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.

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.

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.

Control Of Factor IXa Production By The Formation Of A Factor XIa-Factor IXa Complex

1981 ◽  
Author(s):  
A Jonan ◽  
C W Slattery
Keyword(s):  
Fold Increase ◽  
Factor Ix ◽  
Factor Xii ◽  
Clotting Time ◽  
Contact Phase ◽  
Factor Ixa ◽  
Activity Measurements ◽  
Activity Generation ◽  
Factor Xia ◽  
Dependent Interaction

The activation of factor IX represents a transition step between the “contact phase” and the “phospholipid-dependent phase” of intrinsic blood coagulation. A study was made to see if any of the constituents of either of these phases would influence this activation. Trypsin-activated factor XIa and factor IX were preincubated with various combinations of kaolin, high molecular weight kininogen, factor XII and human brain cephalin before being added to factor IX deficient plasma to determine the clotting time. Of these, only cephalin in the preincubation mixture apparently increased factor IXa activity. Measurements of the rate of activity generation with and without cephalin and with early and late addition of cephalin indicated that cephalin caused a five-fold increase in the maximum obtainable factor IXa activity. Late addition started at lower activity and gradually increased to the maximum. Factor IXa, isolated from factor XIa by means of an immobilized factor Xl-antibody column, responded to cephalin addition by becoming more active and to factor XIa addition by becoming less active. Centrifugation of cephalin-protein mixtures showed that neither factor XIa nor factor IX but only factor IXa was bound to the phospholipid. These data suggest that: 1) the activation of factor IX by factor XIa does not occur on a phospholipid surface; 2) once cleaved, the factor IXa that is produced remains bound to the active site of factor XIa, preventing further factor IX activation until a phospholipid surface is available for the reaction sequence to continue; 3) phospholipid competes with factor XIa for the bound factor IXa and these, in a time-dependent interaction, then form an active complex. This mechanism would prevent the production and dissemination of large amounts of factor IXa until required for further clotting.

Activation of Normal and Abnormal (Genetic Variant) Human Factor IX

1979 ◽  
Author(s):  
R.M. Bertina ◽  
I.K. van der Linden
Keyword(s):  
Human Factor ◽  
Genetic Variant ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Thromboplastin Factor

Isolated human factor IX (single chainjjMW 63,000) has been activated in three ways;a) in the presence of factor XIa and Ca2+ active 2-chain factor IXa (MW 47,000; MWHC 27,500) is formed via an inactive 2-ehain intermediate (MW 63,000, MWHC 44,500);b) in the presence of thromboplastin, factor VII and Ca2+ essentially the same sequence of reactions takes place as sub a;c) in the presence of RVV-X active 2-chain factor IXa (MW 63,000; MWHC 30,0001 is formed The dependance of these three reactions on the Caconcentration has seen studied.A genetic variant of factor IX was found that like PIVKA IX can be separated from normal factor IX by electropheresis in the presence of Ca. Again like PIVKA IX this variant shows a strongly reduced affinity for binding to Al(OH)3. These findings suggested an abnormality in the Cabinding properties of this variant.The activation of the isolated variant factor IX by the foremen!ioned activators will be compared with that of normal factor IX.

Activation of Bovine Factor IX By Bovine Factor XIa . Active Site Titration Of Factor IXa and Development of A spectro-Photometric assay for Factor IX

1981 ◽  
Author(s):  
G Tans ◽  
T Janssen-Claessen ◽  
G v Dieijen ◽  
J Rosing ◽  
H C Hemker
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Factor Ix ◽  
Plasma Samples ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Bovine Factor

Activation of factor IX by factor XIa occurs via an intermediate which has no esterase activity towards synthetic arginine esters or coagulant activity as determined with a clotting assay. Factor IXa can be active site titrated using p-nitrophenyl-p1-guanidinobenzoate (p-NPGB) as a titrant. The rate and equilibrium constants describing the active site titration will be presented. To determine whether the intermediate occurring during factor IX activation by factor XIa has its active site exposed for p-NPGB the time course of activation of factor IX by factor XIa was followed l) by active site titration of the active sites generated, 2) by gel- electrophoretic analysis in the presence of sodium dodecyl sulfate, 3) by a clotting assay for factor IXa and 4) by measurement of factor IXa using a spectrophotomefric assay. It will be shown that the intermediate occurring during activation of factor IX by factor XIa does not interact with p-NPGB indicating that the active site is not available in the intermediate.Factor IXa can be determined spectrophotometrically by measurement of the rate of factor X activation by factor IXa in the presence of phospholipid and CaCl2. The factor Xa generated is measured using the chromogenic substrate S222. Since human factor IX can be activated with bovine factor XIa and since human factor IXa can activate bovine factorX the bovine clotting factors factor XIa and factor X can be used to construct an assay for factor fx in plasma samples. Experiments will be presented in which it is shown that the spec- trophotometric assay for human factor IXa can be used to determine levels of factor IX in plasma samples of healthy individuals, in plasma samples deficient in various clotting factors and in plasma samples from patients under anti-coagulant therapy. The results are in agreement with a factor IX clotting test.

