Calpain activity and Toll-like receptor 4 expression in platelet regulate haemostatic situation in patients undergoing cardiac surgery and coagulation in mice

Abstract

Human platelets express Toll-like receptors (TLR) 4. However, the mechanism by which TLR4 directly affects platelet aggregation and blood coagulation remains to be explored. Therefore, in this study, we evaluated the platelet TLR4 expression in patients who underwent CABG surgery; we explored the correlation between platelet TLR4 expression and the early outcomes in hospital of patients. Additionally, C57BL/6 and C57BL/6-Tlr(LPS-/-) mice were used to explore the roles of platelet TLR4 in coagulation by platelet aggregometry and rotation thromboelastometry. In conclusion, our results highlight the important roles of TLR4 in blood coagulation and platelet function. Of clinical relevance, we also explored novel roles for platelet TLR4 that are associated with early outcomes in cardiac surgery.

Figure 1

CABG surgery with conventional CPB decreases calpain activity in platelets. (a) Total protein was isolated from washed platelets collected from patients who underwent the off-pump technique. The western blot results demonstrate the calpain activity before and after CABG surgery. (b) Total protein isolated from washed platelets collected from patients who underwent conventional CPB. The western blot results demonstrate the calpain activity before and after CABG surgery.

Figure 2

Platelet TLR4 is associated with platelet function in C57BL/6 mice. (a) TLR4 protein expression on the surface of platelets in C57BL/6 and C57BL/6-Tlr ^LPS−/− mice was analyzed by western blot analysis (upper graph) and flow cytometry (button graph). Negative control analyses were performed in the absence of specific PE-conjugated anti-TLR4 antibody (gray outline-hollow graph). C57BL/6 mice (black graph), but not C57BL/6-Tlr ^LPS−/− mice (black outline-hollow graph), express TLR4 protein in platelets. (b) Platelet aggregation was analyzed using aggregometry. The upper graph shows C57BL/6, C57BL/6-Tlr ^LPS−/−, and C57BL/6-Tlr ^LPS−/− mouse platelets with 0.1 U/mL thrombin or PBS treatment. The data at the bottom illustrate the mean ± SD (n = 6). *P < 0.05 indicates a significant difference compared with the C57BL/6 group. (c and d) ROTEM was performed. The thromboelastometry graph shows the curves of EXTEM (c) and FIBTEM (d) in C57BL/6 (black line) and C57BL/6-Tlr ^LPS−/− (red line) mice.