The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

1978 ◽  
Vol 40 (02) ◽  
pp. 350-357
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Molecular Weight ◽  
Dissociation Constant ◽  
Calcium Ions ◽  
Activation Process ◽  
Factor X ◽  
The Real ◽  
High Affinity ◽  
Binding Data ◽  
Sequential Mechanism ◽  
Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

Activation of Bovine Factor X in the Presence of Calcium, Magnesium, Barium or Manganese ion

1978 ◽  
Vol 40 (02) ◽  
pp. 358-367 ◽  
Author(s):  
Robert H Yue ◽  
Menard M Gertler
Keyword(s):  
Metal Ions ◽  
Binding Site ◽  
Metal Ion ◽  
Factor Xa ◽  
Divalent Metal ◽  
Activation Process ◽  
Factor X ◽  
Divalent Metal Ion ◽  
Russell’S Viper ◽  
Bovine Factor

SummaryThe binding of divalent metal ions to bovine factor X, factor Xa and the coagulant protein in Russell’s viper venom was studied by the technique of fluorescence quenching. Titration of factor X with Ca+2, Mg+2 or Ba+2 revealed that these metal ions can bind to factor X. A tightly binding site(s) was observed with Kd of 79 and 98 μM for Ca+2 and Mg+2 respectively. A loosely binding site(s) was evident with Kd of 0.55, 0.50 and 0.35 mM for Ca+2, Mg+2 and Ba+2 respectively. The quenching phenomenon was also observed when Mn+2 was used as titrant but factor X precipitated out when the concentration of Mn+2 was 10 mM. The binding of Ca+2, Mg+2, Ba+2 or Mn+2 to bovine factor Xa or to the purified coagulant fraction of Russell’s viper venom was very weak in each case.In the absence of Ca+2, the coagulation fraction of Russell’s viper venom could not activate bovine factor X. Activation of factor X was achieved when Ca+2 was replaced by either Mg+2, Ba+2 or Mn+2. When the concentration of these ions were 5 mM, the efficiency of factor Xa generation was estimated to be: Ca+2> Mg+2> Ba+2> Mn+2. Higher concentration of Mg+2, Ba+2, or Mn+2 retarded the activation process. However, Ca+2, Mg+2, Ba+2 or Mn+2 has little or no influence on the esterase activity of factor Xa or purified Rusell’s viper venom.The results suggest that complexation of divalent metal ion with factor X is prerequisite in the activation process. The binding of Mg+2, Ba+2 or Mn+2 to these loosely binding sites might have altered the geometrical configuration as well as the electrostatic environment on factor X significantly. Thus, it is more difficult to form the binary complex and a slower generation of factor Xa results. Therefore, divalent metal ion serves as a dual role in the activation of factor X to factor Xa depending upon the ionic concentration.

STUDIES ON THE BINDING OF 125I-LABELLED CORTICOTROPHIN TO ISOLATED RAT ADRENOCORTICAL CELLS

1975 ◽  
Vol 64 (1) ◽  
pp. 175-184 ◽  
Author(s):  
R. A. J. McILHINNEY ◽  
D. SCHULSTER
Keyword(s):  
Dissociation Constant ◽  
Adrenal Glands ◽  
Direct Evidence ◽  
Scatchard Analysis ◽  
Adrenocortical Cells ◽  
Binding Characteristics ◽  
Whole Cells ◽  
High Affinity ◽  
Binding Data ◽  
Normal Rat

SUMMARY Isolated adrenocortical cells, prepared by collagenase disaggregation of normal rat adrenal glands, have been used to study the binding characteristics of 125I-labelled corticotrophin (ACTH) of established biological activity. The binding of the labelled hormone to these whole cells was highly specific, only peptides possessing steroidogenic activity displaced the labelled hormone. Binding was rapid, being complete within 5 min of adding the hormone, and the amount of hormone bound remained constant for up to 20 min thereafter. Over the range 160–10000 pg ACTH/ml, increased binding of the hormone was observed at all concentrations of hormone which stimulated steroidogenesis. However at levels of ACTH which stimulated maximal steroidogenesis there was no saturation of binding. This provides the first direct evidence for the existence of 'spare receptors' for ACTH on whole adrenocortical cells. Scatchard analysis of the binding data suggests that there are two types of receptor for ACTH in this preparation of cells. One receptor is of high affinity (dissociation constant = 2·5 × 10−10 mol/l) about 3000 sites/cell and the other is of lower affinity (dissociation constant = 1 × 10−8 mol/l) with about 30000 sites/cell.

