Objective To explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism.
Methods Venous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 μmol/L CORM-2 group, and 50 μmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 μg/mL) stimulation, and the PRP in iCORM-2 group, 10 μmol/L CORM-2 group, and 50 μmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 μmol/L iCORM-2, 10 μmol/L CORM-2, and 50 μmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein Ⅰ bα (GPⅠbα) and GPⅥ were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test.
Results Compared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPⅠbα and GPⅥ in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with
P values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with
P values above 0.05), while the levels of ATP in platelets in the 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly decreased (with
P values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 μmol/L CORM-2, and 50 μmol/L CORM-2 groups were 0.550±0.060, 1.437±0.214, 1.210±0.108, 0.720±0.010, and 0.670±0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950±0.070, 1.607±0.121, 1.420±0.040, 1.167±0.015, and 0.513±0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with
P values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with
P values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 μmol/L CORM-2 and 50 μmol/L CORM-2 groups were significantly decreased compared with those in LPS group (with
P values below 0.05).
Conclusions LPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.
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