THE MODE OF ACTION OF PENTOSAN POLYSULPHATE IN PLASMA

1987 ◽  
Author(s):  
S Béguin ◽  
H C Hemker
Keyword(s):  
Factor Viii ◽  
Linear Increase ◽  
Factor Ix ◽  
Factor X ◽  
Heparin Cofactor Ii ◽  
Normal Plasma ◽  
Viii And Ix ◽  
Pentosan Polysulphate ◽  
Lag Times ◽  
Prothrombinase Activity

We developed a method which enables as to compute the course of prothrombinase activity in clotting plasma (H.C. Hemker, G.M. Willems, S. Béguin: Thromb. Haemostas. 56, 9-17, 1986) and used this for a study of the effect of pentosan polysulphate (PPS) on thrombin generation.When added to normal plasma in the concentration range of 0-8 μg/ml PPS induces a linear increase of the pseudo first order decay constant of endogenous thrombin like heparin does, 1 ug of PPS being equivalent to 0.045 Aig of heparin. Contrary to heparin this action is partly (∼ 65%) dependent upon AT III and partly (∼ 35%) upon heparin cofactor II.In normal plasma PPS causes an inhibition of both extrinsic and intrinsic prothrombinase formation. Only in the intrinsic system an increase of the lag time of prothrombinase appearance is observed. Unlike heparin, PPS does not inhibit factor IXa induced thrombin formation neither does it inhibit prothrombinase formation in the presence of preactivated factor VIII. The prolongation of the lag times must therefore be ascribed to inhibition by PPS of the activation of factor VIII.The inhibition of extrinsic prothrombinase formation by PPS increases with progressive dilution of thromboplastin and is not seen in haemophilia A or B plasma. This demonstrates the existance of a factor VIII and IX dependent process in extrinsic coagulation that gains in importance when the potency of factgr VII-tissue factor complex decreases, i.e. the Josso pathway.PPS, but also heparin causes an unexplained increase of prothrombinase action in haemophIIic plasma. The same phenomenon may be expected to exist in normal plasma, be it obscured by a concomitant inhibition. This, together with the incomplete inhibition of factor VIII activation by PPS makes that we cannot use this inhibitor as a means to quantitate the Josso pathway. The best estimate that we can obtain is that, in the presence of 2% thromboplastin, the factor IX dependent activation of factor X contributes more then 20% to prothrombinase generation.

scholarly journals In Hemophilia Α Plasma Treated with Emicizumab, Factor IX Activation By Factor VIIa Drives Thrombin Generation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-17
Author(s):  
Dougald Monroe ◽  
Mirella Ezban ◽  
Maureane Hoffman
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Plasma Levels ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Ix ◽  
Factor X ◽  
Dose Dependent

