Studies on the purification and characterization of human factor VIII

Abstract

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.

The Subunit Structure of Normal and Hemophilic Factor VIII

“…Several investigators have shovn that highly purified factor VIII can be prepared by filtering factor VIII concentrates through columns of 2-4% agarose as the final purification step (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). As confirmed in the present studv, this latter purification step results in an approximate 10,000-fold increase in specific activity Nwith respect to normal plasnma.…”

“…This is probably due to the large size of the molecule which prevents its penetrating the usual 5% polyacrylamide gels (5,10,11). In the present study non-reduced factor VIII did not penetrate even the most loosely cross-linked acrylamide gels (3.36%).…”

“…Several invrestigators have shown that highly purified humian factor VIII (antihemophilic factor; AHF') can be eluted near the void volume when factor VIII concentrates are filtered through 2 or 4% agarose colulmins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). These studies suggested that factor VIII h.as a molecular weight of approximately 2 million, but aggregation as a possible explanation for this higlh mlolecular weight cannot be entirely excluded.…”

“…Recently it has been suggested that bovine factor VIII is likewise a glvcoprotein witlh a molecular weiglht in excess of one million and is composed of disulfide linlked subunits, the smallest of w1hiclh miay be about 85,000 in moleculalr weight (10). While some of the physicochemical properties of humain factor VIII have been determiined( (2)(3)(4)(8)(9)(10)(11), the study of its subunit structure has only been recently approached (5,(10)(11)(12). Particularly in light of the immunological evidence that hemophilic plasma contains nonfunctional factor VIII in quantities comparable to those of functional factor VIII in normal plasnma (13)(14)(15)(16)(17)(18), the characterization of the structures 1 Abbreviationis utscd ini this paper: AHF, antihemophilic factor; MWT, molecular weight; s25s, sedimentation coefficient; SDS, sodium dodecyl sulfate; v, partial specific voluime.…”

Studies of the human factor VIII/von Willebrand factor protein. III. Qualitative defects in von Willebrand's disease.

“…The human Factor VIII/von Willebrand Received for publication 28 January 1975 and in revised form 11 June 1975. 'Abbreviations used in this paper: F. VIII/vWF, Faccharacterized as a high molecular weight glycoprotein with a major subunit of 195,000-240,000 mol wt (1)(2)(3)(4)(5)(6). In severe von Willebrand's disease (vWD), the deficiency of procoagulant and vWF activity (as judged by the ristocetin assay) is associated with a marked deficiency or absence of the F.VIII/vWF protein (7)(8).…”