Measurement of factor Xa-antithrombin III in plasma: relationship to prothrombin activation in vivo

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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Unfractionated heparin inhibits thrombin-catalysed amplification reactions of coagulation more efficiently than those catalysed by factor Xa

Biochemical Journal ◽

10.1042/bj2570143 ◽

1989 ◽

Vol 257 (1) ◽

pp. 143-150 ◽

Cited By ~ 69

Author(s):

F A Ofosu ◽

J Hirsh ◽

C T Esmon ◽

G J Modi ◽

L M Smith ◽

...

Keyword(s):

Factor Viii ◽

Antithrombin Iii ◽

Factor Xa ◽

Activate Factor ◽

Factor V ◽

Inhibitory Effects ◽

Exogenous Factor ◽

Additional Support ◽

Prothrombin Activation ◽

Inhibit Factor

We have proposed previously that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent amplification reactions, and that prothrombinase is formed in heparinized plasma only after Factor Xa activates Factor VIII and Factor V. These propositions were based on the demonstration that both heparin and Phe-Pro-Arg-CH2Cl completely inhibited 125I-prothrombin activation for up to 60 s when contact-activated plasma (CAP) was replenished with Ca2+. Furthermore, the addition of thrombin to CAP before heparin or Phe-Pro-Arg-CH2Cl completely reversed their inhibitory effects. Additional support for the above hypotheses is provided in this study by demonstrating that, when the activity of thrombin is suppressed by heparin (indirectly) or by Phe-Pro-Arg-CH2Cl (directly), exogenous Factor Xa reverses the ability of these two agents to inhibit prothrombin activation. Prothrombin activation was initiated by adding Factor Xa (1 nM) or thrombin (1 or 10 nM) simultaneously with CaCl2 to CAP. In the absence of heparin or Phe-Pro-Arg-CH2Cl, prothrombin activation was seen 15 s later in either case. Heparin failed to delay, and Phe-Pro-Arg-CH2Cl delayed for 15 s, prothrombin activation in CAP supplemented with Factor Xa. In contrast, heparin and Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation for at least 45 s in CAP supplemented with 1 nM-thrombin. Heparin failed to delay prothrombin activation in CAP supplemented with 10 nM-thrombin, whereas Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation in this plasma for 45 s. These results suggest that in CAP: (1) Factor Xa can effectively activate Factor VIII and Factor V when the proteolytic activity of thrombin is suppressed; (2) heparin-antithrombin III is less able to inhibit Factor Xa than thrombin; (3) suppression of the thrombin-dependent amplification reactions is the primary anticoagulant effect of heparin.

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Monitoring "mini-intensity" anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F1+2 levels

Blood ◽

10.1182/blood.v79.8.2034.bloodjournal7982034 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2034-2038 ◽

Cited By ~ 1

Author(s):

MM Millenson ◽

KA Bauer ◽

JP Kistler ◽

S Barzegar ◽

L Tulin ◽

...

Keyword(s):

Prothrombin Time ◽

Factor Xa ◽

High Sensitivity ◽

International Normalized Ratio ◽

Target Range ◽

Lower Sensitivity ◽

Sensitivity Index ◽

Plasma Samples ◽

Prothrombin Activation

Treatment with warfarin using a target International Normalized Ratio (INR) range of 1.7 to 2.5 is efficacious for many clinical indications, but the minimal intensity of anticoagulation required for antithrombotic protection has yet to be determined. To evaluate whether patients could be reliably monitored with a less intense regimen, we anticoagulated patients with warfarin for several months using a target INR range of 1.3 to 1.6 as determined by prothrombin time (PT) using a sensitive thromboplastin (Dade IS, International Sensitivity Index [ISI] = 1.3). Plasma measurements of F1+2, a marker of factor Xa action on prothrombin in vivo, were also obtained to determine the suppressive effect of warfarin on hemostatic system activity. Overall, 20 of 21 patients with a history of cerebrovascular events (mean age, 61 years) could be reliably regulated with warfarin in the target INR range. F1+2 levels were significantly suppressed from baseline in all patients, with a mean reduction of 49% (range, 28% to 78%). We found a significant relationship between the extent of suppression of prothrombin activation levels and the baseline measurements. A mean reduction of 65% was observed for those patients with baseline F1+2 greater than or equal to 1.5 nmol/L, but only 38% for baseline F1+2 less than or equal to 0.5 nmol/L. Overall, 68% of plasma samples obtained during stable anticoagulation were within the target INR range. PTs were also determined on all plasma samples with two thromboplastins of lower sensitivity (C+, ISI = 2.09; and automated simplastin, ISI = 2.10). Only 47% and 35% of PT determinations, respectively, were within the target range with these reagents. We conclude that prothrombin activation can be significantly suppressed in vivo with use of warfarin in an INR range of 1.3 to 1.6. This level of anticoagulation can be reliably achieved by monitoring PTs with a thromboplastin of high sensitivity.

