Influence of high molecular weight factor VIII on the measurement of low molecular weight factor VIII procoagulant in different assay systems

Two forms of rabbit factor VIII procoagulant activity, distinguishable by size on gel filtration and ultracentrifugation, have been identified in normal rabbit plasma. These studies have been carried out with citrate-anticoagulated rabbit plasma obtained by cardiac puncture. Two peaks of factor VIII activity were obtained on agarose gel chromatography, using physiologic ionic strength buffers: a high molecular weight peak eluting at the void volume and a second peak eluting with smaller plasma proteins. The presence of high and low molecular weight factor VIII activities was confirmed by sucrose density gradient centrifugation. The two peaks of factor VIII activity remained distinct when proteolytic inhibitors were added to the plasma and eluting buffers. Both the high and low molecular weight factor VIII procoagulant activities were inhibited by antibodies to human and rabbit factor VIII, and both were activated by thrombin. The identification of size heterogeneity of factor VIII in normal rabbit plasma, in the absence of any modification by ionic strength, may permit more satisfactory study of the relationship of factor VIII size to function.

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Characterization of Low Mololecular Weight Factor VIII.

10.1055/s-0039-1687038 ◽

1979 ◽

Author(s):

G. Rock ◽

D. Palmer ◽

E. Kang ◽

G. Jamieson ◽

W. Cruickshank

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Polyacrylamide Gel Electrophoresis ◽

Structural Features ◽

Protein Characterization ◽

Low Molecular Weight ◽

Weight Factor ◽

High Molecular Weight Complex ◽

Peptide Map ◽

Polyethylene Glycol Precipitation

The high molecular weight complex of Factor VIII was isolated from resolubilized cryoprecipitate by polyethylene glycol precipitation followed by chromatography on Bio Gel A15M. Upon rechromatography of this compound in buffer containing 1M NaCl and 1mM benzamidine the low molecular weight sub-unit possessing the procoagulant activity eluted at a volume of 2.3V. SDS polyacrylamide gel electrophoresis of this material in 5% acrylamide gave a single band whose Rf indicated a molecular weight of 150,000. The isoelectric point was determined to be 7.4. A peptide map of pepsin digested, I125 labelled material showed very few peptides which were radioactive and/or fluorescamine positive; as well, there was a relatively large amount of radioactive, non-fluorescamine positive material which was slow moving on pH 2.1 electrophoresis and immobile on chromatography. Amino acid analysis yielded data consistent with the presence of a small amount of protein. Carbohydrate analysis indicated a large amount of neutral hexoses, a very small amount of hexosamines and no detectable sialic acid. These results suggest that the structural features of low molecular weight Factor VIII may account for its anomalous behaviour in standard protein characterization procedures.

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Conditions Affecting the Molecular Weight of Factor VIII.

10.1055/s-0039-1687039 ◽

1979 ◽

Author(s):

G. Rock ◽

E. Tackaberry ◽

D. Palmer

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

High Molecular Weight ◽

Calcium Concentration ◽

Low Molecular Weight ◽

Biochemical Purification ◽

Molecular Weights ◽

Will Self ◽

And Storage ◽

Molecular Weight Form

By purifying Factor VIII while maintaining physiological concentrations of calcium we have recently demonstrated that about 50% of the procoagulant activity is in a very low molecular weight (VLHW) form not associated with the carrier (VIII: RAG). The remainder is carrier associated and elutes at Vo as a high molecular weight (HMW) compound upon Sepharose 6B chromatography. Reduction of the calcium concentration by increasing the amount of citrate added to heparin results in decreasing the ratio of VLMW:HMW from 1:1 in pure heparin to 1:5 in pure citrate. If citrate is replaced with the more strongly chelating EDTA no VLMW is detectable in the plasma. It has also been found that most of the biochemical purification techniques which have been previously used to prepare Factor VIII for study actually result in the aggregation of this VLMW with the carrier to produce the high molecular weight form. This includes: cryoprecipitation, precipitation by polyethylene glycol and storage -80°C. As well, the VLMW material will self-associate upon freezing to produce an aggregate with a molecular weight of 106. However, this material does not cross-react with rabbit antibody directed against VIII: RAG. The data indicate that many of the previously reported biochemical characteristics, including molecular weights, actually describe species which are artifacts of the isolation process rather than those of the physiologically occuring Factor VIII.

