Studies on the purification of antihemophilic factor (factor VIII)

Kass, Lawrence; Ratnoff, Oscar D.; Leon, Myron A.

doi:10.1172/jci105991

Cited by 66 publications

(7 citation statements)

References 26 publications

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“…Taken together, these results imply that thrombin generates the FVIII procoagulant activity that is stabilized by 0.25 M CaCl2 and elutes aberrantly from 4% agarose in that solvent. INTRODUCTION The initial physicochemical characterization of a purified protein with Factor VIII (FVIII)l procoagulant activity appeared to establish that it is a glycoprotein with a mol wt> 1 million daltons and a covalent subunit structure (1)(2)(3)(4)(5)(6)(7)(8)(9). Subsequently it was shown that this glycoprotein also had von Willebrand factor (vWF) activity and it was suggested that the two activities (i.e.…”

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confidence: 99%

Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

Switzer¹,

McKee²

1977

J. Clin. Invest.

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A B S T R A C T When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl2, the protein and vWF activity appear in the void volume, but most ofthe FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl2. To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl2 on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl2. When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl2, the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl2, the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl2. When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIIIVvWF protein was filtered in 0.25 M CaCl2, the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h.

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confidence: 99%

Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

Switzer¹,

McKee²

1977

J. Clin. Invest.

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“…Support for the presence of an a-linked disaccharide unit in factor VIII can be provided by data showing that both human (19) and bovine (16) factor VIII are precipitated by concanavalin A. Since concanavalin A preferentially binds a-D-glucopyranosyl, a-Dmannopyranosyl, and 2-acetamido-2-deoxy-a-D-glucopyranosyl units (20), the presence of these units in factor VIII may be inferred.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of bovine factor viii‐induced guinea pig platelet aggregation by simple sugars and derivatives

Kuwahara

Malik

1977

American J Hematol

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Working with the hypothesis that the binding of carbohydrate groups is important in the induction of platelet aggregation by bovine factor VIII, we studied 36 different sugars and sugar derivatives for their ability t o inhibit the aggregation of guinea pig platelets. Concentrations that produced 5 0% inhibition of the aggregation response ranged from 232 mM (galactose) t o 12 mM (glucosamine and galactosamine).Using these values, we found that several factors affected the ability of sugar structures t o inhibit bovine factor VIII-induced aggregation of guinea pig platelets. In general, galactose and its derivatives were not as effective as glucose and its derivatives. Sugars possessing a-glycosidic linkages tended to be stronger inhibitors than those having &linkages. Polyols were better inhibitors than their corresponding aldoses. D-fucose (129 mM) was a weaker inhibitor than L-fucose (66 mM), but Dand L-arabinitol were not significantly different. N-acetylation of glucosamine reduced its ability t o inhibit aggregation. The number of free hydroxyl groups in a molecule did not appear t o be a significant determinant of its ability t o inhibit aggregation.The results support the hypothesis that a binding site for carbohydrate groups exists either on the platelet membrane or on the bovine factor VIII molecule. The binding of the carbohydrate t o this site is an important step in the induction of aggregation, and this binding site is relatively specific for the carbohydrate structures that it will accept.

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“…Purified preparations of AHF from three normal individuals and of the material produced by two patients with hemophilia were subjected to ultracentrifugation by applying 0.3 ml samples to the surface of 5-20%o sucrose gradients in cellulose nitrate cups (Beckman Instruments, Inc., Palo Alto, Calif.) as previously described (8 …”

Section: Methodsmentioning

confidence: 99%