INHIBITION OF HUMAN BLOOD COAGULATION FACTOR Xa BY α2-MACROGLO-BULIN

1987 ◽  
Author(s):  
Joost C M Meijers ◽  
Pim N M Tijburg ◽  
Bonno N Bouma
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Sodium Dodecyl Sulphate ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Order Rate

The inactivation of activated factor X (factor Xa) by α2 macroglobulin (α2M) was studied. Irreversible inhibition was observed with the initial formation of a reversible enzyme-inhibitor complex The secopd-order rate constant for the reaction was 8.4 × 104 M−1 min−1. The binding ratio was found to be 2 mol factor Xa/ mol α2M. Interaction of factor Xa with α2M resulted in the appearance of four thiolgroups/molecule α2. The apparent second-order rate constants for the appearance of thiolgroups were dependent on the factor Xa concentration. Sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis was used to study complex formation between α2M and factor Xa. Under non-reducing conditions four factor Xa - α2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of α2M, while the second complex (Mr A00000) consisted of the heavy chain of factor Xa molecule and two subunits of α2M. Factor Xa was able to form a bridge between two subunits or α2M, either within one molecule of α2M, or by linking two molecules of The role of the light chain of factor Xa in this process remains to be elucidated. For this purpose, monoclonal antibodies specific for the light chain of factor Xa were prepared. Sodium dodecyl sulphate agarose electrophoresis studies showed that complexes involving more than two molecules of α2M were not formed.

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 Dirofilaria immitis possesses molecules with anticoagulant properties in its excretory/secretory antigens

Parasitology ◽  
2020 ◽  
Vol 147 (5) ◽  
pp. 559-565 ◽  
Author(s):  
Alicia Diosdado ◽  
Fernando Simón ◽  
Rodrigo Morchón ◽  
Javier González-Miguel
Keyword(s):  
Dirofilaria Immitis ◽  
Thrombus Formation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Circulatory System ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Blood Clots ◽  
Key Enzyme

AbstractDirofilaria immitis is a parasitic nematode that survives in the circulatory system of suitable hosts for many years, causing the most severe thromboembolisms when simultaneous death of adult worms occurs. The two main mechanisms responsible for thrombus formation in mammals are the activation and aggregation of platelets and the generation of fibrin through the coagulation cascade. The aim of this work was to study the anticoagulant potential of excretory/secretory antigens from D. immitis adult worms (DiES) on the coagulation cascade of the host. Anticoagulant and inhibition assays respectively showed that DiES partially alter the coagulation cascade of the host and reduce the activity of the coagulation factor Xa, a key enzyme in the coagulation process. In addition, a D. immitis protein was identified by its similarity to the homologous serpin 6 from Brugia malayi as a possible candidate to form an inhibitory complex with FXa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. These results indicate that D. immitis could use the anticoagulant properties of its excretory/secretory antigens to control the formation of blood clots in its immediate intravascular habitat as a survival mechanism.

The Mechanism of Activation of Human Coagulation Factor X

1979 ◽  
Author(s):  
K. Mertens ◽  
R.M. Bertina
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Process Factor ◽  
Molecular Events ◽  
Product Factor

During the coagulation process factor X is converted to a serine protease, factor Xa. The present study concerns the molecular events which occur during the activation of human factor X by Russell’s viper venom and by the purified proteins of the extrinsic and intrinsic activator. Conversion of factor X was detected by amidolytic assays and SDS/polyacrylamide-gel electrophoresis.The results show that all activators convert factor X (MW 72,000) to an active form. In the presence of phospholipid the initially formed factor Xa (MW 54,000) complicates the further sequence of reactions by catalysing a) the conversion of factor Xa to a second active form (MW 50,000), b) the conversion of factor X to an inactive product (MW 59,000) by splitting off a peptide containing the active site serine, and c) the further degradation of the 50,000 and 59,000 components to a smaller component (MW 40,000).Comparison of these data with those reported for bovine factor X suggests that the mechanism of activation of human factor X is more complicated. The inactivation of both factor Xa and factor X by product factor Xa might be considered as important regulatory principles.

