Influence of endothelial cells on platelet aggregation and adhesion in 96-well plates

Nicola J. Pearson, Zetty N.M. Zain, Paul C.J. Armstrong, Martin J. Carrier, Jane A. Mitchell & Timothy D. Warner. The William Harvey Research Institute, Barts & the London, QMUL, EC1M 6BQ, U.K.

Studies have shown that light transmission Born aggregometry can be adapted for use in 96-well plate readers, allowing many aggregatory responses to be followed at the same time (Bednar et al., 1995; Maayani et al., 2001) . Platelet adhesion can be determined colourimetrically as the conversion of p-nitrophenol phosphate to p-nitrophenol by platelet acid phosphatase (Bellavite et al., 1994) . We have combined these techniques to investigate the influence human endothelial cells have on platelet aggregation and adhesion.

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 or absence of confluent monolayers of primary human aortic endothelial cells (HAECs) and incubated for 30min at 37°C before the addition of agonists or vehicle. Plates were then immediately placed in a 96-well plate reader and absorbance determined at 595nm every 15s for 16min with vigorous shaking. Changes in absorbance were converted to % aggregation by reference to the absorbance of PRP and platelet free plasma. At the end of the 16min period plates were washed with saline and platelet adhesion determined by incubating the plate with p-nitrophenol phosphate (0.2mg/ml final); followed by addition of NaOH (2M) and the measurement of absorption at 405nm (Bellavite et al., 1994) . The extent of platelet adhesion was determined relative to the total level of acid phosphatase activity present within an aliquot of unstimulated platelets.

Aggregatory responses to ADP (0.1-30µM), adrenaline (0.001-100µM), arachidonic acid (0.03-3mM), collagen (0.1-30µg/ml), ristocetin (0.2-3mg/ml), TRAP-6 amide (0.1-30µM) or U46619 (0.1-30µM) were not inhibited by the presence of HAECs. Adhesion, however, was reduced in the presence of HAECs. For example, adhesion stimulated by 1mM arachidonic acid was 93±3% without HAECs but 36±7% with HAECs; adhesion stimulated by 1μg/ml collagen was 91±2% in the absence of HAECs but was reduced to 19±5% in their presence.

These data demonstrate that 96-well plate protocols can be adapted to analyse platelet- endothelial cell interactions. This could be particularly useful in determining the mechanisms underlying events, such as nonsteroid drug induced thrombosis, which are hypothesised to be explained by an imbalance in the production of pro- and anti-thrombotic mediators by endothelial cells and platelets.

Bednar , B ., et al.. (1995). Thromb Res, 77, 453-63.
Bellavite , P ., et al.. (1994). Anal Biochem, 216, 444-50.
Maayani , S ., et al .. (2001). Platelets, 12, 83-93.

Research supported by European Community FP6 funding (“Eicosanox”; LSHM-CT-2004-0050333) and the St Bartholomew’s and the Royal London Charitable Foundation.