scholarly journals Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 301-306
Author(s):  
M Ogura ◽  
N Tanabe ◽  
J Nishioka ◽  
K Suzuki ◽  
H Saito
Keyword(s):  
Cell Line ◽  
Plasma Protein ◽  
Culture Medium ◽  
De Novo ◽  
Specific Activity ◽  
Calf Serum ◽  
Protein S ◽  
Immunoblot Analysis ◽  
Functional Assay ◽  
Functional Protein

A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

scholarly journals Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 301-306 ◽  
Author(s):  
M Ogura ◽  
N Tanabe ◽  
J Nishioka ◽  
K Suzuki ◽  
H Saito
Keyword(s):  
Cell Line ◽  
Plasma Protein ◽  
Culture Medium ◽  
De Novo ◽  
Specific Activity ◽  
Calf Serum ◽  
Protein S ◽  
Immunoblot Analysis ◽  
Functional Assay ◽  
Functional Protein

Abstract A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

BIOSYNTHESIS AND SECRETION OF β-THROMBOGLOBULIN BY A HUMAN MEGAKARYOBLASTIC CELL LINE ( MEG-01 )

1987 ◽  
Author(s):  
M Ogura ◽  
N Tanabe ◽  
M Hamaguchi ◽  
T Hotta ◽  
H Saito
Keyword(s):  
Cell Line ◽  
Culture Medium ◽  
De Novo ◽  
Immunoblot Analysis ◽  
De Novo Synthesis ◽  
Serum Free ◽  
Cell Lysates ◽  
Specific Radioimmunoassay ◽  
Immunofluorescent Technique ◽  
Indirect Immunofluorescent

β-Thromboglobulin ( βTG ) is a well known platelet specific a-granular protein, but synthesis of βTG by human megakaryocytes has not been fully proved. A human megakaryoblastic cell line ( MEG-01 ) was investigated for the presence of βJG in the culture medium and cell lysates using a specific radioimmunoassay ( RIA ). The concentration of βTG increased with time in the serum-free culture medium as well as in the cell lysates as shown in the following table.By an indirect immunofluorescent technique using a monospesific rabbit anti serum against human βTG, βTG antigen was detected in MEG-01 cells. Immunoblot analysis of culture medium revealed a single band ( mol wt 8,900 ) that is identical to the band of human plasma βTG. De novo synthesis of βTG was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 h of labeling of the cells with [35S]-methionine as a 8,900 mol wt protein. These results indicate that human megakaryocytes produce βTG, The production of βTG by MEG-01 cells may be useful for the study of megakaryocyte maturation and differentiation.

Functional and Immunologic Protein S in Normal Pregnant Women and in Full-Term Newborns

1989 ◽  
Vol 61 (03) ◽  
pp. 474-478 ◽  
Author(s):  
José A Fernández ◽  
Amparo Estellés ◽  
Juan Gilabert ◽  
Francisco España ◽  
Justo Aznar
Keyword(s):  
Activated Protein C ◽  
Protein S ◽  
Test Sample ◽  
Full Term ◽  
Functional Assay ◽  
Major Protein ◽  
Functional Protein ◽  
Free Protein ◽  
Term Newborns ◽  
Protein S Activity

SummaryTotal and free protein S antigen and C4b-binding protein (C4bp) were determined by rocket immuno-electrophoresis, and functional protein S was assayed by a coagulation method, throughout pregnancy and normal puerperium and in a group of normal full-term newborns (FTN). The functional protein S assay is based on a modification of the APTT, using a mixture of test sample, protein S deficient plasma, activated protein C, phospholipids and calcium. This protein S functional assay is specific for protein S since the APTT prolongation by normal plasma was abolished by incubation of plasma with monospecific, rabbit antiprotein S IgG. The ratios of functional protein S/free protein S antigen in healthy men (n = 13) and women (n = 14) were 1.0 ± 0.13 (mean ± SD) and 1.03 ± 0.20, respectively. During pregnancy there is a decrease in functional protein S and a progressive decrease in total and free protein S antigen, with a functional/free protein S ratio of 0.75 ± 0.28 in the third trimester of pregnancy (n = 16). In early puerperium the functional protein S level was lower than the free protein S antigen level (ratio about 0.5). In the FTN group, the free protein S level was 39% and protein S activity was about 70% that of adults, with a functional/free protein S ratio of 1.84 ± 0.31. C4bp values were 23.5 ± 10.3% in the FTN group, and crossed immunoelectrophoresis showed that in this group the major protein S peak corresponded to free protein S. These results indicate that both in early puerperium and in FTN group, free protein S antigen may not be an adequate parameter for estimating of functional protein S activity. The decrease in functional protein S activity during early puerperium may be connected with the risk of developing thrombotic episodes during the postpartum period.