scholarly journals Some characteristics of the coagulation Factor Xa purified from human serum

1973 ◽  
Vol 135 (4) ◽  
pp. 791-795 ◽  
Author(s):  
Åse Gladhaug Berre ◽  
Bjarne Østerud ◽  
Terje B. Christensen ◽  
Turid Holm ◽  
Hans Prydz
Keyword(s):  
Molecular Weight ◽  
Human Serum ◽  
Gel Electrophoresis ◽  
Preliminary Report ◽  
Analytical Ultracentrifugation ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Apparent Homogeneity

1. Coagulation Factor X was purified from human serum to apparent homogeneity in disc gel electrophoresis, sodium dodecyl sulphate–polacrylamide-gel electrophoresis, immunoelectrophoresis and analytical ultracentrifugation. The method used was a modification of that described by Gladhaug & Prydz (1970). 2. The method permits the isolation of an activated form of Factor X (Xa) which has a molecular weight of about 25000. 3. Factor Xa is a glycoprotein containing about 14% carbohydrate. A preliminary report of the amino acid composition is given.

scholarly journals Isolation and characterization of the factor X Friuli variant

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2108-2116
Author(s):  
DS Fair ◽  
DJ Revak ◽  
JG Hubbard ◽  
A Girolami
Keyword(s):  
Clinical Laboratory ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Normal Rate ◽  
Light Chains ◽  
Factor X ◽  
Laboratory Findings ◽  
Isolation And Characterization

Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange chromatography and compared with normal factor X purified by the same method. Similar molecular weights were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or activated factor X molecules including their respective heavy and light chains. These data indicated that there were no gross structural differences between the normal and variant proteins. Immunochemical assays employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not reveal any structural deviations. Two- dimensional peptide maps indicated that while the light chains of normal and Friuli factor X were very similar, the heavy chains of the native and activated molecules contained a limited number of differences. These data suggested that the defect in factor X Friuli may be a point mutation which lies within the activated heavy chain defined by the 195–424 amino acid sequence. Activation of factor X Friuli in purified systems showed that Russell's viper venom cleaved the molecule at 70% of the normal rate, while the rate of proteolysis of the variant protein was reduced 98% and 75% when incubated with the extrinsic and intrinsic activation complexes, respectively. These data support the clinical laboratory findings and the hypothesis that the defect associated with the Friuli variant may reflect an abnormal interaction between factor X Friuli and the nonproteolytic cofactors of the extrinsic and intrinsic factor X activation complexes. Fluorescence polarization studies suggested that a bound dansylated inhibitor of factor Xa was not oriented to the same extent within the active site of the variant enzyme relative to normal factor Xa until the addition of phospholipid and factor Va. Activated factor X Friuli generated thrombin from prothrombin in a purified system, but at one third the normal rate that was attributed to the Kcat suggesting a secondary effect of this defect.

FVIII Heavy Chain Enhances Tenase Activity Induced By FVIIIa Mimicking Bispesific Antibody, ACE910

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1481-1481
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Takehisa Kitazawa ◽  
Kunihiro Hattori ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Research Funding ◽  
Factor Xa ◽  
Factor X ◽  
Advisory Committees ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Physiological Structure

