scholarly journals Coupled amidolytic assay for factor VII: its use with a clotting assay to determine the activity state of factor VII

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 978-988 ◽  
Author(s):  
U Seligsohn ◽  
B Osterud ◽  
SI Rapaport
Keyword(s):  
Healthy Subjects ◽  
Activity Ratio ◽  
Factor Xa ◽  
Factor Vii ◽  
Test Material ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Transfusion Therapy ◽  
The Mean ◽  
Activity State

Abstract A coupled amidolytic assay for factor VII (VII) has been developed that when used with a clotting assay for VII enables detection of activated VII. In the assay, VII in a test material determines generation of factor Xa in a mixture of purified factor X, tissue factor, and calcium; factor Xa is measured with a chromogenic substrate. Factor VII activity in the coupled amidolytic assay (VIIam) correlated well with VII activity in a one-stage clotting assay (VIIc) in 57 healthy subjects, 5 patients with hereditary VII deficiency, and 11 patients with liver disease. Activation of plasma VII by kaolin, clotting, or cold strikingly increased VIIc but not VIIam levels. Thus the ratio VIIc/VIIam (VII activity ratio) is a measure of VII activation. In 27 warfarin-treated patients the mean VII activity ratio was significantly decreased, reflecting a greater decline in VIIc than in VIIam. This probably stems from partially carboxylated VII being able to act during the 3-min incubation of the amidolytic assay but unable to act rapidly enough to affect the clotting assay. Measurement of VIIc/VIIam should enable evaluation of the activity state of VII in thrombotic disorders and in components for transfusion therapy.

scholarly journals Coupled amidolytic assay for factor VII: its use with a clotting assay to determine the activity state of factor VII

Blood ◽  
1978 ◽  
Vol 52 (5) ◽  
pp. 978-988 ◽  
Author(s):  
U Seligsohn ◽  
B Osterud ◽  
SI Rapaport
Keyword(s):  
Healthy Subjects ◽  
Activity Ratio ◽  
Factor Xa ◽  
Factor Vii ◽  
Test Material ◽  
Chromogenic Substrate ◽  
Factor X ◽  
Transfusion Therapy ◽  
The Mean ◽  
Activity State

A coupled amidolytic assay for factor VII (VII) has been developed that when used with a clotting assay for VII enables detection of activated VII. In the assay, VII in a test material determines generation of factor Xa in a mixture of purified factor X, tissue factor, and calcium; factor Xa is measured with a chromogenic substrate. Factor VII activity in the coupled amidolytic assay (VIIam) correlated well with VII activity in a one-stage clotting assay (VIIc) in 57 healthy subjects, 5 patients with hereditary VII deficiency, and 11 patients with liver disease. Activation of plasma VII by kaolin, clotting, or cold strikingly increased VIIc but not VIIam levels. Thus the ratio VIIc/VIIam (VII activity ratio) is a measure of VII activation. In 27 warfarin-treated patients the mean VII activity ratio was significantly decreased, reflecting a greater decline in VIIc than in VIIam. This probably stems from partially carboxylated VII being able to act during the 3-min incubation of the amidolytic assay but unable to act rapidly enough to affect the clotting assay. Measurement of VIIc/VIIam should enable evaluation of the activity state of VII in thrombotic disorders and in components for transfusion therapy.

Factors Affecting the lnteraction of Tissue Factor/Factor Vll with Factor X in a Heterogeneous Tubular Reactor

1991 ◽  
Vol 65 (02) ◽  
pp. 139-143 ◽  
Author(s):  
Cynthia H Gemmell ◽  
Vincet T Turitto ◽  
Yale Nemerson
Keyword(s):  
Steady State ◽  
Tissue Factor ◽  
Factor Viia ◽  
Transmembrane Protein ◽  
Strong Association ◽  
Tubular Reactor ◽  
Factor Xa ◽  
Phospholipid Bilayer ◽  
Factor Vii ◽  
Factor X

SummaryA novel reactor recently described for studying phospholipiddependent blood coagulation reactions under flow conditions similar to those occurring in the vasculature has been further charactenzed. The reactor is a capitlary whose inner wall is coated with a stable phospholipid bilayer (or two bilayers) containing tissue factor, a transmembrane protein that is required for the enzymatic activation of factor X by factor VIIa. Perfusion of the capillary at wall shear rates ranging from 25 s−1 to 1,200 s−1 with purified bovine factors X and VIIa led to steady state factor Xa levels at the outlet. Assay were performed using a chromogenic substrate, SpectrozymeTMFXa, or by using a radiometric technique. In the absence of Ca2+ or factor VIIa there was no product formation. No difference was noted in the levels of factor Xa achieved when non-activated factor VII was perfused. Once steady state was achieved further factor Xa production continued in the absence of factor VIIa implying a very strong association of factor VIIa with the tissue factor in the phospholipid membrane. In agreement with static vesicle-type studies the reactor was sensitive to wall tissue factor concentration, temperature and the presence of phosphatidylserine in the bilayer.

