-approved compounds with applications in other diseases (probenecid, mefloquine) affect the activity of Panx1 channels in platelets. On one hand, this opens new therapeutic perspectives to selectively modify platelet reactivity. On the other hand, one should consider potential secondary effects of these compounds. In this context, the safe food dye Brilliant Blue FCF used in multiple candies and energy drinks has been described as a selective inhibitor of Panx1⁹. We describe here a protocol for the isolation of human washed platelets and turbidimetric measurements of platelet aggregation adapted to investigate the effect of the Brilliant Blue FCF dye as an antagonist of platelet aggregation.

Protokół

Five unrelated healthy volunteers were recruited for blood sampling for platelet isolation and aggregation tests. Written informed consent was obtained and the protocol was approved by the Central Ethics Committee of the Geneva University Hospitals. All volunteers certified to be healthy and to have not taken any platelet-interfering drugs during at least the 10 days preceding the experiments.

1. Buffer Preparation for Human Blood Collection and Washed Platelet Isolation

1. Prepare a 100 mL aqueous solution of acid-citrate-dextrose (ACD) by dissolving 1.4 g citric acid monohydrate (C₆H₈O₇•H₂O, 66.6 mM), 2.5 g trisodium citrate dihydrate (Na₃C₆H₅O₇•2 H₂O, 85 mM) and 2 g of anhydrous D(+)-glucose. The pH of the solution is about 4.5.
2. Prepare stock solutions for Tyrode's buffer as follows
   1. Prepare stock solution 1 by dissolving 80 g NaCl, 2 g KCl, 10 g NaHCO₃ and 0.58 g NaH₂PO₄*H₂O in 500 mL of distilled H₂O. The respective final concentrations are 2.73 M, 53.6 mM, 238 mM and 8.4 mM. Keep the solution at 4 °C.
   2. Prepare stock solution 2 by dissolving 10.15 g MgCl₂*6 H₂O (100 mM) in 500 mL distilled H₂O. Keep solution at 4 °C.
   3. Prepare stock solution 3 by dissolving 10.95 g (100 mM) CaCl₂*6 H₂O in 500 mL distilled H₂O. Keep solution at 4 °C.
3. Prepare Tyrode's buffer by diluting 2.5 mL of stock solution 1 in a final volume of 50 mL with distilled H₂O. This corresponds to final concentrations of 136.5 mM NaCl, 2.68 mM KCl, 11.9 mM NaHCO₃ and 0.42 mM NaH₂PO₄*H₂O. Adjust the pH to 7.35 and sterilize by filtering with 0.22-μm filters.
4. Prepare Tyrode's albumin 0.35% buffer (TA buffer⁷) by diluting 5 mL of stock solution 1, 1 mL of stock solution 2, 2 mL of stock solution 3, 0.5 mL 1M HEPES, 1.8 mL of 200 g/L human serum albumin and 0.1 g of anhydrous D(+)-glucose in a final volume of 100 mL distilled H₂O.
   1. Adjust the pH to 7.35 with 1N HCl and set the osmolarity to 295 mOsm/L by adding distilled H₂O (10% of total volume). Final concentrations in this solution are: 124 mM NaCl, 2.44 mM KCl, 10.82 mM NaHCO₃, 0.38 mM NaH₂PO₄*H₂O, 0.91 mM MgCl₂*6 H₂O, 1.82 mM CaCl₂*6 H₂O. Keep TA buffer at 37 °C during the whole experiment.

2. Blood Collection

1. Collect 45-50 mL of venous blood, from the antecubital vein using a 19 G needle and no or low tourniquet, into 50 mL tubes containing ACD anticoagulant (1 volume ACD for 6 volumes of blood). Discard the first 1-2 mL of blood to avoid the presence of thrombin and tissue factor.
   1. After collection, mix the blood with the ACD by gently inverting the tube. Incubate the sample for 10 min at 37 °C.

