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.

Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen

1981 ◽  
Vol 45 (01) ◽  
pp. 090-094 ◽  
Author(s):  
Katsuo Sueishi ◽  
Shigeru Nanno ◽  
Kenzo Tanaka
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Electron Microscopic Observation ◽  
Chemotactic Activity ◽  
Lower Molecular Weight ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Rabbit Skin ◽  
Molecular Weights ◽  
Active Fragments

SummaryFibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observation of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction.These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.

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.

A Simple and Practical Method for Isolation of an Early Plasmin Degradation Product of Human Fibrinogen

1977 ◽  
Vol 37 (03) ◽  
pp. 464-470 ◽  
Author(s):  
Kimiteru Takagi ◽  
Tadashi Kawai
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Degradation Product ◽  
Degradation Products ◽  
Practical Method ◽  
Human Fibrinogen ◽  
Simple Method ◽  
Hydrophobic Amino Acids ◽  
Very High

SummaryOne of the earliest plasmin degradation products of human fibrinogen, so-called fragment A, was isolated by a simple method.This peptide has a molecular weight of approximately 22,500, migrating electrophoretically at beta-area, and its amino acid composition shows a very high content of glycine, serine, threonine and proline, and a markedly low content of hydrophobic amino acids. This fragment does not react against anti-fibrinogen; however, the anti-serum of this fragment reacts strongly with fibrinogen.

Effect of Neutral Proteases from Blood Leukocytes on Human Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 768-772 ◽  
Author(s):  
K Bykowska ◽  
J Kaczanowska ◽  
M Karpowicz ◽  
J Stachurska ◽  
J Kopeć
Keyword(s):  
Light Transmission ◽  
Enzyme Concentration ◽  
Human Platelets ◽  
Serotonin Release ◽  
Time Dependent ◽  
Lag Phase ◽  
Blood Leukocytes ◽  
Human Fibrinogen ◽  
Serotonin Secretion ◽  
Neutral Proteases

SummaryTwo highly purified neutral proteases from human leukocytes i.e. elastase-like protease (ELP) and chymotrypsin-like protease (CLP) do not destroy human platelets since no difference was found in 51Cr liberation from control and enzyme-treated platelets. As with pancreatic chymotrypsin (α-CT) ELP does not induce the release of 3H-serotonin while CLP provokes 3H- serotonin secretion, in an enzyme concentration and time dependent fashion. The rate and degree of 3H-serotonin release by CLP is similar to that produced by thrombin. Incubation of platelets at 37° C for 30 min with α-CT or ELP renders them resistant to thrombin-releasing activity. Thrombin did not liberate any additional label from platelets which lost over 60% of serotonin during the preceding incubation with CLP. α-CT and ELP do not aggregate platelets either in the presence or absence of apyrase. CLP does aggregate platelets suspended in Tyrode buffer without apyrase but not in the presence of apyrase (100 mg/1). The action of α-CT, ELP and CLP on washed platelets induces a progressive prolongation of lag phase and a decrease in changes of light transmission during aggregation by thrombin. Similarly to α-CT-treated platelets, those subjected to CLP action aggregate in the presence of human fibrinogen.It is concluded that: (1) neutral proteases possibly contribute to development of defects in platelet function in pathological states associated with liberation of leukocyte content into the circulation, (2) CLP similarly to a-CT, exposes fibrinogen receptors but in contrast to α-CT, CLP aggregates platelets and stimulates serotonin secretion.

The Action of Brinase in Vitro and in Vivo

1975 ◽  
Author(s):  
P. J. Gaffney ◽  
K. Lord ◽  
R. D. Thornes
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Size Range ◽  
Degradation Products ◽  
Molecular Size ◽  
Human Fibrinogen ◽  
Rate Limiting Step ◽  
Rate Limiting

