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

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.

Preparation of Degradation Products of Fibrinogen Free of Plasmin, and Their Effect on Thrombin Activity

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y72-096 ◽

1972 ◽

Vol 50 (7) ◽

pp. 655-661 ◽

Cited By ~ 5

Author(s):

S. Chandra ◽

D. C. Triantaphyllgpoulos

Keyword(s):

Trypsin Inhibitor ◽

Cyanogen Bromide ◽

Antithrombin Iii ◽

Degradation Products ◽

Saline Control ◽

Agarose Gel ◽

Thrombin Activity ◽

Human Thrombin ◽

Glass Tubes ◽

Sepharose 4B

Sepharose 4B, a beaded agarose gel, was first activated with cyanogen bromide and then covalently coupled with pancreatic or soybean trypsin inhibitor. The degradation products of plasmin-lysed fibrinogen (DPF) were filtered through the coupled gel and the contaminating plasmin was removed by binding with the coupled inhibitor.The plasmin-free DPF thus obtained were used to study their effect on the clotting activity of thrombin. Chromatographed human thrombin was incubated in siliconized and nonsiliconized glass tubes with: (a) plasmin-free DPF, (b) albumin, (c) ceruloplasmin, and (d) saline. The activity of thrombin declined rapidly in the saline control, less so in the presence of albumin and ceruloplasmin (especially in siliconized glass tubes), and remained unchanged in the presence of DPF both in siliconized and nonsiliconized tubes. In order to study the effect of plasmin-free DPF on the inactivation of thrombin by antithrombin III, thrombin was first mixed with saline or DPF and then added to heat-defibrinated plasma (supplier of antithrombin III). The residual thrombin activity was subsequently determined at frequent intervals and was found to decline at the same rate both in the presence and in the absence of DPF. These findings demonstrate that DPF protect thrombin from spontaneous inactivation but fail to protect it against inactivation by antithrombin III.

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 ◽

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.

Shedaoenase, a Novel Fibrinogenase from the Venom of Agkistrodon shedaoenthesis Zhao

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00119.x ◽

2005 ◽

Vol 37 (12) ◽

pp. 835-842 ◽

Cited By ~ 8

Author(s):

Hao-Mang Jiao ◽

Li-Xia Yang ◽

Bin Lu ◽

Yu-Qun Wu ◽

Yuan-Cong Zhou

Keyword(s):

Snake Venom ◽

Serine Proteases ◽

Venom Gland ◽

Amino Acid Residues ◽

Terminal Sequence ◽

Human Fibrinogen ◽

Polymerase Chain ◽

Sepharose 4B ◽

Chelating Reagent ◽

Sulphonyl Fluoride

AbstractShedaoenase, a serine protease, was isolated from the venom of Agkistrodon shedaoenthesis Zhao with an apparent molecular mass of 36 kDa. It was purified by affinity chromatography on arginine Sepharose 4B column and anion exchange on Mono Q fast protein liquid chromatography. Shedaoenase preferentially cleaved the Aα-chain of human fibrinogen and slowly digested the Bβ-chain. It also showed arginyl esterase activity using Nα-benzoyl-L-arginine ethyl ester as a substrate, and some synthetic chromogentic substrates, such as Chromozym PL, S-2266, and S-2160, could also be hydrolyzed. The enzyme activity of shedaoenase could be completely inhibited by phenylmethyl sulphonyl fluoride and could be little inhibited by the chelating reagent EDTA. The N-terminal sequence of shedaoenase was determined, and its full-length cDNA encoding a protein of 238 amino acid residues was cloned by reverse transcription-polymerase chain reaction from the total mRNA extracted from the snake venom gland. The deduced primary sequence of shedaoenase shares significant homology with other snake venom serine proteases.

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

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.

ε-Poly-l-Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly-l-Lysine Synthetase

Applied and Environmental Microbiology ◽

10.1128/aem.01201-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4993-5000 ◽

Cited By ~ 13

Author(s):

Yoshimitsu Hamano ◽

Naoko Kito ◽

Akihiro Kita ◽

Yuuki Imokawa ◽

Kazuya Yamanaka ◽

...