Background.Recently a novel bifunctional antibody (emicizumab) that binds both factor IXa (FIXa) and factor X (FX) has been used to treat hemophilia A. Emicizumab has proven remarkably effective as a prophylactic treatment for hemophilia A; however there are patients that still experience bleeding. An approach to safely and effectively treating this bleeding in hemophilia A patients with inhibitors is recombinant factor VIIa (rFVIIa). When given at therapeutic levels, rFVIIa can enhance tissue factor (TF) dependent activation of FX as well as activating FX independently of TF. At therapeutic levels rFVIIa can also activate FIX. The goal of this study was to assess the role of the FIXa activated by rFVIIa when emicizumab is added to hemophilia A plasma. Methods. Thrombin generation assays were done in plasma using 100 µM lipid and 420 µM Z-Gly-Gly-Arg-AMC with or without emicizumab at 55 µg/mL which is the clinical steady state level. The reactions were initiated with low (1 pM) tissue factor (TF). rFVIIa was added at concentrations of 25-100 nM with 25 nM corresponding to the plasma levels achieved by a single clinical dose of 90 µg/mL. To study to the role of factor IX in the absence of factor VIII, it was necessary to create a double deficient plasma (factors VIII and IX deficient). This was done by taking antigen negative hemophilia B plasma and adding a neutralizing antibody to factor VIII (Haematologic Technologies, Essex Junction, VT, USA). Now varying concentrations of factor IX could be reconstituted into the plasma to give hemophilia A plasma. Results. As expected, in the double deficient plasma with low TF there was essentially no thrombin generation. Also as expected from previous studies, addition of rFVIIa to double deficient plasma gave a dose dependent increase in thrombin generation through activation of FX. Interestingly addition of plasma levels of FIX to the rFVIIa did not increase thrombin generation. Starting from double deficient plasma, as expected emicizumab did not increase thrombin generation since no factor IX was present. Also, in double deficient plasma with rFVIIa, emicizumab did not increase thrombin generation. But in double deficient plasma with FIX and rFVIIa, emicizumab significantly increased thrombin generation. The levels of thrombin generation increased in a dose dependent fashion with higher concentrations of rFVIIa giving higher levels of thrombin generation. Conclusion. Since addition of FIX to the double deficient plasma with rFVIIa did not increase thrombin generation, it suggests that rFVIIa activation of FX is the only source of the FXa needed for thrombin generation. So in the absence of factor VIII (or emicizumab) FIX activation does not contribute to thrombin generation. However, in the presence of emicizumab, while rFVIIa can still activate FX, FIXa formed by rFVIIa can complex with emicizumab to provide an additional source of FX activation. Thus rFVIIa activation of FIX explains the synergistic effect in thrombin generation observed when combining rFVIIa with emicizumab. The generation of FIXa at a site of injury is consistent with the safety profile observed in clinical use. Disclosures Monroe: Novo Nordisk:Research Funding.Ezban:Novo Nordisk:Current Employment.Hoffman:Novo Nordisk:Research Funding.

scholarly journals Development of Monospecific Antibodies to Human Factor IX

1977 ◽  
Author(s):  
Cheryl Y. Tiarks ◽  
Chin-Hai Chang ◽  
Liberto Pechet
Keyword(s):  
Neutralizing Antibodies ◽  
Human Factor ◽  
Human Albumin ◽  
Factor Ix ◽  
Hemophilia B ◽  
Red Cross ◽  
Factor X ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

Effect of Stanozolol on Factors VIII and IX and Serum Aminotransferases in Haemophilia

1985 ◽  
Vol 53 (03) ◽  
pp. 386-389 ◽  
Author(s):  
I A Greer ◽  
M Greaves ◽  
R Madhok ◽  
K McLoughlin ◽  
N Porter ◽  
...  
Keyword(s):  
Factor Viii ◽  
Anabolic Steroid ◽  
Anabolic Steroids ◽  
Factor Ix ◽  
Chronic Liver Diseases ◽  
Oral Agent ◽  
Interesting Possibility ◽  
Consistent Change ◽  
Viii And Ix ◽  
Serum Aminotransferases

SummaryThe treatment of haemophilia has been dramatically improved since the introduction of factor VIII and IX concentrates, however these concentrates have brought new problems such as hepatitis and A.I.D.S. An oral agent which could raise endogenous levels of factor VIII and IX would be of great benefit. Danazol, an anabolic steroid, has recently been shown to increase levels of factors VIII and IX in haemophilia. We therefore studied the effect of stanozolol, a closely related anabolic steroid, in 15 patients with haemophilia A or Christmas disease over a 2-4 week period. There was no consistent change in factor VIIIc or factor IX, and fibrinolysis was significantly enhanced. No effect was apparent on the incidence of spontaneous bleeds. However serum aminotransferases which were abnormal in 11 of the 15 patients at the start of the study fell significantly with stanozolol therapy. This raises the interesting possibility that anabolic steroids may be beneficial in patients with chronic liver diseases.

