Human Fibrinogen Binds Edta And Citrate

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
A Vermond ◽  
J Hermans
Keyword(s):  
Calcium Ions ◽  
Equilibrium Dialysis ◽  
Strong Dependence ◽  
Direct Interaction ◽  
Heat Stability ◽  
Binding Studies ◽  
Human Fibrinogen ◽  
Direct Binding ◽  
Carboxy Terminal ◽  
Fibrinogen Molecule

It was shown that EDTA and citrate make the carboxy-terminal parts of the γ-chains of fibrinogen more susceptible to plasmin degradation. This is an effect independent of the calcium-chelating properties of those compounds. Furthermore, EDTA prolongs the thrombin times decreases the heat stability and causes the formation of abnormal clots.This suggests a direct interaction of EDTA (and possibly also of citrate) with the fibrinogen molecule thereby causing a conformational change. The interaction of citrate and EDTA with fibrinogen was measured by direct binding studies (equilibrium dialysis).It was found that at pH 7.5 human fibrinogen binds 3.4 molecules of citrate with Kd = 1.4 x 10-4M or 0.4 molecules of EDTA with Kd = 2.2 x 10-5M. Citrate and EDTA compete for the same binding site(s). No binding of acetate could be demonstrated.The binding of EDTA and citrate shows a strong ^-dependence suggesting a (partly) ionogenic binding between charged areas on the fibrinogen molecule and charged groups on EDTA or citrateOur results support the idea that the binding of EDTA and citrate and the concomitant effect on the plasmin attack are independent of the effect of calcium ions

Factors Influencing The Structure Of Terminal Plasmin Degradation Products Of Human Fibrinogen And Fibrin

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
A Vermond ◽  
F Haverkate
Keyword(s):  
Protective Effect ◽  
Calcium Ions ◽  
Degradation Products ◽  
Iminodiacetic Acid ◽  
Human Fibrinogen ◽  
Factors Influencing ◽  
Separate Effect ◽  
The Media ◽  
Carboxy Terminal ◽  
D Dimer

Experiments have been carried out with fibrinogen and with purified degradation products of fibrinogen and fibrin which demonstrate that the structure of D fragments obtained after prolonged plasmin digestion is influenced by several factors in the media.The previously described protective effect of calcium ions on the γ-chain carboxy-terminals of fibrinogen against plasmin attack is rather independent of the composition of the media (e.g., also observed in 2 M urea and/or high plasmin activities).Several compounds such as EDTA, EGTA, citrate and iminodiacetic acid appear to have a separate effect, which is best observed at low plasmin concentrations and in the absence of Ca2+ . Under these conditions, these compounds appear to make the γ-chain carboxy-terminal ends of the D- and D-dimer fragments more susceptible to plasmin digestion.Finally, as demonstrated by experiments with purified D;E complexes from fibrinogen and with whole fibrinogen digests, the E-moiety of the D:E complexes appears to be capable of protecting the D-moiety against low plasmin concentrations also in the absence of calcium ions.

Binding Studies of Bile Acids using the Native Fluorescence of the Tryptophan Residue Of Bax Protein

2006 ◽  
Vol 26 (3) ◽  
pp. 245-250 ◽  
Author(s):  
Wei Zhang ◽  
Clifford J. Steer ◽  
Kenneth T. Douglas ◽  
Cecilia M. P. Rodrigues
Keyword(s):  
Bile Acids ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Potential Interaction ◽  
Tryptophan Residue ◽  
Binding Studies ◽  
Native Fluorescence ◽  
Bax Protein ◽  
Tryptophan Residues ◽  
Direct Binding

Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA), play a unique role in modulating the apoptotic threshold in cells. The mechanism is thought to involve, in part, inhibition of translocation for Bax from the cytosol to mitochondria. Here, we attempted to use the native fluorescence of the tryptophan residues of Bax to determine whether bile acids bind directly to recombinant Bax protein. The results showed that UDCA had no effect on the tryptophan fluorescence of Bax. Similarly, there was no evidence of direct binding between Bax protein and the more hydrophobic bile acid, deoxycholic acid (DCA). In contrast, the fluorescence change detected for Bax solution titrated against TUDCA in dimethylsulfoxide was greater than that observed with solvent alone. In conclusion, data from fluorescence spectroscopy does not support a direct interaction of UDCA or DCA with Bax protein, whereas it suggests that there may be some potential interaction with TUDCA.

