Towards the development of a recombinant von Willebrand factor-cleaving protease (ADAMTS-13)

Abstract number: OC118

Plaimauer^* B., Zimmermann^* K., Völkl^* D., Mohr^* G., Wernhart^* W., Antoine^* G., Schwarz^* H. P., Lämmle† B., Scheiflinger^* F.

^*Baxter BioScience, Austria; †Central Hematology Laboratory, Inselspital, Switzerland

The multimeric size of von Willebrand factor (VWF) is physiologically regulated primarily by a specific VWF-cleaving protease (VWF-cp) present in human plasma that cleaves the mature VWF subunit between tyrosine-842/methionine-843. Reduced concentration of VWF-cp is thought to be responsible for the maintenance and accumulation of newly secreted ultralarge (UL) VWF multimers in the circulation, which are hemostatically most effective and spontaneously bind to platelets. In the disorder thrombotic thrombocytopenic purpura (TTP) VWF-cp was found to be severely reduced (<5% of normal) resulting in widespread microvascular thrombosis and leading to microangiopathic hemolytic anemia and ischemic organ dysfunction [1]. An inherited, constitutional deficiency and an acquired idiopathic TTP mediated by IgG autoantibodies that inhibit VWF-cp activity have been described. TTP patients are currently treated by replacement therapy with fresh-frozen plasma and concurrent plasma exchange. These treatment regimens are inconvenient and associated with complications. Therefore it would be of great therapeutic value if a recombinant VWF-cp could be developed. Recently several independent groups cloned the gene encoding VWF-cp. Deduction of the protein sequence identified VWF-cp as a new member of the ADAMTS metalloprotease family and designated ADAMTS-13 (Zheng X., J. Biol. Chem. 276, 2001; Levy. GG, Nature, 413, 2001).

Objective  

To characterize the functionality of recombinant ADAMTS-13 (rADAMTS-13) expressed in a mammalian cell line.

Results  

We showed that rADAMTS-13 is recognized by plasma from patients with acquired TTP as well as by monoclonal antibodies directed against the catalytic domain of ADAMTS-13. The recombinant enzyme was found to degrade VWF multimers in a concentration-dependent manner. The specificity of the cleavage site was verified by N-terminal sequencing of the generated cleavage fragments. Purified IgG from TTP patient plasma with inhibiting antibodies completely prevented VWF cleavage. In addition, supplementing congenital deficient plasma with rADAMTS-13 restored the lacking VWF-cp activity dose-dependently and normalized the VWF-processing pattern. New data related to the development of a stably rADAMTS-13 producing cell line will be presented.

Conclusions  

These data provide convincing evidence that ADAMTS-13 is the enzyme responsible for the functional activity of degrading VWF multimers (Plaimauer B, Blood 100, 2002). A recombinant VWF-cp can now be developed as a candidate therapeutic protein for an improved treatment strategy for TTP patients especially as a treatment option for hereditary familial TTP.

1  Furlan, M., Best. Pract. Res. Clin. Haematol. 2001; 14.