Purification of the Antihemophilic Factor by Gel Filtration on Agarose

Paulssen, M.M.P.; Wouterlood, A.C.M.G.B; Scheffers, H.L.M.A

doi:10.1055/s-0038-1651394

Cited by 5 publications

(2 citation statements)

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“…Since the development of large pore gel filtration for the separation of macromolecules, filtration on agarose has been widely used as a final step in the purification of factor VIII (8)(9)(10)(11)(12). In this step, factor VIII is recovered in or near the exclusion volume, suggesting a molecular weight greater than 2 X 106 (8,13,14).…”

Section: Introductionmentioning

confidence: 99%

Studies on the purification and characterization of human factor VIII

Marchesi¹,

Shulman²,

Gralnick³

1972

J. Clin. Invest.

112

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A B S T R A C T Factor VIII (antihemophilic globulin) has been prepared from Hyland method IV AHG and cryoprecipitate using limited chymotryptic digestion followed by Sepharose gel filtration. The activity of factor VIII is unaffected by the digestion procedure, while fibrinogen is converted to large noncoagulable fragments.The purified factor VIII has been found to be a macromolecular glycoprotein with a major subunit of 240,000, as shown by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis. Carbohydrate analysis of factor VIII gave values of 1% sialic acid, 2.8% hexosamine, and 1-2% hexose (mannose, galactose, and fucose). The lipid content was found to be less than 5% of the protein content, and included no detectable phospholipid. The amino acid content is also reported. Immunoelectrophoretic analysis using rabbit antibody to purified factor VIII produced a single precipitin line.The chymotrypsin digestion step facilitates the preparation of factor VIII by reducing the viscosity of fibrinogen in the crude starting material, thereby increasing fivefold the quantity of material which can be processed at one time. It also improves markedly the resolution between factor VIII and fibrinogen on gel filtration.

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Section: Introductionmentioning

confidence: 99%

Studies on the purification and characterization of human factor VIII

Marchesi¹,

Shulman²,

Gralnick³

1972

J. Clin. Invest.

112

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“…Several methods to obtain higher purified factor VIII concentrates are described in the literature [17,18,20], all of which have the dis advantage of a considerable loss of factor VIII during preparation. The question was raised whether a higher purified factor VIII concentrate could be prepared with a high recovery, solely by improvements of the cryoprecipitation technique.…”

Section: Introductionmentioning

confidence: 99%

Contributions to the Optimal Use of Human Blood

Vermeer¹,

Soute²,

Ates³

et al. 1976

Vox Sanguinis

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The influence of different variables on the yield of factor VIII in cryoprecipitate as prepared in the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, was studied. The following conclusions may be drawn: (1) In case blood should be stored, the use of the anticoagulant solution acid citrate dextrose (ACD) is preferable to citrate phosphate dextrose (CPD) or trisodium citrate (TSC). (2) The temperature of stored whole blood should not decrease below 8 °C because of a spontaneous precipitation of factor VIII from blood (and plasma) below this temperature. (3) Cryoprecipitate derived from rapidly frozen plasma (1 min) contains a decreased amount of proteins in comparison with cryoprecipitate prepared from slowly frozen plasma (45 min to 4 h). On the other hand, equal amounts of factor VIII activity were obtained in the precipitate after freezing of plasma at varying rates. (4) Rapid thawing of plasma results in both higher yields of factor VIII procoagulant activity and a higher specific activity of this factor in the resulting cryoprecipitate. (5) The sedimentation of cryoprecipitate is completed after 5 min centrifugation at 1,500 g. (6) At temperatures higher than 8 °C, cryoprecipitated factor VIII starts dissolving into the supernatant plasma or in buffer. (7) Factor VIII in lyophilized cryoprecipitate is stable at room temperature. At elevated temperatures it rapidly looses its activity. (8) Evidence was obtained that the improvements which are introduced in the preparation of factor VIII do not lead to a product which is less stable in vitro as well as in vivo.

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