Size and shape of the fibrinogen molecule: crystalline forms of native and modified human fibrinogen

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.

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Existence of Common Antigenic Determinants in the Aα, Bβ and γ Chains of Some Mammalian Fibrinogens.

10.1055/s-0039-1687463 ◽

1979 ◽

Author(s):

C.S. Cierniewski

Keyword(s):

Amino Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Antigenic Determinants ◽

Terminal Amino Acid ◽

Human Fibrinogen ◽

Passive Hemagglutination ◽

Terminal Amino ◽

Polypeptide Chains ◽

Fibrinogen Molecule

Polypeptide chains Aα, Bβ and γ of porcine fibrinogen were isolated by preparative SDS polyacrylamide gel electrophoresis. Their purity was estimated by electrophoresis in polyacrylamide gel, amino acid composition and N-terminal amino acid analyses. Antisera to the pig polypeptide chains were produced in rabbits and they were employed in immunological comparative studies of porcine, bovine, human and duck fibrinogens. Antisera to the pig Aα chain showed in gel immunodiffusion and passive hemagglutination a strong cross-reaction with porcine, bovine and human fibrinogens. Antisera to the pig βB and γ chains cross-reacted only with porcine and bovine fibrinogens but they did not recognize human fibrinogen, The reaction of antiγ antisera was detectable only by passive hemagglutination test. In all cases antigenic similarity of the analyzed fibrinogens was mainly related to antigenic determinants of the Aα, Bβ and γ chains exposed on the intact fibrinogen molecule. None of analyzed antisera reacted with duck fibrinogen.

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Human Fibrinogen Binds Edta And Citrate

10.1055/s-0038-1652434 ◽

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

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Studies Of The Peptide Fractions Derived From Fibrinogen By Digestion With Trypsin Or Plasmin

10.1055/s-0038-1653058 ◽

1981 ◽

Author(s):

E Mihalyi

Keyword(s):

Proteolytic Enzymes ◽

Random Coil ◽

Small Peptides ◽

Compact Structure ◽

Human Fibrinogen ◽

Clinical Interest ◽

Α Chain ◽

Peptide Segments ◽

Peptide Fractions ◽

Fibrinogen Molecule

Digestion of fibrinogen with a number of proteolytic enzymes yields a series of large fragments with protein-like structure and a substantial amount of peptidic material. Some of the peptides released possess biological activity, therefore, the study of their formation and properties may be of clinical interest. In the present investigations human or bovine fibrinogen was digested by either plasmin or trypsin. Approximately 35% of the fibrinogen molecule is removed as peptides with either enzyme and these are well divided into two groups: 1. large peptides of 15,000 to 40,000 MW and 2. small peptides of less than 10,000 MW. The distribution of material between these groups is approximately the same with either enzyme and with bovine or human fibrinogen, however, the plasmic peptides in either group are larger than the tryptic ones. All the large peptides are derived from the approximately 40,000 MW C-terminal portion of the α-chain. In this segment, according to the sequence of the human α-chain, there are 33 bonds theoretically susceptible to either plasmin or trypsin. Of these, only 3 are attacked at a fast rate by plasmin in the native molecule, resulting in the appearance of peptide segments of 40,000, 32,000 and 26,000 MW. The first two are short lived, reach only about 25% of the stoichiometric amount, and are converted into the last one, which accumulates as a fairly stable end product. Trypsin digestion of the latter involves again 3 susceptible bonds and through an intermediate of 17,000 MW results in 2 fragments, one of 10,000 and another of 7,000 MW. The restricted cleavage of susceptible bonds in these fragments, their intrinsic viscosity and its increase in denaturing media, suggest that these peptides possess a fairly compact structure, i.e., they are not in the random coil state as hitherto suggested.

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Size and Shape of Human Fibrinogen in Solution

10.1055/s-0039-1680592 ◽

1977 ◽

Author(s):

V. Hofmann ◽

P.W. Straub ◽

T. Binkert ◽

E. Serrallach ◽

W. Känzig ◽

...

Keyword(s):

Light Scattering ◽

Dynamic Light Scattering ◽

Analytical Ultracentrifugation ◽

Rotational Diffusion ◽

Axial Ratio ◽

Major Axis ◽

Fluorescence Depolarization ◽

Human Fibrinogen ◽

Size And Shape ◽

Rotational Diffusion Coefficients

In order to obtain information on size and shape of the fibrinogen molecule in solution the translational diffusion coefficient (DT), the rotational diffusion coefficients (DR⊥ and DR//) and the sedimentation coefficients (S) have been measured on human fibrinogen with a clottaoility above 95%. The methods used were dynamic light scattering, nanosecond fluorescence depolarization and analytical ultracentrifugation. Dynamic light scattering yields DT = 2.0 ± 3% x 10-7 cm2sec–1 at a concentration of 7 mg/ml in 0.15 M Tris-NaCl, pH 7.4. DT is strongly dependent on concentration, being 3.4 ± 10% × 10-7 cm2 sec-1 at 0.1 mg/ml. The rotation along the minor axis as measured with the same method is DR = // 1.5 × 106 sec-1 at 0.1 mg/ml. The rotation along the major axis as measured on fibrinogen labeled with dansylchloride is DR// = 1.5 x106 sec–1. S is also strongly dependent on concentration, being 7.9 S at 0.1 mg/ml, 8.1 S at 1 mg/ml and 6.6 S at 10 mg/ml.These results fit with an elongated molecule having an axial ratio of 7. They are compatible with a MW of 340’000 only for concentrations above 2 mg/ml, while at lower concentrations (0.1 mg/ml) they agree with a MW of approximately half the accepted value.