scholarly journals Significance of the intact polypeptide chains of human fibrinogen in ADP-induced platelet aggregation

Blood ◽  
1977 ◽  
Vol 49 (4) ◽  
pp. 635-644
Author(s):  
S Niewiarowski ◽  
AZ Budzynski ◽  
B Lipinski
Keyword(s):  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Terminal Part ◽  
Human Fibrinogen ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Stage 1 ◽  
Polypeptide Chains ◽  
Fibrinogen Molecule

The presence of human fibrinogen in suspensions of washed human platelets is a requirement for ADP-induced platelet aggregation. Digestion of fibrinogen with plasmin destroys this function of the protein. The high solubility fraction of Kabi fibrinogen, fragment X (stage 1) and framgent X (stage 2), are two, eight, and ten times, respectively, less potent in promoting ADP-induced platelet aggregation, as compared with intact fibrinogen. Fragments Y and D and the mixture of reduced and carboxymethylated chains of human fibrinogen do not support ADP-induced platelet aggregation at all. SDS polyacrylamide gel electrophoresis of nonreduced and reduced fibrinogen and its derivatives indicates that the intact fibrinogen molecule is essential for ADP-induced platelet aggregation. It is suggested that the carboxy-terminal part of the Aalpha chain and possibly also the amino-terminal part of the Bbeta chain are required for the platelet aggregation-promoting function of fibrinogen.

scholarly journals Significance of the intact polypeptide chains of human fibrinogen in ADP-induced platelet aggregation

Blood ◽  
1977 ◽  
Vol 49 (4) ◽  
pp. 635-644 ◽  
Author(s):  
S Niewiarowski ◽  
AZ Budzynski ◽  
B Lipinski
Keyword(s):  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Terminal Part ◽  
Human Fibrinogen ◽  
Amino Terminal ◽  
Carboxy Terminal ◽  
Stage 1 ◽  
Polypeptide Chains ◽  
Fibrinogen Molecule

Abstract The presence of human fibrinogen in suspensions of washed human platelets is a requirement for ADP-induced platelet aggregation. Digestion of fibrinogen with plasmin destroys this function of the protein. The high solubility fraction of Kabi fibrinogen, fragment X (stage 1) and framgent X (stage 2), are two, eight, and ten times, respectively, less potent in promoting ADP-induced platelet aggregation, as compared with intact fibrinogen. Fragments Y and D and the mixture of reduced and carboxymethylated chains of human fibrinogen do not support ADP-induced platelet aggregation at all. SDS polyacrylamide gel electrophoresis of nonreduced and reduced fibrinogen and its derivatives indicates that the intact fibrinogen molecule is essential for ADP-induced platelet aggregation. It is suggested that the carboxy-terminal part of the Aalpha chain and possibly also the amino-terminal part of the Bbeta chain are required for the platelet aggregation-promoting function of fibrinogen.

A Plasma Fibrinogen Fraction With High Sialic Acid Content And Eecngated γ-Chains

1981 ◽  
Author(s):  
C Kuyas ◽  
A Haeberli ◽  
P W Straub
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Strong Dependence ◽  
Sialic Acid Content ◽  
Human Fibrinogen ◽  
Single Donor ◽  
Acidic Fraction ◽  
Normal Human ◽  
The One ◽  
Fibrinogen Molecule