scholarly journals The preparation of activated Factor X and its action on prothrombin

1973 ◽  
Vol 131 (4) ◽  
pp. 791-797 ◽  
Author(s):  
Jolyon Jesty ◽  
M. Peter Esnouf
Keyword(s):  
Molecular Weight ◽  
Factor Xa ◽  
Mean Value ◽  
Factor V ◽  
Factor X ◽  
Reaction Products ◽  
Molecular Weights ◽  
Russell’S Viper ◽  
Bovine Factor

The preparation of activated Factor X from reaction mixtures of bovine Factor X and Russell's-viper venom is described. The molecular weight of purified protein varies about a mean value of 40000; this variation is the result of at least two forms of Factor Xa. The action of activated Factor X, together with purified Factor V, was studied on purified prothrombin and the reaction products were isolated. In addition to thrombin, two other polypeptides with molecular weights of 16000 and 19500 were recovered.

Self-Damping Mechanism in Blood Coagulation

1974 ◽  
Vol 32 (01) ◽  
pp. 057-064 ◽  
Author(s):  
Y Nemerson ◽  
S.A Silverberg ◽  
J Jesty
Keyword(s):  
Tissue Factor ◽  
Blood Coagulation ◽  
Calcium Ions ◽  
Control Mechanism ◽  
Factor Vii ◽  
Damping Factor ◽  
Factor V ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Two Systems

SummaryTwo reactions of the extrinsic pathway of coagulation, the activations of Factor X and prothrombin, have been studied in purified systems and shown to be self-damping. Factor X was activated by the tissue factor - Factor VII complex, and prothrombin by two systems: the coagulant protein of Taipan venom, and the physiological complex of activated Factor X, Factor V, lipid, and calcium ions. In each case the yield of enzyme, activated Factor X or thrombin, is a function of the concentration of activator. These and other observations are considered as a basis for a control mechanism in coagulation.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

1989 ◽  
Vol 61 (03) ◽  
pp. 437-441 ◽  
Author(s):  
Cindra Condra ◽  
Elka Nutt ◽  
Christopher J Petroski ◽  
Ellen Simpson ◽  
P A Friedman ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Salivary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Potent Inhibitor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Sds Page ◽  
Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

1987 ◽  
Vol 58 (04) ◽  
pp. 1064-1067 ◽  
Author(s):  
K Kodama ◽  
B Pasche ◽  
P Olsson ◽  
J Swedenborg ◽  
L Adolfsson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Surface Coating ◽  
Antithrombin Iii ◽  
Lower Class ◽  
Biologically Active ◽  
High Affinity ◽  
Heparin Coating ◽  
Continuous Surface ◽  
Approximate Molecular Weight

SummaryThe mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

1983 ◽  
Vol 49 (02) ◽  
pp. 109-115 ◽  
Author(s):  
M Hoylaerts ◽  
E Holmer ◽  
M de Mol ◽  
D Collen
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Life Time ◽  
Low Molecular Weight ◽  
High Affinity ◽  
Plasma Half Life ◽  
Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

Factor X Activation by washed human platelets

1979 ◽  
Author(s):  
E van Wijk ◽  
L Kahlé ◽  
J ten Cate
Keyword(s):  
Human Factors ◽  
Trypsin Inhibitor ◽  
Calcium Ions ◽  
Factor Xa ◽  
Human Platelets ◽  
Soybean Trypsin Inhibitor ◽  
Thrombin Inhibitor ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Factor X Activation

In a system of washed human platelets, Ca2+and purified human factors X anc II, a sufficient amount of thrombin is generated in about 10 minutes to aggregate the platelets. This thrombin is formed through the activation of FX by the platelets. In a system with either FX or FII present, no aggregation occurs. In addition no aggregation is observed when hirudin, a specific thrombin inhibitor, or when soybean trypsin inhibitor, which inhibits factor Xa, are added to the mixture. The formation of factor Xa can be monitored indirectly through the generation of thrombin, in the presence of an excess of prothrombin, using a thrombin sensitive chromogenic substrate. When washed platelets are incubated with FX alone for 10 minutes, no aggregation occurs and after the addition of prothrombin aggregation starts within 6 minutes. These findings confirm that washed platelets possess a factor X activating property. The generation of FXa proceeds in the absence of added Ca2+, whereas in the presence of Ca2+factor Xa activity reaches a maximum in 3 minutes, whereafter the activity progressively decreases. This may be due to the binding of Xa to the platelets in the presence of calcium ions.

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

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