Brinase (an extract of Aspergillus Oryzae) was shown to rapidly digest human fibrinogen in vitro to aggregable degradation products with a molecular size range of 310,000 to 230,000 the latter fragments being more slowly digested to core fragments, Dbr and Ebr. The fibrinogen polypeptide chain susceptibility to Brinase attack was in the order Aα, γ, Bβ, Lysis of the Bβ chain seems to be the rate limiting step in the conversion of the high molecular weight fragments (MW 310,000–230,000) to the core fragments Dbr and Ebr. The conservation of NH2 terminal Tyrosine during fibrinogen digestion and the very transient existence of D dimer fragments during totally crosslinked fibrin lysis suggest that the carboxy end of the γ chain is prone to Brinase attack. The crosslinked α chains of fibrin, while resistant to plasmin, are vigorously digested by Brinase. The plasma of cancer patients being treated with Brinase contained degraded fibrinogen (lacking intact Aα chains) and their aggregates. These aggregates contained some crosslinked γ chains (γ-γ dimers) suggesting that Brinase in vivo exorcises both a lytic and coagulant effect. Thrombin mediated clots in all the plasmas examined contained no crosslinked α chains. Positive plasma ethanol gelation tests can be explained by the presence of the aggregable high molecular weight fragments observed during the in vitro lysis of fibrinogen by Brinase.

Limited Proteolysis of the Purified Aα Chain of Human Fibrinosen by Plasmin

1975 ◽  
Author(s):  
L. Williams ◽  
G. Murano
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Peptide Mapping ◽  
Proteolytic Enzymes ◽  
Degradation Products ◽  
Limited Proteolysis ◽  
Gel Filtration ◽  
Terminal Region ◽  
Low Molecular Weight ◽  
Human Fibrinogen

Based on evidence that a portion of circulating fibrinogen consists of a family of catabolic intermediates formed by proteolytic degradation of the COOH terminal region of Aα chains, we attempted to obtain early degradation products using the purified alkylated Aα chain derivative of human fibrinogen as the substrate and plasmin as the enzyme. Having established optimal conditions, a preparative quantity of material was digested in 0.1 M tris buffer pH = 9.5; time = 4 min; E/S ratio = 1/75 (mole/mole); temp = 37° C. Low molecular weight fragments were separated from the larger species, and further purified by gel filtration on Sephadex G-100. Selected early fragments were analyzed by polycrylamide gel electrophoresis, amino acid composition, peptide mapping and partial N-terminal amino acid sequence. Two of the earliest low molecular weight fragments released by plasmin were derived from the N-terminal region of the Aα chain. Their molecular size was estimated at about 10,000 daltons. One fragment contains fibrinopeptide A; both fragments extend beyond Met-51. Our data indicate that: a) the specificity of plasmin on the purified Aα chain differs from that on intact fibrinogen; or b) proteolytic enzymes other than or in addition to plasmin are responsible for the formation of early catabolic fibrinogen intermediates having a degraded Aα chain.(Supported by USPHS N. I. H. Grant HL 14142.)

Antigenic Properties Of Human Fibrin Plasmic Fragment D-Dimer And Its γ-γ Chain Remnant

1981 ◽  
Author(s):  
C S Cierniewski ◽  
P Nowak ◽  
T Krajewski
Keyword(s):  
Ion Exchange ◽  
Competitive Inhibition ◽  
Degradation Products ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Antigenic Determinants ◽  
Human Fibrinogen ◽  
Antigenic Properties ◽  
D Dimer

Immunochemical analyses of human D-dimer and its γ-γ chain remnant were performed to identify their antigenic markers which would be of value in distinguishing between disseminated intravascular coagulation and primary fibrinogenolysis. Cross-linked fibrin was obtained by direct clotting of the fresh citrated plasma after adding excess CaCl2. The plasmic digests of the cross-linked fibrin were fractionated by ion-exchange chromatography and gel filtration. Thus, high molecular weight D-dimer was purified. The γ-γ peptide remnant was purified by ion-exchange chromatography on CM-52 cellulose from the reduced D- dimer. Purity of both polypeptide fragments was determined by standard techniques. They were used to immunize rabbits. Antisera to D-dimer and γ-γ chain remnant were characterized in binding assays and in equilibrium competitive inhibition assays in order to analyze the expression of their antigenic determinants in intact fibrinogen and its plasmic degradation products. Antisera to D-dimer preferentially bound D-dimer and γ-γ chain remnant and to a lesser extent fragment D and fibrinogen. Similarly, antisera to γ-γ chain remnant bound mostly γ-γ , D-dimer as well as γ poiypeptide chain. There was no binding of the intact human fibrinogen. The results obtained in the competitive inhibition assays employing antisera to D-dimer and γ-γ chain remnant, before and after adsorption with fragment D or γ polypeptide chain respectively, as well as fibrin fragments are discussed in this report.