FACTOR VIII MEDIATES BINDING OF FACTOR IX TO STIMULATED PLATELETS

1987 ◽  
Author(s):  
W Muntean ◽  
B Leschnik
Keyword(s):  
Factor Viii ◽  
Phospholipase C ◽  
Human Platelets ◽  
Final Concentration ◽  
Platelet Lysate ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Platelet Surface ◽  
Human Thrombin

In previous work we have shown that factor VII! binds to phospholipids of the membrane of stimulated platelets and that von Willebrand factor is not required for binding of factor VIII to platelets. Since factor VIII is a cofactor in the activation of factor X by factor IX, we investigated whether factor VIII enhances binding of factor IX to the platelet surface.Factor VIII and factor IX were purified by immunoadsorbent chromatography using specific rabbit antibodies. Washed human platelets (250/nl final concentration) stimulated by human thrombin and collagen were incubated with barbiturate buffer, or with purified factor IX (1 U/ml final concentration), or with factor IX (1 U/ml) in the presence of factor VIII (1 U/ml). Washed and stimulated platelets were also incubated with factor VIII and IX as above in the presence of different amounts of CaCl2. Platelets were then washed again and lysed by sonication. Factor VIII:Ag (immunoradiometric assay) and factor IX:Ag (ELISA) were measured in the platelet lysate prior to and after incubation of the lysate with phospholipase C.Platelet bound IX:Ag was significantly higher after incubation of stimulated platelets with factor IX in the presence of factor VIII than after incubation of platelets with buffer or with factor IX alone. CaCl2 proved to be essential for binding of factor IX to platelets even in the presence of factor VIII, but CaCl2 was not required for binding of factor VIII to platelets. Measurable VIII :Ag and IX:Ag increased significantly after incubation of the platelet lysate with phospholipase C.Our data suggest that factor VIII mediates binding of factor IX to phospholipids or receptors containing phospholipids on the membrane of stimulated platelets and thereby contributes to the assembly of the factor X activating complex on the platelet surface.

Characterization Of The In Vitro “Factor VIII Bypassing Activity”

1981 ◽  
Author(s):  
D L Aronson ◽  
J Bagley
Keyword(s):  
Factor Viii ◽  
Early Stage ◽  
Deae Cellulose ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor V ◽  
Factor X ◽  
Hemostatic Effect ◽  
Prolonged Aptt

The in vitro correction of the prolonged APTT of hemophilic plasma has been ascribed to an uncharacterized entity “Factor VIII Bypassing Activity.” Such products also correct the prolonged APTT plasma deficient in Factor IX, Factor X and Factor XII, but not of Factor V deficient plasma. Correction of the APTT in Factor VIII deficient plasma by early stage coagulants such as Factor XIIa, Kallikrein and Factor IXa is minimal. These results indicate that this in vitro activity acts at the level of either the activation of Factor X or the activation of prothrombin.A coagulant has been prepared from serum by barium precipitation, heparin-agarose, DEAE cellulose and high pressure liquid chromatography (HPLC). The in vitro coagulant properties are similar to “activated” prothrombin complex (Autoplex) and the biologic and chemical properties are identical to activated Factor X.Infusion of the partially purified serum coagulant into normal dogs was well tolerated and, in contrast to Factor IX concentrates, gave no signs of DIC. Infusion into bleeding hemophilic dogs had no hemostatic effect. It is concluded that a major portion of the in vitro potency of activated prothrombin concentrates is due to activated Factor X, a material which when infused has no in vivo hemostatic effect.Acknowledgments - The authors gratefully acknowledge the studies of Dr. Henry Kingdon in hemophilic dogs.

Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly

1999 ◽  
Vol 82 (08) ◽  
pp. 209-217 ◽  
Author(s):  
Patrick Celie ◽  
Joost Kolkman ◽  
Peter Lenting ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Three Dimensional ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor X Activation

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.

