Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

von Willebrand factor propeptide in vascular disorders: A tool to distinguish between acute and chronic endothelial cell perturbation.

Before de novo synthesized von Willebrand factor (vWF) leaves the endothelial cell, it undergoes endoproteolytic cleavage of its propeptide (vW antigen II). The processed vWF and propeptide are either released constitutively or, following activation of the endothelium, released through the regulated pathway. In a recent study (Borchiellini et al, Blood 88:2951, 1996), we showed that the half-life of mature vWF and of its propeptide differ fourfold to fivefold. We postulated that the molar ratio of the propeptide to mature vWF could serve as a tool to assess the extent of endothelial cell activation under physiologic and clinical conditions. To test this hypothesis, we measured mature vWF and propeptide in patients with documented acute and chronic vascular disease, including patients with thrombotic thrombocytopenic purpura (TTP), acute septicemia, and diabetes mellitus. These data were compared with experimental conditions in healthy subjects in which perturbation of the endothelium was simulated by physical exercise or by administration of 1-deamino-8-D-arginine vasopressin (DDAVP) or endotoxin. In all individuals of the latter study group, both vWF and propeptide levels were elevated during the acute phase of the experimentally induced vascular perturbation; at later time points after stimulation, only vWF levels remained elevated. In patients with sepsis and TTP, both vWF and propeptide were elevated several-fold. Thus, this pattern can readily be explained in terms of acute perturbation of the endothelium. In contrast, in patients with diabetes mellitus propeptide levels were only slightly elevated, whereas vWF levels were elevated twofold to threefold. This pattern is a typical feature of chronic, low-grade activation of the endothelium. These observations support our hypothesis that measurement of both propeptide and vWF levels allows to discriminate between chronic and acute phases of endothelial cell activation in vivo. Measurement of only vWF is less indicative in this respect.

Quantitative analysis of von Willebrand factor and its propeptide in plasma in acquired von Willebrand syndrome.

Measurement of the von Willebrand factor (vWF) propeptide, also known as von Willebrand antigen II, has been suggested to be helpful in the discrimination of congenital von Willebrand disease type I from type 2 and in assessing the extent of activation of the endothelium. We performed a quantitative analysis of mature vWF and its propeptide in plasma in 8 patients with acquired von Willebrand syndrome (AvWS) and in 20 normal individuals. Mature vWF levels were significantly lower in AvWS as compared with normal individuals (13.4+/-3.5 vs 35.6+/-3.3 nM, p <0.001). In contrast, propeptide levels were significantly higher in AvWS (11.4+/-1.1 vs 4.7+/-0.2 nM, p <0.001), probably reflecting a compensatory increase in vWF synthesis or increased perturbation of the endothelium in AvWS. After treatment with DDAVP, propeptide and mature vWF levels rose 5-fold in AvWS, whereas propeptide levels were not altered by the infusion of a vWF concentrate or treatment with high dose intravenous immunoglobulins, indicating that plasma propeptide levels are a reliable reflection of vWF synthesis. Measurement of propeptide levels may provide additional information in AvWS as to whether decreased levels of mature vWF in the circulation are due to a decrease in synthesis or due to an accelerated removal of vWF from the circulation.