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

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.

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

Blood ◽

10.1182/blood.v64.6.1163.bloodjournal6461163 ◽

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

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.

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

10.1055/s-0038-1652435 ◽

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.

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Influence of the Carbohydrate Moiety in the Dysfibrinogenemia of Liver Disease

10.1055/s-0039-1684718 ◽

1979 ◽

Cited By ~ 1

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.

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Density subpopulations of rabbit platelets: size, protein, and sialic acid content, and specific radioactivity changes following labeling with 35S-sulfate in vivo

Blood ◽

10.1182/blood.v57.4.741.bloodjournal574741 ◽

1981 ◽

Vol 57 (4) ◽

pp. 741-746 ◽

Cited By ~ 1

Author(s):

ML Rand ◽

JP Greenberg ◽

MA Packham ◽

JF Mustard

Keyword(s):

Sialic Acid ◽

Acid Content ◽

Specific Radioactivity ◽

Sialic Acid Content ◽

Rabbit Platelets

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The isolation of ovomucoid variants differing in carbohydrate composition

Biochemical Journal ◽

10.1042/bj1230399 ◽

1971 ◽

Vol 123 (3) ◽

pp. 399-405 ◽

Cited By ~ 32

Author(s):

J. G. Beeley

Keyword(s):

Sialic Acid ◽

Amino Acid ◽

Acid Content ◽

Deae Cellulose ◽

Structure And Function ◽

Inhibiting Activity ◽

Carbohydrate Composition ◽

Charge Heterogeneity ◽

Sialic Acid Content ◽

And Function

Three major and two minor species of ovomucoid were separated by chromatography on sulphoethyl-Sephadex. The predominant sialic acid-free species was further resolved into three fractions by DEAE-cellulose chromatography. Although all species of ovomucoid had closely similar trypsin-inhibiting activity, immunochemical properties and amino acid composition, they differ in carbohydrate composition. Wide variation was observed in the content of galactose, N-acetylglucosamine and sialic acid. Charge heterogeneity was related, in part, to variation in sialic acid content. The implications of variable carbohydrate composition for the structure and function of ovomucoid are discussed.

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Density subpopulations of rabbit platelets: size, protein, and sialic acid content, and specific radioactivity changes following labeling with 35S-sulfate in vivo

Blood ◽

10.1182/blood.v57.4.741.741 ◽

1981 ◽

Vol 57 (4) ◽

pp. 741-746 ◽

Cited By ~ 43

Author(s):

ML Rand ◽

JP Greenberg ◽

MA Packham ◽

JF Mustard

Keyword(s):

Sialic Acid ◽

Acid Content ◽

Specific Radioactivity ◽

Sialic Acid Content ◽

Rabbit Platelets

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Contribution of Sialic Acid to the Heterogeneity of Human Fibrinogen Polypeptide Chains

10.1055/s-0039-1687462 ◽

1979 ◽

Author(s):

C. Kuyas ◽

A. Haeberli ◽

P.W Straub

Keyword(s):

Sialic Acid ◽

Isoelectric Focusing ◽

Acid Content ◽

Polypeptide Chain ◽

Sialic Acid Content ◽

Human Fibrinogen ◽

Single Donor ◽

The Individual ◽

Polypeptide Chains ◽

Vibrio Cholerae Sialidase

Human fibrinogen, isolated from single donor or from pooled plasma, shows a heterogeneity of the Bऔ- and the γ-polypeptide chains on CM-cellulose chromatography. In order to find out whether this heterogeneity is due to the observed differences in sialic acid content of the variants (2 and 1 residue per chain; Gati et al., J.Biol.Chem.253:1315,1978) pooled or single donor fibrinogen (clottability 93-95%) was incubated 24 hrs with either vibrio cholerae sialidase or buffer. The asialofibrinogen (10% of original sialic acid) was compared with intact fibrinogen. After dithiothreitol reduction and alkylation the chains were separated on CM Sepharose. The individual homogeneous Bऔ- and γ-chains were rechromatographed on CM-cellulose. The γ-chain heterogeneity of normal fibrinogen was absent in asialofibrinogen whereas the Bऔ-chain heterogeneity appeared unaffected. Although the variants were indistinguishable on SOS-PAGE, isoelectric focusing in presence of urea demonstrated heterogeneities of both Bऔ- and γ-chains even in asialofibrinogen. Thus, the differences in sialic acid content of the main polypeptide chain variants of pooled as wel l as single donor human fibrinogen can only explain a small part of the polypeptide chain heterogeneity.

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