Inhibition of TLR-4 Prevents From Sepsis-Related eNOS/NO Disturbance in Human Platelets

Toll-like receptor 4 (TLR-4) has the ability to activate platelet and involve in intravascular coagulation, inflammatory cytokine and oxidative stressors release in sepsis. Nitric oxide (NO) synthesis in platelets is adjusted by iNOS and/or eNOS and contributes to platelet aggregation and adhesion. Our previous study has found that increased platelet aggregation in patients with sepsis was associated with the expression of TLR4 on platelets and NO synthesis in platelets. This study was to investigate whether inhibition of TLR-4 activation on platelets decreases eNOS(endothelial NO synthase)/NO disturbance-related platelet aggregation.

Blood samples were collected from 10 patients with severe sepsis and 10 healthy volunteers as controls. PRP(platelet-rich plasma) and PPP (platelet-poor plasma) were prepared for platelet aggregation, and platelet suspension (at a concentration of 2×10⁸/mL platelets) for next experimemts. After the platelet suspension from healthy volunteers was incubated with L-Arginine (LA, 10 mM) alone, and LA and L-NAME (NΩ-Nitro-L-arginine methyl ester hydrochloride, 25mg/mL, a nonselective NOS inhibitor) for 1 hour, LPS(10.0μg/ml) was respectively added them. In separate experiments, the platelet suspension was preincubated with anti-TLR-4 Abs (10μg/mL) and then repeat above experiments. The levels of serine phosphorylation in eNOS (p-eNOS) and NO production, and platelet aggregation were determined with Western blotting, nitrate concentration analysis and platelet aggregometer, respectively.

The protein levels of p-eNOS and NO production had 2.2-fold and 1.8-fold of increases in platelets from septic patients, and in vitro had 1.8-fold and 1.7-fold of increases in LA-incubated platelets stimulated with LPS as compared to controls. The p-eNOS expression and NO production were inhibited in the presence of L-NAME. Furthermore, thrombin-induced platelet aggregation was markedly promoted by LPS and L-NAME [(61 □ ‘98) %, (80□ ‘100)% versus (40□ ‘72)% of control) ], and the effect of LPS was abolished by LA pretreatment [(54□ ‘76)% ]. Blockade of TLR-4 didn't alter the elevated levels of p-eNOS and NO under LPS challenge, but the inhibition of L-NAME on p-eNOS and NO was significantly decreased. Importantly, blockade of TLR-4 significantly decreased platelet aggregation induced by LPS, and both LPS and L-NAME, but had no effects on LA pretreatment.

These data suggest that inhibition of TLR-4 may play a role in prevention from sepsis-induced platelet aggregation and maintaining platelet eNOS activity in sepsis.

No relevant conflicts of interest to declare.