3. Preparation of Human Washed Platelets

1. Pre-heat the centrifuge to 37 °C. All centrifugation steps below are performed at this temperature.
2. Dispatch the collected blood into 15 mL tubes (5 mL per tube) and centrifuge at 250 x g for 13 min to obtain PRP.
   NOTE: This centrifugation step results in the production of three layers in the sample: 1) The upper layer, composed of plasma, platelets, and a small fraction of white blood cells. 2) The intermediate layer, a portion rich in white blood cells. 3) The bottom layer, which is essentially composed of red blood cells.
3. Collect the PRP by pipetting the upper layer carefully into a new 15 mL tube to maximally prevent contamination with red and white blood cells, and incubate for 10 min at 37 °C.
4. Centrifuge the PRP at 2,200 x g for 12 min (for 5 mL PRP).
   NOTE: This centrifugation step should be performed with low brake or without brake.
5. Remove the supernatant (platelet-poor plasma), and carefully resuspend the pellet with 10 mL of TA buffer containing 2 µL/mL of heparin (5,000 U/mL) and 2.5 µL/mL of 25 μM PGI₂ using a plastic Pasteur pipet. Incubate for 10 min at 37 °C.
6. Add 2.5 µL/mL of 25 μM PGI₂ and centrifuge for 8 min at 1,900 x g (with low brake or without brake).
7. Remove the supernatant and resuspend the pellet with 5 mL TA buffer containing 2.5 µL/mL of 25 μM PGI₂ using a plastic Pasteur pipet. Incubate for 10 min at 37 °C.
8. During the incubation period, pipet 150 µL of the platelet suspension into a 1.5 mL tube and count platelets, using an automatized cell counter (that detects the size of blood cells by measuring the changes in direct-current resistance).
9. After the 10 min incubation, add 2.5 µL/mL of 25 μM PGI₂ to the platelet suspension and immediately centrifuge at 1,900 x g for 8 min.
10. Remove the supernatant and resuspend the pellet to a concentration of 250,000 platelets/µL with an adequate volume of TA buffer (i.e. if the cell count is 500,000 per µL, resuspend in 10 mL TA buffer) containing 32 µL/mL of apyrase at 0.01 U/mL (final concentration 0.32 U/mL).
    NOTE: High concentration of apyrase is used to avoid the desensitization of P2X1 receptors induced by spontaneous secretion of ATP^10,11 in absence of agonists. This is important because collagen-induced responses are induced by fast paracrine/autocrine activation of P2X1 by ATP released from activated platelets. If the platelet signaling pathway does not critically require preservation of P2X1 function, use 0.02 U/mL apyrase. Several studies (reviewed in Mahaut-Smith et al.¹⁰) demonstrated that 0.02 U/mL apyrase avoids ADP receptor P2Y1 desensitization with negligible P2X1 responses.
11. Incubate the cell suspension for at least 30 min at 37 °C before performing the aggregometric measurements. The preparation is stable for 5 to 8 h.

4. Aggregometry

1. Prepare fibrinogen (56 mg/mL) in Tyrode's buffer.
2. Pipet 260 µL of platelet suspension into glass cuvettes (Figure 1A; left cuvette) containing 10 µL of fibrinogen (56 mg/mL) and a magnetic stirring rod, then incubate the suspension for 2 - 3 min at 37 °C in incubation wells present in the aggregometer (Figure 1B and 1C).
3. Pre-incubate with the Panx1 inhibitor Brilliant Blue FCF by adding 10 µL of a 2.8 mM or 28 mM stock solution (final concentration 100 μM and 1 mM, respectively) for 7 min at 37 °C.
4. Calibrate the aggregometer to an assumptive 100% aggregation value by measuring the OD of a cuvette containing 10 µL fibrinogen (56 mg/mL), 10 µL Brilliant Blue FCF (2.8 mM or 28 mM) and TA buffer without platelets.
   1. Place the cuvette in an aggregation well under automatic stirring and press the corresponding button on the keyboard of the computer linked to the aggregometer (i.e. press F1 if aggregation well 1 is used).
      NOTE: This experiment described below includes Brilliant Blue FCF. The compound used for calibration has to be adjusted to the experimental condition.
5. Calibrate the aggregometer to an assumptive 0% aggregation value by using the same platelet sample that will be used for the experiment under automatic stirring.
   1. Place the cuvette in the aggregation well and press the corresponding button on the keyboard of the computer linked to the aggregometer. Wait for about 20-30 s before proceeding. This delay serves to assure that no aggregation happens before adding the agonists.
      NOTE: As any difference in platelet number may have an effect on the measured OD, the 0% calibration step needs to be repeated for each individual measurement.
6. Add 20 µL of desired agonist, such as 15 µg/mL collagen (1 µg/mL final) or 1.125 mM arachidonic acid (75 µM final), into the cuvette. Immediately start the recording under continuous automatic stirring by pressing the corresponding button on the keyboard of the computer linked to the aggregometer.
   NOTE: The addition of the agonist induces platelet activation. Platelet aggregates can clearly be distinguished in the glass cuvette at the end of the experiment (Figure 1A; right cuvette).
7. The recording automatically stops after 6 min. At this point, save the data by clicking on the save icon of the computer.
   NOTE: The calculation of the rate of aggregation is performed by the computer, which expresses the end results of the aggregation process as a percentage.
8. Analyze the data.
   1. For additional extensive information on protocols for the preparation of washed platelets suspensions and turbidimetric measurement of platelet aggregation, refer to other papers authored by experts in the field^12,13.