Abstract Background: ACE910, asymmetric bispecific monoclonal antibodies to activated factor IX (IXa) and factor X, mimics the cofactor function of activated factor VIII (VIIIa) by modulating an optimal position on the tenase assembly. The estimated therapeutic range of ACE910 shows ~30% of thrombin generation in native tenase assembly, supporting that the structure on ACE910-mimicking tenase assembly is different from that on native tenase. Being close to physiological structure consisting from factor IXa, factor X, and factor VIIIa is important for potentiating the clotting function. We examined the effects of factor VIII subunits (light chain, heavy chain, A1 and A2, C2) on ACE910-tenase. Materials/Methods: The factor VIII light chain and heavy chain were isolated from EDTA-treated recombinant factor VIII following chromatography on SP- and Q- Sepharose columns. The A2 and A1 subunits were purified from thrombin-cleaved factor VIII heavy chain by Heparin-, SP- Sepharose columns. Purified factor Xa generation assays was examined with (i) factor VIII subunit (0-40 nM), ACE910 (10 µg/ml), phospholipid (PL) (40 µM), factor IXa (1 nM) and factor X (200 nM), (ii, iii) the A2 or heavy chain (40 nM), ACE910 (10 µg/ml), PL (40 µM), factor IXa and factor X (1 or 0-80 nM, and 0-300 or 200 nM, respectively). These mixtures were reacted for five minutes (i, ii) or one minute (iii). These assays were conducted at 37 °C. Results: (i) The factor Xa generation in ACE910-tenase complex in the absence of factor VIIIa was 10.1±2.2 nM. With the intact heavy chain and A2, amounts of factor Xa were increased dose-dependently, resulting in 1.3- and 1.2-fold increases, respectively. While, the light chain and A1 subunit failed to increase at all. (ii) Vmax for factor X in ACE910-tenase was 173.0±7.0 nM and Km was 31.2±3.9 nM. Vmax obtained with the heavy chain or A2 was 175.9±6.1 or 159.0±6.1 nM, whilst Km was 17.0±2.2 or 31.9±3.5 nM, respectively, indicating that the heavy chain enhanced the binding affinity for factor X in ACE910-tenase. (iii) Vmax for factor IXa in ACE910-tenase was 43.8±2.7 nM and Km was 36.9±4.8 nM. With the heavy chain or A2, Vmax was 46.8±3.0 or 45.0±3.1 nM, and Km was 36.4±3.0 or 32.1±4.9 nM, respectively, indicating that either the heavy chain or A2 did not enhance the catalytic activity and the binding affinity for factor IXa in ACE910-tenase. Conclusion: ACE910-tenase assembly seems to be close to physiological structure by the presence of intact heavy chain interacting with factor X. In addition, ACE910 may substitute the position such as the factor VIII(a) light chain associated with FIXa and FX on ACE910-tenase assembly defecting factor VIII. Disclosures Minami: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Amino Acid Sequence of Trocarin, a Prothrombin Activator FromTropidechis carinatus Venom: Its Structural Similarity to Coagulation Factor Xa

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 621-631 ◽  
Author(s):  
Jeremiah S. Joseph ◽  
Maxey C.M. Chung ◽  
Kandiah Jeyaseelan ◽  
R. Manjunatha Kini
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Blood Coagulation ◽  
Snake Venom ◽  
Heavy Chain ◽  
Light Chain ◽  
Critical Role ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Group Ii

Among snake venom procoagulant proteins, group II prothrombin activators are functionally similar to blood coagulation factor Xa. We have purified and partially characterized the enzymatic properties of trocarin, the group II prothrombin activator from the venom of the Australian elapid, Tropidechis carinatus (rough-scaled snake). Prothrombin activation by trocarin is enhanced by Ca2+, phospholipids, and factor Va, similar to that by factor Xa. However, its amidolytic activity on peptide substrate S-2222 is significantly lower. We have determined the complete amino acid sequence of trocarin. It is a 46,515-Dalton glycoprotein highly homologous to factor Xa and shares the same domain architecture. The light chain possesses an N-terminal Gla domain containing 11 γ-carboxyglutamic acid residues, followed by two epidermal growth factor (EGF)-like domains; the heavy chain is a serine proteinase. Both chains are likely glycosylated: the light chain at Ser 52 and the heavy chain at Asn 45. Unlike other types of venom procoagulants, trocarin is the first true structural homologue of a coagulation factor. It clots snake plasma and thus may be similar, if not identical, to snake blood coagulation factor Xa. Unlike blood factor Xa, it is expressed in high quantities and in a nonhepatic tissue, making snake venom the richest source of factor Xa-like proteins. It induces cyanosis and death in mice at 1 mg/kg body weight. Thus, trocarin acts as a toxin in venom and a similar, if not identical, protein plays a critical role in hemostasis.

