Sequential Formation Of Des-A Fibrin And Des-Ab Fibrin And Binding Of Fibrinogen To Des-Ab Fibrin

1981 ◽  
Author(s):  
G Müller-Berghaus ◽  
J C Bernhard ◽  
H Hofmann ◽  
I Mahn ◽  
F R Ostheimer ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Weak Interaction ◽  
Gel Filtration ◽  
Clot Formation ◽  
Soluble Fibrin ◽  
Low Concentration ◽  
Thrombin Cleavage ◽  
Physico Chemical ◽  
Sequential Formation

Thrombin cleaves the fibrinopeptides A and B from fibrinogen in a consecutive manner, producing first des-A fibrin and consequently, des-AB fibrin. Physico-chemical and in-vivo properties of both types of fibrin were comparatively studied by gel filtration, affinity chromatography and in-vivo behavior. At 37°C and in the presence of fibrinogen and Ca++, des-A fibrin formed soluble fibrin-fibrin dimers. Des-AB fibrin, however, formed oligomers at 37°C. Fibrin-fibrinogen interactions were studied by affinity chromatography. At 37°C, des-A fibrin did not interact with immobilized fibrinogen, whereas des-AB fibrin stuck to it. Small amounts of des-A fibrin iv injected were cleared from the circulation much faster than des-AB fibrin. Large amounts of des-A fibrin injected into animals aggregated to microclots. These results suggest that des-A fibrin in a low concentration (0.01 mg/ml) does not form circulating oligomers and can very fast be cleared from the circulation possibly because of its weak interaction with fibrinogen. Aggregated des-A fibrin, however, is cleaved to form des-AB fibrin. Des-AB fibrin binds fibrinogen making it accessible to further thrombin cleavage. Thus, the threshold of clot formation would be the concentration of des-A fibrin in plasma. The preferential sequence of reactions is: fibrinogen → des-A fibrin → des-A fibrin aggregates → des-AB fibrin aggregates. Des-AB fibrin + fibrinogen → des-AB fibrin-fibrinogen → des-AB fibrin-des-A fibrin etc.

scholarly journals Complex Formation of Covalently Crosslinked Fibrin Oligomers with Agarose Coupled Fibrinogen and Fibrin

1977 ◽  
Author(s):  
R. von Hugo ◽  
R. Hafter ◽  
A. Stemberger ◽  
H. Graeff
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
High Molecular Weight ◽  
Calcium Chloride ◽  
Gel Filtration ◽  
Void Volume ◽  
Soluble Fibrin ◽  
Derivatives Of

Crosslinked high molecular weight derivatives of fibrin (fibrinoligomers) were observed during intravascular coagulation. It was the purpose of this study to investigate the complex formation of fibrin oligomers with fibrinogen and fibrinmonomer. Fibrinogen coupled to agarose (Fg-ag) as well as fi-brinmonomer coupled to agarose (Fm-ag) was used as substrate. Soluble oligomers of fibrin were produced by incubating fibrinogen with thrombin, calcium-chloride, cystein and F XIII. They were separated from fibrinogen by gel filtration. Γ-dimers were demonstrated in fractions from the void volume and the shoulder prior to the fibrinogen peak. These fractions were subjected to affinity chromatography. Crosslinked oligomers of fibrin were not adsorbed on Fg-ag, yet adsorption occured on Fm-ag. This indicates that fibrin oligomers have no affinity to fibrinogen, yet readily form complexes with fibrin. This could mean that in vivo they compete with fibrinogen for the fibrinmonomer part of soluble fibrin monomer complexes, and hence have a tendency to increase in size.

scholarly journals The Effect of Fibrinopeptide B Release on Temperature Dependent Fibrin Association

1977 ◽  
Author(s):  
W. Edgar ◽  
C.R.M. Prentice
Keyword(s):  
Affinity Chromatography ◽  
Clot Formation ◽  
Effect Of Temperature ◽  
Fibrinopeptide A ◽  
Temperature Dependent ◽  
Fibrin Polymerization ◽  
Soluble Fibrin ◽  
Agkistrodon Contortrix ◽  
The Stability ◽  
Sepharose 4B

