Insights into thrombotic thrombocytopenic purpura by monoclonal antibody-based analysis of the Von Willebrand factor cleaving protease, ADAMTS-13

The von Willebrand factor cleaving protease (ADAMTS-13) hydrolyses a peptide bond in the plasma multimeric glycoprotein; von Willebrand factor (VWF). The latter plays an important role in haemostasis by mediating arrest of platelets onto subendothelial collagen. ADAMTS-13 deficiency can cause a rare but deadly disease, called thrombotic thrombocytopenic purpura (TTP). Mutations in the ADAMTS-13 gene cause TTP in an autosomal recessive inherited pattern, although this is extremely rare. More common is the acquired form of TTP in which autoantibodies inhibit the function of the protease. Autoantibodies can arise without a clear pre-clinical cause (idiopathic TTP) or can be secondary to several conditions or pathologies; amongst others pregnancy, HIV infection, allogeneic bone marrow transplantation and sepsis. During this PhD, monoclonal antibodies against ADAMTS-13 were generated in mice using genetic immunization and cell fusion technology. The immunoglobulins were fully characterized and the ones bearing the highest affinity were used to setup an immunosorbent assay. This binding assay was the first to accurately and reproducibly measure antigen titers in human plasma. With the assay we were able to show that acquired TTP patients mainly suffer from antigen depletion, in combination with functional inhibition or not. This is likely due to clearance of the antigen posterior to occupation with the autoimmunoglobulin molecules. Next, plasma levels from normal donors were compared with that from donors in a specific condition or disease. We found that the level of ADAMTS-13 decreases linearly with age in the group of normal donors. Although it is not yet established what consequences lowered ADAMTS-13 levels impose, our findings could contribute to the general observation that elderly are more prone to thrombotic events than youngsters. Our novel assay is of major importance to gain new insights, not only in the pathogenesis of TTP, but also in general cardiovascular disease (e.g. heart attacks). Also here VWF is involved and wherever VWF is, ADAMTS-13 might play a crucial role. Furthermore, this PhD also investigated the physiologic effect of a novel mutation in the ADAMTS-13 gene. The patient bearing this mutation and suffering from congenital TTP was found without a trace of the enzyme in the plasma. We could show that complete retention of the protein (in a heterologous cell line) was responsible for the latter observation.status: publishe