Today, cardiovascular diseases are increasing, which are thought as a major death-cause. Platelet aggregation is essential for normal hemostatic process when blood vessels are injured. However, it can also cause cardiovascular diseases such as thrombosis, atherosclerosis, and myocardial infarction. ...
Today, cardiovascular diseases are increasing, which are thought as a major death-cause. Platelet aggregation is essential for normal hemostatic process when blood vessels are injured. However, it can also cause cardiovascular diseases such as thrombosis, atherosclerosis, and myocardial infarction. Hence, inhibition of platelet aggregation might be a promising target to the development of antithrombotic drugs and an approach for the prevention of cardiovascular diseases. In this study, it was sought to explore the mechanism by which some phytochemicals, including ephigallocatechin-3-gallate (EGCG) and WIB-801CE inhibit platelet function.
A. Inhibitory effects of epigallocatechin-3-gallate on fibrinogen binding via inhibition of αIIb/β3 in collagen-activated human platelets
Because various agonists-induced the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/β3) is involved in intensification of thrombus, evaluated the effect of EGCG on fibrinogen binding and its associated phosphorylation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase-1 (PDK1), and Akt because it is unknown whether EGCG moderates these signaling molecules. EGCG inhibited collagen-induced phosphorylation of PI3K (Tyr458), PDK1 (Ser241) and Akt (Ser473 and Thr308), which was resulted in prevention of fibrinogen binding to αIIb/β3. These were again attenuated by wortmannin, an inhibitor of PI3K and PDK1, and miltefosine, an Akt inhibitor. In special, cyclic adenosine monophosphate (cAMP) stimulator pCPT-cAMP abolished EGCG-attenuated phosphorylation of PI3K (Tyr458) and Akt (Ser473) to reduce the fibrinogen binding to αIIb/β3 in collagen-activated platelets. This is indicated that inhibitory effect of EGCG on phosphorylation of PI3K (Tyr458) and Akt (Ser473) may be caused in cAMP-dependent manner. Cytosolic free Ca2+ ([Ca2+]i) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. EGCG inhibited collagen-elevated [Ca2+]i, which was increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. EGCG increased the phosphorylation of inositol 1,4,5-triphosphate receptor type I (IP3RI) (Ser1756) to inhibit [Ca2+]i mobilization in collagen-stimulated platelet aggregation. In addition, EGCG inhibited collagen-induced phosphorylation of extracellular signal-regulated kinase (ERK) and PI3K, indicating inhibition Ca2+ influx across plasma membrane, and attenuated phosphorylation of p38 and myosin light chain (MLC). Consequently, EGCG decreased collagen-elevated [Ca2+]i, which contributes to inhibition of adenosine triphosphate (ATP) and serotonin release. These results showed that EGCG have a beneficial novel Ca2+-antagonistic effect. Fibrin clot retraction, an index of thrombi formation, was controlled by integrin cytoplasmic domain β3 (Tyr747 and Tyr759) phosphorylation. EGCG inhibited phosphorylation of integrin β3 (Tyr747) and dephosphorylation of integrin β3 (Tyr759). In addition, EGCG dose-dependently decreased the rate of clot retraction, reflecting inhibition of αIIb/β3, without the prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT). These results suggest that the inhition of αIIb/β3 activation by EGCG is resulted from regulation of integrin β3 (Tyr747 and Tyr759) phosphorylation. According to the inhibition of αIIb/β3 activation and [Ca2+]i mobilization achieved by regulation of inside-out and outside-in signaling pathway, the EGCG-mediated antiplatelet effected, then it is thought that they may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.
B. Antithrombotic effect of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo and in vivo
A species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, it was prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo and in vitro antiplatelet and antithrombotic effects. Ex vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. Cordycepin-enriched WIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. Therefore, these results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.
Today, cardiovascular diseases are increasing, which are thought as a major death-cause. Platelet aggregation is essential for normal hemostatic process when blood vessels are injured. However, it can also cause cardiovascular diseases such as thrombosis, atherosclerosis, and myocardial infarction. Hence, inhibition of platelet aggregation might be a promising target to the development of antithrombotic drugs and an approach for the prevention of cardiovascular diseases. In this study, it was sought to explore the mechanism by which some phytochemicals, including ephigallocatechin-3-gallate (EGCG) and WIB-801CE inhibit platelet function.
A. Inhibitory effects of epigallocatechin-3-gallate on fibrinogen binding via inhibition of αIIb/β3 in collagen-activated human platelets
Because various agonists-induced the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/β3) is involved in intensification of thrombus, evaluated the effect of EGCG on fibrinogen binding and its associated phosphorylation of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent kinase-1 (PDK1), and Akt because it is unknown whether EGCG moderates these signaling molecules. EGCG inhibited collagen-induced phosphorylation of PI3K (Tyr458), PDK1 (Ser241) and Akt (Ser473 and Thr308), which was resulted in prevention of fibrinogen binding to αIIb/β3. These were again attenuated by wortmannin, an inhibitor of PI3K and PDK1, and miltefosine, an Akt inhibitor. In special, cyclic adenosine monophosphate (cAMP) stimulator pCPT-cAMP abolished EGCG-attenuated phosphorylation of PI3K (Tyr458) and Akt (Ser473) to reduce the fibrinogen binding to αIIb/β3 in collagen-activated platelets. This is indicated that inhibitory effect of EGCG on phosphorylation of PI3K (Tyr458) and Akt (Ser473) may be caused in cAMP-dependent manner. Cytosolic free Ca2+ ([Ca2+]i) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. EGCG inhibited collagen-elevated [Ca2+]i, which was increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. EGCG increased the phosphorylation of inositol 1,4,5-triphosphate receptor type I (IP3RI) (Ser1756) to inhibit [Ca2+]i mobilization in collagen-stimulated platelet aggregation. In addition, EGCG inhibited collagen-induced phosphorylation of extracellular signal-regulated kinase (ERK) and PI3K, indicating inhibition Ca2+ influx across plasma membrane, and attenuated phosphorylation of p38 and myosin light chain (MLC). Consequently, EGCG decreased collagen-elevated [Ca2+]i, which contributes to inhibition of adenosine triphosphate (ATP) and serotonin release. These results showed that EGCG have a beneficial novel Ca2+-antagonistic effect. Fibrin clot retraction, an index of thrombi formation, was controlled by integrin cytoplasmic domain β3 (Tyr747 and Tyr759) phosphorylation. EGCG inhibited phosphorylation of integrin β3 (Tyr747) and dephosphorylation of integrin β3 (Tyr759). In addition, EGCG dose-dependently decreased the rate of clot retraction, reflecting inhibition of αIIb/β3, without the prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT). These results suggest that the inhition of αIIb/β3 activation by EGCG is resulted from regulation of integrin β3 (Tyr747 and Tyr759) phosphorylation. According to the inhibition of αIIb/β3 activation and [Ca2+]i mobilization achieved by regulation of inside-out and outside-in signaling pathway, the EGCG-mediated antiplatelet effected, then it is thought that they may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.
B. Antithrombotic effect of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo and in vivo
A species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, it was prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo and in vitro antiplatelet and antithrombotic effects. Ex vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. Cordycepin-enriched WIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. Therefore, these results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.
주제어
#Phytochemicals Platelet aggregation Fibrinogen binding αIIb/β3 PI3K/PDK1/Akt [Ca2+]i
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