Synthesis of Factor VIII in Human Hepatocytes in Culture

1988 ◽  
Vol 60 (03) ◽  
pp. 387-391 ◽  
Author(s):  
J Ingerslev ◽  
B Sloth Christiansen ◽  
L Heickendorff ◽  
C Munck Petersen
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Gradient Centrifugation ◽  
Human Fibroblasts ◽  
Human Hepatocytes ◽  
Human Hepatocyte ◽  
Blood Monocytes ◽  
Hep G2 ◽  
Human Hepatoma Cells ◽  
Sds Page

SummaryAlthough several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78–79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all of these cell cultures, vWf : Ag was indetectable or present as trace. Our results suggest that the human hepatocyte is a production site of FVIII.

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Determination of a factor VIII-interactive region within plasmin responsible for plasmin-catalysed activation and inactivation of factor VIII(a)

2010 ◽  
Vol 104 (07) ◽  
pp. 105-117 ◽  
Author(s):  
Katsumi Nishiya ◽  
Kiyotaka Okada ◽  
Osamu Matsuo ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Light Chain ◽  
Catalytic Domain ◽  
Active Form ◽  
Limited Proteolysis ◽  
Sds Page ◽  
A2 Domain ◽  
Domain Independent

SummaryPlasmin, an active form of plasminogen, activates and inactivates factor VIII (FVIII) by limited proteolysis. We have previously identifiedly sine-binding site-independent plasmin-interactive sites on the FVIII A2 domain responsible for cleavages at Arg336 and Arg372, together with lysine-binding site-dependent plasmin sites on the light chain responsible for cleavage at Lys36. We have now characterised FVIII-interactive regions on plasmin. SDS-PAGE analysis demonstrated that a monoclon al antibody (mAb) against kringle (K)5-catalytic domain (K5-CD) of plasmin significantly blocked plasmin-catalysed cleavages at Arg336 and Arg372. K5-CD fragment and this mAb blocked plasmincatalysed activation and inactivation of FVIII(a). Anti-K1–2–3 and anti-K4 mAbs blocked plasmin-catalysed cleavages at Lys36, and K1–2–3 and K4 fragments inhibited plasmin-catalysed inactivation of A11–336 FVIIIa. The K5-CD preferentially bound to the A2 domain (Kd app ; 52 nM), whilst the K1–2–3 and K4 bound to the light chain (Kd app; 75 and 106 nM, respectively) in ELISA. Binding was attributed to the A2 484–509 region and A3 1690–1705/1804–1818 region, respectively. 6-aminohexanoic acid, a lysine analogue, significantly inhibited the light chain/K1–2–3 (and K4) binding by ∼90%, whilst A2/K5-CD binding was moderated by only ∼35%. Furthermore, an anti-CD antibody blocked plasmin-catalysed cleavage by inhibiting the A2/K5-CD interaction. These data demon strate that the K5-CD of plasmin (and plasminogen) interacts with the A2 domain independent of lysine-binding site, whilst interactions of K1–2–3 and K4 with the light chain are lysine-binding site-dependent. Interactions between the K5-CD and A2 likely constitute the major regulatory mechanism for activation and inactivation of FVIII(a) mediated by cleavage at Arg372 and Arg336.

THROMBIN-ACTIVATED FACTOR VIIIa IS COMPOSED OF A NONCOVALENT 73/51 kD DIMER

1987 ◽  
Author(s):  
P J Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Similar Extent ◽  
Factor Viiia ◽  
Polypeptide Patterns ◽  
Human Factor Viii ◽  
Sds Page

