In vitro studies of human platelets

3.3.1.

Isolation of washed human platelets

The isolation of human platelets was performed using multi-step centrifugation methods. The principle behind these methods is to separate blood cells based on their differential sedimentation velocity. Platelets, being smaller in size, settle down at high centrifugation speed as compared to other cells.

Method A

Buffer B 20 mM HEPES, 138 mM NaCl, 2.9 mM KCl, 1 mM

MgCl2.6H20, 0.36 mM NaH2PO4, and pH 6.2

Buffer C Composition same as buffer B except pH 7.4

Both buffers B and C were pre-warmed at 37°C before use.

Blood from healthy volunteers, who did not take any medication, was drawn by cubital venepuncture into a propylene syringe containing tri-sodium citrate (3.13% w/v) in a ratio 1:10 of blood (v/v). The anticoagulant-treated blood was immediately transferred into 50 ml polystyrene tubes and centrifuged at 160x g for 20 minutes (without brake) to separate the platelet rich plasma (PRP) from other blood cells. The top layer of PRP was aspirated carefully without disturbing the buffy coat and the red blood cells layer. The COX inhibitor (aspirin, 1 mM), and ADPase (apyrase, 0.3 U/ml) was added to the PRP and incubated at 37°C for 15 minutes to avoid platelet activation by TxA2 and ADP released during the isolation, respectively. Subsequently, in order to prevent clotting of the PRP induced by Ca2+ released calcium chelators like citric acid (citrate; 9 mM) and EDTA (5 mM) were added to the PRP. The platelets from PRP were isolated by centrifugation at 800x g for 20 minutes (with brake). The platelet pellet was then washed once with buffer B containing apyrase (0.3 U/ml). The volume of buffer B was determined by 10% of the original blood volume. Before the final centrifugation at 800x g for 20 minutes, 10 µl of platelet suspension was mixed with THROMBO PLUS for calculating the number of platelets/µl. The final platelet pellet was resuspended in a volume of buffer C required to obtain the final platelet count of 400,000/µl. Apyrase (0.6 U/ml) and glucose (5 mM) were added prior to the resuspension of platelet pellet.

Method B

ACD buffer 111 mM glucose, 85 mM tri-sodium citrate and 65 mM citric

acid

Tyrode buffer 10 mM HEPES, 137 mM NaCl, 2 mM KCl, 0.3 mM NaH2PO4,

12 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 0.35% (w/v) BSA and pH 7.6

Materials and methods 36

Working buffer Composition same as of Tyrode buffer, but with 0.1% BSA

All buffers were pre-warmed at 37°C before use.

Washed platelet suspension used to study platelet aggregation induced by LPA was prepared according to Cazenave et al (Cazenave JP 1993) and Haserück et al (Haseruck et al. 2004) with some modifications. Tyrode buffer as described by Cazenave et al. was used, except the omission of heparin from first wash buffer and the use of 0.1% albumin concentration in final suspension. The blood anti-coagulated with acid-citrate-dextrose (ACD; 1:6) was centrifuged at 160x g for 20 minutes to prepare PRP. Prostacyclin (PGI2, 25 nM) was added to PRP and incubated at 37°C for 20 minutes in order to prevent the platelet activation during subsequent centrifugation at 1100x g for 8 minutes. The platelets were resuspended in Tyrode buffer (one fifth of initial blood volume) containing PGI2 (1 µM) and incubated at 37°C for 10 minutes and then re-supplemented with PGI2 (1 µM). Subsequently, platelets were resuspended in Tyrode buffer containing PGI2 (1 µM) for a second time and incubated for 10 minutes at 37°C prior to the final centrifugation step (1100x g for 8 minutes). Meanwhile, 10 µl of the platelet suspension was used for calculating the concentration of platelets/µl, in a similar manner as described in method A. Finally, platelets were resuspended in working buffer, and the platelet concentration was adjusted to 400,000/µl. Apyrase (0.02 U/ml) was added to the suspension. The platelet suspension was stored at 37°C under an atmosphere of 5% CO2 and 95% humidity in a cell incubator, in order to avoid pH changes, during experiment.

3.3.2.

Platelet shape change and aggregation by turbidimetric method

This method is based on obstruction or augmentation of the light transmitted through a platelet suspension during activation. During shape change, the turbidity increases due to the discoid- spheroid transformation of discoid platelets and hence the light transmission decreases. During aggregation, an augmentation of the light transmission is observed due to the decrease in number of particulate matters, such as single platelets. Platelet shape change and aggregation were measured in a two-channel LABOR aggregometer. Aliquots of platelet suspension (400,000 platelets/µl) were incubated at 37°C for 5 minutes in translucent plastic cuvettes containing a magnetic stirrer. The platelet suspensions were stirred and further incubated at 37°C for 2 minutes prior to the addition of agonists, antagonists or inhibitors. The change in light transmission was recorded using an analog chart recorder. Unstimulated and stimulated platelet suspensions (100 µl) were transferred from the aggregometer cuvette to an eppendorf cups containing an equal volume of appropriate buffer for performing flow cytometry, biochemical or microscopic studies. During aggregation, the reactions were quenched at a given time point by adding an equal volume of appropriate buffer directly to the platelet suspension.

Materials and methods 37

3.3.3.

Measurement of ATP secretion

The ATP secretion during platelet activation was measured in a PICA® (Platelet Ionized Calcium Aggregometer) lumi-aggregometer that simultaneously monitors aggregation and secretion of ATP within the same platelet sample. This instrument uses the same turbidimetric principle implied for measuring platelet aggregation. In addition, secretion of ATP is measured by coupling ATP to the firefly luciferase system for obtaining luminescence. This luminescence is detected at the right angle to the aggregometer light path. The lumi-aggregometer was pre- adjusted for 37°C temperature, 1000 rpm stirring and for appropriate luminescence gain. Two platelet suspensions of varying concentrations were prepared from the initial platelet suspension (400,000 platelets/µl) for calibrating the instrument. Namely, the platelet rich suspension (PRS) by mixing 15 µl of buffer C to 385 µl of initial platelet suspension and the platelet poor suspension (PPS) by mixing 100 µl of buffer C to 300 µl of platelet suspension. The lumi- aggregometer was set for the base line correction using PRS and PPS. The ATP secreted from dense granules during platelet stimulation with various agonists was measured after addition of 15 µl of Chrono-Lumi luciferase/luciferin reagent. The emitted luminescence converted to electronic signals was recorded using an analog chart reader.