scholarly journals The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type

1988 ◽  
Vol 256 (3) ◽  
pp. 815-820 ◽  
Author(s):  
H Soons ◽  
G Tans ◽  
H C Hemker
Keyword(s):  
Unfractionated Heparin ◽  
Binding Constant ◽  
Kinetic Data ◽  
Maximum Rate ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Pseudo First Order Reaction ◽  
Rate Enhancement ◽  
Factor Xia ◽  
Heparin Fractions

The effect of various well-characterized heparin preparations on the inactivation of human Factor XIa by human antithrombin III was studied. The heparin preparations used were unfractionated heparin and four heparin fractions obtained after anion-exchange chromatography. Inactivation of Factor XIa was monitored with S2366 as chromogenic substrate and followed pseudo-first-order reaction kinetics under all reaction conditions tested. Enhancement of the rate of inhibition of Factor XIa in the presence of unfractionated heparin correlated to the binding of antithrombin III to heparin. From the kinetic data a binding constant of 0.1 microM was inferred. The maximum rate enhancement, achieved at saturating heparin concentrations, was 30-fold. The rate enhancement achieved in the presence of each of the heparin fractions could also be correlated to the binding of antithrombin III to the heparin. The binding constant inferred from the kinetic data varied from 0.10 to 0.28 microM and the number of binding sites for antithrombin III varied from 0.06 to 0.74 site per heparin molecule. The maximum rate enhancements, achieved at saturating heparin concentrations, were strongly dependent on the type of heparin used and varied from 7-fold for fraction A to 41-fold for fraction D. Therefore, although the stimulation of Factor XIa inactivation by antithrombin III could be quantitatively correlated to the binding of antithrombin III to heparin, the heparin-catalysed inhibition of Factor XIa is dependent not only upon the degree of binding of antithrombin III to heparin but also upon the type of heparin to which antithrombin III is bound.

scholarly journals The acceleration of the inhibition of platelet prothrombinase complex by heparin

1986 ◽  
Vol 233 (1) ◽  
pp. 161-165 ◽  
Author(s):  
V Ellis ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Unfractionated Heparin ◽  
Rate Constant ◽  
Nitrous Acid ◽  
Maximum Rate ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Prothrombinase Complex ◽  
High Affinity ◽  
Heparin Fractions

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 × 10(7) M−1 × min−1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 × 10(8) M−1 × min−1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 × 10(7), 1.4 × 10(7) and 9.8 × 10(6) M−1 × min−1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.

scholarly journals Effect of heparin on the inactivation rate of human factor XIa by antithrombin-III

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 940-947 ◽  
Author(s):  
CF Scott ◽  
M Schapira ◽  
RW Colman
Keyword(s):  
Clinical Practice ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Fold Increase ◽  
Factor Ix ◽  
Inactivation Rate ◽  
Amidolytic Activity ◽  
Factor Ixa ◽  
Factor Xia ◽  
Plasma Heparin

Abstract Factor XIa catalyzes an important reaction in the early phase of blood coagulation by converting factor IX to an active enzyme (factor IXa). Although antithrombin-III, an inhibitor of factor XIa, normally accounts for only one-sixth of the plasma inhibitory activity against factor XIa, its effectiveness has been reported to be enhanced by heparin. We have reinvestigated the ability of heparin to potentiate factor XIa inhibition by both purified antithrombin-III and plasma using synthetic tripeptide amide substrates as well as a coagulant assay. No increase in the inactivation rate of factor XIa amidolytic activity by purified antithrombin-III was observed in the presence of therapeutic heparin concentrations (1 U/ml), although inhibition of the amidolytic activity of thrombin by purified antithrombin-III was enhanced at least 20-fold by the same concentration of heparin. Furthermore, despite the ability of heparin (1 U/ml) to increase the inactivation rate of thrombin by plasma, no acceleration of the rate of inhibition of factor XIa by plasma was observed. Similar results were found when the inhibition of factor XIa was monitored with a coagulant assay after first removing the heparin. Only at heparin concentrations of 5 and 10 U/ml, was a 2- and 4-fold increase in the inactivation rate of factor XIa by purified antithrombin III observed. Therefore, in both purified systems as well as plasma, heparin, at concentrations observed in clinical practice, does not accelerate the inactivation rate of human factor XIa by antithrombin-III.

scholarly journals Cleavage and activation of human factor IX by serine proteases

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 821-831
Author(s):  
DL Enfield ◽  
AR Thompson
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
Immunoradiometric Assay ◽  
Single Chain ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

scholarly journals Cleavage and activation of human factor IX by serine proteases

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 821-831 ◽  
Author(s):  
DL Enfield ◽  
AR Thompson
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
Immunoradiometric Assay ◽  
Single Chain ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia

