Fractionation of plasmic fibrinogen digest on lysine-agarose. Isolation of two fragments D, fragment E and simultaneous removal of plasmin

Rupp, C; Sievi, R.; Furlan, Miha

doi:10.1016/0049-3848(82)90285-7

Cited by 12 publications

(5 citation statements)

References 11 publications

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“…To remove bound Ca2+, fibrinogen or albumin solutions were dialyzed in 2 mM EDTA overnight and then in EDTA-free (3 times changed) buffer. Isolated plasmic fragments D, and E from fibrinogen (Rupp et al, 1982) and E|_3 from cross-linked fibrin (Olexa et al, 1981) were prepared and electrophoretically identified as originally described. The following absorbance, at 280 nm (A\*m), coefficients were assumed: fibrinogen or fibrin, 15.5 (Mosesson & Sherry, 1966); albumin, 5.3 (Finlayson, 1975); fragments Db 20.8, and E, 10.2 .…”

Section: Methodsmentioning

confidence: 99%

Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen

Galanakis¹,

Lane²,

Simon³

1987

Biochemistry

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We identified a new property of human albumin. It enhances formation of fine fibril (or leptofibril) structures during fibrin gelation, and by nephelometric and electron microscopic measurements, this property is independent of and synergistic with that of fibrinogen. We examined fibrin aggregation using physiologic temperatures and pH and albumin:fibrin concentration ratios below those at which the known accelerating effect on fibrin aggregation occurs. An albumin concentration dependent decrease in gel turbidity maxima was consistently demonstrable in buffers containing or lacking (2-5 mM) CaCl2. This decrease was shown to be induced by albumin preparations which had been exposed to 2 mM ethylene-diaminetetraacetate disodium salt (EDTA), dialyzed, and tested in EDTA-free buffer. A delay in the onset of aggregation was also shown in calcium-lacking buffers by use of either reaggregating fibrin or fibrinogen aggregated with low (0.01-0.05 unit/mL) thrombin concentrations. Rates of fibrin aggregation as well as those of fibrinopeptide release were not affected by albumin, and the decrease in gel absorbance was demonstrable when solubilized fibrin was reaggregated at all final fibrin concentrations (0.2-4 microM) examined. Computed from wavelength dependence turbidity measurements (1 microM fibrin, I = 0.20), albumin decreased the average mass:length ratio from 8.24 X 10(11) to 4.26 X 10(11) daltons/cm, or from that of an approximately six to a three protofibril-thick strand. It also decreased the mean fibril radius from 48.5 to 36.4 nm but had no effect on fibril density. Electron microscopic measurements of cross-sectional fibril widths, performed on sections of glutaraldehyde-fixed gels, disclosed differences between albumin-containing and control gels which were significant by chi 2 analysis (P greater than 0.001). Fibril groups of 7-20- and 21-40-nm width together comprised 77% of fibrils formed in the presence of albumin (n = 251) compared to 30% of controls (n = 309). Conversely, coarser fibrils of 41-60- and 61-97-nm width together comprised 23% of fibrils formed in the presence of albumin and 70% of controls. This albumin effect was demonstrable by use of different monomeric albumin preparations including defatted, undefatted (unexposed and exposed to 60 degrees C, 10 h), chromatographically [gel exclusion and (diethylaminoethyl)cellulose] pure, S-(carboxymethyl)albumin, and S-(N-ethylsuccinimidyl)albumin. Chromatographically isolated albumin oligomers lacked this property, suggesting that a specific site(s) on albumin was (were) required.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Methodsmentioning

confidence: 99%

Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen

Galanakis¹,

Lane²,

Simon³

1987

Biochemistry

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“…The fragment D3 was isolated as described earlier, 14 with a slight modification. Briefly, 2 mL normal or variant fibrinogen (3 mg/mL) were proteolytically degraded for 4 hours at 37°C by the addition of 1 U/mL plasminogen (Kabi, Stockholm, Sweden) and 200 U/mL streptokinase (Behring, Marburg, Germany) in the presence 10 mM EDTA.…”

Section: Purification Of the Fibrinogen Degradation Fragment D3mentioning

confidence: 99%

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

Bolliger-Stucki

Lord

Furlan

2001

Blood

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Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A␣ R16C and ␥ G165R. As seen previously with other heterozygous A␣ R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A␣ 16 C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the ␣-helix content in the variant D3 fragment. It is concluded that the A␣-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the ␥-chain induced a conformational change in the D3 fragment. IntroductionFibrinogen, a soluble plasma glycoprotein of 340 kd, is made up of 2 copies of 3 different polypeptide chains (A␣ 2 , B␤ 2 ,␥ 2 ), linked together with 29 interchain and intrachain disulfide bridges. The molecule is organized in a dimeric fashion consisting of a central E domain containing the amino termini of all 6 polypeptide chains and 2 outer D domains. In the final stage of blood coagulation, thrombin cleaves the fibrinopeptides A and B in a sequential manner from the amino termini of the A␣-and B␤-chains. Cleavage occurs between residues R16 (single-letter amino acid abbreviations) and G17 of the A␣-chain and residues R14 and G15 of the B␤-chain, exposing the A and B polymerization sites. Resultant monomers join together to form 2-stranded, halfstaggered protofibrils. It has been shown that protofibrils result from longitudinal D-D interactions and noncovalent contacts between the A and the a sites. The a site is formed by residues 329, 330, 340, and 364 in the carboxy-terminal part of the ␥-chain. 1,2 Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers that increase progressively in thickness and develop branch points. The evolving network is finally stabilized with covalent bonds by the activated factor XIII, resulting in a clot resistant to mechanical disruption.Dysfibrinogenemia is a heritable disorder characterized by structural mutations in any of the 3 polypeptide cha...

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“…A recent alternate purification scheme using lysineagarose, which gives good yields of Fragments D,, D,, E and X + Y, may prove to be more efficient. 41 FIGURE 3 shows sodium dodecyl sulfate gel electrophoretic analyses of the purified fragments, which were subsequently examined by electron microscopy. Each appears as a single band by this method of analysis and clearly represents the major species of each fragment present in the whole digest.…”

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confidence: 99%

The Relationship of Fibrinogen Structure to Plasminogen Activation and Plasmin Activity During Fibrinolysis*

Lucas

Fretto

McKee

1983

Annals of the New York Academy of Sciences

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Fractionation of plasmic fibrinogen digest on lysine-agarose. Isolation of two fragments D, fragment E and simultaneous removal of plasmin

Cited by 12 publications

References 11 publications

Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen

Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen

Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

The Relationship of Fibrinogen Structure to Plasminogen Activation and Plasmin Activity During Fibrinolysis*

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