scholarly journals The role of sialic acid in the dysfibrinogenemia associated with liver disease: distribution of sialic acid on the constituent chains

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1196-1202 ◽  
Author(s):  
J Martinez ◽  
KA MacDonald ◽  
JE Palascak
Keyword(s):  
Liver Disease ◽  
Sialic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Thrombin Time ◽  
Beta Chain ◽  
Gamma Chain ◽  
Sialic Acid Content ◽  
Fibrin Monomer

To further evaluate the role of sialic acid in the dysfibrinogenemia associated with liver disease, we studied the effect of removal of excess sialic acid residues from the fibrinogen of five patients with liver disease on the thrombin time and fibrin monomer aggregation. Patient fibrinogens containing 1.4–3.4 residues of sialic acid per molecule in excess of normal controls, with thrombin times 12–22 sec longer than normal and with abnormal fibrin monomer aggregation, were stripped of their excess sialic acid by incubation with Vibrio cholerae neuraminidase, followed by rapid removal of the enzyme by antineuraminidase antibody affinity chromatography. These partially desialylated patient fibrinogens, with a normal number of sialic acid residues remaining, exhibited normal thrombin times and normal fibrin monomer aggregation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced normal, patient, and partially desialylated patient (sialyl-3H)-fibrinogen exhibited 60% of the radioactivity in the B beta chain and 40% in the gamma chain. There was no radioactivity detectable in the A alpha chain. These studies provide additional evidence that the increased sialic acid content of the acquired dysfibrinogenemia of liver disease is responsible for its functional defect and that the excess sialic acid is distributed on the B beta chain and gamma chains of the fibrinogen.

scholarly journals The role of sialic acid in the dysfibrinogenemia associated with liver disease: distribution of sialic acid on the constituent chains

Blood ◽  
1983 ◽  
Vol 61 (6) ◽  
pp. 1196-1202 ◽  
Author(s):  
J Martinez ◽  
KA MacDonald ◽  
JE Palascak
Keyword(s):  
Liver Disease ◽  
Sialic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Thrombin Time ◽  
Beta Chain ◽  
Gamma Chain ◽  
Sialic Acid Content ◽  
Fibrin Monomer

Abstract To further evaluate the role of sialic acid in the dysfibrinogenemia associated with liver disease, we studied the effect of removal of excess sialic acid residues from the fibrinogen of five patients with liver disease on the thrombin time and fibrin monomer aggregation. Patient fibrinogens containing 1.4–3.4 residues of sialic acid per molecule in excess of normal controls, with thrombin times 12–22 sec longer than normal and with abnormal fibrin monomer aggregation, were stripped of their excess sialic acid by incubation with Vibrio cholerae neuraminidase, followed by rapid removal of the enzyme by antineuraminidase antibody affinity chromatography. These partially desialylated patient fibrinogens, with a normal number of sialic acid residues remaining, exhibited normal thrombin times and normal fibrin monomer aggregation. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced normal, patient, and partially desialylated patient (sialyl-3H)-fibrinogen exhibited 60% of the radioactivity in the B beta chain and 40% in the gamma chain. There was no radioactivity detectable in the A alpha chain. These studies provide additional evidence that the increased sialic acid content of the acquired dysfibrinogenemia of liver disease is responsible for its functional defect and that the excess sialic acid is distributed on the B beta chain and gamma chains of the fibrinogen.

scholarly journals Role of Sialic Acid in the Dysfibrinogenemia Associated with Liver Disease

1977 ◽  
Author(s):  
J. Martinez ◽  
J. Palascak ◽  
D. Kwasniak ◽  
S.S. Shapiro
Keyword(s):  
Liver Disease ◽  
Sialic Acid ◽  
Acid Content ◽  
Polyacrylamide Gel Electrophoresis ◽  
Precipitation Method ◽  
Thrombin Time ◽  
Functional Abnormality ◽  
Sialic Acid Content ◽  
Fibrin Monomer