Having recently been able to separate Bβ- and γ-chains of human fibrinogen heterogeneous with respect to sialic acid, we were faced with the question whether a carbohydrate heterogeneity might be present in the original native fibrinoge.Using DEAE 52 cellulose and stepwise elution with phosphate buffer human fibrinogen from pooled or single donor plasma could indeed be separated into 3 populations with different sialic acid content. The most acidic fraction contained 30% more sialic acid (8 moles) than the original fibrinogen (6 moles). After reduction the 3 fibrinogen fractions were analyzed by two-dimensional electrophoresis. The first fraction (15%) contained partly degraded Aα-chains and a pattern of Bβ and γ-chains similar to the one described for the main fraction. The latter (70%) consisted of intact Act-chains, two main Bβ- and two main γ-chains and one additional minor band for each chain, all with the previously described carbohydrate heterogeneity.The third, most acidic fibrinogen fraction (15%) revealed an unusual γ-chain pattern. In addition to the two normal chains, two γ-chains with higher mol. wt. and more acidic IEP were found. The Aα- and Bβ-chains did not differ from those of the original fibrinogen. Again a heterogeneity with respect to sialic acid was visible both in the Bβ- and the normal as wsll as the elongated γ-chains. Desialylation of the elongated γ-chains resulted in only partial normalisation of the IEP.The binding of EDTA and citrate shows a strong ^-dependence suggesting a (partly) ionogenic binding between charged areas on the fibrinogen molecule and charged groups on EDTA or citrateIt has thus been possible to attribute the recently described elongated γ-chain variants to a particular sialic acid rich subfraction of normal human fibrinogen and therefore to conclude that the observed fibrinogen heterogeneity is at least to a considerable extent of inter- rather than intramolecular nature.

Anticoagulant And Calcium-Binding Properties Of High Molecular Weight Derivatives Of Human Fibrinogen, Produced By Plasmin (Fragments X)

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
M Gravesen
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Degradation Products ◽  
Amino Acid Sequences ◽  
Binding Properties ◽  
Human Fibrinogen ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Carboxy Terminal

Early plasmin degradation products (fragments X) of human fibrinogen were prepared and purified on Sepharose 6B-CL. X-fragments were characterized with respect to amino-terminal amino acid sequences, polypeptide-chain composition, anticlotting properties and calcium-binding. Amino-terminal amino acids were alanine and tyrosine. The molecular weights of the chains were about 26,000, 58,000 and 48,000 for Aα-, Bβ- and γ-chains, respectively. Fragments X were about 6 times as potent in anticlotting behaviour as D-fragments prepared in the presence of calcium ions. Calcium-binding properties were similar to those of fibrinogen: No differences were observed between fragments X prepared in the presence of calcium ions and those prepared in the presence of EGTA. Results indicate that the carboxy-terminal parts of the Aα-chains of fibrinogen are not involved in calcium-binding and that differences in chain-remnants as observed in late plasmic degradation products (which depend on the presence of calcium ions or EGTA in the incubation medium) are introduced beyond the stage of fragment X-formation.

Properties of Fibrinogen Cleaved by Jararhagin, a Metalloproteinase from the Venom of Bothrops jararaca

1994 ◽  
Vol 72 (02) ◽  
pp. 244-249 ◽  
Author(s):  
Aura S Kamiguti ◽  
Joseph R Slupsky ◽  
Mirko Zuzel ◽  
Charles R M Hay
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Human Fibrinogen ◽  
Activated Platelets ◽  
Bothrops Jararaca ◽  
Platelet Binding ◽  
Fibrin Monomers ◽  
Fibrinogen Molecule

SummaryHaemorrhagic metalloproteinases from Bothrops jararaca and other venoms degrade vessel-wall and plasma proteins involved in platelet plug and fibrin clot formation. These enzymes also cause proteolytic digestion of fibrinogen which has been suggested to cause defective platelet function. Fibrinogen degradation by jararhagin, a metalloproteinase from B. jararaca, and the effect of jararhagin fibrinogenolysis on both platelet aggregation and fibrin clot formation were investigated. Jararhagin was found to cleave human fibrinogen in the C-terminal region of the Aα-chain giving rise to a 285-290 kDa fibrinogen molecule lacking the Aα-chain RGD 572-574 platelet-binding site. Platelet binding and aggregation of ADP-activated platelets is unaffected by this modification. This indicates that the lost site is not essential for platelet aggregation, and that the remaining platelet binding sites located in the N-terminal portion of Aα chains (RGD 95-97) and the C-terminal of γ chains (dodecapeptide 400-411) are unaffected by jararhagin-digestion of fibrinogen. Fibrin clot formation with thrombin of this remnant fibrinogen molecule was defective, with poor polymerization of fibrin monomers but normal release of FPA. The abnormal polymerization could be explained by the loss of one of the two complementary polymerization sites required for side-by-side association of fibrin protofibrils. Jararhagin-induced inhibition of platelet function, an important cause of haemorrhage in envenomed patients, is not caused by proteolysis of fibrinogen, as had been thought, and the mechanism remains to be elucidated.