Human factor VIII purified from plasma concentrates consists of a series of heterodimers composed of a light chain of 83 kD noncovalently bound to a heavy chain which varies in size from 93 to 170 kD. Previously, we showed that each of the purified heterodimers wasactivated by thrombin to a similar extent. Activation to factor VIIIa was correlated with proteolysis of the light chain generating a73 kD polypeptide and cleavage of the heavy chain(s) generating polypeptides of 51 and 43 kD, whereas subsequent inactivation of factor VIIIa occurred in the absence of further proteolysis (Biochim Biophys Acta 871:268-278, 1986). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced or nonreduced samples showed similar polypeptide patterns indicating that there were no covalent linkages between the 51 and 43 kD chains. However, prior data does not distinguish between a factor VIIIa complex of the 73, 51 and 43 kD polypeptides and a subset of these chains. To identify factor VIIIa, thrombin- treated factor VIII at peak activity was subjected to rapid gel filtration on Superose 12. Factor VIII activity eluted as a single peak representing about 30% of the applied activity after correction for spontaneous inactivation. SDS-PAGE followedby silver staining showed that activity was correlated to fractionscontaining the 73 and 51 kD polypeptides, which co-eluted and which were separated from both the 43 kD fragment and thrombin. Densitometric scans of the stained gel indicated the stoichiometry of the 73:51 kD polypeptides in eachactive fraction to be 1:1. Addition of EDTA(50 mM) to a similar thrombin-factor VIII mixture resulted in rapid inactivation of factor VIIIa. Gel filtration followed by SDS-PAGE analysis of this sample showed that the 73 and 51 kD polypeptides eluted separately and were more included, while the elution position of the 43 kD polypeptide was unchanged. These results suggest that factor VIIIa is represented by a noncovalent dimer consisting of a 73 kD polypeptide derivedfrom the light chain plus a 51 kD polypeptide derived from the heavy chain.

Thrombin Cleavage at Arg1689 Influences Subsequent Cleavage at Arg372 during Activation of Factor VIII

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1021-1021
Author(s):  
Jennifer L. Newell ◽  
Philip Fay
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Specific Activity ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Protein Factor ◽  
Thrombin Cleavage ◽  
Fold Reduction ◽  
Sds Page ◽  
Thrombin Activation

Abstract Hemophilia A is the most common of the severe inherited bleeding disorders and is caused by defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor and is converted to factor VIIIa by thrombin or factor Xa-catalyzed cleavage at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction). Factor VIII cleavage by thrombin is suggested to be an ordered mechanism, with initial cleavage at Arg740 facilitating cleavages at Arg372 and Arg1689 (Newell and Fay, J. Biol. Chem.282: 25376, 2007). The catalytic efficiency for thrombin cleavage at Arg740 is greater than at either Arg1689 or Arg372, while cleavage at Arg372 appears rate-limiting. For this reason, we investigated whether cleavage at Arg1689 influenced catalysis at the Arg372 site. We constructed, stably transfected, and expressed two recombinant factor VIII mutants, Arg1689His and Arg1689Gln, to slow and eliminate, respectively, cleavage at this site. The specific activity of the Arg1689His and Arg1689Gln factor VIII variants were reduced to 47% and 9% the WT factor VIII value, respectively. This result was consistent with impaired cleavage rates of the light chain impacting this parameter. SDS-PAGE and Western blotting analysis of the Arg1689His variant showed a ~150-fold reduction in the rate of cleavage at Arg1689, while the Arg1689Gln variant was resistant to cleavage at this site. Furthermore, the Arg1689His and Arg1689Gln variants showed 2-fold and 7-fold rate reductions, respectively, in the generation of the A2 subunit following thrombin-catalyzed cleavage at Arg372 as compared to WT factor VIII. This result indicated a linkage between cleavage of the light chain and the primary activating cleavage in the factor VIII heavy chain. In monitoring thrombin activation time courses, we observed a 2-fold reduction in the peak activation for the Arg1689His variant and a 12-fold reduction for Arg1689Gln variant compared to wild type factor VIII. To investigate thrombin binding to Arg1689Gln factor VIII, this variant was used as a competitor of thrombin activation of WT factor VIII. The apparent Ki for Arg1689Gln factor VIII (59 nM) was similar to the Km (apparent) of WT factor VIII (55 nM), suggesting that the Arg1689Gln mutation does not impair binding of thrombin to factor VIII. Taken together, these results suggest thrombin cleavage of factor VIII Arg1689 is preferred before cleavage at Arg372 but not required. Since factor Xa also cleaves factor VIII at the same sites as thrombin, we evaluated factor Xa cleavage of factor VIII using SDS-PAGE and Western blot analysis. These results showed that factor Xa cleavage at Arg372, as judged by A2 subunit generation, for both Arg1689 variants was ~2-fold slower than that for WT factor VIII. Moreover, factor Xa cleavage of light chain at the Arg1721 site was reduced by ~2–3 fold for the variants suggesting that factor Xa cleavage at the Arg1689 site is preferred over the Arg1721 site. Together, these results suggest a less selective mechanism for cleavage order using factor Xa as compared with thrombin.

