Coagulation factor Xa is a plasma serine protease that catalyzes prothrombin to thrombin conversion, which, in turn, leads to the generation of the fibrin clot. Of the several parameters that govern the plasma level of factor Xa, control of its catabolism is of crucial importance. However, little is known regarding the mechanisms by which factor Xa is catabolized. In the present study we examine the cellular basis for the uptake and degradation of factor Xa. 125I-Factor Xa was degraded by hepatoma cells and embryonic fibroblasts via a process which required cell surface-bound tissue factor pathway inhibitor (TFPI), a potent inhibitor of factor Xa. Uptake and degradation of cell surface-bound 125I-TFPI was also markedly stimulated in response to factor Xa binding. The intracellular kinetics of 125I-factor Xa and cell surface-bound 125I-TFPI display a strikingly similar pattern, suggesting that factor Xa and cell surface-bound TFPI are taken up as a bimolecular complex. Using cell lines either deficient in low density lipoprotein receptor-related protein, an endocytic receptor that mediates the degradation of uncomplexed TFPI (Warshawsky, I., Broze, G.J., Jr., and Schwartz, A.L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668), or deficient in tissue factor (TF), an integral membrane protein capable of forming quarternary complexes with factor Xa, TFPI, and factor VIIa, we demonstrated that the receptor that mediates the uptake and degradation of factor Xa-TFPI complex was neither low density lipoprotein receptor-related protein nor TF. As the vascular endothelial cell surface retains a substantial pool of TFPI (Sandset, P.M., Alildgaard, U., and Larsen, M.L. (1988) Thromb. Res. 50, 803-813; Novotny, W.F., Brown, S.G., Miletich, J.P., Rader, D.J., and Broze, G.J., Jr. (1991) Blood 78, 387-393), our data suggest that endothelial cell surface TFPI may be actively involved in the clearance of factor Xa from the circulation via mediated uptake and degradation.
Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to + 1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G+C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Spl. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.Factor VII is a vitamin K-dependent glycoprotein that participates in the initiation of the extrinsic pathway of blood coagulation (1). It is synthesized in the liver as a single-chain zymogen. The most abundant form of the zymogen has a prepro leader sequence of 38 amino acids, while a less abundant form has 60 amino acids (2). The zymogen undergoes Nand 0-glycosylation (3, 4), vitamin K-dependent carboxylation of N-terminal glutamic acids to y-carboxyglutamic acids (2), and hydroxylation of Asp63 to 3-hydroxyaspartic acid (5).Factor VII is converted to the two-chain active form, factor VIla, by cleavage of a single peptide bond between Arg'52 and Ile153 (2,6). Factor VIla associates with tissue factor in the presence of calcium ions to initiate the extrinsic pathway of coagulation following vascular damage.Factor VII deficiency is a rare autosomal recessive disorder that causes severe bleeding and occasionally venous thrombosis. The gene for human factor VII is 12.8 kb long (7) and is 2.8 kb upstream of the factor X gene on chromosome 13q34-qter (8). The factor VII gene shares significant organizational similarity with the genes coding for other vitamin K-dependent proteins (9...
ADP-ribosylation factors (ARFs) regulate coatomer assembly on the Golgi as well as recruitment of clathrin adapter proteins and are therefore involved in vesicle budding from the Golgi and vesicular transport. They are also regulators of phospholipase D (PLD) activity. Arfaptin 1 is an ARF binding protein that inhibits PLD activation, vesicular trafficking and secretion. In the present report, we show that arfaptin 1 interacts with`high speed' membranes independently of ARF. However, addition of myristoylated ARF3 (myrARF3) increases the association of arfaptin 1 with the membranes, suggesting that arfaptin 1 and ARF form a complex on the Golgi. Utilizing several deletion mutants of arfaptin 1 it is shown that the association of arfaptin 1 with myrARF3 is mediated via two binding sites on arfaptin 1. These two domains are needed for arfaptin 1 inhibition of PLD activation by myrARF3 in vitro.z 1999 Federation of European Biochemical Societies.
Tea is widely consumed all over the world. Studies have demonstrated the role of tea in prevention and treatment of various chronic diseases including diabetes and obesity, but the underlying mechanism is unclear. PTP1B is a widely expressed tyrosine phosphatase which has been defined as a target for therapeutic drug development to treat diabetes and obesity. In screening for inhibitors of PTP1B, we found that aqueous extracts of teas exhibited potent PTP1B inhibitory effects with an IC50 value of 0.4 to 4 g dry tea leaves per liter of water. Black tea shows the strongest inhibition activities, followed by oolong and then by green tea. Biochemical fractionations demonstrated that the major effective components in tea corresponded to oxidized polyphenolic compounds. This was further verified by the fact that tea catechins became potent inhibitors of PTP1B upon oxidation catalyzed by tyrosinases. When applied to cultured cells, tea extracts induced tyrosine phosphorylation of cellular proteins. Our study suggests that some beneficial effects of tea may be attributed to the inhibition of PTP1B.
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