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Alicia Lajmanovich Gilbert Hudry-Clergeon Jean-Marie Freyssinet Gérard Marguerie 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,678(1):132-136
The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10–40% w/v saccharose in 0.01 M Tris-HCl/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII : C, VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R : AG and a factor VIII related protein still migrated to the bottom of the tube, while VIII : C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII relateda activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII : C to certain active phospholipids. 相似文献
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Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes 总被引:8,自引:1,他引:7 下载免费PDF全文
A Duperray R Berthier E Chagnon J J Ryckewaert M Ginsberg E Plow G Marguerie 《The Journal of cell biology》1987,104(6):1665-1673
Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected. 相似文献
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G Uzan G Courtois C Besmond M Frain J Sala-Trepat A Kahn G Marguerie 《Biochemical and biophysical research communications》1984,120(2):376-383
Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA. 相似文献
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The influence of Ca2+ on the basic reaction between thrombin and fibrinogen was investigated. The results demonstrate that: (a) A Ca2+-dependent dimeric intermediate is formed during the early step of the clotting process. This dimeric intermeidate is shown to be formed by the association of an intact fibrinogen molecule and a fibrin monomer devoid in only the peptide A, (b) Ca2+ enhances the proteolytic step as illustrated by the measure of the kinetics of H+ release at pH 8.6. On the basis of these observations it is proposed that Ca2+ catalyses the proteolysis of fibrinogen by thrombin through the formation of a Ca2+-dependent dimer. 相似文献
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Differential structural requirements for fibrinogen binding to platelets and to endothelial cells 总被引:3,自引:1,他引:2 下载免费PDF全文
L Tranqui A Andrieux G Hudry-Clergeon J J Ryckewaert S Soyez A Chapel M H Ginsberg E F Plow G Marguerie 《The Journal of cell biology》1989,108(6):2519-2527
The cytoadhesins represent a group of RGD receptors that belongs to the integrin superfamily of adhesion molecules. Members of this cytoadhesin family include the platelet GPIIb-IIIa and the vitronectin receptors. These glycoproteins share the same beta-subunit, which is associated with different alpha subunits to form an alpha/beta heterodimer. In the present study, we have analyzed the fine recognition specificy of the cytoadhesins from platelets and endothelial cells for the adhesive protein, fibrinogen. Two sets of synthetic peptides, RGDX peptides and peptides corresponding to the COOH terminus of the fibrinogen gamma chain, were compared for their structure-function relationships in the two cellular systems. The results indicate that: (a) both RGDX and gamma-chain peptides inhibit the binding of fibrinogen to platelets and endothelial cells; (b) a marked influence of the residue at the COOH- and NH2-terminal positions of each peptide set can be demonstrated on the two types; and (c) RGDX and gamma peptides have differential effects on platelets and endothelial cells with respect to fine structural requirements. These results clearly indicate that while the platelet and endothelial cytoadhesins may interact with similar peptidic sequences, they express a different fine structural recognition. 相似文献
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Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion 总被引:30,自引:0,他引:30
M Ginsberg M D Pierschbacher E Ruoslahti G Marguerie E Plow 《The Journal of biological chemistry》1985,260(7):3931-3936
To define regions within fibronectin (Fn) recognized by platelet binding sites, inhibition of Fn binding by an Fn fragment and synthetic peptides has been analyzed. A highly purified 120-kDa chymotryptic fragment, which has cell attachment activity but did not bind to insolubilized heparin or gelatin, inhibited Fn binding to platelets with an ID50 approximately 3 microM. Previous work indicates that fibroblasts attach to an 11.5-kDa subfragment of this 120-kDa fragment, and that one of four 30-residue synthetic peptides containing sequences from this region supports cell attachment. Only the peptide containing the COOH terminus of the 11.5-kDa fragment inhibited Fn binding to platelets, with an ID50 approximately 10 microM and is the peptide which supports fibroblast attachment. Of the smaller peptides studied from this sequence, all peptides containing the Arg-Gly-Asp-Ser sequence, including the tetrapeptide itself, were active in inhibiting Fn binding to platelets (ID50 values approximately 10-20 microM). The same peptides support fibroblast attachment. Those which lacked this sequence including Gly-Asp-Ser-Pro and Thr-Gly-Arg-Gly (immediately adjacent tetrapeptides) lacked both activities. Further evidence for specificity of inhibition was provided by structurally modified peptides in which substitution of a Glu for Asp abolished inhibitory activity and substitution of Lys for Arg or Ala for Gly reduced activity 6- and 8-fold, respectively. In addition, Arg-Gly-Asp-Ser-containing peptides inhibited the rate and extent of thrombin-induced platelet aggregation. These data suggest that the Arg-Gly-Asp-Ser tetrapeptide contains a recognition specificity involved in the binding of Fn to platelets and that platelets share features of this recognition specificity with fibroblasts. 相似文献
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A number of glycyl-tRNA synthetase (glyS) mutants have been isolated as glycine auxotrophs in Salmonella typhimurium. One of the mutants, glyS141, has a glycyl-tRNA synthetase with a Km for glycine that is 700 times higher than the wild-typeKm. Prototrophic revertants glyS141 occur at high spontaneous frequencies (>5 × 10?5). The majority of these revertants contain large tandem duplications including the mutant glyS gene. Some of the duplications cover at least 22% of the chromosome. The duplications overlap with a large duplication isolated previously by a different selection procedure (Straus &; Hoffmann, 1975). Evidence has been obtained which suggests that formation of the duplications may occur by recA-dependent recombination. The Gly+ phenotype of revertants carrying the duplications does not appear to be explainable simply by the increased gene dosage of glyS. 相似文献