Ultrastructural observations of human monocytes infected with HIV-1

1991 ◽  
Vol 49 ◽  
pp. 108-109
Author(s):  
James K. Koehler ◽  
Steven G. Reed ◽  
Joao S. Silva
Keyword(s):  
Gradient Centrifugation ◽  
Virus Production ◽  
Human Monocyte ◽  
Red Cross ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Hiv Replication ◽  
Electron Microscopic ◽  
Ultrastructural Studies ◽  
Hiv 1

As part of a larger study involving the co-infection of human monocyte cultures with HIV and protozoan parasites, electron microscopic observations were made on the course of HIV replication and infection in these cells. Although several ultrastructural studies of the cytopathology associated with HIV infection have appeared, few studies have shown the details of virus production in “normal,” human monocytes/macrophages, one of the natural targets of the virus, and suspected of being a locus of quiescent virus during its long latent period. In this report, we detail some of the interactions of developing virons with the membranes and organelles of the monocyte host.Peripheral blood monocytes were prepared from buffy coats (Portland Red Cross) by Percoll gradient centrifugation, followed by adherence to cover slips. 90-95% pure monocytes were cultured in RPMI with 5% non-activated human AB serum for four days and infected with 100 TCID50/ml of HIV-1 for four hours, washed and incubated in fresh medium for 14 days.

Detection of both Type 1 and Type 2 Plasminogen Activator Inhibitors in Human Monocytes

1990 ◽  
Vol 63 (01) ◽  
pp. 067-071 ◽  
Author(s):  
Joan C Castellote ◽  
Enric Grau ◽  
Maria A Linde ◽  
Nuria Pujol-Moix ◽  
Miquel LI Rutllant
Keyword(s):  
Molecular Mass ◽  
Plasminogen Activator ◽  
Human Peripheral Blood ◽  
Direct Interaction ◽  
Apparent Molecular Weight ◽  
Fibrinolytic System ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Single Chain ◽  
Sds Page

SummaryIncreasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125Ifibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

scholarly journals Differences in hypolipidaemic effects of two statins on Hep G2 cells or human hepatocytes in primary culture

1996 ◽  
Vol 118 (7) ◽  
pp. 1862-1868 ◽  
Author(s):  
Thierry Clerc ◽  
Véronique Sbarra ◽  
Nicole Domingo ◽  
Jean Philippe Rault ◽  
Nicolas Diaconescu ◽  
...  
Keyword(s):  
Primary Culture ◽  
Human Hepatocytes ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
G2 Cells

Mass isotopomer study of the nonoxidative pathways of the pentose cycle with [1,2-13C2]glucose

1998 ◽  
Vol 274 (5) ◽  
pp. E843-E851 ◽  
Author(s):  
Wai-Nang Paul Lee ◽  
Laszlo G. Boros ◽  
Joaquim Puigjaner ◽  
Sara Bassilian ◽  
Shu Lim ◽  
...  
Keyword(s):  
Pentose Phosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Hep G2 ◽  
Tracer Method ◽  
Human Hepatoma Cells ◽  
Glucose Flux ◽  
Isotopomer Analysis ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Mass Isotopomer

We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227–2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24–72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution ( m 1) and 10% contained two 13C substitutions ( m 2). A similar m 1-to- m 2ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose.13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.

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