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LOW-MOLECULAR-WEIGHT FACTOR VIII

The Lancet ◽

10.1016/s0140-6736(73)90626-0 ◽

1973 ◽

Vol 301 (7807) ◽

pp. 827-828 ◽

Cited By ~ 1

Author(s):

F. Nouk-Eldin ◽

MariaBenedetta Donati ◽

Giovanni De Gaetano ◽

Jozef Vermylen

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Low Molecular Weight ◽

Weight Factor

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LOW-MOLECULAR-WEIGHT FACTOR VIII

The Lancet ◽

10.1016/s0140-6736(73)92219-8 ◽

1973 ◽

Vol 301 (7804) ◽

pp. 661-662 ◽

Cited By ~ 25

Author(s):

A.L. Bloom ◽

J.C. Giddings ◽

I.R. Peake

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

Low Molecular Weight ◽

Weight Factor

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Factor VIII Is A Calcium-Binding Protein

10.1055/s-0038-1652970 ◽

1981 ◽

Author(s):

W H Cruickshank ◽

E S Tackaberry ◽

D S Palmer ◽

G A Rock

Keyword(s):

Molecular Weight ◽

Factor Viii ◽

High Molecular Weight ◽

Column Chromatography ◽

Calcium Binding ◽

Essential Role ◽

Polyacrylamide Gel Electrophoresis ◽

Basic Structure ◽

Low Molecular Weight ◽

High Molecular Weight Complex

The high molecular weight complex of Factor VIII is known to dissociate into two subunits when chromatographed in a high ionic strength solution containing either calcium or sodium salts. Calcium also has an essential role in maintaining Factor VIII activity during storage (AABB 1980). These two findings suggest ah integral involvement of calcium in the basic structure-function relationship of the Factor VIII molecule. Consequently, we have examined the binding of 45Ca to both the low molecular weight (LMW) procoagulant subunit of Factor VIII and the high molecular weight (HMW) complex using PAGE and column chromatography on Sepharose 4B. When the HMW complex of Factor VIII was isolated from cryoprecipitate by standard chromatographic procedures, incubated with 45CaCl2 and subjected to polyacrylamide gel electrophoresis, the resultant autoradiogram demonstrated that all of the 45Ca was associated with the HMW material which did not enter the gel. When this HMW Factor VIII was incubated with 45Ca and then dissociated by column chromatography in a buffer containing 0.25 M CaCl2, all of the 45Ca was associated with the material eluting in the 2.3 Vo region. This corresponds to the elution volume of the LMW, procoagulant subunit of Factor VIII. Dialysis against buffer was not effective in removing the 45Ca. The data indicate that calcium is tightly associated with the low molecular weight subunit of Factor VIII and presents further corroborative evidence that calcium has an essential role in determining both the chemical and physical properties of the Factor VIII molecule.

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Autosomal Factor VIII Deficiency in Rabbits: Size Variations of Rabbit Factor VIII.

10.1055/s-0039-1682596 ◽

1977 ◽

Author(s):

R.E. Benson ◽

W.J. Dodds

Keyword(s):

Molecular Weight ◽

New Zealand ◽

Factor Viii ◽

High Molecular Weight ◽

Gel Filtration ◽

Low Molecular Weight ◽

Antigenic Relationship ◽

Genetic Studies ◽

Coagulant Activity ◽

Autosomal Inheritance

Many rabbits from our Flemish Giant-Chinchilla colony have moderate to severely reduced levels of factor VIII coagulant activity (FVIII-C). Some have shown prolonged bleeding after venipunctures and gastrointestinal and intramuscular hemorrhages. Genetic studies indicate autosomal inheritance. Gel filtration of plasma from these rabbits by the method of Rick et al. (Blood, 49, 209, 1977) at 25°C, pH 6.8 revealed two distinct peaks of FVIII-C; the majority of activity eluting as high molecular weight (HMW) material at the void volume (V°) followed by a much smaller low molecular weight (LMW) peak eluting close to that of fibrinogen. By contrast, filtration of plasma from New Zealand (NZ) rabbits produced threefold greater protein at the V° and equal amounts of HMW and LMW FVIII-C. Increasing the pH to 7.4 had little effect on FVIII-C recovery, although filtration at 4°C virtually abolished the HMW FVIII-C peak of NZ plasma. Rat antiserum (AS) to rabbit HMW FVIII-C, absorbed with precipitate low in FVIII-C, detected precipitating antigen in both HMW and LMW fractions. After absorption with rabbit fibrinogen, the AS no longer detected HMW V° material. The antigenic relationship between HMW and LMW FVIII-C and fibrinogen thus remains unclear. The differences in amount of HMW protein and the ratio of HMW to LMW FVIII-C suggest that in comparison to NZ rabbits our animals have a variant factor VIII molecule as well as low FVIII-C.

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