Effects of bacterial lipopolysaccharides on platelet function: inhibition of weak platelet activation

Abstract

Platelets are anucleate blood cells with reported roles in hemostasis and immune responses, which possess a functional receptor for bacterial lipopolysaccharides (LPSs), the well-known inducers of inflammation. However, LPSs effects on platelets are contradictory. Here we aim to investigate mechanisms of platelet functioning in the presence of LPS and to find the cause of the discrepancy in the previously published data. Cell activity was analyzed by flow cytometry, western blotting, and aggregometry. Thrombus growth was assessed by fluorescent microscopy. LPS' activity was checked by their capability to induce PMN activation. However, LPSs did not substantially affect either thrombus growth in flow chambers, irreversible platelet aggregation, or platelet responses to strong activation. Platelet aggregation in response to 1 μM of ADP was significantly inhibited by LPSs. Flow cytometry analysis revealed that platelet activation responses to weak stimulation were also diminished by LPSs, while VASP phosphorylation was weakly increased. Additionally, LPSs were capable of inhibition of ADP-induced P2-receptor desensitization. Incubation of platelets with a pan-PDE inhibitor IBMX significantly enhanced the LPSs-induced platelet inhibition, implying cAMP/cGMP dependent mechanism. The discrepancy in the previously published data could be explained by LPSinduced weak inhibition of platelet activation and the prevention of platelet desensitization. Lipopolysaccharides (LPSs) are the components of the outer membrane of the gram-negative bacteria 1 , which are recognized by the toll-like receptor 4 (TLR4) 2. LPS are among the most potent mediators of bacteria-induced sepsis and associated disseminated intravascular coagulation (DIC) 3. TLR4 was detected on the surface of myeloid cell line: granulocytes, monocytes, dendric cells 4. TLR4 is also expressed on the surface of platelets 5,6-anucleate blood cells, responsible for hemostasis 7. In order to recognize LPS, TLR4 requires such cofactor proteins as CD14, myeloid-differentiation factor 2 (MD2), and LPS binding protein (LBP) 8. Upon ligation and dimerization, TLR4 induces the formation of a "myddosome" signaling complex, which initiates nuclear factor kappa-B (NF-κB) intracellular signaling cascade 2,8,9. In anucleate platelets, the most important part of this cascade is the inhibitor of NF-κB (IκB), which has been reported to mediate granule secretion and other signalling events 8. LPS mediated TLR4 activation is claimed to enhance platelet stimulation by ADP and CRP 6,10,11. LPS also were shown to induce increased oxygen consumption by platelet mitochondria 5,12. LPS driven platelet activation is a potential mediator of neutrophil extracellular traps (NETosis) in vivo 8. Finally, it was reported that LPS are capable of inducing synthesis of IL1β in platelets 13. Platelet reactivity to LPS could be caused by activation of

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“…Due to differences in the experimental protocols, the influence of LPS on platelet activity cannot be clearly defined. However, Martyanov et al showed that LPS restored the response to ADP in desensitized platelets by activating platelet TLR4 while inhibiting platelet activation by potentiating cAMP/cGMP pathways [24]. This could explain why the most pronounced increase in the PECAM-1/thrombus ratio observed in the LPS + ASA 3 mg/kg group (Figures 1b and 2d) was opposite to the tendency observed in control mice (Figure 2b).…”

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