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Fibrinogenolysis: Studies on its Degradation Products

10.1055/s-0039-1689149 ◽

1975 ◽

Author(s):

J. Bouvier ◽

R. Braude ◽

R. Altman

Keyword(s):

Degradation Products ◽

The Other ◽

Small Peptides ◽

Human Fibrinogen ◽

Terminal Fragment ◽

Antigen Antibody ◽

Fibrinogen Molecule

Human fibrinogen is degradated by plasmin into four generically accepted major products namely X, Y, D and E, and three lower molecular wheight fragments called A, B and C. A new early fragment named E’ is described when plasmin degradation of fibrinogen was analysed in agar and poliacrylamide electrophoresis, Sheidegger inmunoelectrophoresis and Laurell cross antigen-antibody electrophoresis. In the most early moment of proteolysis the electrophoretic position of this fragment is similar to there of fibrinogen X and Y fragments, but it migrate faster toward the anode when further digests were analysed. It seem to appear together of fragment X and, after loosing small peptides, will finally reach in the gel, the terminal fragment E position. On this basis, we suggest that from each fibrinogen molecule derivate two fragments E; one released from fragment Y and the other from the fragment E’ here described. Some modifications of the fibrinogen degradation scheme are postulated.

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Significance Of Tryptophan Residues In Fibrinogen In Fibrin Polymer Formation

10.1055/s-0038-1652706 ◽

1981 ◽

Author(s):

A Matsushima ◽

Y Saito ◽

K Okano ◽

Y Inada

Keyword(s):

Functional Significance ◽

Inhibitory Effect ◽

Complete Loss ◽

Human Fibrinogen ◽

Stepwise Manner ◽

Polymer Formation ◽

Fibrin Monomer ◽

Tryptophan Residues ◽

A Chain ◽

Fibrinogen Molecule

Human fibrinogen and its fragment were modified with 2-hydroxy-5-nitrobenzyl bromide(HNBB) or with H2O2-dioxane to see the functional significance of tryptophan residues in the fibrinogen molecule. Tryptophan residues were modified with HNBB in a stepwise manner. Modification of two out of the total 64 tryptophan residues in the molecule did not reduce the polymerization activity with thrombin but further modification of the next two residues led to a complete loss of the polymerization activity. The latter two tryptophan residues modified were in Fragment E and the position may be either Trp 33 or Trp 41 in the a-chain in the fibrinogen molecule.Fragment D inhibited the association activity of fibrin monomer. This inhibitory effect was lost completely by the modification of approximately six out of the total 21 tryptophan residues in the fragment with H2O2-dioxane. These resul ts indicate the significance of tryptophan residues in Fragments D and E of fibrinogen molecule in fibrin polymer formation.

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

Blood ◽

10.1182/blood.v49.4.635.bloodjournal494635 ◽

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.

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The Acelerating Effect of Fibrinogen and Early Fibrinogen Degradation Products on Erythrocyte Sedimentation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666914 ◽

1979 ◽

Vol 42 (02) ◽

pp. 757-763 ◽

Cited By ~ 4

Author(s):

N A Marsh

Keyword(s):

Sedimentation Rate ◽

Degradation Products ◽

Biological Activities ◽

Red Cells ◽

Proteolytic Digestion ◽

Human Fibrinogen ◽

Erythrocyte Sedimentation ◽

Carboxyl Terminal ◽

Fibrinogen Molecule ◽

Human Red Cells

SummaryThe fibrinogen molecule has a number of biological activities and some of these are shared by the larger degradation products. This study was carried out to investigate the erythrocyte sedimentation-accelerating property of fibrinogen and how this property might be modified by proteolytic digestion with plasmin.The sedimentation rate of washed human red cells suspended in saline, was found to be directly proportional to the concentration of added purified human fibrinogen, down to 250 mg%, below which there was no difference from saline controls. Plasmic digestion of fibrinogen yielding fragment X, did not reduce the accelerating affect on erythrocyte sedimentation, indicating that the intact carboxyl terminal end of the Aα chain is unnecessary for this phenomenon. Further digestion to fragment Y reduced the effect slightly but digestion to fragments D and E abolished the accelerating effect completely.

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A structural study of modified fibrinogen and fibrin

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077396 ◽

1983 ◽

Vol 41 ◽

pp. 764-765

Author(s):

E. A. Hewat ◽

L. Tranqui ◽

R. H. Wade

Keyword(s):

Structural Study ◽

Molecular Form ◽

Negative Staining ◽

Principal Plane ◽

Molecular Axis ◽

Detailed Model ◽

Crystal Forms ◽

Crystalline Forms ◽

Fibrinogen Molecule

A knowledge of the form of the fibrinogen molecule and the structure of fibrin are clearly essential to the understanding of their role in the coagulation of vertebrate blood. The elongated multinodular form of the molecule is well established, but its detailed form and the structure of the fibrin fibre have remained controversial. Our approach to this problem has been firstly to examine the form of the molecule in several types of microcrystals of proteolytically modified fibrinogen. Tilting experiments enabled identification of the same molecular form in two different crystalline forms (P21212 and P21). Two projections of the molecule at 0° and at 37° to the 2-fold molecular axis were obtained from the two principal plane projections of the two crystal forms. This information enabled a rough determination of the relative heights of the different subdomains of the molecule. Hence we are able to propose a more detailed model of the fibrinogen molecule than has previously been reported.Secondly, to clarify the existence of any differences between the native and the modified fibrinogen molecule, we examined individual molecules using negative staining and shadowing techniques.

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