Characterisation of the pro-aggregatory effects of Gram positive or Gram negative bacteria in human platelet rich plasma

Ferhana Ali1, Timothy Warner2, Jane Mitchell1
1National Heart & Lung Institute, London, United Kingdom, 2William Harvey Research Institute, London, United Kingdom

Platelets are non-nuclear cell fragments that are involved in innate immunity and inflammation. Platelets express pattern recognition receptors including Toll-like receptors (TLR) 2, 4 and 9 (Cognasse et al., 2005) that are utilised by tissues to sense Gram positive and Gram negative bacteria. However, reports regarding the effects of TLR activation or bacteria on platelet activation are conflicting (Ward et al., 2005; Matera et al., 1992 ). In the current study we have investigated the effects of Gram positive bacteria, S.aureus, or Gram negative bacteria, E.coli, on the aggregation of human platelets. We have gone on to characterise the nature of the aggregatory response induced by bacteria using aspirin, the stable prostaglandin I2 mimetic treprostinil sodium, and the nitric oxide donor SIN-1, the three of which inhibit platelet activation by different mechanisms.

Platelet rich plasma (PRP) was prepared and 100μl added to the individual wells of 96-well plates. Aggregation was determined by measuring the changes in absorbance of individual wells at 595nm for 16 minutes at 37°C.

S.aureus and E.coli (109cfu/ml) stimulated aggregation by 61±1% and 62±7%, respectively (n=3). Addition of 10-5M ADP increased the aggregations to S.aureus and E.coli by 24% and 29%, respectively (n=3). Similar increases were seen following the addition of arachidonic acid (3x10-5M – 3x10-3M) or the thromboxane A2 mimetic, U46619 (10-7M – 10-5M) (n=3). Treprostinil sodium inhibited the aggregatory responses to the bacteria in a concentration-dependent manner, with log IC50 values of -8.0±1.6 and -8.8±0.5 against S.aureus and E.coli, respectively. In contrast, bacterial-induced aggregation was not inhibited by aspirin (n=3). As for treprostinil sodium, SIN-1 inhibited bacterial-induced platelet aggregation, with log IC50 values of -5.9±0.1 and -6.3±0.2 against S.aureus and E.coli respectively.

Our findings that S.aureus and E.coli stimulate platelet aggregation and that these aggregations are inhibited by SIN-1 or treprostinil sodium, but not aspirin imply that this aggregation is not mediated by thromboxane A2. This may have important implications in the treatment of bacterial diseases in which platelets play key roles such as sepsis.

Cognasse F et al. (2005) Immunol Cell Biol 83(2): 196-98
Matera C et al. (1992) J Med 23(5): 327-38
Ward JR et al (2005) Thromb Haemost 94(4): 831-38

This work was funded by the CRC, Royal Brompton Hospital, London and European Community FP6 funding (LSHM-CT-2004-0050333; “Eicosanox”)