Characterization of protein unable to bind von Willebrand factor in recombinant factor VIII products

SummaryRecombinant factor VIII SQ (r-VIII SQ), ReFacto®, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118.We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore® instrument. The apparent association rate constant was 4 × 106 M−1s−1. Two dissociation rate constants were found, 1 × 10−2s−1 and 4 × 10−4s−1. The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

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Characterization of factor VIII/von Willebrand factor concentrates using a modified method of von Willebrand factor multimer analysis

Haemophilia ◽

10.1046/j.1365-2516.1998.0040s3025.x ◽

1998 ◽

Vol 4 (S3) ◽

pp. 25-32 ◽

Cited By ~ 36

Author(s):

Metzner ◽

Hermentin ◽

Cuesta-Linker ◽

Langner ◽

MÜller ◽

...

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Modified Method ◽

Von Willebrand ◽

Willebrand Factor ◽

Von Willebrand Factor Multimer

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Factor VIII, a Cofactor for Accelerating Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease.

Blood ◽

10.1182/blood.v110.11.766.766 ◽

2007 ◽

Vol 110 (11) ◽

pp. 766-766

Author(s):

Wenjing Cao ◽

Sriram Krishnaswamy ◽

Peter Lenting ◽

X. Long Zheng

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Physiological Condition ◽

Proteolytic Cleavage ◽

Recombinant Factor Viii ◽

Inactivation Kinetics ◽

Maximal Effect ◽

Cofactor Activity ◽

Von Willebrand ◽

Willebrand Factor

Abstract von Willebrand factor (VWF)-cleaving protease (ADAMTS13), a 195-kDa plasma glycoprotein, preferentially cleaves “unusually large” VWF multimers and generates a range of VWF multimers with sizes from approximately 500,000 Daltons to 20 million Daltons. However, the molecular basis how ADAMTS13 prefers the larger VWF multimers to the smaller ones under physiological condition remains unknown. We hypothesize that coagulation factor VIII, which binds VWF in high affinity, may alter the susceptibility of VWF to ADAMTS13 protease. In the present study, a plasma-derived or recombinant VWF at final concentration of 150 nM was incubated for 3 min with 50 nM of recombinant ADAMTS13 in the absence and the presence of various concentrations of recombinant factor VIII (0–40 nM) in 50 mM HEPES, pH 7.5 containing 150 mM NaCl and 0.5 mg/ml bovine serum albumin under constant vortex-induced mechanic shear stress at rotation rate of 2,500 rpm. The specific cleavage products (the dimer of 176 kDa) were determined by Western blotting with rabbit anti-VWF IgG, followed by a fluorescence (IRDye800)-labeled anti-rabbit IgG and an infrared fluorescent image system. We showed that the rate of proteolytic cleavage of VWF by ADAMTS13 was markedly accelerated by an addition of recombinant factor VIII into the reaction. This augmenting effect was factor VIII concentration-dependent. The maximal augmentation in proteolytic cleavage of VWF by ADAMTS13 in the presence of 20 nM of recombinant factor VIII was approximately 10 fold over the baseline in the absence of factor VIII. The concentration of factor VIII that achieved half of the maximal effect on proteolytic cleavage of both plasma and recombinant VWF was nearly identical (approximately 3.0 nM). The B domain-deleted factor VIII appeared as efficacious as wild type factor VIII in accelerating the proteolytic cleavage of plasma-derived VWF by ADAMTS13, suggesting that the large, central B-domain of factor VIII is not required for its cofactor activity. Moreover, after being activated by thrombin, the activated factor VIII retained its cofactor activity initially, but rapidly lost its ability to enhance the proteolysis of VWF by ADAMTS13 within minutes, which was correlated with the dissociation (or inactivation) kinetics of the activated factor VIII heterotrimer. These data demonstrate that both factor VIII and activated factor VIII may be the cofactors that regulate ADAMTS13 protease function under physiological condition. The findings may provide a novel insight into how the larger VWF multimers are more susceptible to ADAMTS13 protease, likely because the larger VWF multimers are able to bind more factor VIII. The findings may also help design an optimal therapeutic regimen for treatment of thrombotic thrombocytopenic purpura.

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A new von Willebrand factor (vWF) defect in a patient with factor VIII (FVIII) deficiency but with normal levels and multimeric patterns of both plasma and platelet vWF. Characterization of abnormal vWF/FVIII interaction

Blood ◽

10.1182/blood.v75.1.20.20 ◽

1990 ◽

Vol 75 (1) ◽

pp. 20-26 ◽

Cited By ~ 101

Author(s):

C Mazurier ◽

J Dieval ◽

S Jorieux ◽

J Delobel ◽

M Goudemand

Keyword(s):

Factor Viii ◽

Von Willebrand Factor ◽

Laboratory Data ◽

Bleeding Diathesis ◽

Excessive Bleeding ◽

New Variant ◽

History Of ◽

Von Willebrand ◽

Willebrand Factor

Abstract The patients with inherited bleeding diathesis related to quantitative, structural, and/or functional abnormalities of von Willebrand factor (vWF) are said to have von Willebrand's disease (vWD). We report here the clinical and laboratory features of a 50-year-old woman with a life- long history of excessive bleeding. Her particular laboratory data are factor VIII (FVIII) deficiency, subnormal bleeding time, and the presence of all plasma and platelet vWF multimers in normal amounts. Infused with FVIII/vWF concentrate, she showed a persistent increase in FVIII that led us to discard hemophilia A carrier or “acquired hemophilia” diagnoses. vWF devoid of FVIII purified from normal and patient's plasma by immunoaffinity on anti-vWF monoclonal antibody (MoAb) was immobilized onto polystyrene tubes that were further incubated with purified normal FVIII. The bound FVIII was evidenced using radiolabeled anti-FVIII MoAb. The data showed that the patient's vWF, in contrast to vWF purified from normal plasma, was unable to bind FVIII. Furthermore, no inhibitor of FVIII/vWF interaction was evidenced in incubating purified normal vWF with the patient's plasma before the addition of FVIII and anti-FVIII MoAb. These results support the concept that the bleeding diathesis of this patient appears to be due mainly to her abnormal vWF preventing FVIII/vWF interaction. This abnormality, which is not yet described in present classification of vWD, could be considered as a new variant of vWD.

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