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Monitoring "mini-intensity" anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F1+2 levels

Blood ◽

10.1182/blood.v79.8.2034.2034 ◽

1992 ◽

Vol 79 (8) ◽

pp. 2034-2038 ◽

Cited By ~ 39

Author(s):

MM Millenson ◽

KA Bauer ◽

JP Kistler ◽

S Barzegar ◽

L Tulin ◽

...

Keyword(s):

Prothrombin Time ◽

Factor Xa ◽

High Sensitivity ◽

International Normalized Ratio ◽

Target Range ◽

Lower Sensitivity ◽

Sensitivity Index ◽

Plasma Samples ◽

Prothrombin Activation

Abstract Treatment with warfarin using a target International Normalized Ratio (INR) range of 1.7 to 2.5 is efficacious for many clinical indications, but the minimal intensity of anticoagulation required for antithrombotic protection has yet to be determined. To evaluate whether patients could be reliably monitored with a less intense regimen, we anticoagulated patients with warfarin for several months using a target INR range of 1.3 to 1.6 as determined by prothrombin time (PT) using a sensitive thromboplastin (Dade IS, International Sensitivity Index [ISI] = 1.3). Plasma measurements of F1+2, a marker of factor Xa action on prothrombin in vivo, were also obtained to determine the suppressive effect of warfarin on hemostatic system activity. Overall, 20 of 21 patients with a history of cerebrovascular events (mean age, 61 years) could be reliably regulated with warfarin in the target INR range. F1+2 levels were significantly suppressed from baseline in all patients, with a mean reduction of 49% (range, 28% to 78%). We found a significant relationship between the extent of suppression of prothrombin activation levels and the baseline measurements. A mean reduction of 65% was observed for those patients with baseline F1+2 greater than or equal to 1.5 nmol/L, but only 38% for baseline F1+2 less than or equal to 0.5 nmol/L. Overall, 68% of plasma samples obtained during stable anticoagulation were within the target INR range. PTs were also determined on all plasma samples with two thromboplastins of lower sensitivity (C+, ISI = 2.09; and automated simplastin, ISI = 2.10). Only 47% and 35% of PT determinations, respectively, were within the target range with these reagents. We conclude that prothrombin activation can be significantly suppressed in vivo with use of warfarin in an INR range of 1.3 to 1.6. This level of anticoagulation can be reliably achieved by monitoring PTs with a thromboplastin of high sensitivity.

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Adverse Effect of Heparin on Thrombin Inactivation

10.1055/s-0039-1689220 ◽

1975 ◽

Author(s):

E. Marciniak

Keyword(s):

