Antiplatelet and Antithrombotic Activities of Lespedeza cuneata via Pharmacological Inhibition of Integrin αIIbβ3, MAPK, and PI3K/AKT Pathways and FeCl3-Induced Murine Thrombosis

Figure 8

Western blot analysis to assess protein phosphorylation under Lespedeza cuneata stimulation. Various quantities of L. cuneata extracts with 1 mM CaCl₂ were preincubated using washed platelets for 1 min at 37°C before collagen stimulation for 5 min with constant stirring. By adding a lysis buffer, platelet aggregation stopped, and the protein concentration was calculated using the BCS assay (PRO-MEASURE; iNtRON Biotechnology). In a 10% SDS-PAGE, total platelet proteins were isolated, and then they were transferred to PVDF membranes. Membranes were blocked with 5% skim milk, probed with the appropriate antibodies (phospho-ERK, phospho-JNK, phospho-p38MAPK, phospho-PI3K, phospho-Akt, etc.), and then observed using enhanced chemiluminescence. , , and compared with the collagen.