Abstract Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

Effect of heparin on the inactivation rate of human factor XIa by antithrombin-III

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 940-947
Author(s):  
CF Scott ◽  
M Schapira ◽  
RW Colman
Keyword(s):  
Clinical Practice ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Fold Increase ◽  
Factor Ix ◽  
Inactivation Rate ◽  
Amidolytic Activity ◽  
Factor Ixa ◽  
Factor Xia ◽  
Plasma Heparin

Factor XIa catalyzes an important reaction in the early phase of blood coagulation by converting factor IX to an active enzyme (factor IXa). Although antithrombin-III, an inhibitor of factor XIa, normally accounts for only one-sixth of the plasma inhibitory activity against factor XIa, its effectiveness has been reported to be enhanced by heparin. We have reinvestigated the ability of heparin to potentiate factor XIa inhibition by both purified antithrombin-III and plasma using synthetic tripeptide amide substrates as well as a coagulant assay. No increase in the inactivation rate of factor XIa amidolytic activity by purified antithrombin-III was observed in the presence of therapeutic heparin concentrations (1 U/ml), although inhibition of the amidolytic activity of thrombin by purified antithrombin-III was enhanced at least 20-fold by the same concentration of heparin. Furthermore, despite the ability of heparin (1 U/ml) to increase the inactivation rate of thrombin by plasma, no acceleration of the rate of inhibition of factor XIa by plasma was observed. Similar results were found when the inhibition of factor XIa was monitored with a coagulant assay after first removing the heparin. Only at heparin concentrations of 5 and 10 U/ml, was a 2- and 4-fold increase in the inactivation rate of factor XIa by purified antithrombin III observed. Therefore, in both purified systems as well as plasma, heparin, at concentrations observed in clinical practice, does not accelerate the inactivation rate of human factor XIa by antithrombin-III.

Binding of Heparin Fractions to von Willebrand Factor: Effect of Molecular Weight and Affinity for Antithrombin III

1994 ◽  
Vol 71 (01) ◽  
pp. 141-146 ◽  
Author(s):  
D Baruch ◽  
Nadine Ajzenberg ◽  
Cécile Denis ◽  
Paulette Legendre ◽  
Jean-Claude Lormeau ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Antithrombin Iii ◽  
Heparin Binding ◽  
Agarose Beads ◽  
Heparin Interaction ◽  
Factor Effect ◽  
Heparin Fractions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo investigate the influence of the structure of heparin on its binding to vWF, we compared heparin fractions of different molecular weight (MW) or affinity for antithrombin III (ATIII). We studied the interaction of purified 125I-vWF or plasma vWF, labeled with a pool of 125I-monoclonal antibodies to vWF, with unfractionated heparin immobilized on agarose beads. Fractions were compared as competitors of these interactions and their effect was quantitated by their half-maximal inhibition (IC50). When the MW of the fractions decreased, especially below 7500, their IC50 increased, indicating that the affinity of the fractions for vWF decreased with their MW. Using heparin-derived oligosaccharides, we also demonstrated that a minimal chain length of 18 monosaccharides was required for heparin binding to vWF. In addition, different fractions with low affinity for ATIII were compared as competitors of 125-vWF binding to heparin-agarose. Despite a very low content of ATIII binding sites, some fractions retained a low IC50. Thus, heparin interaction with vWF is independent of the presence of the ATIII binding site and is mostly dependent on the length of the heparin chain. These data suggest that unfractionated heparin is a more potent inhibitor of vWF-dependent functions than low MW heparin fractions.

HEPARIN CATALYZED FACTOR XIa INHIBITION BY ANTITHROMBIN III

1987 ◽  
Author(s):  
H soons ◽  
T Jansen-claessen ◽  
G C Tans ◽  
H C Hemker
Keyword(s):  
Rate Constant ◽  
Active Sites ◽  
Time Course ◽  
Antithrombin Iii ◽  
Order Reaction ◽  
Pseudo First Order Reaction ◽  
First Order ◽  
At Iii ◽  
Factor Xia ◽  
Pseudo First Order