scholarly journals Interaction of the Putative Human Cytomegalovirus Portal Protein pUL104 with the Large Terminase Subunit pUL56 and Its Inhibition by Benzimidazole-d-Ribonucleosides

2005 ◽  
Vol 79 (23) ◽  
pp. 14660-14667 ◽  
Author(s):  
Alexandra Dittmer ◽  
John C. Drach ◽  
Leroy B. Townsend ◽  
Anke Fischer ◽  
Elke Bogner
Keyword(s):  
Dna Replication ◽  
Human Cytomegalovirus ◽  
Unit Length ◽  
Large Subunit ◽  
Intracellular Localization ◽  
Direct Interaction ◽  
Portal Protein ◽  
Specific Association ◽  
Carboxy Terminal

ABSTRACT Herpesvirus DNA replication leads to unit length genomes that are translocated into preformed procapsids through a unique portal vertex. The translocation is performed by the terminase that cleaves the DNA and powers the insertion by its ATPase activity. Recently, we demonstrated that the putative human cytomegalovirus (HCMV) portal protein, pUL104, also forms high-molecular-weight complexes. Analyses now have been performed to determine the intracellular localization and identification of interaction partners of pUL104. In infected cells, HCMV pUL104 was found to be predominantly localized throughout the nucleus as well as in cytoplasmic clusters at late times of infection. The latter localization was abolished by phosphonoacetic acid, an inhibitor of viral DNA replication. Immunofluorescence revealed that pUL104 colocalized with pUL56, the large subunit of the HCMV terminase. Specific association of in vitro translated pUL104 with the carboxy-terminal half of GST-UL56C was detected. By using coimmunoprecipitations a direct interaction with pUL56 was confirmed. In addition, this interaction was no longer detected when the benzimidazole-d-nucleosides BDCRB or Cl4RB were added, thus indicating that these HCMV inhibitors block the insertion of the DNA into the capsid by preventing a necessary interaction of pUL56 with the portal. Electron microscopy revealed that in the presence of Cl4RB DNA is not packaged into capsids and these capsids failed to egress from the nucleus. Furthermore, pulsed-field gel electrophoresis showed that DNA concatemers synthesized in the presence of the compound failed to be processed.

scholarly journals Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis

1999 ◽  
Vol 277 (4) ◽  
pp. L684-L693 ◽  
Author(s):  
Christine L. Zanella ◽  
Cynthia R. Timblin ◽  
Andrew Cummins ◽  
Michael Jung ◽  
Jonathan Goldberg ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Direct Interaction ◽  
Mrna Levels ◽  
Binding Studies ◽  
Therapeutic Implications ◽  
Asbestos Fibers ◽  
Epidermal Growth

We examined the mechanisms of interaction of crocidolite asbestos fibers with the epidermal growth factor (EGF) receptor (EGFR) and the role of the EGFR-extracellular signal-regulated kinase (ERK) signaling pathway in early-response protooncogene (c- fos/c- jun) expression and apoptosis induced by asbestos in rat pleural mesothelial (RPM) cells. Asbestos fibers, but not the nonfibrous analog riebeckite, abolished binding of EGF to the EGFR. This was not due to a direct interaction of fibers with ligand, inasmuch as binding studies using fibers and EGF in the absence of membranes showed that EGF did not adsorb to the surface of asbestos fibers. Exposure of RPM cells to asbestos caused a greater than twofold increase in steady-state message and protein levels of EGFR ( P < 0.05). The tyrphostin AG-1478, which inhibits the tyrosine kinase activity of the EGFR, but not the tyrphostin A-10, which does not affect EGFR activity, significantly ameliorated asbestos-induced increases in mRNA levels of c- fos but not of c- jun. Pretreatment of RPM cells with AG-1478 significantly reduced apoptosis in cells exposed to asbestos. Our findings suggest that asbestos-induced binding to EGFR initiates signaling pathways responsible for increased expression of the protooncogene c- fos and the development of apoptosis. The ability to block asbestos-induced elevations in c- fos mRNA levels and apoptosis by small-molecule inhibitors of EGFR phosphorylation may have therapeutic implications in asbestos-related diseases.

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