The activation and inactivation of human factor VIII by thrombin: effect of inhibitors of thrombin

Abstract The activation and inactivation of human factor VIII by thrombin have been investigated by the use of thrombin inhibitors. The addition of inhibitors to nonactivated factor VIII blocks activation by thrombin. In contrast, their addition to factor VIII activated with thrombin does not block inactivation, but causes an initial period of decay that is more rapid than in the absence of inhibitor. This effect was seen only with protease inhibitors that inhibit thrombin. After the initial decay, low levels of factor VIII coagulant activity persist in the presence of inhibitors, but an assay specific for activated factor VIII showed this to be largely a result of the persistence of nonactivated factor VIII. Only in the case of reversible inhibition is activated factor VIII present in this plateau phase. Possible mechanisms that would account for these observations were studied by iterative computer simulation of model reactions. Two classes were considered: (formula: see text). The experimental results are inconsistent with the first mechanism, which predicts that thrombin indicators should stabilize activated factor VIII (VIIIt). Alternative mechanisms were studied where activation is thrombin-dependent, but inactivation is a first- order reaction (mechanism 2). This family of mechanisms includes those where VIIIt is an VIII. thrombin complex. Simulation of the addition of thrombin inhibitors to such model systems shows the initial rapid decay of activity characteristic of the experimental observations and predicts qualitatively the different effects of reversible and irreversible inhibitors that are observed in the plateau phase. These results argue strongly against a two-cleavage model for the activation and inactivation of factor VII by thrombin and support a one-cleavage model in which inactivation is due to first-order decay. In addition, they provide a plausible mechanistic explanation for the fact that serine protease inhibitors appear to inhibit thrombin-activated factor VIII.

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The activation and inactivation of human factor VIII by thrombin: effect of inhibitors of thrombin

Blood ◽

10.1182/blood.v57.3.476.bloodjournal573476 ◽

1981 ◽

Vol 57 (3) ◽

pp. 476-482

Author(s):

MB Hultin ◽

J Jesty

Keyword(s):

Factor Viii ◽

Protease Inhibitors ◽

Human Factor ◽

Initial Period ◽

Model Systems ◽

Thrombin Inhibitors ◽

Factor Vii ◽

Plateau Phase ◽

First Order ◽

Human Factor Viii

The activation and inactivation of human factor VIII by thrombin have been investigated by the use of thrombin inhibitors. The addition of inhibitors to nonactivated factor VIII blocks activation by thrombin. In contrast, their addition to factor VIII activated with thrombin does not block inactivation, but causes an initial period of decay that is more rapid than in the absence of inhibitor. This effect was seen only with protease inhibitors that inhibit thrombin. After the initial decay, low levels of factor VIII coagulant activity persist in the presence of inhibitors, but an assay specific for activated factor VIII showed this to be largely a result of the persistence of nonactivated factor VIII. Only in the case of reversible inhibition is activated factor VIII present in this plateau phase. Possible mechanisms that would account for these observations were studied by iterative computer simulation of model reactions. Two classes were considered: (formula: see text). The experimental results are inconsistent with the first mechanism, which predicts that thrombin indicators should stabilize activated factor VIII (VIIIt). Alternative mechanisms were studied where activation is thrombin-dependent, but inactivation is a first- order reaction (mechanism 2). This family of mechanisms includes those where VIIIt is an VIII. thrombin complex. Simulation of the addition of thrombin inhibitors to such model systems shows the initial rapid decay of activity characteristic of the experimental observations and predicts qualitatively the different effects of reversible and irreversible inhibitors that are observed in the plateau phase. These results argue strongly against a two-cleavage model for the activation and inactivation of factor VII by thrombin and support a one-cleavage model in which inactivation is due to first-order decay. In addition, they provide a plausible mechanistic explanation for the fact that serine protease inhibitors appear to inhibit thrombin-activated factor VIII.

