In subsequent experiments, we used collagen as an agonist to explore the inhibitory mechanisms of sim vastatin in platelet activation. Triflavin is an Arg Gly Asp containing antiplatelet peptide purified from Trimeresurus flavoviridis snake venom. Triflavin inhibits platelet aggregation through direct interference with fibrinogen binding to the IIbB3 integrin. high throughput screening There is now a multitude of evi dence suggesting that the binding of fibrinogen to the IIbB3 integrin is the final common pathway for agonist induced platelet aggregation. Therefore, we further eval uated whether or not simvastatin directly binds to the platelet IIbB3 integrin, leading to interruption of platelet aggregation induced by collagen. In this study, the relative intensity of the fluorescence of FITC triflavin bound directly to collagen activated platelets was relatively higher than that of negative control.

Simvastatin did not significantly affect FITC Inhibitors,Modulators,Libraries triflavin binding to the IIbB3 integrin in platelet suspensions indicating Inhibitors,Modulators,Libraries that the inhibitory effect of simvastatin on platelet aggregation does not involve binding to the plate let IIbB3 integrin. Free cytoplasmic Ca 2 concentrations in human plate Inhibitors,Modulators,Libraries lets were measured by the Fura 2 AM loading method. As shown in Figure 1D, collagen evoked a marked increase in i, and this increase was markedly inhib ited in the presence of simvastatin Effects of simvastatin on TxA2, PLC��2, and PKC activation As shown in Figure 2A, resting platelets produced rela tively little TxB2 compared to collagen activated platelets.

Simvastatin concentration dependently inhibited TxB2 formation in platelets stimulated by colla gen. PLC hydrolyzes phosphatidylinositol 4,5 bisphosphate to generate two secondary messen gers inositol Inhibitors,Modulators,Libraries 1,4,5 trisphosphate and diacylglycerol. DAG activates PKC, inducing protein phos phorylation and ATP release. Phosphorylation is one of the key mechanisms regulating the activity of PLC. The immunoblotting analysis revealed that treatment with simvastatin markedly abolished the phosphorylation of PLC��2 stimulated by collagen. Stimulation of platelets with a number of different agonists Inhibitors,Modulators,Libraries induced acti vation of PKC, which then phosphorylated p47 proteins. In this study, phosphorylation experiments were per formed to examine the role of simvastatin in PKC activa tion in human platelets.

When collagen or PDBu was added to human platelets, a protein with an apparent of p47 was predomi nately phosphorylated compared to resting platelets. selleck chemicals llc Simvastatin inhibited p47 phosphorylation stimulated by collagen but not by PDBu. Effect of simvastatin on collagen induced MAPK phosphorylation To further investigate the inhibitory mechanisms of sim vastatin in platelet activation stimulated by collagen, we further detected MAPK signaling molecules including p38 MAPK, JNKs, and ERKs.