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

The overall goal of this procedure is to describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. This method can help answer key questions in the field of human platelet biology such as the effects of pannexin-1 inhibition on agonist-induced platelet aggregation. The main advantage of this technique is that it is rapid and easy to perform while it allows reliable and reproducible monitoring of aggregation responses.

Light transmission aggregometry is not only used for research purposes but it is also used for the investigation of platelet function in patients with bleeding disorders. To begin, dissolve 1.4 grams of citric acid monohydrate, 2.5 grams of trisodium citrate dihydrate, and two grams of anhydrous D-glucose in distilled water to prepare 100 milliliters of an ACD solution with a pH of approximately 4.5. Then prepare Tyrode's buffer by diluting 2.5 milliliters of stock solution one in a final volume of 15 milliliters of distilled water.

Adjust the pH to 7.35 by adding one normal hydrogen chloride and filter sterilize the solution using a 0.22 micron filter. Finally, add five milliliters of stock solution one, one milliliter of stock solution two, two milliliters of stock solution three, 0.5 milliliters of one molar heaps, 1.8 milliliters of 200 grams per liter human serum albumin, and 0.1 gram of anhydrous d-glucose to a final volume of 100 milliliters distilled water to prepare the TA buffer. Using one normal hydrogen chloride, adjust the pH to 7.35 and set the osmolarity to 295 milliosmoles per liter with distilled water.

Fill 50 milliliter tubes with one volume of ACD anticoagulant per anticipated six volumes of blood to be collected. Introduce a 19 gauge needle into an antecubital vein of a healthy donor. Discard the initial one to two milliliters of the withdrawn blood as it may contain thrombin and tissue factor and collect the following 45 to 50 milliliters.

Gently invert the tube to mix the blood with the ACD solution. Then incubate the sample in an oven at 37 degrees Celsius for 10 minutes. To prepare platelet-rich plasma, transfer five milliliter aliquots of the collected blood into 15 milliliter tubes and centrifuge the samples at 250 times g in 37 degrees Celsius for 13 minutes.

When centrifuged, aspirate the top layer of the samples, avoiding any red and white blood cell contamination. Then transfer the obtained platelet-rich plasma into a new 15 milliliter tube and incubate it at 37 degrees Celsius for 10 minutes. Centrifuge platelet-rich plasma at 2, 200 times g for 12 minutes.

Next, remove the supernatant and using a plastic Pasteur pipette, carefully resuspend the pellet in 10 milliliters of the TA buffer containing two microliters per milliliter of heparin and 2.5 microliters per milliliter of 25 micromolar prostacyclin in Tris-HCL buffer. Incubate the sample for 10 minutes at 37 degrees Celsius. Then add 2.5 microliters per milliliter of 25 micromolar prostacyclin and centrifuge the sample at 1, 900 times g for eight minutes.

After spinning, remove the supernatant and with a plastic Pasteur pipette, resuspend the pellet in five milliliters of the TA buffer containing 2.5 microliters per milliliter of 25 micromolar prostacyclin. Incubate the platelets at 37 degrees Celsius for 10 minutes one more time. While the sample is being incubated, transfer 150 microliters of the platelet suspension into a 1.5 milliliter tube and count the platelets using an automated cell counter.

After incubation, add 2.5 microliters per milliliter of 25 micromolar prostacyclin to the platelet suspension and immediately centrifuge the sample at 1, 900 times g for eight minutes. Remove the supernatant and resuspend the pellet to achieve a density of 250, 000 platelets per microliter using an adequate volume of the TA buffer containing apyrase at 0.32 units per milliliter. Before the aggregametric measurements, incubate the cell suspension at 37 degrees Celsius for at least 30 minutes.

To proceed with aggregometry, dissolve fibrinogen in Tyrode's buffer to obtain a 56 milligram per milliliter solution. Pipette 260 microliters of the platelet suspension into glass cuvettes containing 10 microliters of the fibrinogen solution and a magnetic stirring rod. Then incubate the suspensions for two to three minutes at 37 degrees Celsius in an incubation well within the aggregometer.

Next, add 10 microliters of a 2.8 millimolar brilliant blue FCF stock solution prepared in distilled water and pre-incubate the suspension at 37 degrees Celsius for seven minutes. To calibrate the aggregometer, prepare a cuvette containing a solution of 10 microliters fibrinogen, 10 microliters brilliant blue FCF solution, and the TA buffer without platelets that corresponds to 100%aggregation. Place the cuvette in an aggregometer channel with automatic stirring turned on.

Indicate the experimental channel in the software controlling the apparatus and measure the optical density of the solution. Then under constant stirring conditions, calibrate the aggregometer using a platelet sample with no agonist added corresponding to 0%aggregation. Place the cuvette in the aggregometer and indicate the experimental channel in the software controlling the apparatus.

Wait for about 20 to 30 seconds to ensure no aggregation occurs. Then add 20 microliters of the desired agonist into the cuvette. Immediately start recording aggregation data.

Proceed with a readout for six minutes and save the data when completed. Presented here are the aggregation curves obtained for collagen-stimulated platelets that were incubated with either water or different concentrations of brilliant blue FCF. The low brilliant blue FCF concentration did not influence the platelet response to collagen while brilliant blue FCF at one millimolar completely abolished the collagen-induced platelet aggregation.

Finally, the platelet inhibiting activity of brilliant blue FCF was shown to be specific towards platelets activated by collagen stimulation whereas it did not influence the aggregation of platelets activated with arachidonic acid as demonstrated by five independent measures taken in healthy volunteers. Once mastered, this technique can be done in four to six hours, depending on the number of tests, if it is performed properly. After watching this video, you should have a good understanding of how to perform turbidimetry-based aggregation tests on washed platelets isolated from human blood.

Don't forget that working with human blood can be hazardous and particular precautions such as wearing protective gloves should always be taken while performing this procedure.