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

1972 ◽  
Vol 50 (7) ◽  
pp. 655-661 ◽  
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.

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.

Is Quantitative Determination of Fibrin(ogen) Degradation Products and Thrombin-Antithrombin III Complexes Useful to Diagnose Deep Venous Thrombosis in Outpatients?

1989 ◽  
Vol 62 (04) ◽  
pp. 1043-1045 ◽  
Author(s):  
Paul F M M van Bergen ◽  
Eduard A R Knot ◽  
Jan J C Jonker ◽  
Auke C de Boer ◽  
Moniek P M de Maat
Keyword(s):  
Quantitative Determination ◽  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Antithrombin Iii ◽  
Degradation Products ◽  
Family Doctor ◽  
Diagnostic Value ◽  
Impedance Plethysmography ◽  
Fibrin Degradation Products

SummaryWe studied the diagnostic value of recently introduced ELISA’s for the determination of thrombin-antithrombin III (TAT) complexes, fibrin degradation products (FbDP), fibrinogen degradation products (FgDP) and total degradation products (TDP) for deep venous thrombosis (DVT) in plasma of 239 consecutive outpatients, suspected for DVT by their family doctor. DVT was confirmed by impedance plethysmography in 60 patients. Using the 95th percentile range of 42 healthy volunteers the sensitivity for the detection of DVT was: 37% for TAT, 95% for TDP, 92% for FbDP and 90% for FgDP. Specificity was: 88% for TAT, 16% for TDP, 20% for FbDP and 25% for FgDP.We conclude that these assays are of little value in the diagnosis of DVT in outpatients.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

scholarly journals Active fragments obtained by cyanogen bromide cleavage of ovomucoid

1976 ◽  
Vol 155 (2) ◽  
pp. 345-351 ◽  
Author(s):  
J G. Beeley
Keyword(s):  
Molecular Weight ◽  
Trypsin Inhibitor ◽  
Inhibitory Activity ◽  
Cyanogen Bromide ◽  
Peptide Chain ◽  
Terminal Portion ◽  
Trypsin Inhibitory Activity ◽  
Methionine Residues ◽  
Active Fragments

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.

Determination of Ochratoxin A in Feed by Immunoaffinity Cleanup and Liquid Chromatography

2013 ◽  
Vol 96 (3) ◽  
pp. 599-602 ◽  
Author(s):  
Ping Ding ◽  
Ziyou Mi ◽  
Yali Hou ◽  
Yigang He ◽  
Jianhua Xie
Keyword(s):  
Liquid Chromatography ◽  
Detection Limit ◽  
Ochratoxin A ◽  
Cyanogen Bromide ◽  
Polyclonal Antibodies ◽  
Immunoaffinity Column ◽  
Low Levels ◽  
Sepharose 4B ◽  
Feed Samples

Abstract A method using LC was developed for determination of ochratoxin A (OTA) in feeds. The extracted samples were cleaned up by an immunoaffinity column prepared by covalently coupling polyclonal antibodies against OTA to cyanogen bromide-activated Sepharose 4B. The eluates were determined by LC with fluorescence detection. Recoveries of OTA from fortified samples of 1–10 μg/kg levels ranged from 84.3 to 90.0%, with CVs of 3.3–7.8%. The detection limit was 0.045 μg/kg based on an S/N of 3:1. A total of 65 feed samples were screened for OTA with the proposed method. The results showed that only nine samples were contaminated with OTAs at low levels. The presented method was successfully applied to quantify OTAs in real feed samples.

Effects of the Synthetic Thrombin Inhibitor Argatroban on Fibrin- or Clot-Incorporated Thrombin: Comparison with Heparin and Recombinant Hirudin

1994 ◽  
Vol 72 (03) ◽  
pp. 381-386 ◽  
Author(s):  
Christopher N Berry ◽  
Christine Girardot ◽  
Catherine Lecoffreo ◽  
Catherine Lunven
Keyword(s):  
Enzymatic Activity ◽  
Antithrombin Iii ◽  
Thrombin Inhibitor ◽  
Chromogenic Substrate ◽  
Inhibitory Effects ◽  
Amidolytic Activity ◽  
Recombinant Hirudin ◽  
Plasma Clot ◽  
Human Thrombin ◽  
Ic50 Values

SummaryThe inhibitory effects of argatroban on clot- or fibrin-bound human thrombin were studied using the thrombin-specific chromogenic substrate S2238 (200 μM). These effects were compared to those of recombinant hirudin (rHV2 Lys 47) and the heparin/antithrombin III complex. Argatroban concentration-dependently inhibited the cleavage of S2238 by a thrombin solution, which had been titrated to give the same change in OD405 nm as fibrin-bound thrombin, with an IC50 of 1.1 μM with 90% inhibition at 8 μM. rHV2 Lys 47 and heparin had IC50 values of 1.2 nM and 0.003 U/ml respectively under these conditions. However, when the compounds were tested against fibrin-bound thrombin, argatroban had an IC50 of 2.8 μM with 65% inhibiton at 8 μM, whereas rHV2 Lys 47 had an IC50 of 23 nM (with only 56% inhibition at 200 nM), and heparin had an IC50 of 0.5 α 0.38 U/ml (with only 58% inhibition at 5 U/ml); i. e. the two compounds were 19 and 168 times less active against fibrin-bound thrombin than against thrombin in solution. The differences between the inhibitory effects of the compounds against thrombin bound to a plasma clot were even more striking in that the IC50 of argatroban was increased from 1.1 (vs. thrombin in solution) to 2.7 μM, while, although rHV2 Lys 47 and heparin had IC50 values of 2.8 nM and 0.004 U/ml against thrombin in solution, they had little (32% inhibition by 4 pM rHV2 Lys 47) or no effect (even at 5.0 U/ml heparin) against the amidolytic activity of a plasma clot. We conclude that argatroban could present advantages over hirudin and heparin in the treatment of pathologies where the enzymatic activity of clot-bound thrombin may play a significant role.

