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.

Activation of Human Coagulation Factor VIII by Activated Factor X, the Common Product of the Intrinsic and the Extrinsic Pathway of Blood Coagulation

1982 ◽  
Vol 47 (02) ◽  
pp. 096-100 ◽  
Author(s):  
K Mertens ◽  
R M Bertina
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Extrinsic Factor ◽  
Extrinsic Pathway ◽  
Factor Ixa ◽  
The Common

SummaryThe intrinsic activation of human factor X has been studied in a system consisting of purified factors and in plasma. In both these systems factor Xa stimulated the activation of factor X by factor IXa plus factor VIII This is due to the activation of factor VIII by factor Xa. When this factor Xa is formed via the extrinsic pathway, the extrinsic factor X activator functions as a stimulator of the intrinsic factor X activator.

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.

Isolation and Properties of the Abnormal Factor X Induced by Vitamin K Antagonist (PIVKA-X)

1975 ◽  
Author(s):  
M. J. Lindhout ◽  
B.H.M. Kop-Klaassen
Keyword(s):  
Vitamin K ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Term Treatment ◽  
Vitamin K Antagonist ◽  
Factor X ◽  
Short Term ◽  
Short Term Treatment ◽  
Ca Ions

The abnormal factor X induced by vitamin K antagonist could be isolated from the plasma obtained from a cow under short term treatment with Marcoumar®. A 3,500 fold purification was achieved at a 10% yield. A single band was seen on polyacrylamide gel electrophoresis. The protein was immunologically identical to normal factor X.After incubation with an activator of factor X from Russell’s Viper Venom (RVV), PIVKA-X shows esterase and proteolytic activity. Although PIVKA-XRVv has no activity in a one-stage factor Xa assay, it seems that the active site is intact, as it could be titrated with p-nitrophenyl-p-guanidinobenzoate (p-NPGB).In the absence of phospholipids and Ca++-ions both factor Xa and PIVKA-XRVv activate prothrombin at the same low rate. Identical prothrombin subunits are obtained in both cases.

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 The contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

1984 ◽  
Vol 223 (3) ◽  
pp. 607-615 ◽  
Author(s):  
K Mertens ◽  
R M Bertina
Keyword(s):  
Human Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Activate Factor ◽  
Factor X ◽  
Formation Kinetic ◽  
Factor Ixa ◽  
Fold Stimulation

The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.

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.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

scholarly journals Proteolytic inactivation of blood coagulation factor IX by thrombin

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1302-1308 ◽  
Author(s):  
W Kisiel ◽  
KJ Smith ◽  
BA McMullen
Keyword(s):  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Light Chains ◽  
Factor X ◽  
Amino Terminal ◽  
Coagulation Factor Ix ◽  
Human Factor Ix ◽  
Coagulant Activity

Coagulation factor IX is a vitamin K-dependent glycoprotein that circulates in blood as a precursor of a serine protease. Incubation of human factor IX with human alpha-thrombin resulted in a time and enzyme concentration-dependent cleavage of factor IX yielding a molecule composed of a heavy chain (mol wt 50,000) and a doublet light chain (mol wt 10,000). The proteolysis of factor IX by thrombin was significantly inhibited by physiological levels of calcium ions. Under nondenaturing conditions, the heavy and light chains of thrombin- cleaved factor IX remained strongly associated, but these chains were readily separated by gel filtration in the presence of denaturants. Amino-terminal sequence analyses of the isolated heavy and light chains of thrombin-cleaved human factor IX indicated that thrombin cleaved peptide bonds at Arg327-Val328 and Arg338-Ser339 in this molecule. Comparable cleavages were observed in bovine factor IX by bovine thrombin and occurred at Arg319-Ser320 and Arg339-Ser340. Essentially, a complete loss of factor IX procoagulant activity was associated with its cleavage by thrombin. Furthermore, thrombin-cleaved factor IX neither developed coagulant activity after treatment with factor XIa nor inhibited the coagulant activity of native factor IX. These data indicate that thrombin cleaves factor IX near its active site serine residue, rendering it incapable of activating factor X. Whether or not this reaction occurs in vivo is unknown.

Two parallel prothrombin activator systems in Australian rough-scaled snake, Tropidechis carinatus

2005 ◽  
Vol 93 (01) ◽  
pp. 40-47 ◽  
Author(s):  
Md. Abu Reza ◽  
Sanjay Swarup ◽  
Manjunatha Kini
Keyword(s):  
Blood Coagulation ◽  
Blood Clotting ◽  
Cdna Sequence ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Venom Gland ◽  
Factor X ◽  
Specific Expression ◽  
Life Saving ◽  
Sequence Encoding

SummaryIt is uncommon for similar pathways/systems to be involved in highly divergent functions within single organisms. Earlier, we have shown that trocarin D, a venom prothrombin activator, from the Australian rough-scaled snake Tropidechis carinatus, is structurally and functionally similar to the blood coagulation factor Xa (FXa). The presence of a haemostatic system in these snakes implies that they have two parallel prothrombin activating systems: one in the plasma, that participates in the life saving process of blood clotting and the other in their venom, where it acts as a toxin. Here, we report the complete cDNA sequence encoding the blood coagulation factor X (FX) from the liver of T. carinatus. Deduced T. carinatus FX sequence shows ~80% identity with trocarin D but ~50% identity with the mammalian FX. Our present study confirms the presence of two separate genes – one each for FX and trocarin D, that code for similar proteins in T. carinatus snake. These two genes have different expression sites and divergent uses suggesting that snake venom prothrombin activators have probably evolved by the duplication of the liver FX gene and subsequently marked for tissue-specific expression in the venom gland.

scholarly journals Active site-specific immunoassays

Blood ◽  
1990 ◽  
Vol 76 (4) ◽  
pp. 755-766 ◽  
Author(s):  
KG Mann ◽  
EB Williams ◽  
S Krishnaswamy ◽  
W Church ◽  
A Giles ◽  
...  
Keyword(s):  
Active Site ◽  
Serine Proteases ◽  
Solid Phase ◽  
Activated Protein C ◽  
Factor Xa ◽  
Immune Recognition ◽  
Factor X ◽  
Zymogen Activation ◽  
Prothrombinase Complex ◽  
Active Site Histidine

Abstract This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl- epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.

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