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.

scholarly journals Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 645-651 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Mechanism Of Inhibition ◽  
Peptide Release ◽  
Release Assay

Abstract We have extended earlier studies (Blood 66:204, 1985) of a mechanism of inhibition of factor VIIa/tissue factor activity that requires a plasma component (called herein extrinsic pathway inhibitor or EPI) and factor Xa. An activated peptide release assay using 3H-factor IX as a substrate was used to evaluate inhibition. Increasing the tissue factor concentration from 20% to 40% (vol/vol) overcame the inhibitory mechanism in normal plasma but not in factor VII-deficient plasma supplemented with a low concentration of factor VII. A second wave of factor IX activation obtained by a second addition of tissue factor to plasma with a normal factor VII concentration was almost abolished by supplementing the reaction mixture with additional EPI and factor X. Factor Xa's active site was necessary for factor Xa's contribution to inhibition, but preliminary incubation of factor Xa with EPI in the absence of factor VIIa/tissue factor complex or of factor VIIa/tissue factor complex in the absence of EPI did not replace the need for the simultaneous presence of factor Xa, factor VIIa/tissue factor, calcium, and EPI in an inhibitory reaction mixture. Inhibition of factor VIIa/tissue factor was reversible; both tissue factor and factor VIIa activity could be recovered from a dissociated, inhibited factor VIIa/tissue factor complex. EPI appeared to bind to a factor VIIa/tissue factor complex formed in the presence of factor Xa but not to a factor VIIa/tissue factor complex formed in the absence of factor Xa.

scholarly journals Pathways in the activation of human coagulation factor X

1980 ◽  
Vol 185 (3) ◽  
pp. 647-658 ◽  
Author(s):  
K Mertens ◽  
R M Bertina
Keyword(s):  
Heavy Chain ◽  
Active Form ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Terminal Region ◽  
Factor Vii ◽  
Factor X ◽  
Catalytically Active ◽  
A Site ◽  
Polypeptide Chains

Purified human Factor X (apparent mol.wt. 72000), which consists of two polypeptide chains (mol.wt. 55000 and 19000), was activated by both Russell's-viper venom and the purified physiological activators (Factor VII/tissue factor and Factor IXa/Factor VIII). They all convert Factor X to catalytically active Factor Xa (mol.wt. 54000) by cleaving the heavy chain at a site on the N-terminal region. In the presence of Ca2+ and phospholipid, the Factor Xa formed catalyses (a) the cleavage of a small peptide (mol.wt. 4000) from the C-terminal region of the heavy chain of Factor Xa, resulting in a second active form (mol.wt. 50000), and (b) the cleavage of a peptide containing the active-site serine residue (mol.wt. 13000) from the C-terminal region of the heavy chain of Factor X, resulting in an inactivatable component (mol.wt. 59000). A nomenclature for the various products is proposed.

scholarly journals In vivo studies of the role of factor VII in hemostasis

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1197-1200
Author(s):  
AR Giles ◽  
S Tinlin ◽  
L Brosseau ◽  
H Hoogendoorn
Keyword(s):  
Alternative Pathway ◽  
Factor Xa ◽  
In Vivo Studies ◽  
Factor Vii ◽  
High Dose ◽  
Factor X ◽  
Clotting Factors ◽  
Dose Warfarin ◽  
Factor X Activation

The effect of both congenital and acquired factor VII deficiency on the cuticle bleeding time (CBT) was evaluated in dogs. The CBT has been previously documented to be a sensitive indicator of factor VIII:C deficiency in hemophilic dogs. Serial CBT determinations were made on normal dogs treated with high-dose warfarin. At 48 hours post- treatment, the CBT was normal, although the factor VII level was less than 1%, whereas the levels of factors II, IX, and X were 44%, 25%, and 17%, respectively. At 120 hours the CBT became abnormal when all vitamin K-dependent clotting factors had dropped to less than 18%. Administration of a plasma concentrate of factors II, IX, and X corrected the CBT, despite the factor VII level remaining at less than 1%. Similar studies in a congenitally factor VII-deficient dog (factor VII less than 2%) confirmed that this deficiency state was not associated with an abnormality of the CBT. Administration of heparin to both normal and factor VII-deficient animals was associated with prolongation of the CBT, but the heparin dose required in the normal animals was substantially higher than in the factor VII-deficient animals. These data do not suggest that factor VII/VIIa has an exclusive role in generating factor Xa, either directly or indirectly, by way of factor IXa generation, in vivo. However, the increase in heparin sensitivity of the factor VII-deficient animals does suggest that factor VII/VIIa may, in some circumstances, present a significant alternative pathway of factor X activation, although the activation pathway involved cannot be determined from the studies performed.

scholarly journals Initiation of the extrinsic pathway of coagulation by human and rabbit alveolar macrophages: a kinetic study

