Modulation of human platelet activation and in vivo vascular thrombosis by columbianadin: regulation by integrin αIIbβ3 inside-out but not outside-in signals

Abstract

Background: Columbianadin (CBN) is one of the main coumarin constituents isolated from Angelica pubescens. The pharmacological value of CBN is well demonstrated, especially in the prevention of several cancers and analgesic activity. A striking therapeutic target for arterial thrombosis is inhibition of platelet activation because platelet activation significantly contributes to these diseases. The current study examined the influence of CBN on human platelet activation in vitro and vascular thrombotic formation in vivo. Methods: Aggregometry, immunoblotting, immunoprecipitation, confocal microscopic analysis, fibrin clot retraction, and thrombogenic animals were used in this study. Results: CBN markedly inhibited platelet aggregation in washed human platelets stimulated only by collagen, but was not effective in platelets stimulated by other agonists such as thrombin, arachidonic acid, and U46619. CBN evidently inhibited ATP release, intracellular ([Ca 2+ ]i) mobilization, and P-selectin expression. It also inhibited the phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), Akt (protein kinase B), and mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase [ERK] 1/2 and c-Jun N-terminal kinase [JNK] 1/2, but not p38 MAPK) in collagen-activated platelets. Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the CBN-mediated inhibition of platelet aggregation. CBN had no significant effect in triggering vasodilator-stimulated phosphoprotein phosphorylation. Moreover, it markedly hindered integrin α IIb β 3 activation by interfering with the binding of PAC-1; nevertheless, it had no influences on integrin α IIb β 3-mediated outside-in signaling such as adhesion number and spreading area of platelets on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Additionally, CBN did not attenuate FITC-triflavin binding or phosphorylation of proteins, such as integrin β 3 , Src, and focal adhesion kinase, in platelets spreading on immobilized fibrinogen. In experimental mice, CBN increased the occlusion time of thrombotic platelet plug formation. Conclusion: This study demonstrated that CBN exhibits an exceptional activity against platelet activation through inhibition of the PLCγ2-PKC cascade, subsequently suppressing the activation of Akt and ERKs/JNKs and influencing platelet aggregation. Consequently, this work provides solid evidence and considers that CBN has the potential to serve as a therapeutic agent for the treatment of thromboembolic disorders.

Effectiveness of Columbianadin, a Bioactive Coumarin Derivative, in Perturbing Transient and Persistent INa

“…The principal results of the present work are that (a) the presence of CBN could depress I Na in a concentration-, time-, and state-dependent manner in GH 3 cells, (b) this compound differentially inhibited the peak and late amplitudes of I Na activated by rapid membrane depolarization with effective IC 50 of 14.7 and 2.8 µM, respectively, (c) it shifted the midpoint of I Na inactivation curve along the voltage axis to a negative potential, despite inability to alter the activation curve of the current, (d) it suppressed peak I Na in a tonic and use-dependent manner, (e) subsequent addition of CBN was capable of depressing the Tef-induced increase in the I Na(P) amplitude activated by the upright isosceles-triangular ramp at either upsloping or downsloping limb, and (f) it mildly inhibited the amplitude of I K(DR) , I K(erg) , or I h , and (g) in HL-1 cardiomyocytes, and CBN was effective at depressing I Na as well as at decreasing the τ inact(S) value of the current. The experimental results enable us to reflect that CBN-mediated change in the amplitude and gating kinetics of ionic currents presented herein tends to be upstream of its action either on cytosolic NOD1/Nf-κB activation [ 2 , 5 , 31 ] or on the activity of antioxidant enzymes [ 14 ], and that it could conceivably participate in the adjustments on different functional activities in electrically excitable cells (e.g., GH 3 or HL-1 cells) occurring in vivo.…”

“…f. biserrate Shan et Yuan ( Angelicae Pubescentis Radix or “Duho [Dú huó]” in China). This compound is a coumarin-type compound (an angular dihydrofuranocoumarin), which is increasingly recognized to have various biological activities that include analgesic, anti-inflammatory, and antineoplastic effects [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. This compound has been reported to induce changes in cellular proliferation or apoptosis [ 12 , 14 ].…”

Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets

“…This method was applied as described previously [52]. In brief, luciferase/luciferin and Fura 2-AM were used to detect ATP release and calcium mobilization, respectively.…”

Columbianadin Dampens In Vitro Inflammatory Actions and Inhibits Liver Injury via Inhibition of NF-κB/MAPKs: Impacts on ∙OH Radicals and HO-1 Expression

“…Transportation and absorption kinetics studies revealed that CBN easily entered into the blood and circulated mainly in hepatic tissue [ 17 ]. Our recent study also reported the antiplatelet effect of CBN via different molecular mechanisms and suggested that CBN could act as a potential drug to treat thromboembolic disorders [ 18 ]. Although several basic biological and anti-inflammatory properties of CBN have been explored, the underlying mechanisms of the anti-inflammatory and liver protective effects are unclear.…”