IntroducationFactor VIII and von Willebrand factor are plasma glycoproteins whose deficiency or structural defects cause hemophilia A and von Willebrand disease, respectively (1). These diseases are the most common inherited bleeding disorders of man. Factor VIII and vWF are synthesized by different cell types and circulate in plasma as a tightly bound complex. Factor VIII is synthesized in the liver (2), and functions as a cofactor for activated factor IX in the intrinsic activation of factor X on a membrane surface (3). vWF is synthesized in endothelial cells (4, 5) and megakaryocytes (6). vWF has a dual role in hemostasis: it promotes platelet adhesion to subendothelium after vessel injury (7, 8) and it acts as a carrier protein of factor VIII (1).The distinction between factor VIII and vWF was unclear for many years, because severe Von Willebrand disease is associated with factor VIII deficiency and because early preparations of factor VIII concentrates contained vWF and were therefore effective in correcting the platelet adhesion defects in patients with von Willebrand disease (9). Since factor VIII and vWF form a tightly bound non-covalent complex in plasma, both proteins are copurified when isolated from plasma, unless special measures are taken (1). The stoichiometry of factor VIII and vWF in plasma is approximately 1:50 and factor VIII and monomeric vWF have similar molecular weights of approximately 240 kDa. Therefore, vWF represents 98% of the molecular mass of the factor VIII-vWF complex (10) and almost all the antibodies raised against the complex react to vWF. In the 1980’s, factor VIII and vWF have each been purified to homogeneity and the genes for these proteins have been cloned. This set the stage for studies with purified proteins which have elucidated structure-function relationships for both proteins. Also, the interaction between both proteins could be studied using proteolytic fragments, small peptides, and monoclonal antibodies. In the last few years, the construction of recombinant mutants and fragments of both factor VIII (11-13) and vWF (14-16) has proven to be a powerful tool in the elucidation of the structure and function of both proteins.Binding of factor VIII to vWF is essential for the survival of factor VIII in vivo (17, 18). The underlying mechanism is probably that factor VIII bound to vWF is protected from phospholipid dependent proteolysis by activated protein C and factor Xa (19, 20). The binding site for factor VIII has been located at the amino terminus of vWF (21, 22). A tryptic fragment containing this binding site was not sufficient to protect factor VIII against activated protein C-mediated degradation according to some groups (23, 24). In contrast, a recent study using comparable vWF fragments showed protection of factor VIII equivalent to mature vWF (16).In 1989, a new variant of von Willebrand disease was discerned (type Normandy or 2N), distinct from the more than 20 subtypes known, characterized by a mutant vWF that is structurally and functionally normal, except that it does not bind to and stabilize factor VIII (25, 26). Since then, several mutations in the factor VIII binding site on vWF have been found (27). A number of reports have shown that factor VIII binds vWF via a high affinity binding site on its light chain (28-30). Two recent studies suggest that this binding site consists of two separate binding sites (31, 32).This review summarizes current knowledge on the interaction between factor VIII and vWF. Emphasis will be laid on the biological importance of, and the domains involved in binding, and on the stoichiometry and kinetics of complex formation.

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid-phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.

TMPRSS6 variants that affect protein function result in impaired matriptase-2 function and consequently uninhibited hepcidin production, leading to iron refractory iron deficiency anemia (IRIDA). This disease is characterized by microcytic, hypochromic anemia and serum hepcidin values that are inappropriately high for body iron levels. Much is still unknown about its pathophysiology, genotype-phenotype correlation, and optimal clinical management. We describe 14 different TMPRSS6 variants, of which 9 are novel, in 21 phenotypically affected IRIDA patients from 20 families living in the Netherlands; 16 out of 21 patients were female. In 7 out of 21 cases DNA sequencing and multiplex ligation dependent probe amplification demonstrated only heterozygous TMPRSS6 variants. The age at presentation, disease severity, and response to iron supplementation were highly variable, even for patients and relatives with similar TMPRSS6 genotypes. Mono-allelic IRIDA patients had a milder phenotype with respect to hemoglobin and MCV and presented significantly later in life with anemia than bi-allelic patients. Transferrin saturation (TSAT)/hepcidin ratios were lower in IRIDA probands than in healthy relatives. Most patients required parenteral iron. Genotype alone was not predictive for the response to oral iron. We conclude that IRIDA is a genotypically and phenotypically heterogeneous disease. The high proportion of female patients and the discrepancy between phenotypes of probands and relatives with the same genotype, suggest a complex interplay between genetic and acquired factors in the pathogenesis of IRIDA. In the absence of inflammation, the TSAT/hepcidin ratio is a promising diagnostic tool, even after iron supplementation has been given.Am. J. Hematol. 91:E482-E490,

The binding of factor VIII to von Willebrand factor (vWF) is essential for the protection of factor VIII against proteolytic degradation in plasma. We have characterized the binding kinetics of human factor VIII with vWF using a centrifugation binding assay. Purified or plasma vWF was immobilized with a monoclonal antibody (MoAb RU1) covalently linked to Sepharose (Pharmacia LKB Biotechnology, Uppsala, Sweden). Factor VIII was incubated with vWF-RU1-Sepharose and unbound factor VIII was separated from bound factor VIII by centrifugation. The amount of bound factor VIII was determined from the decrease of factor VIII activity in the supernatant. Factor VIII binding to vWF-RU1-Sepharose conformed to the Langmuir model for independent binding sites with a Kd of 0.46 +/- 0.12 nmol/L, and a stoichiometry of 1.3 factor VIII molecules per vWF monomer at saturation, suggesting that each vWF subunit contains a binding site for factor VIII. Competition experiments were performed with a recombinant vWF (deltaA2-rvWF), lacking residues 730 to 910 which contain the epitope for MoAB RU1. DeltaA2-rvWF effectively displaced previously bound factor VIII, confirming that factor VIII binding to vWF-RU1-Sepharose was reversible. To determine the association rate constant (k(on)) and the dissociation rate constant (k(off)), factor VIII was incubated with vWF-RU1-Sepharose for various time intervals. The observed association kinetics conformed to a simple bimolecular association reaction with k(on) = 5.9 +/- 1.9 x 10(6) M(-1) s(-1) and k(off) = 1.6 +/- 1.2 x 10(-3) s(-1) (mean +/- SD). Similar values were obtained from the dissociation kinetics measured after dilution of preformed factor VIII-vWF-RU1-Sepharose complexes. Identical rate constants were obtained for factor VIII binding to vWF from normal pooled plasma and to vWF from plasma of patients with hemophilia A. The kinetic parameters in this report allow estimation of the time needed for complex formation in vivo in healthy individuals and in patients with hemophilia A, in which monoclonally purified or recombinant factor VIII associates with endogenous vWF. Using the plasma concentration of vWF (50 nmol/L in monomers) and the obtained values for K(on) and K(off), the time needed to bind 50% of factor VIII is approximately 2 seconds.

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