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1583-1590 ◽  
Author(s):  
MP McGee ◽  
R Wallin ◽  
FB Wheeler ◽  
H Rothberger
Keyword(s):  
Alveolar Macrophage ◽  
Alveolar Macrophages ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Activation Rate ◽  
Factor X Activation

We examined assembly and expression of the factor X activating complex on human and rabbit alveolar macrophages. Kinetic parameters of the factor X activating reaction were determined by functional titrations of factors VII and X with macrophage tissue factor (TF) added. We found rapid activation of factor X to Xa on alveolar macrophage surfaces. Detection of rapid factor Xa formation on macrophages required addition of exogenous factors VII and X. At plasma concentrations of the purified factors, factor Xa was formed on freshly isolated macrophages at approximately 5.4 pmol/min/10(6) cells. After macrophage maturation in culture for 20 hours with LPS (endotoxin) added, the factor X activation rate was increased two- to sixfold. The km' (apparent km) of TF-factor VII enzymatic complexes assembled on alveolar macrophages for factor X were (258 +/- 55 and 475 +/- 264 nmol/L for human and rabbit cells, respectively). The km' did not change during macrophage maturation in culture, but V'max (apparent Vmax) was consistently increased. The K1/2 of human factor VII (concentrations giving half maximal rates of factor X activation) for the interaction with human and rabbit alveolar macrophage TF were 0.191 +/- 0.096 and 1.7 +/- 0.7 etamol/L, respectively. The K1/2 were not significantly changed after maturation, whereas rates of Xa formation at saturation with factor VII were increased. The fast rates of factor X activation observed at physiologic concentrations of plasma-derived factors VII and X indicate that TF on alveolar macrophages is likely to provide sites for binding of factor VII and activation of factor X in vivo during clotting reactions associated with alveolar edema and inflammation.

Expression and Assembly of Procoagulant Complexes by Human Pleural Mesothelial Cells

1994 ◽  
Vol 71 (05) ◽  
pp. 587-592 ◽  
Author(s):  
Anuradha Kumar ◽  
Kathleen B Koenig ◽  
Alice R Johnson ◽  
Steven Idell
Keyword(s):  
Tissue Factor ◽  
Factor Xa ◽  
Mesothelial Cells ◽  
Factor Vii ◽  
Dependent Manner ◽  
Factor X ◽  
Specific Concentration ◽  
Pleural Mesothelial Cells ◽  
Direct Binding ◽  
Thrombin Formation

SummaryMany pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor Vila to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

scholarly journals Studies of the activation of factor VII bound to tissue factor [see comments]

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3738-3748 ◽  
Author(s):  
LV Rao ◽  
T Williams ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Tissue Injury ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
Ovarian Carcinoma Cell Line

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I- factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.

MECHANISMS OF PROCOAGULANT GENERATION BY ALVEOLAR MACROPHAGES DURING MATURATION

1987 ◽  
Author(s):  
Maria McGee ◽  
Henry Rothberger
Keyword(s):  
Alveolar Macrophages ◽  
Cultured Cells ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Complex Assembly

During maturation in vivo and in vitro alveolar macrophages generate procoagulant(s) capable of activating the extrinsic pathway. It is generally agreed that at least part of the activity is due to TF (tissue factor). However, whether or not macrophages also generate functional factor VII or X is controversial. To characterize procoagulant activity increases, we measured kinetic parameters defining interactions between components of the TF-VII complex on membranes of alveolar macrophages either freshly isolated or cultured in serum free medium. In incubation mixtures with fixed concentrations of macrophages and added factor VII, the rate of factor Xa formation (measured by S-2222 hydrolysis) approached a maximum as factor X concentration was increased. Estimated concentrations of factor X yielding 1/2 maximal activation rates, (apparent Km) were 127.1±26 nM and 99.7±34 nM for fresh and cultured cells, respectively. Vmax (maximal velocities) were 1.21±0.24 and 8.9±5 nM Xa/min/106 cells. When concentrations of added factor X were kept constant, the rate of factor X activation increased as the added factor VII concentration was increased. For fresh and cultured cells, the respective apparent Kd were 1.810.7 and 1.410.25 nM. Maximal rates observed with X concentration fixed at 108 nM were 0.46±10.06 and 5.7±1.6 nM Xa/min/106 cells. In the absence of either added factor X or added factor VII, no factor Xa generation was detected in fresh or cultured cells, during 10-20 min incubation periods used for kinetic studies. The observed increase in Vmax without changes in apparent Km and Kd indicate that gains in procoagulant activity during macrophage maturation are due to increases in the number of functional binding sites for factor VII, without significant generation of functional vitamin K dependent factors (VII and X) by the cells. The data also indicate that maturation does not alter the rate behaviour of the TF-VII enzymatic complex on macrophage membranes. Mechanisms of complex assembly that we observed on macrophage membranes are similar to those described for the TF-VII complex assembly on purified systems.

Sign in / Sign up

Export Citation Format

Share Document