scholarly journals Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1081-1081
Author(s):  
Oblaise Mercury ◽  
Lucy Liu ◽  
Ayman Ismail ◽  
Ming Zhang ◽  
Qi Lu ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Culture Medium ◽  
Specific Activity ◽  
Protein S ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Hek293 Cells ◽  
Affinity Column ◽  
Factor X

Abstract Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity. Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content. Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column. Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible). Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. Disclosures Mercury: Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

Resistance to Activated Protein C in Nine Thrombophilic Families: Interference in a Protein S Functional Assay

1993 ◽  
Vol 70 (06) ◽  
pp. 1067-1071 ◽  
Author(s):  
E M Faioni ◽  
F Franchi ◽  
D Asti ◽  
E Sacchi ◽  
F Bernardi ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Abnormal Behavior ◽  
Gene Marker ◽  
Functional Assay ◽  
Polymorphism Analysis ◽  
Functional Protein ◽  
Response Curves ◽  
First Degree Relatives

SummaryNine thrombophilic patients who had had previous diagnoses of functional protein S deficiency were reinvestigated. The functional protein S assays gave dose-response curves that were not parallel to those of the reference plasma. The same pattern was true for approximately half of the first-degree relatives of the propositi. When protein S was extracted from the plasma of the patients by immunoabsorption, it had a normal ratio of functional activity to immunologic concentration. Restriction fragment length polymorphism analysis, informative in one family, showed no linkage between the protein S gene marker and the abnormal behavior of the protein S functional assay. All the propositi and 23/36 first-degree relatives were resistant to the prolongation of activated partial thromboplastin time induced by activated protein C. Furthermore, there was striking concordance in all patients and relatives between the abnormal pattern of the protein S functional assay and resistance to activated protein C. We conclude that a plasma-based functional protein S assay is sensitive to activated protein C resistance and this may lead to spuriously low results in the assay. In agreement with the results of others, this study indicates that resistance to activated protein C is a frequent hemostatic defect in selected thrombophilic populations.

scholarly journals Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein.

1992 ◽  
Vol 175 (6) ◽  
pp. 1697-1705 ◽  
Author(s):  
J D Lee ◽  
K Kato ◽  
P S Tobias ◽  
T N Kirkland ◽  
R J Ulevitch
Keyword(s):  
Membrane Protein ◽  
Cell Line ◽  
Plasma Protein ◽  
Binding Protein ◽  
Protein S ◽  
Response Curves ◽  
Membrane Expression ◽  
Glycosyl Phosphatidylinositol ◽  
Stimulation Of ◽  
Lps Binding

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.

scholarly journals An abnormal plasma distribution of protein S occurs in functional protein S deficiency

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 504-508 ◽  
Author(s):  
PC Comp ◽  
D Doray ◽  
D Patton ◽  
CT Esmon
Keyword(s):  
Binding Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Free Form ◽  
Functional Assay ◽  
Type I ◽  
Protein S Deficiency ◽  
Functional Protein ◽  
S Deficiency ◽  
Disease Protein

Protein S is a natural anticoagulant present in the plasma that serves as a cofactor for activated protein C. Patients deficient in protein S are subject to recurrent venous thrombotic disease. Protein S deficiency differs from other plasma protein deficiencies in that deficient patients often have normal or only mildly reduced levels of protein S in their plasma as detected by conventional immunologic methods but have markedly reduced functional protein S levels. This apparent discrepancy is due to the presence of two forms of protein S in plasma. The protein S is present free and in a complex with C4b- binding protein. The free form is functionally active, whereas the bound form is not. Examination by crossed immunoelectrophoresis of 31 functionally protein S-deficient individuals from seven families reveals that 29 of the 31 have all or most of their protein S complexed to C4b-binding protein with little or no free protein and have correspondingly low levels of protein S functional activity (type I deficiency). Two related protein S-deficient individuals show a different type of distribution with little or no protein S, either bound or free (type II deficiency). The detection and classification of protein S-deficient individuals requires the application of both a functional assay and an assessment of protein S distribution between bound and free forms.