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The Fractionation Properties of Human Factor VIII (Antihaemophilic Factor)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654503 ◽

1960 ◽

Vol 04 (02) ◽

pp. 253-260 ◽

Cited By ~ 1

Author(s):

Franco Gobbi

Keyword(s):

Factor Viii ◽

Human Factor ◽

Maximum Yield ◽

Factor Vii ◽

Precipitation Method ◽

Salting Out ◽

Human Factor Viii ◽

Plasma Factor ◽

Two Factors ◽

Thromboplastic Activity

SummaryThe fractionation properties of human Factor VIII (antihaemophilic factor, AHF, antihaemophilic globulin) have been studied using a plasma of congenital afibrinogenaemia as a starting material.From a fibrinogen-free plasma, Factor VIII does not precipitate with ethanol at a final concentration of 8%; on the contrary the maximum yield is reached at an ethanol concentration of 25%.With a precipitation method carried out by a one to ten dilution of plasma with distilled water and acidification by N/10 hydrochloric acid to a pFI 5.2, Factor VIII does not precipitate with the euglobulin fraction; when normal plasma is used, such a precipitation is almost complete.With the salting-out fractionation method by ammonium sulphate, Factor VIII precipitates at a concentration between 25 and 33% of saturation either from fibrinogen-free and from normal human plasma.A non-specific thromboplastic activity appears in the fractions prepared by every method. This activity, which is probably due to the activation of seric accelerators, is easily removed by Al(OH)s adsorption. Thus, in order to insure the specificity of Factor VIII assays, the preliminary adsorption of the fractions is indispensable before testing their antihaemophilic activity.Fibrinogen and Factor VIII have different and definite precipitation patterns. When these two factors are associated the fractionation properties of AHF appear quite modified, showing a close similarity to those of fibrinogen. This fact can explain the technical difficulties encountered in the attempt to purify the antihaemophilic factor, and the lack of reproducible procedures for removing fibrinogen without affecting Factor VII.

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The Effects of Injection of Human Factor VIII Antibody Into Rabbits

Blood ◽

10.1182/blood.v42.4.509.509 ◽

1973 ◽

Vol 42 (4) ◽

pp. 509-521 ◽

Cited By ~ 2

Author(s):

S. M-C. Shen ◽

D. I. Feinstein ◽

S. I. Rapaport

Keyword(s):

Factor Viii ◽

Human Factor ◽

Rabbit Model ◽

Kinetic Studies ◽

Factor Vii ◽

Factor V ◽

Initial Values ◽

Human Factor Viii ◽

Hemolytic Complement Activity ◽

Antigen Antibody

Abstract Rabbits were injected with an immunoglobulin fraction of human serum containing a factor VIII antibody. Factor VIII levels fell abruptly, persisted below 10% of a rabbit plasma standard for 12 hr, and returned to normal by 120-168 hr. The factor VIII antigen-antibody reaction did not result in Intravascular clotting as evaluated by kinetic studies with 125I-fibrinogen. However, small falls in factor V and factor VII levels were observed over a 6-hr period after the injection. Platelets fell to about one-half of initial values within 15 min, rose to 80% of initial levels over 2 hr, and subsequently declined to 65%-70% of initial levels. WBC levels fell to below 20% of initial values 2 hr after the injection but returned to about 75% of initial values by 6 hr. Total hemolytic complement activity was unaffected. Animals made granulocytopenic with nitrogen mustard and animals with hereditary C'6 deficiency behaved similarly to normal animals. One may conclude that the injection of human factor VIII antibody into rabbits produces a rabbit model with impaired intrinsic coagulation suitable for studies of the mechanism of endotoxin-induced intravascular clotting.

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Survival of Radiolabelled Human Factor VIII in Vivo

10.1055/s-0039-1680462 ◽

1977 ◽

Author(s):

J. Over ◽

J. J. Sixma ◽

A. M. C. Trieschnigg ◽

H. A. A. Vlooswijk ◽

M.H.M. Doucet-de Bruïne ◽

...

