Analysis of proteolytic von Willebrand factor (VWF) fragments in patients with VWF-related diseases

J. Irle1, K. Schwierczek1, H. Rossmann1, T. Falter1, C. von Auer1, A. Trinchero1, K. Lackner1, Z. Ruggeri2, B. Lämmle1, K. Jurk1 (1Mainz, Germany, 2La Jolla, CA, USA)

Vascular wall biology and disorders
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: VWF circulates in plasma as a series of multimers of Mr 500 kD up to 20´000 kD, consists of disulfide-linked subunits of 250 kD and physiologically contains small amounts of 140 kD and 176 kD fragments resulting from ADAMTS13-induced cleavage. Some forms of Von Willebrand disease (VWD) type 2A, i.e. subtype IIA of old classification, show increased VWF sensitivity to ADAMTS13 with enhanced proteolysis and loss of large multimers. Thrombotic thrombocytopenic purpura (TTP), caused by severe acquired or hereditary ADAMTS13 deficiency, displays hyperadhesive unusually large VWF multimers which cause microvascular platelet clumping. It is unclear whether some rescue proteases, such as plasmin, may at least partially regulate VWF multimeric size. We aimed to develop a method to analyze and quantitate intact and proteolytically cleaved VWF subunits and to characterize the fragments by immunoblotting.

Methods: Plasma-VWF was immunoadsorbed in the presence of a universal protease inhibitor to CNBr-activated Sepharose 4B-beads coupled with polyclonal rabbit anti-VWF IgG, reduced and alkylated and subjected to SDS-PAGE and immunoblotting using polyclonal HRP-conjugated anti-VWF antibodies. Quantification of intact VWF subunits and ADAMTS13 cleaved bands was achieved using dilutions of rhVWF, before and after ADAMTS13-induced proteolysis. In addition, we studied rhVWF cleavage by plasmin to characterize the generated fragments.

Results: We show that the efficacy of VWF immunoadsorption is independent of the degree of VWF cleavage by ADAMTS13. A plasma sample of an individual patient with VWD type 2A (subtype IIA) displayed increased concentrations of 140 and 176 kD bands reflecting enhanced ADAMTS13-mediated VWF cleavage. Preliminary data in a patient with acquired severely ADAMTS13-deficient TTP and another with hereditary TTP in remission (3-10% residual ADAMTS13 activity) unexpectedly showed similar 140 and 176 kD bands. Using monoclonal antibodies towards the N- and C-terminal domains of VWF no evidence for plasmin-induced cleavage fragments in the patient with hereditary TTP was obtained.

Conclusion: The technique for the analysis of in vivo proteolysis of VWF has been established. Further study of various patient groups with presumably deficient or enhanced ADAMTS13-induced VWF cleavage and the search for different fragments resulting from alternative protease-induced VWF proteolysis is ongoing.