• Biomolecules & Therapeutics
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• 제31권1호
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• pp.59-67
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• 2023

Thrombin is a serine protease that participates in a variety of biological signaling through protease-activated receptors. Intestinal myofibroblasts play central roles in maintaining intestinal homeostasis. In this study, we found that thrombin-induced apoptosis is mediated by the calcium-mediated activation of cytosolic phospholipase A₂ in the CCD-18Co cell. Thrombin reduced cell viability by inducing apoptosis and proteinase-activated receptor-1 antagonist attenuated thrombin-induced cell death. Endogenous ceramide did not affect the cell viability itself, but a ceramide-mediated pathway was involved in thrombin-induced cell death. Thrombin increased intracellular calcium levels and cytosolic phospholipase A₂ activity. The ceramide synthase inhibitor Fumonisin B1, intracellular calcium chelator BAPTA-AM, and cytosolic phospholipase A₂ inhibitor AACOCF₃ inhibited thrombin-induced cell death. Thrombin stimulated arachidonic acid release and reactive oxygen species generation, which was blocked by AACOCF₃, BAPTA-AM, and the antioxidant reagent Trolox. Taken together, thrombin triggered apoptosis through calcium-mediated activation of cytosolic phospholipase A₂ in intestinal myofibroblasts.

• Bulletin of the Korean Chemical Society
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• 제33권9호
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• pp.3048-3054
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• 2012

The use of aspirin is widely recommended for the prevention of heart attacks owing to its ability to inhibit platelet activation by irreversibly blocking cyclooxygenase 1. However, aspirin also affects the fibrinolytic and hemostatic pathways by mechanisms that are not well understood, causing severe hemorrhagic complications. Here, we investigated the ability of aspirin and aspirin metabolites to inhibit thrombin-activatable fibrinolysis inhibitor (TAFI), the major inhibitor of plasma fibrinolysis. TAFI is activated via proteolytic cleavage by the thrombin-thrombomodulin complex to TAFIa, a carboxypeptidase B-like enzyme. TAFIa modulates fibrinolysis by removing the C-terminal arginine and lysine residues from partially degraded fibrin, which in turn inhibits the binding of plasminogen to fibrin clots. Aspirin and its major metabolites, salicylic acid, gentisic acid, and salicyluric acid, inhibit TAFIa carboxypeptidase activity. Salicyluric acid effectively blocks activation of TAFI by thrombin-thrombomodulin; however, salicylates do not inhibit carboxypeptidase N or pancreatic carboxypeptidase B. Aspirin and other salicylates accelerated the dissolution of fibrin clots and reduced thrombus formation in an in vitro model of fibrinolysis. Inhibition of TAFI represents a novel hemostatic mechanism that contributes to aspirin's therapy-associated antithrombotic activity and hemorrhagic complications.

• The Korean Journal of Physiology and Pharmacology
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• 제19권6호
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• pp.549-555
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• 2015

We attempted to investigate molecular mechanisms underlying phenotypic change of vascular smooth muscle cells (VSMCs) by determining signaling molecules involved in chemokine production. Treatment of human aortic smooth muscle cells (HAoSMCs) with thrombin resulted not only in elevated transcription of the (C-C motif) ligand 11 (CCL11) gene but also in enhanced secretion of CCL11 protein. Co-treatment of HAoSMCs with GF109230X, an inhibitor of protein kinase C, or GW5074, an inhibitor of Raf-1 kinase, caused inhibition of ERK1/2 phosphorylation and significantly attenuated expression of CCL11 at transcriptional and protein levels induced by thrombin. Both Akt phosphorylation and CCL11 expression induced by thrombin were attenuated in the presence of pertussis toxin (PTX), an inhibitor of Gi protein-coupled receptor, or LY294002, a PI3K inhibitor. In addition, thrombin-induced production of CCL11 was significantly attenuated by pharmacological inhibition of Akt or MEK which phosphorylates ERK1/2. These results indicate that thrombin is likely to promote expression of CCL11 via PKC/Raf-1/ERK1/2 and PTX-sensitive protease-activated receptors /PI3K/Akt pathways in HAoSMCs. We propose that multiple signaling pathways are involved in change of VSMCs to a secretory phenotype.

• Bulletin of the Korean Chemical Society
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• 제29권11호
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• pp.2209-2214
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• 2008

• Archives of Pharmacal Research
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• 제25권6호
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• pp.879-884
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• 2002

Previous study showed that an amidrazonophenylalanine derivative, LB30057, which has high water solubility, inhibited the catalytic activity of thrombin potently by interaction with the active site of thrombin. In the current investigation, we examined whether LB30057 inhibited platelet aggregation and vascular relaxation induced by thrombin. Treatment with LB30057 to plateletrich plasma (PRP) isolated from human blood resulted in a concentration-dependent inhibition of thrombin-induced aggregation. Values for $IC_{50}$ and $IC_{100}$ were $54{\pm}4$ nM and $96{\pm}3$ nM, respectively. This inhibition was agonist (thrombin) specific, since $IC_{50}$ values for collagen and ADP were \much greater than those for thrombin. In addition, concentration-dependent inhibitory effects were observed on the serotonin secretion induced by thrombin in PRP. Consistent with these findings, thrombin-induced increase in cytosolic calcium levels was inhibited in a concentration-dependent manner. When LB30057 was treated with aortic rings isolated from rats, LB30057 resulted in a concentration-dependent inhibition of thrombin-induced vascular relaxation. All these results suggest that LB30057 is a potent inhibitor of platelet aggregation and blood vessel relaxation induced by thrombin.