scholarly journals The effect of platelets in the activation of human blood coagulation factor IX by factor XIa

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 140-148
Author(s):  
H Soons ◽  
T Janssen-Claessen ◽  
HC Hemker ◽  
G Tans
Keyword(s):  
Platelet Activation ◽  
Inhibitory Activity ◽  
Human Factor ◽  
Human Platelets ◽  
Factor Ix ◽  
Dependent Manner ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Bovine Factor

We report here the effect of activated human platelets on the activation of human factor IX by human factor XIa. Factor IXa formed during activation was determined via its ability to activate bovine factor X. To increase sensitivity, phospholipids and bovine factor VIIIa were present in the assay. The kinetic parameters of the factor IX activation were determined in the presence of 10 mmol/L CaCl2. The Km for factor IX was 0.30 mumol/L and kcat was 2.4 s-1. Activated human platelets inhibited factor IX activation by factor XIa in a dose- dependent manner, whereas unstimulated platelets had no effect. Factor IX activation was inhibited for more than 90% at a platelet concentration of 4 X 10(8)/mL, whereas concentrations of less than 10(6)/mL had no influence. The inhibitory effect could be induced by thrombin, collagen, calcium ionophore A 23187, and adrenalin. The appearance of inhibitory activity could be blocked by the addition of the prostacyclin analogue ZK 36374 at any time during platelet activation. Stirring during platelet activation was not necessary. These results suggest that the inhibition is caused by a release reaction. This was confirmed by centrifugation experiments that showed that the inhibitory activity could be recovered from the supernatant of the activated platelets. The inhibitory activity was destroyed upon boiling and was susceptible to trypsin digestion. Passage of platelet supernatant over ACA 22 showed that the inhibitory activity eluted with an apparent molecular weight of less than 1,200,000 but greater than 669,000. The inhibition of factor XIa was reversible. These data suggest that platelets release an antiprotease of factor XIa that reversibly inhibits factor XIa. Lineweaver-Burk analysis showed that the inhibitor caused both an increase in Km for factor IX and a decrease in kcat of factor IXa formation by factor XIa.

scholarly journals The effect of platelets in the activation of human blood coagulation factor IX by factor XIa

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 140-148 ◽  
Author(s):  
H Soons ◽  
T Janssen-Claessen ◽  
HC Hemker ◽  
G Tans
Keyword(s):  
Platelet Activation ◽  
Inhibitory Activity ◽  
Human Factor ◽  
Human Platelets ◽  
Factor Ix ◽  
Dependent Manner ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Bovine Factor

Abstract We report here the effect of activated human platelets on the activation of human factor IX by human factor XIa. Factor IXa formed during activation was determined via its ability to activate bovine factor X. To increase sensitivity, phospholipids and bovine factor VIIIa were present in the assay. The kinetic parameters of the factor IX activation were determined in the presence of 10 mmol/L CaCl2. The Km for factor IX was 0.30 mumol/L and kcat was 2.4 s-1. Activated human platelets inhibited factor IX activation by factor XIa in a dose- dependent manner, whereas unstimulated platelets had no effect. Factor IX activation was inhibited for more than 90% at a platelet concentration of 4 X 10(8)/mL, whereas concentrations of less than 10(6)/mL had no influence. The inhibitory effect could be induced by thrombin, collagen, calcium ionophore A 23187, and adrenalin. The appearance of inhibitory activity could be blocked by the addition of the prostacyclin analogue ZK 36374 at any time during platelet activation. Stirring during platelet activation was not necessary. These results suggest that the inhibition is caused by a release reaction. This was confirmed by centrifugation experiments that showed that the inhibitory activity could be recovered from the supernatant of the activated platelets. The inhibitory activity was destroyed upon boiling and was susceptible to trypsin digestion. Passage of platelet supernatant over ACA 22 showed that the inhibitory activity eluted with an apparent molecular weight of less than 1,200,000 but greater than 669,000. The inhibition of factor XIa was reversible. These data suggest that platelets release an antiprotease of factor XIa that reversibly inhibits factor XIa. Lineweaver-Burk analysis showed that the inhibitor caused both an increase in Km for factor IX and a decrease in kcat of factor IXa formation by factor XIa.

scholarly journals The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1226-1231 ◽  
Author(s):  
TB McNeely ◽  
MJ Griffith
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Factor X ◽  
Clotting Time ◽  
Intrinsic Pathway ◽  
Blood Coagulation Factors ◽  
Factor Ixa ◽  
Factor X Activation

Abstract The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

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