A Turbidimetric Analysis to Monitor Collagen-Induced Aggregation of Pretreated Human Platelets

Begin with glass cuvettes containing pre-treated, human platelet suspension containing non-activated platelets and fibrinogens — a plasma glycoprotein.

The cuvettes include a stirring device for uniform mixing.

Place the control cuvette in an aggregometer that measures the aggregation by assessing turbidity.

Introduce collagen — an agonist for platelet aggregation, interacting with platelet's surface receptors and activating downstream signaling cascades.

This results in ATP release via the Pannexin 1 channel — a platelet surface channel, initiating platelet activation and shape change, leading to a depression in the aggregation curve.

Fibrinogen further crosslinks activated platelets, promoting collagen-induced platelet aggregation, altering turbidity, and causing a gradual rise in the aggregation curve.

Treat the test cuvette with a high concentration of an inhibitor of the Pannexin 1 channel that blocks this channel.

Add collagen to the cuvette. Inhibitor interactions prevent ATP release and hinder collagen-induced platelet aggregation, evident from unchanged turbidity and a flat aggregation curve.

To prepare platelet-rich plasma, transfer 5-milliliter aliquots of the collected blood into 15-milliliter tubes, and centrifuge the samples at 250 times g and 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 2 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 5 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 milliliters solution. Pipette 260 microliters of the platelet suspension into glass cuvettes containing 10 microliters of the fibrinogen solution into 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 the readout for 6 minutes, and save the data when completed.