The Subunit Structure of Normal and Hemophilic Factor VIII

Abstract

A B S T R A C T Human factor VIII froml nornmals anid hemophiliacs was partially purified by ethaniol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4%Xc agarose or ion1 exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatographv step. Highly purified factor VIII was not dissociated by 6 -MI guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by P-mercaptoethanol and analyzed by sodium dodecyl sulfate polvacrylamide gel electrophoresis, a single subunit species with an estimiiated 19-5,000 molecular weight was found for both normcal an(d hemiiophilic factor VIII. By sedimiientation equilibrium analysis, the normal factor V-III subunit was homogeneous and had an estimated nmolecular wveight of 202,000. The subunit polypeptides from niormal or hemoplilic factor VIII contained carbohy\drate. Each was homogeneous by isoelectric focusing. Immilllunodiffusion of purified normal and hemoplhilic factor VIII against rabbit aintiserum to purified normiial liuman factor VIII slhowed a single line of precipitationi. V-erv low concenitr-ations of purified humiian thrombin initially increased the activitv of normal factor VIII about threefold and tlhen progressively destroyed activity by 3 h. Only-minimal activation occurred with hemiiophilic factor VIII. Both the activation and inactivation of normal alnd hemo-

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

“…The protein and procoagulant elution patterns of normal cryoprecipitate have been previously described (2)(3)(4)(5)(6). A low level of protein associated with the bulk of the procoagulant activity begins to elute at the void volume (Fig.…”

“…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).…”

Substructure of human von Willebrand factor.

“…SDSgel electrophoresis of reduced samples of purified vWf yields a single subunit of -240,000 mol wt (8,15,16). Recent characterization of the cyanogen bromide fragments of carboxymethylated vWf (17) provides additional strong evidence that the circulating forms ofvWfare polymers composed ofa single type ofsubunit.…”

Studies on human antihemophilic factor. Evidence for a covalently linked subunit structure.

“…Human antihemophilic factor (Factor VIII) eluted near the void volume when plasma or Factor VIII concentrates were chromatographed on 2-4% agarose in dilute, neutral buffers (1)(2)(3)(4)(5)(6)(7). The s25 .…”

“…value of 16.3 for Factor VIII in the potent dispersing reagent guanidine hydrochloride appeared to rule out a noncovalently linked subunit structure (4). Recent studies of purified Factor VIII showed that it is a glycoprotein with a molecular weight of about one million (6) and that it is composed of disulfide-bound subunits which contain carbohydrate (5)(6)(7)(8). Sodium dodecyl sulfate gel electrophoresis, sedimentation equilibrium ultracentrifugation, and isoelectric focusing experiments performed in our laboratory (7) indicated that these covalently linked subunits are in fact identical with respect to size and electrical charge.…”