PI3-Kinase Inhibitor LY294002 Repressed the Expression of Thrombin-Activatable Fibrinolysis Inhibitor in Human Hepatoma HepG2 Cells

Abstract

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase B-like proenzyme biosynthesized in the liver and released into the blood circulation. Activated TAFI (TAFIa) has been implicated as an important player in maintaining the balance between blood coagulation and fibrinolysis. In the present study, regulation of TAFI (CPB2) gene expression was investigated using cultured human hepatoma HepG2 cells. HepG2 cells were treated with the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the levels of TAFI antigen and CPB2 mRNA were measured. HepG2 cells treated with LY29400 decreased their release of TAFI antigen into the conditioned medium (CM). In parallel, there were decreased levels of CPB2 mRNA and TAFI antigen in the cells. However, CPB2 gene promoter activity was not influenced by treatment of the cells with LY294002. The half-life of the CPB2 transcript was shortened by treatment with LY294002 compared with control. The present results suggest that the PI3K inhibitor LY294002 suppresses expression of TAFI, a prothrombotic factor, by decreasing the stability of CPB2 transcripts.Key words thrombin-activatable fibrinolysis inhibitor (TAFI); carboxypeptidase; LY294002; Akt; HepG2 cell Thrombin-activatable fibrinolysis inhibitor (TAFI) is a zymogen of carboxypeptidase B (CPB)-like proenzyme that is synthesized in the liver and released into circulating plasma. 1)The active form of TAFI (TAFIa) is generated by cleavage at Arg92 of the proenzyme by the proteolytic activity of thrombin, plasmin, and trypsin. TAFIa exhibits CPB-like activity that specifically cleaves basic amino acids such as arginine and lysine residues located in the carboxyl (C)-termini of peptides. One of the TAFIa substrates is a partially degraded fibrin that contains a C-terminal lysine that provides a high affinity binding sites for tissue type plasminogen activator (tPA) and plasminogen in tPA-dependent plasminogen activation. Thus, TAFIa inhibits fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin, thereby inhibiting development of the positive feedback mechanism in the fibrinolytic cascade.2,3) Indeed, TAFIa attenuates spontaneous fibrinolysis of batroxobin-induced fibrin deposition in rat lungs. 4) TAFIa inhibitors enhance endogenous fibrinolysis, resulting in antithrombotic effects.5,6) Additionally, TAFIa has been shown to remove the C-terminal arginine residues from bradykinin as well as the anaphylatoxins C3a and C5a, thus indicating a role for the TAFI pathway in the vascular response to inflammation and the regulation of fibrinolysis. 7,8)Recent reports showed that a high level of TAFI expression in circulating plasma was a risk factor for thrombotic disorders. Thus, it was shown that elevated TAFI antigen in circulating plasma was a risk factor for premature peripheral arterial disease 9) and recurrent venous thromboembolism. 10) Moreover, TAFI antigen and functional protein levels in human plasma are significantly higher in the acute phase of ischemic stroke.11,12) Importantly,...

Puerarin attenuates locomotor and cognitive deficits as well as hippocampal neuronal injury through the PI3K/Akt1/GSK-3β signaling pathway in an in vivo model of cerebral ischemia

“…The kinetic analysis of puerarin revealed a half-life of 46.9 minutes [ 40 ]. LY294002 is a strong inhibitor of PI3Kthat is the major mode of activation of Akt; thus, it can efficiently inhibit PI3K/Akt activities and has been widely used in studies regarding the PI3K/Akt pathway [ 41 , 42 ]. The puerarin +LY group was administered with puerarin (100 mg/kg) and LY294002 (10 mg/kg, dissolved in DMSO) intraperitoneally at the same time as the other groups.…”

The inhibitory effect of tachyplesin I on thrombosis and its mechanisms

“…It indicates that PI3K is a new and promising anti‐thrombosis target. The PI3K inhibitors that have an anti‐thrombotic effect include LY294002 (Masuda, Yazawa, Makino, & Takada, ), wortmannin, salvianolic acid A (Joshi, Kumar, & Kumar, ), AZD 6482 (Győri et al., ) and TGX221 (Feng et al., ). In our study, the results showed that tachyplesin I can affect the phosphorylation of Akt in platelet by Western blot analysis.…”