Antibody to C1 Domain of Factor VIII Alters Interaction of Factor Xase Complex with Factor X.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1738-1738
Author(s):  
Gary E. Gilbert ◽  
Anu Bhimavarapu ◽  
Patricia Price ◽  
Marc Jacquemin
Keyword(s):  
Factor Viii ◽  
Factor Ix ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Apparent Affinity ◽  
Factor Ixa ◽  
Factor Viiia ◽  
C1 Domain ◽  
Gla Domain

Abstract The role of the C1 domain in function of factor VIII has not been clearly defined. In contrast, functional interactions have been identified for the three A domains and the C2 domain. We hypothesized that the C1 domain of factor VIII participates in both phospholipid binding and interaction with factor X and/or factor IXa. We evaluated inhibition of the factor Xase complex by LE2E9, a human inhibitor IgG4k mAb against C1. We utilized altered catalytic activity of the factor Xase complex in a defined assay to report the inhibition by LE2E9. Inhibition by LE2E9 was also evaluated when soluble phosphatidylserine replaced vesicles to support the factor Xase complex and when Gla-domainless factor X was the substrate. The deglycosylated form of LE2E9 was also evaluated to better define the mechanism through which LE2E9 exerts its effect. We found that LE2E9 bound to factor VIIIa with an apparent KD of 0.5 nM. The apparent affinity of factor VIIIa for sonicated phospholipid vesicles of phosphatidylserine:phosphatidylethanolamine:phosphatidylcholine 4:20:76 increased 3-fold in the presence of LE2E9. The apparent affinity of factor VIIIa for factor IXa was not significantly changed. The KM of the factor VIIIa-factor IXa complex was 20 ± 2 nM with LE2E9 vs. 40 ± 2 nM without. LE2E9 decreased the Vmax by 77 ± 6% indicating that the affinity of factor X for the factor Xase complex is increased while the rate of cleavage is decreased. When Gla-domainless factor X was used as the substrate for the factor Xase complex, LE2E9 did not inhibit activity indicating that inhibition occurs via an interaction that involves the factor X Gla domain. When the factor VIIIa-factor IX complex was supported by dihexanoyl phosphatidylserine rather than phospholipid vesicles the inhibition of Vmax was 47% indicating that the inhibitory effect does not require a phospholipid bilayer. Deglycosylated LE2E9 did not significantly change the KM but decreased the Vmax by 22% while both antibodies bound to factor VIII with the same affinity. These results suggest that LE2E9 inhibition relates largely to interaction of a carbohydrate moiety with factor VIII or factor X rather than binding the core C1 epitope. We conclude that LE2E9 decreases the KM, and the Vmax for the factor VIIIa-factor IXa complex, but only when the factor X Gla domain is present. These results suggest that in the factor Xase complex the C1 domain of factor VIII is intimately associated with the Gla domain of factor X and that interaction between these domains enhances the kcat for the factor VIIIa-factor IXa complex.

Identification Of The Thrombin-Binding Site On Factor VIII Regulating Arg372 Cleavage In The Factor VIII Heavy Chain

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3570-3570
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Koji Yada ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Solid Phase ◽  
Factor Ix ◽  
Cross Linking ◽  
Dependent Manner ◽  
Factor X ◽  
Dose Dependent