scholarly journals Activation of Human Factor X

1977 ◽  
Author(s):  
Carolyn L. Orthner ◽  
Sam Morris ◽  
David P. Kosow
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Gel Electrophoresis ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Coagulation Cascade ◽  
Molecular Forms ◽  
Factor X

Factor X is the zymogen of the proteolytic coagulation enzyme Factor Xa. Since the activation of Factor X to Factor Xa may be a rate limiting step of the coagulation cascade we have begun investigations of the mechanism of this reaction. Human Factor X has been purified 6000-fold from human plasma and the final product is over 95% pure as judged by Polyacrylamide gel electrophoresis. Human Factor X has a monomeric molecular weight of 75,000 and consists of two chains held together by a disulphide bridge. The molecular weight of the heavy chain is 56,000 and that of the light chain is 17,500. The venom coagulant protein of V. russelli (RVV-X) activates human Factor X by cleaving the heavy chain. When fully activated, human Factor Xa shows two bands on Polyacrylamide gel electrophoresis indicating that human Factor Xa like the bovine enzyme has two molecular forms.The kinetic mechanism of the activation reaction has been investigated utilizing the chromogenic Factor Xa substrate Bz-Ile-Glu-Gly-Arg-p-Nitroanilide (S-2222). The reaction has an absolute requirement for Ca; Mg cannot substitute for Ca, however Mg can increase the Vmax of Xa formation in the presence of suboptimal concentrations of Ca. Both Ca and Mg effects exhibit positive cooperativity. Our data indicate that human Factor X has at least three cooperative metal binding sites some of which are specific for Ca.

scholarly journals Isolation and characterization of the factor X Friuli variant

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2108-2116 ◽  
Author(s):  
DS Fair ◽  
DJ Revak ◽  
JG Hubbard ◽  
A Girolami
Keyword(s):  
Clinical Laboratory ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Normal Rate ◽  
Light Chains ◽  
Factor X ◽  
Laboratory Findings ◽  
Isolation And Characterization

Abstract Factor X Friuli was isolated from plasma by immunoaffinity and ion exchange chromatography and compared with normal factor X purified by the same method. Similar molecular weights were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the intact or activated factor X molecules including their respective heavy and light chains. These data indicated that there were no gross structural differences between the normal and variant proteins. Immunochemical assays employing either polyclonal or 46 monoclonal antibodies (MoAbs) did not reveal any structural deviations. Two- dimensional peptide maps indicated that while the light chains of normal and Friuli factor X were very similar, the heavy chains of the native and activated molecules contained a limited number of differences. These data suggested that the defect in factor X Friuli may be a point mutation which lies within the activated heavy chain defined by the 195–424 amino acid sequence. Activation of factor X Friuli in purified systems showed that Russell's viper venom cleaved the molecule at 70% of the normal rate, while the rate of proteolysis of the variant protein was reduced 98% and 75% when incubated with the extrinsic and intrinsic activation complexes, respectively. These data support the clinical laboratory findings and the hypothesis that the defect associated with the Friuli variant may reflect an abnormal interaction between factor X Friuli and the nonproteolytic cofactors of the extrinsic and intrinsic factor X activation complexes. Fluorescence polarization studies suggested that a bound dansylated inhibitor of factor Xa was not oriented to the same extent within the active site of the variant enzyme relative to normal factor Xa until the addition of phospholipid and factor Va. Activated factor X Friuli generated thrombin from prothrombin in a purified system, but at one third the normal rate that was attributed to the Kcat suggesting a secondary effect of this defect.

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