Measuring the role for Met endosomal signaling in tumorigenesis.

Met is a receptor tyrosine kinase, often overexpressed or mutated in human cancer. Upon activation by its ligand, the hepatocyte growth factor, Met controls several cell functions such as proliferation, migration, and survival through the activation of multiple pathways. Upon ligand binding, Met rapidly internalizes and continues to signal from endosomal compartments prior to its degradation.

A major determinant of the immunogenicity of factor VIII in a murine model is independent of its procoagulant function.

A main complication of treatment of patients with hemophilia A is the development of anti-factor VIII (fVIII) antibodies. The immunogenicity of fVIII potentially is a function of its procoagulant activity, which may result in danger signals that drive the immune response. Alternatively, intrinsic structural elements in fVIII may be particularly immunogenic.

A direct role for Met endocytosis in tumorigenesis.

Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration.

The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice.

Inhibitory antibodies to factor VIII (FVIII inhibitors) are the most significant complication in the management of haemophilia A. The immunogenicity of FVIII may be driven in part by structural determinants within the FVIII molecule itself. Regions of nonidentity between human and porcine FVIII possibly could drive differential immune responses.

Nonclassical anti-C2 domain antibodies are present in patients with factor VIII inhibitors.

The antihuman factor VIII (fVIII) C2 domain immune response in hemophilia A mice consists of antibodies that can be divided into 5 groups of structural epitopes and 2 groups of functional epitopes. Groups A, AB, and B consist of classical C2 antibodies that inhibit the binding of fVIII to phospholipid and von Willebrand factor. Groups BC and C contain nonclassical C2 antibodies that block the activation of fVIII by thrombin or factor Xa.

Antihuman factor VIII C2 domain antibodies in hemophilia A mice recognize a functionally complex continuous spectrum of epitopes dominated by inhibitors of factor VIII activation.

The diversity of factor VIII (fVIII) C2 domain antibody epitopes was investigated by competition enzyme-linked immunosorbent assay (ELISA) using a panel of 56 antibodies. The overlap patterns produced 5 groups of monoclonal antibodies (MAbs), designated A, AB, B, BC, and C, and yielded a set of 18 distinct epitopes. Group-specific loss of antigenicity was associated with mutations at the Met2199/Phe2200 phospholipid binding beta-hairpin (group AB MAbs) and at Lys2227 (group BC MAbs), which allowed orientation of the epitope structure as a continuum that covers one face of the C2 beta-sandwich.

Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope.

Approximately 25% of patients with hemophilia A develop inhibitory antibodies after treatment with factor VIII. Most of the inhibitory activity is directed against epitopes in the A2 and C2 domains. Anti-A2 inhibitory antibodies recognize a 25-residue segment bounded by R484-I508.

Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion.

Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII.

Expression and characterization of recombinant murine factor VIII.

Hemophilia A is the inherited bleeding disorder that results from mutation of blood coagulation factor VIII (fVIII). Described here is the generation of a regulated expression system producing recombinant murine fVIII. Murine B-domainless fVIII was expressed at a peak level of 4 units/106 cells/24 h in serum-free media.

High level expression of recombinant porcine coagulation factor VIII.

Recombinant human factor VIII expression levels, in vitro and in vivo, are significantly lower than levels obtained for other recombinant coagulation proteins. Here we describe the generation, high level expression and characterization of a recombinant B-domain-deleted porcine factor VIII molecule. Recombinant B-domain-deleted porcine factor VIII expression levels are 10- to 14-fold greater than recombinant B-domain-deleted human factor VIII levels by transient and stable expression in multiple cell lines.