The Effects of Injection of Human Factor VIII Antibody Into Rabbits

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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Intravascular Clotting After Endotoxin in Rabbits With Impaired Intrinsic Clotting Produced by a Factor VIII Antibody

Blood ◽

10.1182/blood.v42.4.523.523 ◽

1973 ◽

Vol 42 (4) ◽

pp. 523-534 ◽

Cited By ~ 4

Author(s):

S. M-C. Shen ◽

S. I. Rapaport ◽

D. I. Feinstein

Keyword(s):

Factor Viii ◽

Rabbit Model ◽

Saline Group ◽

Factor Vii ◽

Test Material ◽

Control Group ◽

Inert Material ◽

Factor V ◽

Mean Values ◽

Human Factor Viii

Abstract A rabbit model in which intrinsic clotting was selectively impaired by injection of a human factor VIII antibody was used to evaluate the mechanism of endotoxin-induced intravascular clotting in cortisone-treated rabbits. Three groups of animals were studied: a control group given factor VIII antibody followed by saline; a second control group given an inert material followed by endotoxin; and an experimental group given factor VIII antibody followed by endotoxin. The following parameters were measured: 125I-fibrinogen kinetics, fibrinogen levels, factor VIII, factor VII, factor V, WBC, platelets, and hematocrit. The kidneys were examined for deposition of fibrin. Mean values for factor VIII at the time of injection of the second test material and mean values for fibrinogen consumed in the 6 hr after the second injection were as follows: antibody-saline group, 8.5% and 11.0 mg/kg; control material-endotoxin group, 90% and 29.6 mg/kg; and antibody endotoxin group, 7.0% and 32.7 mg/kg. Factor V, factor VII, granulocytes, and platelets fell in both groups of animals given endotoxin. One animal in each group given endotoxin developed gross renal cortical necrosis. These data establish that selective impairment of the intrinsic clotting reactions does not reduce the amount of clotting induced by a single injection of endotoxin in the cortisone-treated rabbit.

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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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Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Blood ◽

10.1182/blood.v63.2.486.486 ◽

1984 ◽

Vol 63 (2) ◽

pp. 486-489 ◽

Cited By ~ 202

Author(s):

CA Fulcher ◽

JE Gardiner ◽

JH Griffin ◽

TS Zimmerman

Keyword(s):

Factor Viii ◽

Protein C ◽

Human Factor ◽

Activated Protein C ◽

Light Chains ◽

Factor V ◽

Human Factor Viii ◽

Thrombin Activation ◽

A Site ◽

Polypeptide Chains

Abstract Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79–80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79–80,000 (or 71–72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.

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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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Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650218 ◽

1996 ◽

Vol 75 (01) ◽

pp. 036-044 ◽

Cited By ~ 3

Author(s):

Thomas L Ortel ◽

Karen D Moore ◽

Mirella Ezban ◽

William H Kane

Keyword(s):

Factor Viii ◽

Heavy Chain ◽

Human Factor ◽

Secreted Protein ◽

Factor V ◽

Minimal Effect ◽

Repetitive Domain ◽

Human Factor Viii ◽

A1 Domain ◽

A2 Domain

SummaryFactor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.

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Immunological Evidence that Human Factor VIII is Composed of Two Linked Moieties

10.1055/s-0039-1680432 ◽

1977 ◽

Author(s):

J. Koutts ◽

J.-M. Lavergne ◽

D. Meyer

Keyword(s):