We have described an abnormal fibrinogen in 6 patients with liver disease who had prolonged plasma thrombin times due to impaired fibrin monomer aggregation. To investigate the role of sialic acid in this functional abnormality, fibrinogen was purified from normal and patient plasmas by the glycine precipitation method. Sialic acid content of the fibrinogens was measured by the thriobarbituric acid assay after acid hydrolysis. Normal fibrinogen had 6.1 ± 0.5 residues per molecule of fibrinogen, whereas patient fibrinogen sialic acid content ranged between 7.5 and 10 residues per molecule. The reduced fibrinogen demonstrated normal mobility of Aα, B3 and γ chains on SDS Polyacrylamide gel electrophoresis when stained for protein and, similar to normal fibrinogen, only the Bβ and γ chains stained with PAS. The degree of prolongation of the thrombin times of the purified patient fibrinogens appeared to correlate with the increase in the fibrinogen sialic acid. The effect on fibrin monomer aggregation of decreasing patient fibrinogen sialic acid content was studied. Partially desialated patient fibrinogen was prepared by treating the protein with Vibrio cholerae neuraminidase for varying periods of time. Partial removal of sialic acid from patient fibrinogen resulted in normalization of the thrombin time and improvement in fibrin monomer aggregation. Thrombin times ranged from 31.5 to 49.5 seconds prior to removal of excess sialic acid compared to 20.5 to 25.5 seconds post removal. These findings indicate that the dysfibrinogenemia associated with liver disease is biochemically characterized by increased sialic acid content and removal of this sialic acid results in a functional normalization of the protein.

Influence of the Carbohydrate Moiety in the Dysfibrinogenemia of Liver Disease

1979 ◽  
Author(s):  
J. Martinez ◽  
J.E. Palascak
Keyword(s):  
Liver Disease ◽  
Sialic Acid ◽  
Acid Content ◽  
Similar Distribution ◽  
Thrombin Time ◽  
Sialic Acid Content ◽  
Human Fibrinogen ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Functional Defect

Human fibrinogen is a glycoprotein which contains 6 sialic acid residues per molecule. Enzymatic removal of sialic acid modifies its functional properties as indicated by shortening of the thrombin time due to enhanced asialofibrin monomer polymerization. The abnormal fibrinogen of liver disease contains an Increased amount of sialic acid and is functionally characterized by impaired fibrin monomer polymerization. The prolongation of its thrombin time correlates with its increased sialic acid content. Enzymatic cleavage of the excess sialic acid results in normalization of the thrombin time and the fibrin monomer polymerization. Quantitative labelling of sialic acid with (3H) demonstrates increased labelling of the abnormal fibrinogen compared to normal fibrinogen reflecting the sialic acid content of the abnormal molecule. The radioactivity of the labelled normal and abnormal fibrinogens after reduction and SDS-PAGE was limited to the Bβ and γ chains with 60% of the radioactivity in the Bβ chain and 40% in the γ chain. A similar distribution of radioactivity was found after removal of the excess sialic acid from the respective chains. β-galactose was also increased in the abnormal fibrinogen and paralleled the increase in sialic acid. These studies indicate that sialic acid is distributed normally on the chains of the abnormal fibrinogen of liver disease, but its increased content is responsible for the functional defect of the protein.

Correction of the Delayed Fibrin Aggregation of Fetal Fibrinogen by Partial Removal of Sialic Acid

1979 ◽  
Author(s):  
D.K. Galanakis ◽  
M.W. Mosesson
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Acid Value ◽  
Full Term ◽  
Thrombin Time ◽  
Sialic Acid Content ◽  
Vibrio Cholera ◽  
The Mean ◽  
Fraction I ◽  
Free Sialic Acid

Human umbilical oord fibrinogen characteristically displays delayed fibrin aggregation under conditions of relatively high ionic strength. This delay is greater in fibrinogen obtained from premature (p) (24–35 weeks gestation) infants, as compared with that from full term (FT) infants. We compared the sialic acid content of (fraction I-2) fetal (P and FT) with adult (A) fibrinogen, obtained from pooled plasma. The mean sialic acid μg/100mg protein) values were: P, 818 (±135 SD, range 621–1030); FT, 720 (±212, range 505–1280); A, 626 (±110, range 487-806). One P fibrinogen preparation (thrombin time 22.6 seconds) was partially desialated (resulting sialic acid value 490) by incubation with Vibrio cholera neuraminidase, dialyzed , and precipitated with ethanol to remove free sialic acid. The thrombin time of the resulting preparation was 15.4 (range 15.2–15.8), compared to 16.1 (range 15.5–16.4) for untreated A fibrinogen. The results suggest that the delayed fibrin aggregation of fetal fibrinogen is attributable to its relatively high sialic acid content. Moreover, the intermediate sialic acid (and thrombin time) values of FT (as compared to those of p) fibrinogen intimate the presence of a mixture of adult and fetal fibrinogen in full term cord blood.

