Interaction of Thrombin with Antithrombin III and α2Macrogeobulin

1979 ◽  
Author(s):  
A. Takada ◽  
Y. Takada
Keyword(s):  
Complex Formation ◽  
Antithrombin Iii ◽  
Thrombin Activity ◽  
Presence And Absence ◽  
Hydrolysis Of

When one unit of thrombin was added to recalcified diluted plasma, more thrombin activity was shown in the presence of heparin than in its absence, but no difference was shown after two hour incubation. When one unit of thrombin was added to diluted plasma without the addition of Ca++, no difference in thrombin activities was shown in the presence and absence of heparin. When highly purified α2macroglobulin (α2M) and antithrombin III (ATIII) were used, thrombin activity was initially enhanced in the presence of either ATIII or ATIII, and quick inactivation of thrombin by ATIII regardless of the presence of heparin was observed. Electrophoresis shows that migrating patterns of ATIII depended upon amounts of heparin added to plasma, and ATIII migrated more to the anode with larger amounts of heparin. Thrombin-ATIII complex formed quickly in the undiluted recalcified plasma in the presence of heparin, but little complex formation was shown in the absence of heparin. When α2M was mixed with thrombin, and the mixture was added to TLMe at intervals, hydrolysis of TLMe was enhanced initially, then decreased quickly. α2M-thrombin complex seemed to be not as effective as free thrombin in the capacity to hydrolyze TLMe in contrast to α2M-trypsin or α2M-plasmin complex. α2M may be a primary inhibitor of thrombin in the plasma in the absence of heparin. In the presence of heparin, ATIII seems to be a primary inhibitor of thrombin.

scholarly journals Inactivation of α- and β-thrombin by antithrombin-III, α2-macroglobulin and α1-proteinase inhibitor

1977 ◽  
Vol 167 (2) ◽  
pp. 393-398 ◽  
Author(s):  
R Machovich ◽  
A Borsodi ◽  
G Blaskó ◽  
S A Orakzai
Keyword(s):  
Complex Formation ◽  
Incubation Time ◽  
Proteinase Inhibitor ◽  
Antithrombin Iii ◽  
Inhibitor Concentration ◽  
Heparin Concentration ◽  
Thrombin Activity ◽  
Α2 Macroglobulin

Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.

Inactivation of α- and β-Thrombin by Antithrombin-III and Heparin

1976 ◽  
Vol 36 (03) ◽  
pp. 503-508 ◽  
Author(s):  
Raymund Machovich ◽  
György Blaskó ◽  
Anna Borsodi
Keyword(s):  
Heat Treatment ◽  
Complex Formation ◽  
Blood Circulation ◽  
Antithrombin Iii

SummaryInactivation of α- and β-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b).It was found that the inactivation rates of α- and β-thrombin by antithrombin were different, namely α-thrombin was more sensitive to antithrombin than β-thrombin. Heparin facilitated the complex formation between α-thrombin and antithrombin-III, whereas β-thrombin inactivation was only slightly affected.Furthermore, heparin protected α-thrombin against the inactivating effect of heat, while β-thrombin lost its activity during the heat treatment.These findings suggest that the formation of β-thrombin in blood circulation may have an important role in thrombosis predisposition.

Schiff base equilibria. II. Beryllium complexes of N-n-butylsalicylideneimine and the hydrolysis of the Be2+ ion

1965 ◽  
Vol 18 (5) ◽  
pp. 651 ◽  
Author(s):  
RW Green ◽  
PW Alexander
Keyword(s):  
Aqueous Solution ◽  
Schiff Base ◽  
Complex Formation ◽  
Distribution Coefficient ◽  
Dilute Aqueous Solution ◽  
The Stability ◽  
Ph Range ◽  
Hydrolysis Of ◽  
Beryllium Complexes ◽  
Equilibria In Aqueous Solution

The Schiff base, N-n-butylsalicylideneimine, extracts more than 99.8% beryllium into toluene from dilute aqueous solution. The distribution of beryllium has been studied in the pH range 5-13 and is discussed in terms of the several complex equilibria in aqueous solution. The stability constants of the complexes formed between beryllium and the Schiff base are log β1 11.1 and log β2 20.4, and the distribution coefficient of the bis complex is 550. Over most of the pH range, hydrolysis of the Be2+ ion competes with complex formation and provides a means of measuring the hydrolysis constants. They are for the reactions: Be(H2O)42+ ↔ 2H+ + Be(H2O)2(OH)2, log*β2 - 13.65; Be(H2O)42+ ↔ 3H+ + Be(H2O)(OH)3-, log*β3 -24.11.

pH Effects in trypsin catalysis

1969 ◽  
Vol 47 (21) ◽  
pp. 4021-4029 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Complex Formation ◽  
Ph Dependence ◽  
Specific Rotation ◽  
Ph Effects ◽  
Ph Values ◽  
A Value ◽  
Available Information ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Covalent Complex

