Impact of Toll-like receptor 4 signalling in platelet activation

T. Vallance, D. Widera, S. Vaiyapuri. School of Pharmacy, University of Reading, Reading, United Kingdom.

Introduction: Platelets play a vital role in haemostasis and thrombus formation (1). Moreover, platelets are involved in innate immunity via multiple receptors including Toll-like receptor 4 (TLR4) which binds bacterial lipopolysaccharides (LPS) (1,2). TLR4 can signal through both the MyD88-dependent and -independent pathways (1). TLR4 has previously been shown to be expressed and functional on platelets (2). As both the MyD88-dependent and -independent pathways couple to transcription factors (and platelets lack a nucleus), we hypothesise that these transcription factors have non-genomic roles in the modulation of platelet activation. In this study, we examined whether the LPS obtained from distinct bacterial species may modulate platelet function differently.

Method: For aggregation assays, platelet-rich plasma (PRP) incubated with increasing concentrations of LPS from one of three different species (Escherichia coli [E. coli], Salmonella minnesota [S. minnesota], or Rhodobacter sphaeroides [R. sphaeroides]) for five minutes was stimulated with 0.1μg/mL cross-linked collagen-related peptide (CRP-XL). Light transmission was measured through PRP by optical aggregometry and an increase was used to signify the level of aggregation. Additionally, flow cytometry was used to assess the level of fibrinogen binding with FITC-conjugated anti-fibrinogen antibodies. IMD0354 (IKKβ inhibitor) or BX795 (IKKδ/TBK1 inhibitor) were incubated with PRP for five minutes prior to addition of LPS. CRP-XL was added at 0.5μg/mL where necessary.

Results: The results demonstrate that E. coli LPS increased CRP-XL-induced PRP aggregation by 25% whereas LPS from either S. minnesota or R. sphaeroides decreased aggregation elicited by CRP-XL (by 30% or 40% respectively). Further experiments suggest that treatment of PRP with E. coli LPS increases fibrinogen binding to platelets whereas S. minnesota LPS or R. sphaeroides LPS decreased fibrinogen binding to 50% of control. To investigate whether these diverse effects were due to divergent signalling pathways downstream of TLR4, IMD0354 and BX795 were used to inhibit the MyD88-dependent pathway or -independent pathway respectively. The effects of E. coli LPS were partially reduced following IMD0354 treatment and the effects of S. minnesota LPS were inhibited by the presence of BX795. R. sphaeroides LPS, a competitive inhibitor of TLR4, was unaffected by either of the IKK inhibitors. Data were analysed via one-way ANOVA and each experiment had n=3.

Conclusions: These data suggest that different LPS chemotypes preferentially activate one TLR4 signalling pathway over another in platelets, to promote pro- or anti-thrombotic/inflammatory effects.

References:

(1) Cognasse et al. (2015) Frontiers in Immunology 6: 1-15

(2) Andonegui et al. (2005) Blood 106: 2417-2423