Fibrinogenolysis: Studies on its Degradation Products

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.

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

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.

Immunoelectrophoretic Studies on the Kinetics of Plasmic Degradation of Human Fibrinogen

1977 ◽  
Vol 37 (01) ◽  
pp. 136-143 ◽  
Author(s):  
Roberto Braude ◽  
Raúl Altman ◽  
Jorge Rouvier ◽  
Nélida Aguilar
Keyword(s):  
Electrophoretic Mobility ◽  
Human Fibrinogen ◽  
In Vitro Degradation ◽  
Terminal Fragment ◽  
Antigen Antibody ◽  
Kinetics Of

SummaryIn vitro degradation of fibrinogen results in the appearance of an early fragment, different in its electrophoretic mobility to that of fragments X, Y, D and E.It reacts with antifibrinogen antiserum, does not react with anti-D antiserum and it is anti-genically related to fragment E.Compared with terminal fragment E, it migrates more slowly towards the anode, it appears earlier and in a greater concentration at the beginning, decreasing and disappearing during degradation.The antigen antibody crossed electrophoresis was used in these experiments, but to distinguish the fragments from already known X, Y or even fibrinogen, the applying of an intermediate gel with anti D was of great value.

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

1979 ◽  
Vol 42 (02) ◽  
pp. 757-763 ◽  
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.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Abstract Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

scholarly journals Generation of Fibrinogen Degradation Products (FDP’s) by Neutral Proteases from Human Granulocytes and their Importance for the Blood Clotting System

1977 ◽  
Author(s):  
M. Gramse ◽  
Ch. Bingenheimer ◽  
R. Egbring ◽  
K. Havemann
Keyword(s):  
Molecular Weight ◽  
Blood Clotting ◽  
Degradation Products ◽  
Enzyme Concentration ◽  
Antigenic Determinants ◽  
Clotting System ◽  
Human Fibrinogen ◽  
Human Granulocytes ◽  
Antigen Antibody ◽  
Neutral Proteases

The effect of neutral proteases from human granulocytes (elastase-like and chymotrypsin-like protease) on purified human fibrinogen was investigated. Detectable by two-dimensional and Polyacrylamide electrophoresis, dependence of fibrinogen degradation of enzyme concentration and incubation time was found. Molecular weight of the FDP’s was estimated by gelchromatografy. Some greater FDP’s showed the antigenic determinants of fibrinogen and of the split products D and E. High molecular weight FDP’s inhibited fibrinogen clotting. Generated at high enzyme concentrations and longer incubation times, FDP’s lost their ability to clot themselves and to interfere with fibrin polymerization.As these fibrinogen degradating enzymes are released by the influence of antigen-antibody-complexes and endotoxine, it can be assumed that clotting defects in patients with acute leukemia, septicemia a. o. are caused by the effects of the proteases on fibrinogen and the accumulation of FDP’s.

The Intrinsic Fluorescence of Human Fibrinogen and Its Fragments D and E

1982 ◽  
Vol 48 (01) ◽  
pp. 021-023 ◽  
Author(s):  
M A Kowalska ◽  
C S Cierniewski
Keyword(s):  
Conformational Changes ◽  
Fluorescence Intensity ◽  
Degradation Products ◽  
Tryptophan Fluorescence ◽  
Intrinsic Fluorescence ◽  
Excitation Spectra ◽  
Ph Titration ◽  
Human Fibrinogen ◽  
Tryptophan Residues ◽  
Fibrinogen Molecule

SummaryThe tryptophan fluorescence of fibrinogen and its final degradation products - fragment D and E - were compared. Fibrinogen and its derivatives exhibit identical emission and excitation spectra. Their fluorescence intensity is influenced to a different extent by pH titration and temperature.Our studies showed that tryptophan residues of core fragments D and E are much more exposed to quenching effects of acrylamide and ions than intact fibrinogen, which may be caused by conformational changes occurring over the domains during plasmin digestion of fibrinogen molecule.

scholarly journals Distinctive Neoantigenic Marker Associated with a Plasmic Derivative (Fragment D-D) of Cross Linked Human Fibrin

1977 ◽  
Author(s):  
A.Z. Budzynski ◽  
V.J. Marder ◽  
M.E. Parker ◽  
P. Shames
Keyword(s):  
Degradation Product ◽  
Molecular Basis ◽  
Factor Xiii ◽  
Degradation Products ◽  
The Other ◽  
Human Fibrinogen ◽  
Assay Sensitivity ◽  
Double Antibody

Fragment D-D is a plasmic degradation product of crosslinked fibrin consisting of 2 Fragment D molecules linked by Factor XIII-induced ε(γ-glutamyl)lysine bonds. In this study, the antigenic markers of Fragment D-D were compared with those of Fragment D obtained from fibrinogen and non-crosslinked fibrin, as well as with those of fibrinogen Fragments X, Y and E. An antiserum against Fragment D-D raised in chickens and absorbed with fibrinogen and Fragment D showed immunoprecipitation with Fragment D-D but not with fibrinogen or with the other derivatives. In a double antibody radioimmunoassay system the antiserum bound 125-I labeled Fragment D-D but not labeled fibrinogen or Fragment D from fibrinogen and non-crosslinked fibrin. The binding of labeled Fragment D-D was inhibited by non-labeled Fragment D-D at concentrations of 10-10,000 ng/ml and was equally reliable in a plasma milieu. Purified plasmic degradation products of human fibrinogen inhibited binding less effectively. The increased assay sensitivity for Fragment D-D appears to be related to specific neoantigenic sites associated with the overall structure of this degradation product and provides molecular basis for the differentiation between fibrinogenolysis and thrombolysis.

