Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

The Glycosaminoglycan of Recombinant Human Soluble Thrombomodulin Affects Antithrombotic Activity in a Rat Model of Tissue Factor-Induced Disseminated Intravascular Coagulation

1992 ◽  
Vol 67 (03) ◽  
pp. 366-370 ◽  
Author(s):  
Katsuhiko Nawa ◽  
Teru Itani ◽  
Mayumi Ono ◽  
Katsu-ichi Sakano ◽  
Yasumasa Marumoto ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Disseminated Intravascular Coagulation ◽  
Rat Model ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Soluble Thrombomodulin ◽  
Intravascular Coagulation ◽  
Platelet Counts ◽  
Recombinant Human Soluble Thrombomodulin

SummaryPrevious studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTMp) or absence (rsTMa) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTMp was more potent than rsTMa for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTMp and 5.0 h for rsTMa. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTMa, 0.07 mg/kg rsTMp, and 7 U/ kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTMa, 3 mg/kg rsTMp or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTMa, 43-fold for rsTMp and 13-fold for heparin. These results suggest that rsTMp is a potent anticoagulant to inhibit the platelet reduction when injected prior to the induction of DIC in rats.

Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface

2002 ◽  
Vol 362 (3) ◽  
pp. 585-595 ◽  
Author(s):  
Irina V. BALYASNIKOVA ◽  
Eric H. KARRAN ◽  
Ronald F. ALBRECHT ◽  
Sergei M. DANILOV
Keyword(s):  
Transmembrane Domain ◽  
Umbilical Vein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integral Membrane Protein ◽  
Converting Enzyme ◽  
Ovary Cells ◽  
Angiotensin I Converting Enzyme ◽  
Hydrophobic Transmembrane Domain ◽  
Umbilical Vein Endothelial Cells

Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.

Overexpression of GADD34 Enhances Production of Recombinant Human Antithrombin III in Chinese Hamster Ovary Cells

2008 ◽  
Vol 106 (6) ◽  
pp. 568-573 ◽  
Author(s):  
Takeshi Omasa ◽  
Takashi Takami ◽  
Tomoshi Ohya ◽  
Eriko Kiyama ◽  
Tetsuji Hayashi ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Antithrombin Iii ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells

scholarly journals Structural requirements for palmitoylation of surfactant protein C precursor

2002 ◽  
Vol 361 (3) ◽  
pp. 663-671 ◽  
Author(s):  
Anja ten BRINKE ◽  
Arie B. VAANDRAGER ◽  
Henk P. HAAGSMAN ◽  
Anja N. J. A. RIDDER ◽  
Lambert M. G. van GOLDE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein C ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Chinese Hamster Ovary Cells ◽  
Membrane Surface ◽  
Chinese Hamster ◽  
Surfactant Protein ◽  
Structural Requirements ◽  
Surfactant Protein C

Pulmonary surfactant protein C (SP-C) propeptide (proSP-C) is a type II transmembrane protein that is palmitoylated on two cysteines adjacent to its transmembrane domain. To study the structural requirements for palmitoylation of proSP-C, His-tagged human proSP-C and mutant forms were expressed in Chinese hamster ovary cells and analysed by metabolic labelling with [3H]palmitate. Mutations were made in the amino acid sequence representing mature SP-C, as deletion of the N- and C-terminal propeptide parts showed that this sequence by itself could already be palmitoylated. Substitution of the transmembrane domain by an artificial transmembrane domain had no effect on palmitoylation. However, an inverse correlation was found between palmitoylation of proSP-C and the number of amino acids present between the cysteines and the transmembrane domain. Moreover, substitution by alanines of amino acids localized on the N-terminal side of the cysteines had drastic effects on palmitoylation, probably as a result of the removal of hydrophobic amino acids. These data, together with the observation that substitution by alanines of the amino acids localized between the cysteines and the transmembrane domain had no effect on palmitoylation, suggest that the palmitoylation of proSP-C depends not on specific sequence motifs, but more on the probability that the cysteine is in the vicinity of the membrane surface. This is probably determined not only by the number of amino acids between the cysteines and the transmembrane domain, but also by the hydrophobic interaction of the N-terminus with the membrane. This may also be the case for the palmitoylation of other transmembrane proteins.

Regulation of intestinal Cl−/HCO3− exchanger SLC26A3 by intracellular pH

2009 ◽  
Vol 296 (6) ◽  
pp. C1279-C1290 ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Kazuhito Suruga ◽  
Yukari Yamashita
Keyword(s):  
Intracellular Ph ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Functional Coupling ◽  
Intestinal Epithelial ◽  
Transport Activity ◽  
Ovary Cells ◽  
Nacl Absorption ◽  
Reverse Mode

SLC26A3, a Cl−/HCO3− exchanger, is highly expressed in intestinal epithelial cells, and its mutations cause congenital chloride diarrhea. This suggests that SLC26A3 plays a key role in NaCl absorption in the intestine. Electroneutral NaCl absorption in the intestine is mediated by functional coupling of the Na+/H+ exchanger and Cl−/HCO3− exchanger. It is proposed that the coupling of these exchangers may occur as a result of indirect linkage by changes of intracellular pH (pHi). We therefore investigated whether SLC26A3 is regulated by pHi. We generated a hemagglutinin epitope-tagged human SLC26A3 construct and expressed it in Chinese hamster ovary cells. Transport activities were measured with a fluorescent chloride-sensitive dye dihydro-6-methoxy- N-ethylquinolinium iodide (diH-MEQ). pHi was clamped at a range of values from 6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse mode of Cl−/HCO3− and Cl−/NO3− exchange. None of these exchange modes induced membrane potential changes. At constant external pH 7.4, Cl−/HCO3− exchange was steeply inhibited with pHi decrease between 7.3 and 6.8 as opposed to thermodynamic prediction. In contrast, however, Cl−/NO3− exchange was essentially insensitive to pHi within physiological ranges. We also characterized the pHi dependency of COOH-terminal truncation mutants. Removal of the entire COOH-terminal resulted in decrease of the transport activity but did not noticeably affect pHi sensitivity. These results suggest that Cl−/HCO3− exchange mode of human SLC26A3 is controlled by a pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that SLC26A3 activity may be regulated via Na+/H+ exchanger 3 (NHE3) through the alteration of pHi under physiological conditions.

Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression

2008 ◽  
Vol 100 (2) ◽  
pp. 317-324 ◽  
Author(s):  
Tomoshi Ohya ◽  
Tetsuji Hayashi ◽  
Eriko Kiyama ◽  
Hiroko Nishii ◽  
Hideo Miki ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Antithrombin Iii ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells
Sign in / Sign up

Export Citation Format

Share Document