INTERACTION BETWEEN CULTURED ENDOTHELIAL CELLS AND ABNORMAL ANTITHROMBIN III "TOYAMA"

1987 ◽  
Author(s):  
N Sakuragawa ◽  
S Saitoh ◽  
K Takahashi
Keyword(s):  
Endothelial Cells ◽  
Room Temperature ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Binding Activity ◽  
Endothelial Cell Culture ◽  
Antithrombin Activity ◽  
Thrombin Activity ◽  
Cultured Endothelial Cells ◽  
At Iii

Purpose: Abnormal antithrombin III(AT-III)Toyama showed non-affinity to heparin and heparinoid to show loss of immediate antithrombin activity. On the endothelial cells, there are heparinoids including heparan sulfate. We investigated on the interaction between cultured endothelial cells and abnormal AT-III"Toyama" from the viewpoint of antithrombin activity.Materials and methods: (1) Endothelial cell culture:^125I-labelled normal and abnormal AT-III were placed on the washed endothelial cultured cells in 0.2 ml of RPMI-1640 medium for 15 min at 37°C. The medium was suctioned off and the cell layer was washed with Hank's balanced salt solution. The cells were incubated with 1 ml of heparin(3 ug/ml) for 15 min at 4°C. The radioactivity in the supernatant was counted, and represented AT-III which bound to the cells surface. (2) Antithrombin activity: 0.23 ml of thrombin solution^ U/ml) and 0.03 ml of normal or abnormal AT-III plasma were mixed, and incubated on the cultured cell surface for 5 min at room temperature. The residual thrombin activity was assayed by 0.3 ml of the substrate (S-2238) solution(0.8mM)for 5 min. After these procedures,2 ml of 2% citric acid solution was added to stop the reaction, and 0D(405 nm) was recorded.Results: Abnormal AT-III showed reduced binding-activity to cultured cells to one fifth compared with normal AT-III, and the residual thrombin activity in the abnormal was higher compared with that in normal plasma.Conclusion: Abnormal AT-III showed less binding activity to the cultured endothelial cells, and less thrombin neutralizing activity to show thrombogenic tendency.

scholarly journals Elastase-mediated fibrinogenolysis by chemoattractant-stimulated neutrophils occurs in the presence of physiologic concentrations of antiproteinases.

1987 ◽  
Vol 166 (6) ◽  
pp. 1836-1850 ◽  
Author(s):  
J I Weitz ◽  
A J Huang ◽  
S L Landman ◽  
S C Nicholson ◽  
S C Silverstein
Keyword(s):  
Trypsin Inhibitor ◽  
Proteinase Inhibitor ◽  
Degradation Products ◽  
Close Contact ◽  
Soybean Trypsin Inhibitor ◽  
Low Concentrations ◽  
Cell Substrate ◽  
Fibrinogenolytic Activity ◽  
Coated Surface

Plasma levels of the HNE-derived fibrinopeptide A alpha 1-21 reflect in vivo enzyme activity. To provide a possible explanation for the presence of circulating A alpha 1-21 in individuals with normal plasma antiproteinase concentrations we investigated whether PMN-associated HNE is more resistant to inhibition than the free enzyme. PMN were stimulated to migrate across 125I-fibrinogen-coated nitrocellulose filters in response to 10(-7) M FMLP, and the extent of fibrinogenolysis was determined by measuring release of A alpha 1-21 and 125I-labeled fibrinogen degradation products. The fibrinogenolytic activity of migrating PMN was then compared with that of free HNE present in PMN lysates or secreted by PMN stimulated with FMLP. Whereas the fibrinogenolytic activity of soluble HNE was completely inhibited by low concentrations (1%) of plasma or serum and macromolecular antiproteinase (alpha 1 proteinase-inhibitor and soybean trypsin-inhibitor), even in the presence of undiluted plasma or serum the activity of the migrating PMN was incompletely blocked (81-85%). Further, concentrations of alpha 1 proteinase-inhibitor and soybean trypsin-inhibitor that totally inhibited free HNE activity also incompletely blocked (88-89%) the fibrinogenolytic activity of migrating PMN, indicating that FMLP-stimulated PMN demonstrate significant fibrinogenolytic activity in the presence of antiproteinases as small as 20,000 mol wt. A specific low molecular weight HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl), however, totally blocked PMN-mediated fibrinogenolysis without affecting intracellular HNE activity, HNE secretion from PMN, or PMN migration in response to FMLP. These findings support the hypothesis that PMN migrating on a fibrinogen-coated surface form zones of close contact with fibrinogen, thus preventing access of plasma antiproteinases to HNE released at the cell-substrate interface. The occurrence of this phenomenon in vivo would explain the presence of circulating A alpha 1-21 in individuals with normal antiproteinase concentrations.

scholarly journals Chromatography of transforming deoxyribonucleic acid on methylated albumin and by gel filtration

1974 ◽  
Vol 137 (3) ◽  
pp. 543-546
Author(s):  
G. R. Barker ◽  
P. Hodges
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
High Molecular Weight ◽  
Deoxyribonucleic Acid ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Transforming Activity ◽  
Different Strains ◽  
Two Peaks

1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.

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