A human factor VIIa concentrate and its effects in the hemophilic A dog.

Properties of a new concentrate of FVII/FVIIa, obtained by adsorption onto an inorganic adsorbent followed by a chromatographic step onto Q-Sepharose using a by-product of routine fractionation as starting material, are described. This fraction contains only small amounts of the other components of the prothrombin complex but it is enriched in Proteins C and S. This preparation is essentially free of FVIIICag. It has been submitted to animal experiments including those carried out on normal and hemophilic A dogs. A normalization of the buccal bleeding time was seen after injection of a minimal dose of 4 uFVIIa/kg. No adverse reaction was observed at any dose employed. This concentrate might thus be indicated for the treatment of Haemophilia A patients with inhibitors. Its viral inactivation has been achieved.

[Purification of human fibronectin by precipitation and immunoadsorption]. / Purification de la fibronectine humaine par précipitation et immunoadsorption.

Two types of human plasma fibronectin concentrate were obtained by simple, large scale, precipitation methods. Concentrate 1 contains 90% pure fibronectin in a mainly dimeric form. This easy, high yield method provides a functional fibronectin suitable for intravenous therapeutic purposes in deficient patients. Concentrate 2 contains 95-98% pure fibronectin with minimal amounts of fibronectin monomer and degradation products. It seems well adapted to serum free cultures of human cells. A third preparation of an apparently pure fibronectin has also been obtained by immuno-adsorption with a Sepharose 4B column to which has been coupled an anti-fibronectin monoclonal antibody. The capture of dimer, monomers and degradation products by this antibody and their release at pH 2.4 has been demonstrated by gel transfer on nitrocellulose sheet revealed by another monoclonal anti-fibronectin antibody labelled with peroxidase.

Acquired haemophilia: functional study of antibodies to Factor VIII.

Factor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR:Ag levels were found in all patients. VIII:WF levels were 50% of those of VIIIR:Ag, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers. Antibody function has been characterized by kinetics of VIII:C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed: 1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (greater than 1). 2) Antibodies from other patients, usually with lower titres, inactivated VIII:C according to complex order kinetics, were not saturable, and had a less steep affinity slope (less than 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

Immunochemical characterization of antibodies to factor VIII in hemophilic and nonhemophilic patients.

Plasma samples from eighteen patients (9 hemophilic, 9 nonhemophilic) with antibodies to Factor VIII were purified by protein A gel chromatography. This procedure separates the IgG3 from the IgG1, IgG2, and IgG4 subclasses. The partially purified anti--Factor VIII fractions were typed for their heavy and light chain composition by immunoneutralization assay. Residual anti--Factor VIII activity was revealed by inhibition of fibrin formation in agarose gel plates. The combination of these two techniques permitted the study of a relatively large number of partially purified samples, at several dilutions of each, against each anti-heavy or anti-light chain antiserum obtained from various sources. A high degree of subclass and light chain restriction was found in the antibodies from the hemophilic patients, eight were predominantly of IgG4 kappa type, the ninth was IgG4 but of mixed light chains. In contrast, a heterogeneous distribution was found in the ten nonhemophilic patients: two IgG4 kappa, one IgG4 lambda, one IgG4 with both kappa and lambda, two a mixture of IgG1 and IgG4 with only kappa light chains, and three a mixture of IgG1 and IgG4 as well as both light chains.