Permeability Enhancing And Chemotactic Activities Of Lower Molecular Weight Degradation Products Of Human Fibrinogen

1981 ◽  
Author(s):  
K Tanaka ◽  
K Sueishi
Keyword(s):  
Molecular Weight ◽  
Vascular Permeability ◽  
Degradation Products ◽  
Chemotactic Activity ◽  
Lower Molecular Weight ◽  
Boyden Chamber ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Rabbit Skin ◽  
Vascular Walls

Fibrinogen degradation products were investigated for leukocyte chemotactic activity and enhancement of vascular permeability. Fragments X, Y, D and E, and non-dialysable lower molecular weight degradation products were fractionated from plasmin digests of human fibrinogen by gel filtration, zone electrophoresis and ion exchange chromatography. They were assayed for chemotactic activity for human granulocytes by the modified Boyden chamber and for vascular permeability increasing activity in rabbit skin injected intravenously with pontamine blue.Both activities increased progressively with plasmin digestion of fibrinogen. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D and E. The low molecular weight break down product with chemotactic activity showed the highest activity in rabbit skin in vivo as well. Granulocytic infiltration was most prominent 6-12 hr after intradermal injection and apparent even at an intradermal dose of 0.5/μg. Permeability enhancement of the active fraction reached to its muximum level at 5-10 min and was dose-dependent. Electron microscopic observations of the small blood vessels in rabbit skin correlated an increased permeability with the formation of characteristic gaps between adjacent endothelial cells and their contraction. Carbon particles used as a tracer passed into the vascular walls through these intercellular gaps.These observations add more support for the hypothesis that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions associated with deposits of fibrin and/or fibrinogen.

Chicken Fibrinogen and Human Fibrinogen

1973 ◽  
Vol 30 (01) ◽  
pp. 072-083
Author(s):  
Doris Ménaché ◽  
Nicole Cesbron ◽  
Marie-Claude Guillin ◽  
Nicole Schlegel
Keyword(s):  
Red Cells ◽  
Antigenic Determinants ◽  
Human Fibrinogen ◽  
Agarose Gels ◽  
Cross Absorption ◽  
Precipitation Reactions ◽  
Antigen Antibody

SummaryIn order to investigate the immunological features of chicken fibrinogen and to compare them with human fibrinogen, monospecific rabbit antichicken and antihuman fibrinogen antisera have been prepared. Both antisera agglutinated human tanned red cells coated with human fibrinogen. Inhibition of the reaction was obtained by prior incubation of rabbit antichicken fibrinogen antiserum with either human or chicken fibrinogen. Using precipitation reactions in agarose gels, precipitating antigen-antibody complexes were obtained between chicken fibrinogen and rabbit antihuman fibrinogen antiserum and vice versa only if the antisera were concentrated or if an increase in the sensitivity of the reaction was achieved using counter Immunoelectrophoresis. Cross absorption of rabbit antichicken fibrinogen antiserum with human fibrinogen did not abolish the precipitating reaction with chicken fibrinogen. In the same way, cross absorption of rabbit antihuman fibrinogen antiserum with chicken fibrinogen did not abolish the precipitating reaction with human fibrinogen.These results indicate that chicken and human fibrinogen share common antigenic determinants; they also indicate additional antigenic determinants in both fibrinogens.

Quantitation of the Coagulation Inhibition in Vivo Due to Breakdown Products of Fibrin

1970 ◽  
Vol 23 (03) ◽  
pp. 477-485 ◽  
Author(s):  
P. S Mitchell ◽  
F. K Beller
Keyword(s):  
Blood Clotting ◽  
Fibrinogen Level ◽  
Degradation Products ◽  
Thrombin Time ◽  
Clotting Time ◽  
Human Fibrinogen ◽  
Quantitative Basis ◽  
Coagulation Inhibition

SummaryDegradation products of human fibrinogen were prepared by in vitro lysis of fibrin clots by urokinase activation and injected into rabbits on a quantitative basis. The dose necessary to anticoagulate the animal was equal to 2½ to 3 times the animals’ fibrinogen level. The effect on whole blood clotting time and thrombin time lasted for approximately 2 hrs. The “r” time of the TEG returned to normal after 1 hr while the “Max” value remained abnormal for more than 120 min. Degradation product E was shown to clear more rapidly than D by immunochemical techniques. The overall T ½ clearance was found to be approximately 12 hrs.

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