A quantitative automated immunoassay for fibrinogen/fibrin degradation products.

1985 ◽  
Vol 31 (9) ◽  
pp. 1468-1473 ◽  
Author(s):  
R S Schifreen ◽  
G S Cembrowski ◽  
D C Campbell ◽  
A R Craig ◽  
N D Demyanovich ◽  
...  
Keyword(s):  
Degradation Products ◽  
Clinical Information ◽  
Polyclonal Antiserum ◽  
Sample Collection ◽  
Human Fibrinogen ◽  
Automated Assay ◽  
Fibrin Degradation Products ◽  
Collection Tube ◽  
Automated Immunoassay ◽  
Fibrinogen Molecule

Abstract We describe a prototype quantitative automated assay for fibrin and fibrinogen degradation products, a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) in the Du Pont aca discrete clinical analyzer. This assay involves a latex particle reagent with covalently bound fibrinogen and a polyclonal antiserum raised in rabbits against human fibrinogen. A special secondary sample-collection tube quantitatively removes fibrinogen from citrated plasma and inhibits further fibrinolysis, independent of heparin concentration. The assay range is 0-100 mg/L, in fibrinogen equivalents. The CV for the assay is less than 10% when performed with the aca. Nonclottable fibrin and fibrinogen fragments are measured by the assay, the greatest sensitivity being directed at the E domain of the fibrinogen molecule. We illustrate with case studies the potential of this assay for providing clinical information not obtainable with currently available qualitative and semi-quantitative assays.

The αEC domain of human fibrinogen-420 is a stable and early plasmin cleavage product

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2297-2303 ◽  
Author(s):  
Dianne Applegate ◽  
Lara Stoike Steben ◽  
Kathe M. Hertzberg ◽  
Gerd Grieninger
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cleavage Product ◽  
The Other ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
Western Blots ◽  
Sds Page

Human fibrinogen-420, (Eβγ)2, was isolated from plasma and evaluated for its ability to form clots and for its susceptibility to proteolysis. Clotting parameters, including cross-linking of subunit chains, of this subclass and of the more abundant fibrinogen-340 (βγ)2, were found to be similar, suggesting little impact of the unique EC domains of fibrinogen-420 on coagulation. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmic digestion patterns revealed production from fibrinogen-420 of the conventional fibrinogen degradation products, X, Y, D, and E, to be comparable to that from fibrinogen-340 in all respects except the presence of at least 2 additional cleavage products that were shown by Western blot analysis to contain the EC domain. One was a stable fragment (ECX) comigrating with a 34-kd yeast recombinant EC domain, and the other was an apparent precursor. Their release occurred early, before that of fragments D and E. Two bands of the same mobility and antibody reactivity were found in Western blots of plasma collected from patients with myocardial infarction shortly after the initiation of thrombolytic therapy.

COMPLEX FORMATION,BETWEEN THROMBIN AND FIBRINOGEN OR FIBRINOGEN DEGRADATION PRODUCTS (FDP)

1987 ◽  
Author(s):  
E Kaczmarek ◽  
J McDonagh
Keyword(s):  
Complex Formation ◽  
Degradation Products ◽  
Amino Acid Residues ◽  
Amino Groups ◽  
Human Fibrinogen ◽  
Reducing Conditions ◽  
Lysine Residues ◽  
Human Thrombin ◽  
Sepharose 4B ◽  
Fibrinogen Molecule

To identify the part of the fibrinogen molecule which interacts with thrombin binding of human thrombin to plasmic FDP was analyzed.125I-thrombin was incubated with FDP, purified fibrinogen fragment D or fragment E in the presence of 0.2% glutaraldehyde. Incubation mixtures were analyzed by SDS-PAGE and autoradiography. Under non-reducing conditions, the autoradiogram from the thrombin and fibrinogen fragment D incubation showed only one dark band, the molecular weight (Mr) of which was identical to that of thrombin, indicating no complex formation between thrombin and fragment D. With thrombin and fibrinogen fragment E, two dark bands were observed: the electrophoretic mobility of the first was the same as that of thrombin and the Mr of the second was equal to the sum of the Mr of thrombin and fragment E. This shows that human thrombin Forms a complex with fibrinogen fragment E. Hence, we can conclude that only the N-terminal part of the fibrinogen molecule is necessary for interaction with thrombin. Under reducing conditions, the complex of thrombin with fragment E produced four bands on gel electrophoresis. One was thrombin; the remaining three were complexes of thrombin with fragment E chain remnants. To investigate this further, carboxymethylated human fibrinogen chains Aα, Bβ and γ were purified and coupled to Sepharose 4B. 125I-thrombin was applied on the three columns. Nearly all radioactivity was bound to the three affinity columns and was eluted with higher NaCl concentration. We can infer that complex formation between thrombin and fibrinogen requires interaction between thrombin and all three fibrinogen chains. To find which thrombin amino acid residues are responsible for interaction with fibrinogen, human thrombin was coupled to Affi-Gel 102 and Affi-Gel 202 through thrombin's carboxyl and amino groups, respectively. We observed binding of fibrinogen and fibrinogen fragment E only to Affi-Gel 102 column, indicating that lysine residues and perhaps the N-terminal of the thrombin molecule interact with fibrinogen. When thrombin was bound to the gel through its amino groups, there was no interaction between thrombin and fibrinogen or fragment E.

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