Contributions of Asn2198, Met2199, and Phe2200 in the factor VIII C2 domain to cofactor activity, phospholipid-binding, and von Willebrand factor-binding

2003 ◽  
Vol 89 (05) ◽  
pp. 795-802 ◽  
Author(s):  
Deborah Lewis ◽  
Mary Pound ◽  
Thomas Ortel
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Hydrophobic Surface ◽  
C2 Domain ◽  
Mutant Proteins ◽  
Phospholipid Binding ◽  
Cofactor Activity ◽  
The Individual ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe crystal structure of the factor VIII C2 domain consists of a β-sandwich core from which β-hairpins and loops extend to form a hydrophobic surface. The hydrophobic surface includes M2199 and F2200 at the tip of the 1st β-hairpin. To determine the individual contributions of residues N2198, M2199, and F2200 to phospholipid and von Willebrand factor (vWF) binding properties of factor VIII, we prepared mutant proteins with single alanine substitutions. We found that single mutations at N2198 and M2199 had relatively little impact on cofactor activity, or phospholipid and vWF binding. However the F2200A mutant had slightly lower cofactor activity at subsaturating phospholipid concentrations. Competitive ELISAs suggested that F2200 plays a more important role in both phospholipid-binding and vWF-binding than N2198 and M2199. All mutant proteins were still recognized by a monoclonal antibody and two factor VIII inhibitors that neutralized cofactor activity and blocked factor VIII binding to phospholipids.Presented in part at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France, 6-12 July 2001, and the 43rd Annual Meeting of the American Society of Hematology, Orlando, Florida, 7-11 December 2001

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

scholarly journals Selective absence of large forms of factor VIII/von Willebrand factor in acquired von Willebrand's syndrome. Response to transfusion

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 600-606 ◽  
Author(s):  
D Meyer ◽  
D Frommel ◽  
MJ Larrieu ◽  
TS Zimmerman
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Procoagulant Activity ◽  
Factor Viii Related Antigen ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor ◽  
Ristocetin Cofactor Activity

Abstract A previously healthy elderly man with mucocutaneous bleeding was found to have a benign monoclonal IgG gammapathy associated with criteria for severe von Willebrand disease (Factor VIII procoagulant activity, Factor-VIII-related antigen, and ristocetin cofactor activity, less than 10% of normal). Associated qualitative abnormalities of factor VIII/von Willebrand factor were demonstrated by radiocrossed immunoelectrophoresis and immunoradiometric assay. The late clinical onset and negative family history are in favor of an acquired form of vWD. The monoclonal gammapathy and abnormalities of factor VIII/von Willebrand factor have been stable over a 10-yr period. No inhibitor to Factor VIII procoagulant activity, ristocetin cofactor activity, or Factor-VIII-related antigen could be demonstrated. Following transfusion of cryoprecipitate (with a normal cross immunoelectrophoretic pattern), there was a rapid removal of the large forms of Factor.-VIII-related antigen, paralleled by a decay of ristocetin cofactor activity. The transfusion study of this patient with acquired von Willebrand disease type II (variant of von Willebrand disease) serves to emphasize the relationship between polydispersity of Factor VIII/von Willebrand Factor and functional heterogeneity.

Common inhibitory effects of human anti-C2 domain inhibitor alloantibodies on factor VIII binding to von Willebrand factor

1995 ◽  
Vol 91 (3) ◽  
pp. 714-721 ◽  
Author(s):  
Midori Shima ◽  
Hiroaki Nakai ◽  
Dorothea Scandella ◽  
Ichiro Tanaka ◽  
Yoshikatu Sawamoto ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
C2 Domain ◽  
Inhibitory Effects ◽  
Von Willebrand ◽  
Willebrand Factor

A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor

Blood ◽  
2000 ◽  
Vol 95 (1) ◽  
pp. 156-163 ◽  
Author(s):  
Marc Jacquemin ◽  
Abdellah Benhida ◽  
Kathelijne Peerlinck ◽  
Benoı̂t Desqueper ◽  
Luc Vander Elst ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Rare Complication ◽  
Cell Epitope ◽  
Human Antibody ◽  
Mutation Site ◽  
C1 Domain ◽  
Cofactor Activity ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The occurrence of factor VIII (fVIII) inhibitory antibodies is a rare complication of fVIII substitution therapy in mild/moderate hemophilia A patients. fVIII mutations in certain regions such as the C1 domain are, however, more frequently associated with inhibitor, for reasons which remain unclear. To determine whether inhibitors could map to the mutation site, we analyzed at the clonal level the immune response of such a patient with an inhibitor to wild-type but not self-fVIII and an Arg2150His substitution in the C1 domain. Immortalization of the patient B lymphocytes provided a cell line producing an anti-fVIII IgG4κ antibody, LE2E9, that inhibited fVIII cofactor activity, following type 2 kinetics and prevented fVIII binding to von Willebrand factor. Epitope mapping with recombinant fVIII fragments indicated that LE2E9 recognized the fVIII C1 domain, but not the Arg2150His-substituted C1 domain. Accordingly, LE2E9 did not inhibit Arg2150His fVIII activity. These observations identify C1 as a novel target for fVIII inhibitors and demonstrate that Arg2150His substitution alters a B-cell epitope in the C1 domain, which may contribute to the higher inhibitor incidence in patients carrying such substitution. (Blood. 2000; 95:156-163)

Factor VIII’s C Domains Bind to Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1715-1715
Author(s):  
Ting-Chang Hsu ◽  
Kathleen P. Pratt ◽  
Arthur R. Thompson
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Factor Viii ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
C2 Domain ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The C domains of factor VIII contain the primary binding site for the cofactor, activated factor VIII, to interact with the phospholipid membranes, including those on the platelet surface. Isolated C2 domain has been shown to bind to phosphotidyl-L-serine-rich lipids and platelets; under flow cytometry, binding to activated platelets was confirmed. For comparison, C1C2, expressed in E.coli, was prepared with up to mg quantities isolated. Fresh, gel-filtered platelets were then studied in a flow cytometer either with or without activation by the thrombin receptor peptide, SFLLRN-amide. Depending upon the conditions, up to 80% of the platelets could be stained with a monoclonal antibody to C2 (ESH8) that is known not to compete with lipid or von Willebrand factor binding. The results were confirmed using a S2296C mutant C1C2 where the free suflhydryl group was either biotinylated and detected by fluorescein labeled streptavidin or directly labeled with fluorescein. As shown in the figure, essentially all platelets bound directly fluorescein labeled C1C2. Using standardized, labeled microbeads, it was estimated that there are 7000–10,000 binding sites per platelet. After platelet activation, the number of platelets binding C1C2 increased with all three detecting systems but only by 15–30%. In contrast, binding of isolated C2, as determined either by ESH8 or as a C2296 biotinylated species, was much lower when the same molar amounts were added, and was primarily detectable following platelet activation. C1C2 binding appeared independent of von Willebrand factor as platelets from two unrelated subjects with severe, type 3 von Willebrand disease gave the same patterns on flow cytometry as seen in platelets from normal subjects. ESH4, a monoclonal antibody known to inhibit binding of C2 to lipid membranes effectively competed C1C2 binding to platelets. Although an indirect alteration the C2 domain conformation cannot be excluded, results support a direct role of C1 in enhancing platelet binding. Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets

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

Blood ◽  
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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