Linear relationship between collagen-induced platelet aggregation and thromboxane A2 production

PAUL ARMSTRONG1, ZETTY ZAIN1, NICOLA PEARSON1, IVANA VOJNOVIC1, JANE MITCHELL2, TIMOTHY WARNER1
1WILLIAM HARVEY RESEARCH INSTITUTE, LONDON, UNITED KINGDOM, 2NATIONAL HEART AND LUNG INSTITUTE, LONDON, UNITED KINGDOM

There has long been discussion concerning the relationship between platelet activation and thromboxane (Tx) A2 formation (Born & Patrono, 2006). Adapting the Born method for use in 96-well plate readers (Bednar et al., 1995; Maayani et al., 2001) has allowed us to previously demonstrate a linear relationship between platelet aggregration and thromboxane A2 production when arachidonic acid is used as an agonist (Armstrong et al, 2007). Here we have continued to study whether this relationship exists with collagen as the agonist, taking into account that thromboxane (Tx) signalling is only necessary for aggregation when lower concentrations of collagen are used (Cho et al, 2003).

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 collagen (1 or 3μg/ml) 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 platelet TxA2 formation.

Platelet aggregation (72±1% of maximum and 84±1% of maximum in response to collagen 1μg/ml and 3μg/ml respectively) and TxA2 production were both inhibited in concentration-dependent manners by aspirin. The log M IC50 values for aspirin as an inhibitor of collagen-induced platelet aggregation (−5.5±0.3 and −4.7±0.3, respectively) and TxA2 production (−5.3±0.5 and −5.2±0.7, respectively) were not significantly different (n=8 for all). Furthermore, under all conditions there was a direct linear relationship between concentrations of TxA2 and platelet aggregation.

Here we clearly demonstrate a linear relationship between platelet aggregation and TxA2 production in this model of aspirin and low concentration collagen interaction. Such a relationship has important implications for aspirin and NSAID therapy.

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