Adverse Effect ◽

Antithrombin Iii ◽

Factor Xa ◽

Binding Properties ◽

Inhibitory Capacity ◽

Antithrombin Iii Deficiency ◽

Final Amount ◽

Native Plasma

In the presence of heparin thrombin, although fast inactivated, impairs the inhibitory capacity of antithrombin III, in result of which the final amount of neutralized enzyme markedly decreases. This adverse effect of heparin was found during the reaction of purified thrombin with both purified human antithrombin III and native plasma hepariniz purified thrombin with both purified human antithrombin III and native plasma heparinized in vitro or in vivo. In the absence of heparin, at concentration equal to that in normal plasma antithrombin III binds 450 Iowa units of thrombin; in the presence of heparin (at 1 unit concentration) this binding is reduced to 145 thrombin units. A fast depletion of inhibitory capacity is also evident after a stepwise addition of thrombin in small installments into a medium containing antithrombin III and heparin. Portions of enzyme initially added disappear with great velocity; subsequent additions, however, accumulate building up a high thrombin level not seen in the absence of heparin. The escalation of thrombin is reversely proportional to the reacting antithrombin III level, thus especially noticeable in antithrombin III deficient plasma. Residual thrombin left in the presence of heparin disappears at a fast rate upon a new addition of antithrombin III. No decrease in anticoagulant properties of heparin is observed during these interactions. Binding of factor Xa to antithrombin III which reacted with thrombin and heparin is also decreased or abolished.These results indicate that in the presence of heparin thrombin not only exposes rapidly a binding site on the inhibitor, but also causes a further change leading to the deletion of antithrombin III binding properties. This may explain adverse, thrombotic effect of heparin sporadically seen in vivo, and suggests that heparin should be applied with caution in patients with antithrombin III deficiency.

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HEPARIN IS NOT AN EFFICIENT INHIBITOR OF THE FACTOR Xa-DEPENDENT ACTIVATION OF FACTOR V AND FACTOR VIII

10.1055/s-0038-1642931 ◽

1987 ◽

Author(s):

F A Ofosu ◽

G J Modi ◽

M R Buchanan ◽

J Hirsh ◽

M A Blajchman

Keyword(s):

Factor Viii ◽

Antithrombin Iii ◽

Specific Inhibitor ◽

Factor Xa ◽

Factor V ◽

Factor X ◽

Inhibitory Effects ◽

Efficient Inhibitor ◽

System 1 ◽

Prothrombin Activation

We have previously proposed that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent activation of factor V and factor VIII. This observation was based on the demonstration that therapeutic concentrations of heparin or 1μM of the thrombin specific inhibitor, phe-pro-arg CH2Cl (PPACK) completely inhibited the activation of prothrombin when contact-activated plasma (CAP) was recalcified for up to 1 min. Under similar conditions, heparin and PPACK only partially inhibited the activation of factor X. Moreover, the addition of thrombin (lOnM) to CAP 1 min before that of heparin or PPACK reversed their inhibitory effects. We now provide further support for our hypothesis by showing that when the activity of thrombin is suppressed by heparin or PPACK, efficient activation of radiolabelled prothrombin occurs only when the factor Xa then present activates factor V and factor VIII. We compared the effects of HEP of PPACK on the following four systems for initiating the activation of prothrombin: (1) CAP; (2) CAP + lOnM thrombin; (3) CAP + InM Xa and (4) unactivated plasma + InM Xa + InM Va + coagulant phospholipids. In each system, the enzymes were added 1 min before the heparin or PPACK. In the absence of heparin or PPACK, all four systems generated the same amount of thrombin activity in 45s. Complete inhibition of prothrombin activation by heparin and PPACK was observed only in system 1 which did not contain exogenous thrombin or factor Xa. No inhibition by heparin or PPACK was observed when thrombin or factor Xa was added to CAP in systems (2) and (3). Only partial inhibition was observed in system (4) which contained exogenous prothrombi-nase complex. Factor Xa thus provides an effective by-pass mechanism for the activation of factor VIII and factor V in plasma containing therapeutic concentrations of heparin. Our data provide further evidence that the heparin-antithrombin III system is not effective in inactivating factor Xa. These results support the hypothesis that in unactivated normal plasma, the primary anticoagulant effect of heparin is the inhibition of the thrombin-dependent activation of factor V and factor VIII.

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Antithrombotic Effects of Synthetic Pentasaccharide with High Affinity for Plasma Antithrombin III in Non-Human Primates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649640 ◽

1993 ◽

Vol 70 (04) ◽

pp. 631-635 ◽

Cited By ~ 32

Author(s):

Yves Cadroy ◽

Stephen R Hanson ◽

Laurence A Harker

Keyword(s):

