Linear relationship between platelet aggregation and thromboxane A2 production

Paul C.J. Armstrong, Zetty N.M. Zain, Nicola J. Pearson, Ivana Vojnovic, David Bishop-Bailey, Jane A. Mitchell & Timothy D. Warner. The William Harvey Research Institute, Barts & The London, QMUL, EC1M 6BQ, U.K.

There has long been discussion concerning the relationship between platelet activation and thromboxane (Tx) A2 formation (Born & Patrono, 2006). Many previous studies in this area have used the traditional Born light transmission method of platelet aggregometry. However this method has technical restraints, in particular the time required to produce replicate responses. Adapting the Born method for use in 96-well plate readers (Bednar et al., 1995; Maayani et al., 2001) allows us to measure concurrently platelet aggregration in response to agonists over a broad range of concentrations. Samples can also be retained for assay of TxB2, a measure of TxA2 formation. This approach allows us to determine more accurately platelet responses over full agonist and inhibitor concentration response curves.

Human blood was collected by venepuncture into tri-sodium citrate (3.8% w/v final) and centrifuged to obtain platelet rich plasma (PRP). The PRP was then added to the wells of 96-well plates in the presence of increasing concentrations of aspirin (0.01-1000µM) or vehicle and incubated for 30min at 37°C before the addition of arachidonic acid (AA, 0.03-1mM) or vehicle. Plates were then immediately placed in a 96-well plate reader and absorbance determined at 595nm every 15s for 16min between vigorous shaking. Changes in absorbance were converted to % aggregation by reference to the absorbances of PRP and platelet free plasma. At the end of the aggregatory responses plasma samples were removed and retained for analysis by radioimmunoassay of the content of TxB2 as a measure of TxA2 formation.

Maximum arachidonic acid-induced platelet aggregation (15±6%, 64±8% and 87±5% at arachidonic acid concentrations of 0.1, 0.3 and 1mM, respectively) and TxA2 production were both inhibited in concentration-dependent manners by aspirin. The log M IC50 values for aspirin as an inhibitor of arachidonic acid-induced platelet aggregation (-5.4±0.9, -5.3±0.1, and -5.3±0.1, respectively) and TxA2 production (-5.2±0.2, -5.4±0.3, -5.0±1.5, respectively) were not significantly different (n=8 for all).

These data demonstrate the utility of measuring platelet aggregation and TxA2 formation in 96-well plates. Furthermore, this technique clearly demonstrates a linear relationship between platelet aggregation and TxA2 production in this model of aspirin and arachidonic acid interaction.

Bednar, B., et al. (1995). Thromb Res, 77, 453-63.
Born, G. & Patrono, C. (2006). Br J Pharmacol, 147 Suppl 1, S241-51.
Maayani, S., (2001). Platelets, 12, 83-93.

This research was supported by European Community FP6 funding (“Eicosanox”; LSHM-CT-2004-0050333) and the Medical Research Council.