scholarly journals The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 335-343 ◽  
Author(s):  
GJ Jr Broze ◽  
LA Warren ◽  
WF Novotny ◽  
DA Higuchi ◽  
JJ Girard ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Factor Vii ◽  
Diisopropyl Fluorophosphate ◽  
Plasma Factor ◽  
Coagulation Inhibitor ◽  
Catalytically Active

Abstract Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 335-343 ◽  
Author(s):  
GJ Jr Broze ◽  
LA Warren ◽  
WF Novotny ◽  
DA Higuchi ◽  
JJ Girard ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Factor Vii ◽  
Diisopropyl Fluorophosphate ◽  
Plasma Factor ◽  
Coagulation Inhibitor ◽  
Catalytically Active

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS).

The Effect of Antithrombin III on the Activity of the Coagulation Factors VII, IX and X

1976 ◽  
Vol 35 (02) ◽  
pp. 295-304 ◽  
Author(s):  
B Østerud ◽  
M Miller-Andersson ◽  
U Abildgaard ◽  
H Prydz
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Factor Vii ◽  
Native Form ◽  
Factor Ixa ◽  
Plasma Factor

SummaryAntithrombin III, purified to homogeneity according to Polyacrylamide gel disc electrophoresis and immunoelectrophoresis, inhibited the activity of purified factor IXa and Xa, whereas factor VII was not inhibited either in the active or in the native form.Antithrombin III is the single most important inhibitor of factor Xa in plasma. Factor Xa does not, however, reduce the activity of antithrombin III against thrombin.

Lipoprotein Associated Coagulation Inhibitor, Factor VII, Antithrombin III, and Monocyte Tissue Factor Following Surgery

1991 ◽  
Vol 66 (05) ◽  
pp. 534-539 ◽  
Author(s):  
Steven D Carson ◽  
William D Haire ◽  
George J Broze ◽  
William F Novotny ◽  
Samuel J Pirrucello ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Malignant Disease ◽  
Antithrombin Iii ◽  
Peripheral Blood Monocyte ◽  
Factor Vii ◽  
Normal Ranges ◽  
At Iii ◽  
Coagulation Inhibitor ◽  
Temporal Occurrence ◽  
Diagnosis Of Infection

SummaryFifteen patients undergoing major surgical procedures were evaluated for lipoprotein associated coagulation inhibitor (LACI) antigen, factor VII (F VII), antithrombin III (AT III), and peripheral blood monocyte tissue factor (TF) activity immediately before surgery and on following days. A peak in monocyte TF activity occurred between the first and fifth days after surgery in 10 of the patients, while LACI, F VII, and AT III levels dropped in a qualitatively parallel manner in 8 of these patients. LACI, F VII, and AT III levels decreased after surgery in two additional patients even though TF activity also decreased after surgery in these patients. In the remaining 3 patients who developed infections during the study, TF activity rose within 2 days of the diagnosis of infection in addition to the postoperative peak. In two of these patients, LACI levels increased dramatically near the end of the study period without concomitant changes in F VII and AT III. Overall, the presurgical TF levels in disrupted monocytes varied 52-fold and the maximal TF activity varied 24-fold among patients. The TF response following surgery is therefore heterogenous in both temporal occurrence and magnitude of the postsurgical peak. The patients also varied considerably in the presurgical levels of monocyte TF activity. A possible association between the level of presurgical TF activity and the magnitude of the postsurgical peak was noted. Thirteen of the patients had malignant disease, and, as a group, had presurgical levels of LACI (83 ± 26; mean ± SD), F VII (124 ± 22) and AT III (111 ± 14) within the normal ranges established with pooled reference plasma and similar values for three normal donors studied over the same period (AT III 121 ± 6, LACI 95 ± 4, and F VII 112 ± 29). No cause-effect relationship could be established between changes in TF activity and changes in the other factors measured. Patient- or therapy-specific variables may determine postoperative variations in LACI, F VII, AT III, and monocyte TF.

Plasma factor VII and thrombin–antithrombin III levels indicate increased tissue factor activity in sickle cell patients

1992 ◽  
Vol 81 (4) ◽  
pp. 539-544 ◽  
Author(s):  
Joseph Kurantsin-Mills ◽  
Frederick A. Ofosu ◽  
Toufic K. Safa ◽  
Robert S. Siegel ◽  
Lawrence S. Lessin
Keyword(s):  
Tissue Factor ◽  
Sickle Cell ◽  
Antithrombin Iii ◽  
Factor Vii ◽  
Factor Activity ◽  
Tissue Factor Activity ◽  
Plasma Factor

scholarly journals Immunoaffinity purification of bovine factor VII

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 393-398 ◽  
Author(s):  
R Bach ◽  
J Oberdick ◽  
Y Nemerson
Keyword(s):  
Factor Viia ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Internal Standard ◽  
Factor Xa ◽  
Factor Vii ◽  
Single Polypeptide Chain ◽  
Bovine Plasma ◽  
The One

Abstract Factor VII has been purified to homogeneity from bovine plasma by a procedure that includes affinity purification on an immunoadsorbent column. Recovery was determined by both coagulant assay and liquid scintillation counting, using 3H-factor VII as an internal standard. The purification factor calculated by both methods was approximately 120,000-fold, with a final yield of approximately 18%. Homogeneity was assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The material migrated as a single polypeptide chain of 53,000 daltons, and following activation by factor Xa, the one-chain zymogen was quantitatively converted to two-chain factor VIIa. Conversion of affinity-purified factor VII to factor VIIa resulted in up to a 119-fold activation of the coagulant activity, which is 2.7–4 times greater than the activatability reported for factor VII prepared by other methods. Zur et al. calculated that pure factor VII, uncontaminated by traces of factor VIIa, would be activated 123-fold upon conversion to factor VIIa. The close agreement between observed activatability of affinity-purified factor VII and the theoretical prediction suggests that we have isolated factor VII essentially free of factor VIIa. The purification data from three lots of bovine plasma yield an estimate for the plasma concentration of factor VII from 10.1 nM to 18.5 nM.