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Human Factor ◽

Survival Curve ◽

Human Albumin ◽

Factor Vii ◽

Human Factor Viii ◽

Coagulant Activity ◽

A Subunit

Bennett and Ratnoff(1972) reported that in haemophiliacs the coagulant activity of factor VIII(F VIII:C) disappeared faster than the factor VII-related antigen (F VIIIR:AG). We decided to investigate this phenomenon with radiolabelled factor VIII. This also allowed us to study the behaviour of various factor VIII fractions after infus i on. Human factor VIII was purified from cryoprecipitate, labelled with 125 I by “the lactoperoxydase-glucose oxydase method, mixed with a human albumin solution and sterilized by filtration through a Millipore filter. F VIII:C,F VIIIR:AG,F VIIIR:WF assays, gelchromatography, crossed Immunoelectrophoresis and PAGE studies were carried out. No significant changes due to the procedure were observed. The radiolabel in the plasma after in vivo administration was for more than 95% bound to F VIIIR:AG as demonstrated with immunoadsorption with an antiserum prepared against a subunit of factor VIII purified by PAGE. About 1.5–6% of free 125 I was also present. Yield and survival were studied in 6 normal volunteers. The yield was somewhat lower (79%) than expected after transfusion. The survival curve was biphasic showing half lives of hours and 20 hours. In 4 haemophiliacs the yield and disappearance were essentially similar. The F VIII:C in these patients who also received cryoprecipitate of 20 donors disappeared with the same kinetics as the radiolabel. Evidence was obtained indicating that the highest molecular weight forms of factor VIII disappeared more rapidly, while losing the F VIIIR:WF, than the lower molecular weight forms. Radio label in cryoprecipitate showed a biphasic, more rapid disappearance ; label in cryosupernatant plasma disappeared slower.

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Impaired Prothrombin Consumption in Bernard-Soulier Syndrome Is Corrected In Vitro by Human Factor VIII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655972 ◽

1997 ◽

Vol 77 (02) ◽

pp. 383-386 ◽

Cited By ~ 8

Author(s):

S Bellucci ◽

J P Girma ◽

M Lozano ◽

D Meyer ◽

J P Caen

Keyword(s):

Factor Viii ◽

Human Factor ◽

Platelet Membrane ◽

Giant Platelets ◽

Prolonged Bleeding Time ◽

Human Factor Viii ◽

Von Willebrand ◽

Willebrand Factor ◽

Bernard Soulier Syndrome

SummaryThe Bernard-Soulier syndrome (BSS) is characterized by thrombocytopenia with giant platelets, a prolonged bleeding time with defective platelet adhesion to the subendothelium related to a defect in platelet membrane glycoprotein lb (GPIb) and a decreased prothrombin consumption. The mechanism of the latter abnormality remains unknown. In this study, we showed that this defect was corrected by the addition of purified human factor VIII (FVIII) to blood from four patients with BSS. The correction of prothrombin consumption was almost complete at concentrations between 1.5 and 3 IU/ml of FVIII procoagulant activity (VIII.'C) and partially abolished by a monoclonal antibody which neutralizes VIII:C. This correction was specific for FVIII and was not observed after addition of purified human FIX. It was obtained, in the same magnitude range, with FVIII complexed to von Willebrand factor (vWF) but not with free vWF. These data provide a new insight into the knowledge of the physiological interaction between the platelet membrane and the vWF-FVIII complex facilitating plasma coagulation activation and may lead to helpful therapeutic advances.

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Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)68793-9 ◽

1988 ◽

Vol 263 (13) ◽

pp. 6352-6362 ◽

Cited By ~ 9

Author(s):

R J Kaufman ◽

L C Wasley ◽

A J Dorner

Keyword(s):

Factor Viii ◽

Mammalian Cells ◽

Human Factor ◽

Human Factor Viii

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Immunopurification of Human Factor VIII/vWF Complex from Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647497 ◽

1988 ◽

Vol 59 (03) ◽

pp. 364-371 ◽

Cited By ~ 10

Author(s):

Odile Mejan ◽

Vincent Fert ◽

Maryléne Delezay ◽

Michel Delaage ◽

Rose Cheballah ◽

...

Keyword(s):

Factor Viii ◽

Human Factor ◽

Specific Activity ◽

Coupling Method ◽

Purification Process ◽

Solid Supports ◽

Coupling Methods ◽

Human Factor Viii ◽

Alkaline Conditions

SummaryIn this study we describe a process for immunopurification of FVIII/vWF complex directly from plasma. A mAb against vWF has been selected that is able to bind, under physiologic conditions, the FVIII/vWF complex and to release it in slightly alkaline conditions while preserving its activity.After investigating the influence of solid supports and of coupling methods on the recovery of active FVIII we produced an immunoadsorbent by immobilisation of the selected mAb onto a Sephacryl S-1000 support using a benzoquinone coupling method. With this immunoadsorbent we developed a purification process directly from plasma with an excellent recovery (50%) of both FVIII and vWF activities. The product obtained is very enriched (the FVIII: C specific activity is 20 IU/mg of protein) and is stable after lyophilization.

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