Antithrombin Iii ◽

Thrombus Formation ◽

Factor Xa ◽

High Dose ◽

Antithrombin Activity ◽

Model Combining ◽

Baboon Model ◽

Dependent Inactivation ◽

Antithrombotic Effects

SummaryThe pentasaccharide (PS) comprising the minimal heparin structure capable of binding with antithrombin III (ATIII) and exhibiting anti-factor Xa (anti-fXa) activity in plasma without producing detectable antithrombin activity, has been evaluated for its relative antithrombotic and antihemostatic effects in a baboon model combining both platelet-rich and fibrin-rich thrombosis. Thrombosis was produced in a two-component thrombogenic device incorporated into an exteriorized femoral arteriovenous (AV) shunt in baboons; the proximal component constituted a segment of collagen-coated tubing and induced platelet-rich arterial-type thrombus, while the distal component consisted of an expanded chamber producing static and disturbed flow and initiated fibrin-rich venous-type thrombosis. Thrombus formation was measured as the deposition of 111In-platelets and the accumulation of 125I-fibrin. PS was administered intravenously to maintain plasma anti-fXa activity at three different levels: a) low dose (LD) 0.3 ± 0.1 U/ml; b) intermediate dose (ID) 0.6 ± 0.1 U/ml; and c) high dose (HD) 5.6 ± 0.4 U/ml.In untreated Controls, thrombus formed rapidly, reaching a plateau by 40 min of 2.3 ± 0.2 × 109 platelets and 0.62 ± 0.04 mg fibrin deposited on the collagen segments, and 1.9 ±0.4 × 109 platelets and 3.3 ± 0.4 mg fibrin accumulated in the chambers. PS at HD abolished the formation of fibrin-rich thrombus in the chambers and decreased platelet-rich thrombus on collagen by half (p <0.01), while the ID reduced fibrin-rich thrombus in the chambers by about half (p <0.01) but had no effect on platelet-rich thrombus forming on the segments of collagen-coated tubing (p >0.5). Despite its lack of antithrombin activity, PS also decreased plasma fibrinopeptide A levels in a dose-response manner. However, PS had no effect on platelet hemostatic function in vivo, as measured by template bleeding time (BT) determinations (p >0.5). Despite the ability of PS-ATIII complex to inactivate soluble fXa, the complex lacked significant inhibitory activity for fXa immobilized to thrombus formed in vivo. Thus, PS-dependent inactivation of soluble fXa produces antithrombotic effects, primarily for venous-type thrombosis, that are equipotent to Standard heparin on a gravimetric basis, but more sparing of platelet hemostatic function.

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The Relationship between Antithrombin III, Progressive Antithrombin Activity and Anti Xa Activity in Plasma

10.1055/s-0039-1689221 ◽

1975 ◽

Author(s):

Katherine Whigham ◽

P. W. Howie ◽

C. D. Forbes ◽

C. R. M. Prentice

Keyword(s):

Antithrombin Iii ◽

Factor Xa ◽

Normal Subjects ◽

Strong Positive Correlation ◽

Positive Correlation ◽

Antithrombin Activity ◽

Α2 Macroglobulin ◽

Immunological Assays ◽

The Relationship

In 30 normal subjects, progressive antithrombin activity, as measured by the rate of thrombin neutralisation in ancrod-defibrinated plasma, was compared with antithrombin III, as measured by radial immunodiffusion. No significant correlation was found between the two methods of antithrombin measurement (r = —0.101). Similarly, no correlation was found between progressive antithrombin activity and immunological measurements of α2 macroglobulin and α1 antitrypsin. These results were not changed by using thrombin purified by Amberlite 1RC50 chromatography in place of commercial thrombin in the clotting test. There was, however, a strong positive correlation between the measurements of progressive antithrombin activity using the commercial and purified forms of thrombin (r = 0.78, p < 0.001). In contrast, there was a positive correlation between antithrombin III and anti-factor Xa activity in plasma (r = 0.48, p < 0.01). There was no correlation between plasma anti Xa activity and α2 macroglobulin or α1 antitrypsin.These results suggest that plasma antithrombin activity is a measure of the activities of several plasma proteins and that antithrombin III may not be the major determinant of antithrombin activity. There is little evidence that immunological assays of antithrombin III reflect total thrombin inhibitory capacity as measured by the biological assay. Caution must be exercised in extrapolating from immunological measurements of antithrombin III to antithrombin activity in-vivo.