scholarly journals An abnormal plasma distribution of protein S occurs in functional protein S deficiency

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 504-508 ◽  
Author(s):  
PC Comp ◽  
D Doray ◽  
D Patton ◽  
CT Esmon
Keyword(s):  
Binding Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Free Form ◽  
Functional Assay ◽  
Type I ◽  
Protein S Deficiency ◽  
Functional Protein ◽  
S Deficiency ◽  
Disease Protein

Abstract Protein S is a natural anticoagulant present in the plasma that serves as a cofactor for activated protein C. Patients deficient in protein S are subject to recurrent venous thrombotic disease. Protein S deficiency differs from other plasma protein deficiencies in that deficient patients often have normal or only mildly reduced levels of protein S in their plasma as detected by conventional immunologic methods but have markedly reduced functional protein S levels. This apparent discrepancy is due to the presence of two forms of protein S in plasma. The protein S is present free and in a complex with C4b- binding protein. The free form is functionally active, whereas the bound form is not. Examination by crossed immunoelectrophoresis of 31 functionally protein S-deficient individuals from seven families reveals that 29 of the 31 have all or most of their protein S complexed to C4b-binding protein with little or no free protein and have correspondingly low levels of protein S functional activity (type I deficiency). Two related protein S-deficient individuals show a different type of distribution with little or no protein S, either bound or free (type II deficiency). The detection and classification of protein S-deficient individuals requires the application of both a functional assay and an assessment of protein S distribution between bound and free forms.

A Functional Protein S and Microlatex Immunoassay for Protein S and C4b-Binding Protein on the Automated Coagulation Laboratory (ACL) 300 Plus

1996 ◽  
Vol 2 (4) ◽  
pp. 268-275 ◽  
Author(s):  
Shinichiro Hirokawa ◽  
Eberhard F. Mammen
Keyword(s):  
Light Scattering ◽  
Functional Activity ◽  
Binding Protein ◽  
Protein S ◽  
Enzyme Linked Immunosorbent Assay ◽  
Functional Assay ◽  
Functional Protein ◽  
Time Saving ◽  
Normal Ranges ◽  
Protein S Activity

Protein S can be determined by functional or immunological assays. Electroimmunodiffusion (EID) or enzyme immunoassays (enzyme-linked immunosorbent assay; ELISA) are the commonly employed techniques for measuring protein S and C4b-binding protein (C4b- BP) immunologically. Procedures for these assays are time-consuming and labor-intensive. The introduction of microlatex immunoassays (LIATEST system; Diagnos tica Stago, Asnieres-Sur-Seine, France) has provided an alternative for rapid and reliable immunological determi nation. We have placed the microlatex immunoassay for total and free protein S (TPS, FPS) and C4b-BP, using the light-scattering mode, on the Automated Coagulation Laboratory (ACL) 300 Plus (Instrumentation Laboratory, Lexington, MA, U.S.A.). We also placed a functional activity assay of protein S (STACLOT protein S; Amer ican Bioproducts, Parsippany, NJ, U.S.A.) on the ACL 300 Plus. The performance characteristics for the assays yielded a within-run coefficient of variance (CV) of 2.5- 4.6% ( n = 13) for TPS, 4.0-4.8% ( n = 13) for FPS, 1.9- 3.0% ( n = 11) for C4b-BP, and 2.3-5.9% for protein S activity. The interrun CV was 2.1-5.7% ( n = 24), 3.7- 7.0% ( n = 12), 2.6-7.0% ( n = 16), and 4.0-8.4% ( n = 27), respectively. Analytical recovery was 94-109, 97-100, 91-103, and 99-103%, respectively. The normal ranges determined on plasmas from 30 healthy individuals were 113 ± 37 (mean ± 2 SD) for TPS, 106 ± 35 for FSP, 111 ± 22 for C4b-BP, and 107 ± 34 for protein S activity. The results for the microlatex immunoassay and either the EID or the ELISA methods showed excellent correla tions for FPS and C4b-BP; the correlations between LIATEST and either EID or ELISA for TPS were also relatively high. The functional activity of protein S cor related well with FPS. Microlatex immunoassays, using the light-scattering mode for TPS, FPS, or C4b-BP, and the functional assay of protein S can be adapted on the ACL 300 Plus system with a high accuracy and reproduc ibility and with considerable time saving.

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