Keyword(s):

Chain Length ◽

Catalytic Mechanism ◽

Random Mutagenesis ◽

Transmembrane Domains ◽

Polymerization Reaction ◽

Amino Acid Residues ◽

Amino Groups ◽

Content Type ◽

Peptide Synthetases ◽

Lysine Residues

ABSTRACTε-Poly-l-lysine (ε-PL), consisting of 25 to 35l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced byStreptomyces albulusNBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.

A Quantitative Thrombin Time for Determining Levels of Hirudin and Hirulog™

10.1055/s-0038-1649637 ◽

1993 ◽

Vol 70 (04) ◽

pp. 608-616 ◽

Cited By ~ 19

Author(s):

Thomas J Reid ◽

Barbara M Alving

Keyword(s):

Whole Blood ◽

Degradation Products ◽

Thrombin Time ◽

Recombinant Hirudin ◽

Human Fibrinogen ◽

Standard Curve ◽

Fibrin Degradation Products ◽

Low Concentrations ◽

Human Thrombin

SummaryThe anticoagulant effect of recombinant hirudin (rHir) and HirulogTM has been monitored in patients with the activated partial thromboplastin time. Accurate monitoring with this test cannot be achieved if plasmas contain heparin, lupus anticoagulants, low concentrations of fibrinogen or other factors, or elevated fibrinogen-fibrin degradation products (FDP). We have therefore developed a simple, rapid, sensitive clot-based method, the quantitative thrombin time (QTT), to measure levels of rHir and HirulogTM in patient plasma (or whole blood). The QTT is performed by mixing a 1:10 dilution of patient plasma (50 μl) with human fibrinogen (50 μl, 128 mg/dl) at 37° C; the clotting time is initiated by adding human thrombin (50 μl, 5-7.5 U/ml). The concentration of HirulogTM or rHir in plasma can be determined by comparing the QTT in patient plasma with a Standard curve that is generated by adding different concentrations of anticoagulant to pooled normal plasma. Studies with whole blood using the same procedure yield similar results. In the absence of HirulogTM or rHir, the baseline QTT is the same in normal and abnormal plasmas (fibrinogen <150 mg/dl and FDP as high as 1024 μg/ml, elevated FDP alone, lupus anticoagulant, or heparin <0.9 U/ml). When known concentrations of either rHir or HirulogTM are added to abnormal plasmas, the mean observed concentrations as determined by the QTT deviate from the expected values by less than 10% (range 0-19%). The data indicate that the QTT is a simple, rapid, and accurate test for the determination of levels of rHir and HirulogTM in plasma or whole blood.

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

10.1055/s-0038-1657207 ◽

1982 ◽

Vol 48 (01) ◽

pp. 021-023 ◽

Cited By ~ 1

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 ◽

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.

A quantitative automated immunoassay for fibrinogen/fibrin degradation products.

Clinical Chemistry ◽

10.1093/clinchem/31.9.1468 ◽

1985 ◽

Vol 31 (9) ◽

pp. 1468-1473 ◽

Cited By ~ 10

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 ◽

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.

Properties of Fibrinogen Cleaved by Jararhagin, a Metalloproteinase from the Venom of Bothrops jararaca

10.1055/s-0038-1648847 ◽

1994 ◽

Vol 72 (02) ◽

pp. 244-249 ◽

Cited By ~ 48

Author(s):

Aura S Kamiguti ◽

Joseph R Slupsky ◽

Mirko Zuzel ◽

Charles R M Hay

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Fibrin Clot ◽

Clot Formation ◽

Human Fibrinogen ◽

Activated Platelets ◽

Bothrops Jararaca ◽

Platelet Binding ◽

Fibrin Monomers ◽

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.

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

10.1055/s-0038-1650136 ◽

1981 ◽

Vol 45 (01) ◽

pp. 090-094 ◽

Cited By ~ 52

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.