The effect of temperature on soluble fibrin complexes was studied using Biogel filtration and chromatography on fibrinogen-sepharose 4B at 20°C and 37°C. Complexes were formed by plasmin digestion of non-crosslinked fibrin produced by thrombin, ancrod, thrombin in 2M urea, or ancrod plus Agkistrodon contortrix venom. Thrombin removes fibrinopeptides A and B, ancrod removes fibrinopeptide A, while A. contortrix enzyme removes first fibrinopeptide B, followed by A. Complexes containing neither fibrinopeptide A or B, formed by digestion of fibrin produced by thrombin, or by ancrod plus Agkistrodon contortrix, were stable at 37°C. In contrast, complexes which retained fibrinopeptide B, formed from fibrin produced by ancrod or by thrombin in 2M urea, were unstable at 37°C. Fibrin polymerization was necessary for the stability of fibrin complexes. Complexes from plasmin digests of fibrin produced by ancrod plus A. contortrix enzyme in 2M urea, where no clot formation occurred, were unstable at 37°C. Using affinity chromatography, plasmin digests of thrombin-fibrin bound to fibrinogen-sepharose at 37°C, whereas those from ancrod-fibrin did not. A second set of polymerization sites in fibrinogen are proposed, distinct from the N-DSK and carboxyterminal sites. These are activated by removal of fibrinopeptide B and require clot formation.

Physico-Chemical Studies On Oligosaccharides From Procine Mucosal Heparin

1981 ◽  
Author(s):  
J Choay ◽  
J C Lormeau ◽  
M Petitou ◽  
G Torri ◽  
P Sinay
Keyword(s):  
Nitrous Acid ◽  
Additional Data ◽  
Gel Filtration ◽  
Biologically Active ◽  
Low Molecular Weight ◽  
Nmr Analysis ◽  
Structure Activity ◽  
Physico Chemical ◽  
Chemical Studies

Our recent studies have revealed that low molecular weight antithrombotic (in vivo) and anti Xa fractions and fragments can be obtained from porcine mucosal heparin (PMH) using three different processes: 1) Direct alcoholic fractionations, 2) depolymerization with nitrous acid and 3) by bacterial heparinase digestion. Ihe crude products are further fractionated by anti thrombin-III affinity chromatography and gel filtration. Physiocochemical studies were carried on each fragment employing nitrous acid degradation, colorimetric and NMR analysis. A potent antithrombotic octasaccharide (h-ULMF) is isolated from heparinase depolymerized fractions and in the NMR analysis gave some unexpected signals one of which was consistant to a 3-0-sulfated glucosamine unit (unsaturated sulfated uronic acid/N-Sulfo-Glucosamine/Iduronic acid/N- Acetyl-G1ucosamine/Glucouronic acid/N-Sulfoglucosami ne/Sulfated Idouronic acid/N-Sulfo-Glucosamine). ULMFs obtained by extraction and clemical degradation were also found to exhibit similar signals. Further degradation of h-ULMF-8 resulted in formation of smaller fragments with biological activity. Our results suggest that a smaller oligosaccharide fragment contained in the biologically active octasaccharide molecule is primarily responsible for the antithrombotic actions. Furthermore these studies provide additional data on the structure activity relationship for the antithrombotic actions of heparin, its fraction and fragments.

scholarly journals Macrophage-derived neutrophil chemotactic factor is involved in the neutrophil recruitment inhibitory activity present in the supernatants of LPS-stimulated macrophages

1996 ◽  
Vol 5 (2) ◽  
pp. 116-120 ◽  
Author(s):  
B. M. Tavares-Murta ◽  
F. Q. Cunha ◽  
M. Dias-Baruffi ◽  
M. C. Roque-Barreira ◽  
S. H. Ferreira
Keyword(s):  
Affinity Chromatography ◽  
Inhibitory Activity ◽  
Neutrophil Migration ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Neutrophil Recruitment ◽  
Chemotactic Factor ◽  
Tnf Α ◽  
Neutrophil Chemotactic Factor