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Characterization of an inducible endothelial cell prothrombin activator

Blood ◽

10.1182/blood.v88.8.2989.bloodjournal8882989 ◽

1996 ◽

Vol 88 (8) ◽

pp. 2989-2994 ◽

Cited By ~ 5

Author(s):

L Liu ◽

GM Rodgers

Keyword(s):

Thrombin Generation ◽

Interleukin 1 ◽

Factor Xa ◽

Apparent Molecular Weight ◽

Factor V ◽

Factor X ◽

Exogenous Factor ◽

Km Value ◽

Prothrombin Activation

In vivo prothrombin activation is thought to occur via a factor Xa/factor V-dependent mechanism. We investigated whether human venous endothelial cells (EC) could be induced to express a prothrombin activator. EC treated with lipopolysaccharide (LPS) or interleukin-1 activated prothrombin in the absence of exogenous factors Xa and V. This activity resided in the membrane fraction of EC and was not inhibited by an antibody to factor V. The apparent Km value was 3.3 +/- 0.3 mumol/L. Comparative studies of thrombin generation using a model system of phospholipid and factors Xa/V versus LPS-treated EC were performed to quantitate the effects of known inhibitors to factor Xa. The factor Xa inhibitor DEGR-chloromethyl ketone and an antibody to factor X inhibited prothrombin activation. However, the EC activator did not hydrolyze a factor Xa chromogenic substrate, and recombinant tick anticoagulant peptide did not suppress activity of the prothrombin activator. The apparent molecular weight of the EC activator was approximately 30 kD. Exogenous factor V enhanced the activity of the EC activator, such that in the presence of factor V, the apparent K(m) value was 1.28 +/- 0.10 mumol/L. Additionally, LPS-treated EC activated exogenous factor V. This activator has several characteristics of a previously described inducible murine monocyte prothrombin activator and may contribute to thrombin generation associated with pathologic stimuli.

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Dual Effect of Platelet Factor Four on the Activities of Factor Xa

Blood ◽

10.1182/blood.v112.11.1034.1034 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1034-1034

Author(s):

Martine Marie Fiore ◽

Ian J Mackie

Keyword(s):

Factor Xa ◽

Fold Increase ◽

Peptide Hydrolysis ◽

Dual Effect ◽

Platelet Factor ◽

Paradoxical Effects ◽

Heparin Activity ◽

High Concentrations ◽

Prothrombin Activation

Abstract Platelet Factor 4 (PF4) is a cationic molecule that binds to heparin with high affinity and neutralises the activity of the latter. Our recent studies indicate that heparin can promote an interaction of fXa with PF4 since neutralization of heparin activity by PF4 was dependent on the concentration of protease. To examine the contribution of PF4 in protease function, fXa activity was determined in chromogenic assays. Upon preincubation with fXa and heparin, PF4 (at a concentration of 100 nM) decreased the kcat of S2765 peptide hydrolysis 4-fold and that of prothrombin activation about 2-fold. These results suggested an effect of PF4 on the primary specificity of the protease. In fact, PF4 exerted a mild effect (30 % decrease) on the Na+ dependence of fXa, consistent with linkage between Na+ and S1. PF4 preincubation with fXa also prevented the binding of the S1 probe p-aminobenzamidine (pAB) while simultaneous addition of PF4 and pAB diminished the contribution of PF4. In the presence of excess fVa (relative to fXa), kinetic parameters measuring fXa amidolytic activity in the presence of PF4 were restored to control values in the absence of PF4. Interestingly, high concentrations of PF4 (> 1 μM) totally restored fXa activity toward peptidyl substrate and strongly enhanced prothrombin activation, indicating a dual effect of PF4 on fXa activities. The inhibitory contribution of PF4 during prothrombin activation was due to a three-fold decreased affinity of fXa for fVa while enhancement of prothrombin activation was accompanied by a three-fold increase in fVa-dependent cofactor activity. Thus, the effects of PF4 possibly involved a region of the heparin/fVabinding exosite that is linked to the S1 and Na+ sites. These findings suggest that PF4 is a probe of fVa-dependent changes occurring in the active site of fXa and provide an explanation for the in vivo paradoxical effects of PF4 reported in the literature.

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