• Journal of Pharmaceutical Investigation
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• 제39권5호
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• pp.373-379
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• 2009

This study was conducted to develop a pharmacokinetic-pharmacodynamic (PK/PD) model of a direct thrombin inhibitor, argatroban to predict the concentration-effect profiles in rats. Argatroban was i.v. injected to rats at 0. 2, 0.8 and 3.2 mg/kg doses (n = 4-5 per dose), and plasma drug levels were determined by a validated LC/MS/MS assay. The pharmacokinetics of argatroban was linear over the i.v. dose range studied. The thrombin time (TT) and the activated partial thromboplastin time (aPTT) were measured in rat plasma and they were found to linearly increase with increasing the dose. A 2-compartment pharmacokinetic model linked with an indirect response pharmacodynamic model was successfully utilized to evaluate the drug concentration-response relationship.

• Bulletin of the Korean Chemical Society
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• 제30권1호
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• pp.77-82
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• 2009

Thrombin activatable fibrinolysis inhibitor (TAFI) also known as plasma procarboxypeptidase B or U is a 60 kD glycoprotein, which is the major modulator of fibrinolysis in plasma. TAFI is a proenzyme, which is activated by proteolytic cleavage to an active carboxypeptidase B-like enzyme (TAFIa, 35.8 kD) by thrombin/thrombomodulin and plasmin. Modulation of fibrinolysis occurs when TAFIa enzymatically removes C-terminal lysine residues of partially degraded fibrin, thereby inhibiting the stimulation of tissue plasminogen activator (t-PA) modulated plasminogen activation. TAFIa undergoes a rapid conformational change at $37{^{\circ}C}$ to an inactive isoform called TAFIai. Potato tuber carboxypetidase inhibitor (PTCI) was shown to specifically bind to TAFIa as well as TAFIai. In this study, a novel immunoassay TAFIa/ai ELISA was used for quantitation of the two TAFI activation isoforms TAFIa and TAFIai. The ELISA utilizes PTCI as the capture agent and a double antibody sandwich technique for the detection. Low levels of TAFIa/ai antigen levels were detected in normal plasma and elevated levels were found in hemophilia A plasmas. TAFIa/ai antigen represents a novel marker to monitor fibrinolysis and TAFIa/ai ELISA may be a valuable assay for studying the role of TAFI in normal hemostasis and in pathological conditions.

• 생약학회지
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• 제36권4호통권143호
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• pp.263-272
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• 2005

To develop safe and new anticoagulation agents from natural resources, the inhibitory activities of 291 methanol extracts, which were prepared from different parts of 197 medicinal and wild plants, against human thrombin were evaluated. Based in anti-coagulation activity determined by thrombin time and activated partial thromboplastin time, the extract of leaf of Myrica rubra was finally selected. The extract of M. rubra showed a strong thrombin inhibitory activity (above 1,819%) at 0.5 mg/ml as a final concentration, whereas aspirin showed 337% inhibition at concentration of 1.5 mg/ml. The activity of the extract remained more than 85% and 60% by heat treatment at $100^{\circ}C$ for 30min, and acid treatment at pH 2 for 60 min, respectively. Our results suggested that the extract of Myrica rubra could be the potential source as thrombin inhibitor.

• The Korean Journal of Physiology and Pharmacology
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• 제15권4호
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• pp.211-216
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• 2011

Glioblastoma multiforme is one of the most common and aggressive tumors in central nervous system. It often possesses characteristic necrotic lesions with hemorrhages, which increase the chances of exposure to thrombin. Thrombin has been known as a regulator of MMP-9 expression and cancer cell migration. However, the effects of thrombin on glioma cells have not been clearly understood. In the present study, influences of thrombin on glioma cell migration were examined using Boyden chamber migration assay and thrombin-induced changes in MMP-9 expression were measured using zymography, semi-quantitative RT-PCR, and Western blotting. Furthermore, underlying signaling pathways by which thrombin induces MMP-9 expression were examined. Thrombin-induced migration and MMP-9 expression were significantly potentiated in the presence of wortmannin, a PI3K inhibitor, whereas MAPK inhibitors suppressed thrombin-induced migration and MMP-9 expression in C6 glioma cells. The present data strongly demonstrate that MAPK and PI3K pathways evidently regulate thrombin-induced migration and MMP-9 expression of C6 glioma cells. Therefore, the control of these pathways might be a beneficial therapeutic strategy for treatment of invasive glioblastoma multiforme.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1995년도 한국생물과학협회 학술발표대회
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• pp.299.2-299
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• 1995

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