In a previous study, we demonstrated the presence of a neutrophil recruitment inhibitory factor (NRIF) in the supernatants of LPS-stimulated macrophages. Recently, the purification of a 54 kDa protein, identified as the macrophage-derived neutrophil chemotactic factor (MNCF) was reported. Since NRIF and MNCF are obtained under the same conditions, and, since the intravenous administration of TNF-α and IL-8 inhibits neutrophil migration, we have investigated whether MNCF could be responsible for this inhibitory activity. After affinity chromatography of the macrophage supernatants on a D-galactose column, the inhibitory activity was recovered in both the unbound (D-gal−) and bound (D-gal+) fractions, with MNCF being found in the D-gal+fraction. Further gel filtration of the latter on Superdex 75 yielded a single peak containing both activities. In a cytotoxicity assay, most of the TNF found in the crude supernatants was recovered in the D-gal−fraction. Furthermore, the incubation of the D-gal−fraction with anti-TNF-α plus anti-IL-8 antisera partially prevents its inhibitory effect on neutrophil migration, but had no effect on the D-gal+activity. Overall, these results suggest that the D-gal−inhibitory effect is partially mediated by TNF-α and IL-8, and that MNCF accounts for the inhibition of neutrophil migrationin vivoby the D-gal+fraction.

Characterization of the human proliferating cell nuclear antigen physico-chemical properties: aspects of double trimer stability

2006 ◽  
Vol 84 (5) ◽  
pp. 669-676 ◽  
Author(s):  
Stanislav N. Naryzhny ◽  
Leroi V. DeSouza ◽  
K.W. Michael Siu ◽  
Hoyun Lee
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Wide Spectrum ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Nuclear Antigen ◽  
Physico Chemical ◽  
The Stability ◽  
Proliferating Cell

Its toroidal structure allows the proliferating cell nuclear antigen (PCNA) to wrap around and move along the DNA fiber, thereby dramatically increasing the processivity of DNA polymerization. PCNA is also involved in the regulation of a wide spectrum of other biological functions, including epigenetic inheritance. We have recently reported that mammalian PCNA forms a double trimer complex, which may be critically important in coordinating DNA replication and other cellular functions. To gain a better understanding of the stability of PCNA complexes, we characterized the physico-chemical properties of the PCNA structure by in vivo and in vitro approaches. The data obtained by gel filtration and nondenaturing gel electrophoresis of native PCNA molecules confirm our previous observations, obtained using formaldehyde crosslinking, in which PCNA exists in the cell as a double trimer. We have also found that optimal pH (pH 6.5–7.5) is critical for the stability of the PCNA structure. The presence or absence of ATP, dithiothreitol, and Mg2+ does not affect the stability of the PCNA trimer or double trimer. However, 0.02% SDS can effectively inhibit PCNA double trimer, but not single trimer, formation. Interestingly, glycerol and ammonium sulfate significantly destabilize both PCNA trimer and double trimer structures.

Lysine-Binding Heterogeneity of Lp(a): Consequences for Fibrin Binding and Inhibition of Plasminogen Activation

1992 ◽  
Vol 68 (02) ◽  
pp. 185-188 ◽  
Author(s):  
C Bas Leerink ◽  
Pieter F C C M Duif ◽  
Joke A Gimpel ◽  
Wouter Kortlandt ◽  
Bonno N Bouma ◽  
...  
Keyword(s):  
Risk Factor ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Apolipoprotein B ◽  
Independent Risk Factor ◽  
Gel Filtration ◽  
Plasminogen Activation ◽  
High Homology

SummaryLipoprotein(a) [Lp(a)] is recognized as an independent risk factor for atherosclerosis. Lp(a) consists of a LDL-like moiety with an additional glycoprotein, apo(a), linked to apolipoprotein B-100. Apo(a) has a high homology with plasminogen (Pg). In vivo, Pg is activated on a fibrin surface by tissue Pg activator (tPA). We prepared Lp(a) from plasma by sequential ultracentrifugation followed by lysine-sepharose affinity chromatography. We found that a changing (donor dependent) fraction of the Lp(a) did not bind to lysine-sepharose. This fraction, designated Lp(a)lys–, was further purified using gel filtration. Bound Lp(a) [Lp(a)lys+] was eluted with 0.2 M EACA. Apo(a) isoforms in both fractions were identical. In contrast Lp(a)lys+ inhibited Pg activation by tPA in vitro (IC50% 20 mg/1), whereas Lp(a)lys– did not. In addition Lp(a)lys– did not bind to CNBr-digested fibrinogen whereas Lp(a)lys+ did (K d, app = 0.2 nM). Therefore we conclude that a changing donor dependent fraction of human plasma Lp(a) does not inhibit Pg activation in vitro and does not bind to CNBr-digested fibrinogen.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

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