Nobuya Tanaka

Suppression of plasminogen activator inhibitor 1 release by fibrin from human umbilical vein endothelial cells

The effect of fibrin stimulation on the fibrinolytic potential in cultured human umbilical vein endothelial cells (HUVEC) was investigated in the normal state and aged state. The amount of antigen of the two fibrinolytic factors, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), was determined using ELISA and the ABC method, respectively. When a fibrin clot was overlayered on the normal HUVEC, the secretion of t-PA or PAI-1 from the HUVEC was greatly changed. That is, PAI antigen was decreased 3-fold and t-PA antigen increased slightly in the conditioned medium. On the other hand, when the aged HUVEC were stimulated by a fibrin clot, PAI antigen was increased 3-fold and t-PA antigen did not change in the conditioned medium. When the level of fibrinolytic activity in the conditioned medium was expressed as the molar ratio of PAI and t-PA (PAI/t-PA), the value in the fibrin-stimulated normal HUVEC was markedly reduced (a 3.5-fold decrease) when compared with that of the non-stimulated normal HUVEC, reflecting a profibrinolytic state. On the other hand, the value in the fibrin-stimulated aged HUVEC was markedly increased (a 5-fold increase) when compared with that of the non-stimulated aged HUVEC, reflecting an antifibrinolytic state. Actinomycin D- or cycloheximide-treated HUVEC showed no response to the fibrin stimulation. We conclude that the level of HUVEC-mediated fibrinolytic activity was regulated mainly by the production and secretion of PAI from the HUVEC to protect against the generation of thrombi. In the aged HUVEC, the regulatory mechanism acts in an opposite manner and a thrombotic process may be induced.

Monoclonal antibody interferes with fibrin binding of t-PA

Tissue-type plasminogen activator (t-PA) has a high affinity for fibrin, which is in contrast to urokinase-type plasminogen activator (u-PA). The relation between the structure and function of t-PA was investigated using monoclonal antibodies and plasmin digested t-PA fragments. The results obtained indicated that the three dimensional structure of kringle 2 is necessary for t-PA to develop its fibrin affinity. The monoclonal antibodies which interfered with the fibrin binding ability of native t-PA had two separate epitopes, one in kringle 2 and other in the N-terminal region of the light chain of t-PA.

Fibrinolytic factors, matrix metalloprotease-1, and tissue inhibitor of metalloproteinase-1 in gastric carcinoma

Abstract The invasion and metastasis of carcinoma cells require the action of tumor-associated proteases and their inhibitors, which may also may be involved in the stages of carcinogenesis. In the present study, we evaluated the levels of fibrinolytic factors, urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1), and u-PA specific receptor (u-PAR) as well as matrix metalloprotease-1 (MMP-1) and its tissue inhibitor of metalloprotease-1 (TIMP-1) in extracts of human gastric carcinoma tissues and their corresponding normal mucosas. The antigen levels of u-PA, PAI-1, and TIMP-1 were significantly higher in carcinomas than normal mucosas, while those of u-PAR were not changed as much. Thus, the increase of u-PAR ligand u-PA and its inhibitor PAI-1 may be important in carcinoma tissues. In addition, the amidolytic activity with S-2444 of carcinomas was significantly higher than that of normal tissue indicating that u-PA but not t-PA was involved in the fibrinolytic activity of carcinoma tissues. However, the relationships between any two factors among these components were not clear. From these findings, it is also confirmed that u-PA and PAI-1 are the major regulators of human gastric cancer cells. In addition, since both MMP-1 and TIMP-1 were found to be higher in the carcinomas than the normal mucosas, it is suggested that this matrix enzyme and inhibitor are also up-regulated in carcinomas, the pathway of which is independently linked to fibrinolytic system.