Over the past 100 years, Salvia miltiorrhiza f. alba (Lamiaceae) (RSMA) roots have been used to cure thromboangiitis obliterans (TAO) in local clinics. This study aimed to confirm the anti-thrombotic efficacy of 12 phenolic acids obtained from RSMA and to clarify the possible underlying mechanisms. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that most of the phenolic acids markedly inhibited PAI-1 protein and mRNA levels but increased t-PA protein and mRNA levels in TNF-α-induced EA.hy926 cells (P < 0.05 or 0.001), with lithospermic acid displaying the strongest effect. In vitro anticoagulation and antiplatelet aggregation assays showed that lithospermic acid and salvianolic acid B significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), decreased fibrinogen concentration (FIB), and inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rat blood. Both lithospermic acid and salvianolic acid B markedly down-regulated the expression of factor Xa and factor IIa on the external surface of EA.hy926 cells and demonstrated significant anti-factor IIa and anti-factor Xa activity using chromogenic substrates in vitro. Western blot results revealed that both lithospermic acid and salvianolic acid B also significantly inhibited the expression of TF, p-p65, p-p38, and pJNK proteins induced by TNF-α. These results indicated that all of the phenolic acids appeared to have some anti-thrombotic activity, with salvianolic acid B and lithospermic acid markedly decreasing the chance of thrombosis by regulating the NF-κB/JNK/p38 MAPK signaling pathway in response to TNF-α.
Keywords: Anti-thrombotic effect; NF-κB, JNK, p38 MAPK; Phenolic acid; Salvia miltiorrhiza f. alba; TNF-α-induced EA.hy926 cells.