Wyniki

The aggregometer software automatically produces the aggregation curves and gives the values for maximal aggregation in percentage. The values can be copied to a data analysis software in order to perform statistical analysis and visualize maximal aggregation values in form of bar charts. Optionally, each individual point of the aggregation curves can be exported successively into a spreadsheet software and then to statistical software (e.g. GraphPad) in order to visualize the cu...

Dyskusje

There is great interest in finding new drugs capable of modulating platelet function in order to prevent thrombosis without enhancing the risk of bleeding. For this purpose, in vitro laboratory tests which can reliably and reproducibly monitor aggregation responses in human platelets are absolutely necessary. Turbidimetric aggregometry is an easy technique to perform. However, some precautions need to be kept in mind. The measurements need to be performed under continuous stirring as the aggregation process is l...

Materiały

Name	Company	Catalog Number	Comments
citric acid monohydrate (C6H8O7*H2O)	Roth	5949-29-1	danger of eye damage/irritation
trisodium citrate dihydrate (Na3C6H5O7*2H2O)	Sigma-Aldrich	S1804	-
D(+)-glucose	Sigma-Aldrich	G8270	-
Sodium chloride (NaCl)	Sigma-Aldrich	S9888	-
Potassium chloride (KCl)	Sigma-Aldrich	P9541	-
Sodium bicarbonate (NaHCO3)	Sigma-Aldrich	S6014	-
Sodium dihydrogenophosphate monohydrate (NaH2PO4*H2O)	Sigma-Aldrich	S9638	-
Magnesium chloride hexahydrate (MgCl2*6H2O)	Sigma-Aldrich	M9272	-
Calcium chloride hexahydrate (CaCl2*6H2O)	Sigma-Aldrich	442909	danger of eye damage/irritation
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (Hepes)	ThermoFisher Scientific	15630	-
human serum albumin	CSL Behring	00257/374	-
hydrochloric acid	Sigma-Aldrich	320331	Corrosive and irritative for the respiratory system. Can cause severe skin and eye damages.
Eppendorf 5810 R	Fisher Scientific	-	-
heparin	Drosspharm AG/SA	20810
prostacyclin I2 (PGI2)	Cayman	18220	-
apyrase from potatoes	Sigma-Aldrich	A6535	-
fibrinogen (Haemocomplettan)	CSL Behring	HS 73466011	-
thrombo-aggragometer SD-Medical	SD-Innovation	TA8V	-
Brilliant blue FCF (Erioglaucine disodium salt)	Sigma-Aldrich	80717	Harmful to aquatic life with long lasting effects (Avoid release to the environnement)
collagen	Horm, Nycomed		-
arachidonic acid	Bio/Data corporation	C/N 101297	-
cell counter Sysmex KX-21N	Sysmex Digitana	-	-
HEPES	Gibco	15630-056	-
glass cuvettes	SD-Innovation	THCV1000	-
magnetic stirrers	SD-Innovation	THA100	-