Characterization of Low Mololecular Weight Factor VIII.

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.

Factor VIII Is A Calcium-Binding Protein

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.

LOW-MOLECULAR-WEIGHT FACTOR VIII

The Lancet ◽  
1973 ◽  
Vol 301 (7810) ◽  
pp. 1000-1001 ◽  
Author(s):  
A.L. Bloom ◽  
J.C. Giddings ◽  
I.R. Peake ◽  
HarveyJ. Weiss ◽  
B.N. Bouma ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Low Molecular Weight ◽  
Weight Factor

scholarly journals Rabbit factor VIII: identification of size heterogeneity

Blood ◽  
1977 ◽  
Vol 49 (2) ◽  
pp. 209-217
Author(s):  
ME Rick ◽  
DE Wampler ◽  
LW Hoyer
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Weight Factor ◽  
Rabbit Plasma ◽  
Factor Viii Activity ◽  
Size Heterogeneity ◽  
Two Peaks

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.

LOW-MOLECULAR-WEIGHT FACTOR VIII

The Lancet ◽  
1973 ◽  
Vol 301 (7807) ◽  
pp. 827-828 ◽  
Author(s):  
F. Nouk-Eldin ◽  
MariaBenedetta Donati ◽  
Giovanni De Gaetano ◽  
Jozef Vermylen
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Low Molecular Weight ◽  
Weight Factor

LOW-MOLECULAR-WEIGHT FACTOR VIII

The Lancet ◽  
1973 ◽  
Vol 301 (7804) ◽  
pp. 661-662 ◽  
Author(s):  
A.L. Bloom ◽  
J.C. Giddings ◽  
I.R. Peake
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Low Molecular Weight ◽  
Weight Factor

Visualization of low molecular weight factor VIII purified in the presence of benzamidine

1980 ◽  
Vol 17 (3-4) ◽  
pp. 337-346 ◽  
Author(s):  
E.P. Kang ◽  
W.H. Cruickshank ◽  
G. Rock
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Low Molecular Weight ◽  
Weight Factor

scholarly journals Rabbit factor VIII: identification of size heterogeneity

Blood ◽  
1977 ◽  
Vol 49 (2) ◽  
pp. 209-217 ◽  
Author(s):  
ME Rick ◽  
DE Wampler ◽  
LW Hoyer
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Weight Factor ◽  
Rabbit Plasma ◽  
Factor Viii Activity ◽  
Size Heterogeneity ◽  
Two Peaks

Abstract 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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