Factor Viii ◽

Human Factor ◽

Solid Phase ◽

Molecular Size ◽

Detectable Effect ◽

Prolonged Incubation ◽

Human Factor Viii ◽

Single Protein ◽

Antigen Antibody ◽

Ristocetin Cofactor

Whether the three measurable parameters of factor VIII (procoagulant activity, VIII:C; ristocetin cofactor activity, VIIIR:WF; and factor VIII related antigen, VIIIR:AG) reside on a single protein remains disputed. A solid phase immunoadsorption system, in which homologous antibodies to VIII:C arising in haemophiliacs were insolubilized onto Sepharose, was used to examine the action of such antibodies and the inter-relationship between VIII:C, VIIIR:WF and VIIIR:AG. Homologous antibodies were shown to bind specifically VIII:C and to induce a spontaneous separation of VIII:C from VIIIR:WF/VIIIR:AG. The bond between VIII:C and the homologous antibodies bound to Sepharose appeared to be very stable and could not be broken with the usual antigen-antibody dissociating agents. Following prolonged incubation with antibody-sepharose, concentrated VIIIR:WF/VIIIR:AG (20 u/ml), completely devoid of VIII:C and inhibitor-neutralizing activity, was obtained. The loss of VIII:C had no detectable effect on the molecular size, antigenicity or electrophoretic mobility of the original molecule. The concentrated VIIIR:WF/VIIIR:AG was used to absorb heterologous antisera raised against factor VIII. Specific heterologous antisera to VIII:C, no longer neutralizing VIIIR:WF nor precipitating with VIIIR:AG, were obtained. Immunization of rabbits with VIIIR:WF/VIIIR:AG resulted in antisera which potently neutralized VIIIR:WF and precipitated with VIIIR:AG but also weakly neutralized VIII:C. These antibodies, like 4 other heterologous antibodies to Factor VIII studied, did not neutralize VIII:C which had been dissociated from VIIIR:WF/VIIIR:AG.The results indicate that VIII:C and VIIIR:WF/VIIIR:AG are two different, but linked entities.

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THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

10.1055/s-0038-1643887 ◽

1987 ◽

Author(s):

Richard J Jenny ◽

Debra D Pittman ◽

John J Toole ◽

Ronald W Kriz ◽

Randal J Kaufman ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Untranslated Region ◽

Human Factor ◽

Leader Peptide ◽

Cdna Clones ◽

Factor V ◽

Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

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Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Blood ◽

10.1182/blood.v63.2.486.bloodjournal632486 ◽

1984 ◽

Vol 63 (2) ◽

pp. 486-489 ◽

Cited By ~ 1

Author(s):

CA Fulcher ◽

JE Gardiner ◽

JH Griffin ◽

TS Zimmerman

Keyword(s):

Factor Viii ◽

Protein C ◽

Human Factor ◽

Activated Protein C ◽

Light Chains ◽

Factor V ◽

Human Factor Viii ◽

Thrombin Activation ◽

A Site ◽

Polypeptide Chains

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79–80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79–80,000 (or 71–72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.

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The Development and Characterisation of Antibodies to Human Factor VIII in Haemophilic Dogs

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651124 ◽

1987 ◽

Vol 57 (03) ◽

pp. 314-321 ◽

Cited By ~ 10

Author(s):

Janet D Littlewood ◽

T W Barrowcliffe

Keyword(s):

Factor Viii ◽

Human Factor ◽

Kinetic Studies ◽

Cross Reaction ◽

High Responder ◽

Type Ii ◽

Human Factor Viii ◽

Factor Viii Concentrates ◽

Low Responder

SummaryFour haemophilic dogs received infusions of human factor VIII concentrates, and developed inhibitors to human F VIII. These inhibitors cross-reacted with canine F VIII with parallel increases and decreases in titre. Cross-reaction was also found to porcine F VIII but changes in titre did not correlate with anti-human and anti-canine titres. These inhibitors were found to be immunoglobulins, and antibodies were detected against other proteins found in concentrates. Kinetic studies showed that in all four dogs the F VIII inhibitors were Type II antibodies. One of the dogs behaved as a “high-responder”, whilst another was more analogous to a “low-responder” patient. Phospholipid protection experiments in vitro demonstrated that some antibodies could be prevented from inhibiting F VIII, and porcine F VIII was particularly well protected against inhibition.

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

Blood ◽

10.1182/blood.v57.3.476.476 ◽

1981 ◽

Vol 57 (3) ◽

pp. 476-482 ◽

Cited By ~ 37

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

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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