Purification of Human Brain Tissue Factor

1988 ◽  
Vol 59 (03) ◽  
pp. 400-403 ◽  
Author(s):  
Sungzong Kang ◽  
Julian Niemetz
Keyword(s):  
Human Brain ◽  
Tissue Factor ◽  
Brain Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Vii ◽  
Intrinsic Pathway ◽  
Human Brain Tissue ◽  
Agarose Beads

SummaryTissue factor (thromboplastin or Factor III), a glycoprotein cofactor, is required for Factor VII to express its catalytic activity, thereby initiating the extrinsic as well as intrinsic pathway of blood coagulation. Human brain tissue factor was purified 2,500-fold to 98% homogeneity from 2% Tiiton X-100 extraction of acetone dried brain powder with an overall yield of 36%. The method was based upon affinity chromatography utilizing the high affinity binding of tissue factor to Factor VII noncovalently complexed to immobilized anti-Factor VII-agarose beads. The apparent molecular weight of the purified tissue factor is 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point is 4.8–5.1 by column chromatofocussing and flat bed agarose isoelectric focussing.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569 ◽  
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

Abstract A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

scholarly journals Immunoaffinity purification of bovine factor VII

Blood ◽  
1984 ◽  
Vol 63 (2) ◽  
pp. 393-398
Author(s):  
R Bach ◽  
J Oberdick ◽  
Y Nemerson
Keyword(s):  
Factor Viia ◽  
Affinity Purification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Internal Standard ◽  
Factor Xa ◽  
Factor Vii ◽  
Single Polypeptide Chain ◽  
Bovine Plasma ◽  
The One

Factor VII has been purified to homogeneity from bovine plasma by a procedure that includes affinity purification on an immunoadsorbent column. Recovery was determined by both coagulant assay and liquid scintillation counting, using 3H-factor VII as an internal standard. The purification factor calculated by both methods was approximately 120,000-fold, with a final yield of approximately 18%. Homogeneity was assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The material migrated as a single polypeptide chain of 53,000 daltons, and following activation by factor Xa, the one-chain zymogen was quantitatively converted to two-chain factor VIIa. Conversion of affinity-purified factor VII to factor VIIa resulted in up to a 119-fold activation of the coagulant activity, which is 2.7–4 times greater than the activatability reported for factor VII prepared by other methods. Zur et al. calculated that pure factor VII, uncontaminated by traces of factor VIIa, would be activated 123-fold upon conversion to factor VIIa. The close agreement between observed activatability of affinity-purified factor VII and the theoretical prediction suggests that we have isolated factor VII essentially free of factor VIIa. The purification data from three lots of bovine plasma yield an estimate for the plasma concentration of factor VII from 10.1 nM to 18.5 nM.

Antibody-Induced Acute Factor X Deficiency: Clinical Manifestations and Properties of the Antibody

1994 ◽  
Vol 72 (03) ◽  
pp. 363-371 ◽  
Author(s):  
L Vijaya Mohan Rao ◽  
Ariella Zivelin ◽  
Ignacio Iturbe ◽  
Samuel I Rapaport
Keyword(s):  
Tissue Factor ◽  
Light Chain ◽  
Clinical Manifestations ◽  
Factor Xa ◽  
Factor Vii ◽  
Factor X ◽  
Factor X Deficiency ◽  
Sds Page ◽  
Plasma Factor ◽  
Tissue Factor Pathway

SummaryA patient is described with serious bleeding due to a transient selective deficiency of factor X. Crossed immunoelectrophoresis of patient’s plasma with anti-factor X antibody revealed an abnormal factor X arc suggestive of the presence of plasma factor X/anti-factor X immune complexes. A similar abnormal arc was obtained on adding the patient’s IgG to normal plasma. Immunoblotting of factor X after reduced SDS-PAGE revealed that the patient’s IgG bound to the light chain of intact factor X but not Gla-domainless factor X. The patient’s IgG inhibited activation of factor X by Vila/tissue factor (TF), by IXa/VIIIa/phospholipid complex, and by Russell’s viper venom. The IgG failed to inhibit the proteolytic activity of factor Xa towards a chromogenic substrate. However, under reaction conditions of limited factor Xa availability, the IgG could be shown to impair hemostatic functions of factor Xa that require the participation of its light chain: activation of prothrombin by prothrombinase; activation of factor VII bound to TF; and inhibition of VIIa/TF activity by factor Xa/tissue factor pathway inhibitor complexes. A few earlier patients have been described with transient, selective factor X deficiency and serious bleeding, but in only one was evidence obtained of an antibody against factor X. It will be of interest to learn whether use of the techniques described in this report will permit the identification of immunoglobulin with similar binding and functional properties in future patients with this rare syndrome.

Sign in / Sign up

Export Citation Format

Share Document