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6 to 10. The substrate concentrations varied from 1.7 × 10−3 to 4.3 × 10−2 M. From the rates were calculated, at each pH, values of [Formula: see text] (corresponding to [Formula: see text]), [Formula: see text] (corresponding to [Formula: see text]) and [Formula: see text] The specific levorotation of trypsin was measured and found to vary with pH in the pH region 5–11, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At pH 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of [Formula: see text] in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in [Formula: see text] at high pH corresponding to a value of [Formula: see text] whereas [Formula: see text] showed sigmoid pH-dependence. An interpretation of these results that is consistent with all available information is that a group of [Formula: see text] (presumably the —NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

scholarly journals Targeting PirAvp and PirBvp Toxins of Vibrio parahaemolyticus with Oilseed Peptides: An In Silico Approach

Antibiotics ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1211
Author(s):  
Joe-Hui Ong ◽  
Wey-Lim Wong ◽  
Fai-Chu Wong ◽  
Tsun-Thai Chai
Keyword(s):  
Complex Formation ◽  
In Silico ◽  
Vibrio Parahaemolyticus ◽  
Water Solubility ◽  
Toxin Binding ◽  
Binding Peptides ◽  
Acute Hepatopancreatic Necrosis Disease ◽  
Hydrolysis Of ◽  
Tryptic Hydrolysis ◽  
Dual Target

Acute hepatopancreatic necrosis disease (AHPND), caused by PirAvp- and PirBvp-releasing Vibrio parahaemolyticus strains, has resulted in massive mortality in shrimp aquaculture. Excessive use of antibiotics for AHPND management has led to antibiotic resistance, highlighting the urgency to search for alternatives. Using an in silico approach, we aimed to discover PirAvp/PirBvp-binding peptides from oilseed meals as alternatives to antibiotics. To search for peptides that remain intact in the shrimp digestive tract, and therefore would be available for toxin binding, we focused on peptides released from tryptic hydrolysis of 37 major proteins from seeds of hemp, pumpkin, rape, sesame, and sunflower. This yielded 809 peptides. Further screening led to 24 peptides predicted as being non-toxic to shrimp, fish, and humans, with thermal stability and low water solubility. Molecular docking on the 24 peptides revealed six dual-target peptides capable of binding to key regions responsible for complex formation on both PirAvp and PirBvp. The peptides (ISYVVQGMGISGR, LTFVVHGHALMGK, QSLGVPPQLGNACNLDNLDVLQPTETIK, ISTINSQTLPILSQLR, PQFLVGASSILR, and VQVVNHMGQK) are 1139–2977 Da in mass and 10–28 residues in length. Such peptides are potential candidates for the future development of peptide-based anti-AHPND agents which potentially mitigate V. parahaemolyticus pathogenesis by intercepting PirAvp/PirBvp complex formation.

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.

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.

The Thrombin Protecting Function of a Complement Inhibitor in Human Plasma

1979 ◽  
Author(s):  
E.R. Podack ◽  
J.G. Curd ◽  
J.H. Griffin ◽  
H.J. Müller-Eberherd
Keyword(s):  
Complex Formation ◽  
Antithrombin Iii ◽  
Membrane Attack Complex ◽  
Protein S ◽  
Thrombin Time ◽  
Two Dimensional ◽  
Complement Inhibitor ◽  
Functional Studies ◽  
Sds Page ◽  
At Iii

S-protein (S) is a newly discovered 80,000 MW plosma glycoprotein. It functions as an inhibitor of the membrane attack complex of complement. We now wish to report that S also functions as thrombin protecting factor in coagulation; S forms a reversible complex with thrombin which is more resistant to inactivation by antithrombin III (AT III) than thrombin alone. An S-thrombin complex and on S-throm-bin-AT III complex were formed in clotted plasma and with isolated proteins as demonstrated by two dimensional Immunoelectrophoresis. Functional studies measuring the esterolytic or clotting activity of thrombin showed that S in the presence and absence of heparin decreased the rate of inactivation of thrombin by AT III. Similar results were observed using plasma. For example, in the presence of 0.04 u/ml heparin and 1.6 u/ml thrombin, the thrombin time of plasma depleted in S was 150 sec. as opposed to 15 sec. when the plasma was reconstituted with purified S. That this effect of S was due to a decreased inactivation of thrombin by AT III was demonstrated directly by SDS-PAGE analysis of plasma containing 125l-thrombin. In the presence of S the rate of formation of the 95,000 dalton 125I-thrombin-AT III complex was markedly decreased compared to the rate of complex formation in the S-depleted plasma. These data suggest that S may modulate the interactions of thrombin and AT III.

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