The inactivation of human factor XIa by human antithrombin III (AT III) was studied under pseudo-first order reaction conditions (excess AT III) both in the absence and presence of heparin. The time course of inhibition was followed using SDS-PAGE. After electrophoresis proteins were blotted onto nitrocellulose and stained either for glycoprotein or for AT III using antibodies against AT III. Concomittant with factor XIa inactivation two new slower migrating bands became visible on the blots. One of these, representing the intermediate complex consisting of one AT III complexed with one of the active sites present in factor XIa, appeared as a transient band. Complete inactivation resulted in a single band representing the complex of factor XIa with two AT III molecules. This indicates that inhibition of factor XIa by AT III can be described as:Quantitative analysis of the time course of inactivation was accomplished by measurement of the disappearance of factor XIa amidolytic activity towards the chromogenic substrate S2366. Pseudo first order reaction kinetics were observed throughout. The time course of inactivation and the distribution of the reaction products observed upon gelelectrophoresis are best explained assuming a mechanism of inactivation in which the two active sites present in factor XIa are inhibited in random order (i.e. independent of each other) with the same rate constant of inhibition (k1= k2)- The rate constant of inactivation for the active sites in factor XIa was found to be 1,000 M-1 s-1 in the absence of heparin and 34,900 M-1 s-1 in the presence of saturating amounts of heparin.- From the kinetic data a binding constant Kd of 0.11 uM was inferred for the binding of AT III to heparin. Experiments with four well characterized heparin fractions indicate, that the actual magnitude of the rate enhancement of factor XIa inactivation is, however, not only due to the binding of AT III to heparin, but also depends on the type of heparin to which the AT III is bound.

scholarly journals The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides

1987 ◽  
Vol 243 (1) ◽  
pp. 31-37 ◽  
Author(s):  
T W Barrowcliffe ◽  
S J Havercroft ◽  
G Kemball-Cook ◽  
U Lindahl
Keyword(s):  
Protective Effect ◽  
Unfractionated Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Free Enzyme ◽  
Factor V ◽  
High Affinity ◽  
Heparin Fractions ◽  
Heparin Oligosaccharides ◽  
Bovine Factor

The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results.

Inactivation of Factor XIa in Vivo: Studies in Chimpanzees and in Humans

1996 ◽  
Vol 76 (04) ◽  
pp. 549-555 ◽  
Author(s):  
Walter A Wuillemin ◽  
C Erik Hack ◽  
Wim K Bleeker ◽  
Bart J Biemond ◽  
Marcel Levi ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Life Time ◽  
In Vivo Studies ◽  
Clinical Samples ◽  
Plasma Samples ◽  
C1 Inhibitor ◽  
Elimination Kinetics ◽  
Factor Xia ◽  
Best Parameter

SummaryC1-inhibitor (C1Inh), antithrombin III (ATIII), α1-antitrypsin (a1AT), and α2-antiplasmin (a2AP) are known inhibitors of factor XIa (FXIa). However, their precise contribution to FXIa inactivation in vivo is not known. We investigated FXIa inactivation in chimpanzees and assessed the contribution of these inhibitors to FXIa inactivation in patients with presumed FXI activation.Chimpanzees were infused with FXIa and the various FXIa-FXIa inhibitor complexes formed were measured. Most of FXIa was complexed to C1Inh (68%), followed by a2AP (13%), a1AT (10%), and ATIII (9%). Analysis of the plasma elimination kinetics revealed a half-life time of clearance (t1/2) for the FXIa-FXIa inhibitor complexes of 95 to 104 min, except for FXIa-a1AT, which had a t1/2 of 349 min. Due to this long t1/2, FXIa-a1AT complexes were predicted to show the highest levels in plasma samples from patients with activation of FXI. This was indeed shown in patients with disseminated intravascular coagulation, recent myocardial infarction or unstable angina pectoris. We conclude from this study that in vivo C1Inh is the predominant inhibitor of FXIa, but that FXIa-a1 AT complexes due to their relatively long t1/2 may be the best parameter to assess FXI activation in clinical samples.

Factor V from Human Plasma

1961 ◽  
Vol 05 (01) ◽  
pp. 001-020
Author(s):  
Douglas M. Surgenor ◽  
Nancy A. Wilson ◽  
Anne S. Henry
Keyword(s):  
Human Serum ◽  
Human Plasma ◽  
Kinetic Data ◽  
Human Factor ◽  
Kinetic Studies ◽  
Partial Purification ◽  
Factor V ◽  
Two Stage ◽  
Plasma Factor ◽  
Tissue Thromboplastin

SummaryA method is described for the partial purification of a human plasma factor which accelerates the conversion of prothrombin to thrombin in the presence of tissue thromboplastin. This factor may be dried from the frozen state, and may be kept in stable dry form for long periods of time. The quantitative assay of this activity is done in a classical two-stage prothrombin system using tissue thromboplastin and calcium. From its properties, it is concluded that this activity corresponds to factor V, labile factor and plasma Ac-globulin.Chemical and kinetic studies reveal that human factor V is active in plasma and is destroyed by thrombin. Human serum has little or no factor V activity.These results thus fail to support the postulated activation of factor V during clotting. All of the kinetic data are consistent with an enzymatic role for factor V in the formation of tissue prothrombin activator (thromboplastin).

Sign in / Sign up

Export Citation Format

Share Document