Abstract Factor VIII is activated by cleavage at Arg372, Arg740, and Arg1689 by thrombin. Activated factor VIII (VIIIa) forms the tenase complex and markedly amplifies the activation of factor X as a cofactor of factor IX. We had demonstrated that thrombin interacts with factor VIII through the residues 392-394 and 484-509 in the A2 domain and the C2 domain, and each association regulates cleavage at Arg740, Arg372, and Arg1689, respectively (Nogami K, JBC 2000, 2005; BJH 2008). The A2 residues 484-509 partially contribute to cleavage at Arg372 by thrombin, however, the major thrombin binding-site(s) regulating cleavage at Arg372 is unclear. Thrombin recognizes macromolecular substrates and cofactors through either or both of two anion-binding exosite I and II (ABE-I and -II), which are characterized by a high density of solvent-exposed basic residues. ABE-I binds to fibrinogen and hirudin (residues 54-65), whilst ABE-II is primarily characterized as the heparin-binding exosite. The A1 domain of factor VIII also binds to thrombin through the ABE-I (Nogami K. JBC 2005). In this study, we attempted to identify the thrombin-binding region on A1, and focused on the A1 residues 340-350, involving the clustered acidic residues and similar sequences of hirudin (residues 54-65). A synthetic peptide corresponding to the A1 residues 340-350 with sulfated Tyr346 (340-350-S(+)) was prepared to investigate factor VIII interaction with thrombin. Activation of factor VIII (100 nM) by thrombin (0.4 nM) with various concentrations of peptide was evaluated by measurement of factor VIIIa activity in a one-stage clotting assay. A 340-350-S(+) peptide showed a dose-dependent inhibition (by ∼60%) of thrombin-catalyzed activation, and the IC50 was 75 µM. A non-sulfated peptide also showed a modest inhibition by ∼40% (IC50 >400 µM), however. An experiment using thrombin substrate S-2238 demonstrated that P340-350-S(+) did not affect the thrombin activity. The effect of 340-350-S(+) peptide on the thrombin-catalyzed cleavage of heavy chain was further examined by SDS-PAGE/western blotting.The peptide significantly blocked the cleavage at Arg372 in a timed- and dose-dependent manner (IC50; 150 µM), whilst of interest the cleavage at Arg740 was little affected. A non-sulfated peptide also delayed the cleavage at Arg372, with a modest fast cleavage compared to sulfated one. The peptide did not inhibit factor FXa-catalyzed reaction to factor VIII. Direct binding of 340-350-S(+) peptide to thrombin was examined by a surface resonance plasmon (SPR)-based assay and by the zero-length cross-linking reagent EDC. In SPR-based solid phase assay, thrombin bound to immobilized 340-350-S(+) peptide with high affinity (Kd; 1.13 nM). EDC cross-linking fluid phase assay similarly revealed that formation of EDC cross-linking product between the biotinylated 337-350-S(+) peptide and thrombin were observed, and this cross-linking was completely inhibited by non-labeled 340-350-S(+) peptide (IC50; 1.0 µM). Taken together, we demonstrated that the A1 residues 340-350 (NEEAED(sY)DDDL) involving sulfated Tyr346 contained the thrombin binding-site responsible for the proteolytic cleavage at Arg372 in factor VIII. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Production of a High-Titred Sheep Antiserum Against Human Factor IX and Immunological Demonstration of Factor IX Aggregates

1977 ◽  
Author(s):  
K.H. Ørstavik ◽  
A.M. Vennerød
Keyword(s):  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Rabbit Antiserum ◽  
Factor Ix ◽  
Factor X ◽  
Weak Band ◽  
Normal Plasma ◽  
Human Factor Ix ◽  
Plasma Factor

Plasma factor IX was purified from a factor IX concentrate by a five step procedure including absorption onto aluminium hydroxide, affinity chromatography on heparin-coupled Sepharose 4B, preparative disc gel electrophoresis, affinity chromatography on an immunosorbent column with rabbit antiserum against factor X and chromatography on DE-52 cellulose. The pooled fractions had a specific activity of approximately 250 U/mg protein. A sheep was immunized with pooled and concentrated fractions. An antiserum was produced which gave one main precipitin band and occasionally an additional weak band against normal plasma in double immunodiffusion. At a dilution of 1/100-1/200 the antiserum neutralized 90% of the factor IX activity in an equal volume of normal plasma.Polyacrylamide disc gel electrophoresis of the fractions from DE-52 cellulose revealed one major and three minor bands with lower electrophoretic mobility and intensity. The three minor bands disappeared on disc gel electrophoresis in the presence of 10 M urea. When the disc electrophoresis gel was submitted to electrophoresis into anagarose gel containing the sheep antiserum or a previously characterized rabbit antiserum against factor IX, four precipitin arcs corresponding to the four bands were seen. A reaction of identity was seen between the four arcs. This study demonstrates that a highly potent antiserum may be produced against factor IX in sheep.

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