Interaction of low density lipoproteins with arterial proteoglycans The role of charge and sialic acid content

1985 ◽  
Vol 55 (1) ◽  
pp. 93-105 ◽  
Author(s):  
Germán Camejo ◽  
Aura López ◽  
Flor López ◽  
Jorge Quiñones
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Sialic Acid Content

scholarly journals Effects of variant gamma chains and sialic acid content of fibrinogen upon its interactions with ADP-stimulated human and rabbit platelets

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1163-1168 ◽  
Author(s):  
EJ Harfenist ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Sialic Acid ◽  
Acid Content ◽  
Deae Cellulose ◽  
Adenosine Diphosphate ◽  
Gamma Chain ◽  
Sialic Acid Content ◽  
Human Fibrinogen ◽  
Rabbit Platelets ◽  
Chain Composition ◽  
Normal Human

Abstract When platelets are stimulated with adenosine diphosphate (ADP), fibrinogen binds to receptors on the platelet membrane, and the platelets aggregate. The primary platelet recognition sites of human fibrinogen are reported to be at the COOH-terminal ends of the gamma chains, with secondary sites in the A alpha chains. Normal human fibrinogen, which consists of three pairs of disulfide-bonded peptide chains, (A alpha, B beta, gamma)2, is heterogeneous with respect to sialic acid content and also contains a small proportion of molecules with a variant gamma chain (designated gamma'), elongated by a peptide extension at the COOH-terminus of the normal gamma chain. We separated fibrinogen into three fractions by chromatography on DEAE cellulose and tested the interactions of these fractions with ADP-stimulated human and rabbit platelets. Two fractions had the normal chain composition, (A alpha B beta, gamma)2, but different sialic acid contents (6.6 and 7.2 mol/mol), and the third fraction had the chain composition (A alpha, B beta)2 gamma gamma' and a sialic acid content of 7.2 mol/mol, which is similar to that of one of the normal fractions. In binding and aggregation experiments, we detected no significant differences between the reactions of the first two fractions, but ADP-stimulated platelets bound only 50% as much of 125I-fibrinogen from the fraction with the gamma' chains and also aggregated less extensively in the presence of this fraction. We conclude that the sialic acid content of fibrinogen does not significantly affect its interactions with platelets, but the elongated gamma' chains bind less effectively to ADP-stimulated platelets, and thus reduce the ability of fibrinogen to support aggregation. This may result from a conformational change caused by the gamma' extension or from the deletion of a portion of the normal gamma chain recognition site.

scholarly journals Expression in Escherichia coli of the human fibrinogen B beta chain and its cleavage by thrombin

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1202-1206 ◽  
Author(s):  
MG Bolyard ◽  
ST Lord
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Dependent Manner ◽  
Beta Chain ◽  
Gamma Chain ◽  
Human Fibrinogen ◽  
E Coli ◽  
Sds Page

Abstract The human fibrinogen B beta chain was expressed in Escherichia coli to study the functions of fibrinogen associated with this subunit. Recombinant B beta chains were expressed at 100 ng/mL in an IPTG- dependent manner. A first cistron sequence, inserted into the expression vector 5′ to the B beta chain cDNA, was required to express the protein. Recombinant B beta chains were expressed within five minutes after induction with IPTG and were soluble in physiologic buffers. The recombinant B beta chains migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a rate identical to B beta chains from fibrinogen treated with N-glycanase. Recombinant B beta chains were cleaved by thrombin, as demonstrated by the loss of cross-reactivity with a monoclonal antibody (MoAb) specific for the undigested B beta 1–42 fragment. The levels of expression of the B beta chain were much lower than those reported previously for the gamma chain of fibrinogen expressed in a similar vector in E coli. However, these levels are sufficient to allow further characterization of this fibrinogen subunit.

Studies of the heterogeneity of human pituitary LH by fast protein liquid chromatography

1985 ◽  
Vol 105 (3) ◽  
pp. 405-413 ◽  
Author(s):  
A. Stockell Hartree ◽  
J. B. Lester ◽  
R. C. Shownkeen
Keyword(s):  
Liquid Chromatography ◽  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Fast Protein Liquid Chromatography ◽  
Human Pituitary ◽  
Sialic Acid Content ◽  
Receptor Binding Activity

ABSTRACT The Pharmacia fast protein liquid chromatography system was employed to fractionate a purified preparation of human LH (hLH) on the anion exchanger Mono Q at pH 7·8 into 14 sub-fractions. Each of the sub-fractions was characterized by its behaviour on polyacrylamide gel electrophoresis, sodium dodecyl sulphate (SDS) gel electrophoresis, LH receptor binding activity and sialic acid content. All sub-fractions contained sialic acid, were active in binding to LH receptors, and exhibited components typical of hLH subunits on SDS gel electrophoresis. None of the sub-fractions was homogeneous with respect to charge. There is evidence that part of the heterogeneity results from the presence in some molecules of an internal proteolytic cleavage within the β-subunit, and fractions enriched in species containing such cleavages were prepared by this method. J. Endocr. (1985) 105,405–413

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