ADAMTS-13 Activity and Antigen in Commercial Von Willebrand Factor Concentrates.
Authors: Scharrer Inge , Böhm Martina 
Published: April 2020

The plasma metalloprotease ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type I motif - 13) cleaves the Von Willebrand factor (VWF) at the peptide bond Tyr1605-Met1606 and thereby controls the hemostatic activity of the VWF. We tested three commercial VWF concentrates with respect to their ADAMTS-13 activity and antigen contents. ADAMTS-13 activity in the VWF concentrates was tested by measuring the capacity of the concentrates for auto-proteolysis both in the presence and in the absence of neutralising ADAMTS-13 auto-antibodies. To achieve this, the concentrates were reconstituted to a final VWF concentration of 100 I.E. U VWF:Ristocetin Cofactor activity (VWF:RCo). The VWF solutions were diluted with 5 mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples or Imidazole buffer as plasma replacement.The residual VWF:RCo was subsequently measured using the BC von Willebrand Reagent from Dade Behring (Marburg, Germany). The residual VWF:RCo in the presence of plasma from a patient with acquired TTP (ADAMTS-13 activity: &lt;6.25% due to high levels of neutralising ADAMTS-13 auto-antibodies) was then compared to the residual VWF:RCo in the mixtures containing normal plasma, Imidazole buffer or plasma from a patient with congenital TTP (ADAMTS-13 activity: &lt;6.25% due to mutations in the ADAMTS-13 gene). ADAMTS-13 antigen was measured by a commercial ADAMTS-13 ELISA from American Diagnostica (Stamfort, USA).

Residual VWF:RCo after proteolysis of VWF concentrates in the presence of Normal plasma, Imidazole buffer and plasma from patients with congenital and acquired TTP, respectively VWF concentrate Normal plasma Imidazole buffer Congenital TTP Acquired TTP A 7% 106% 111% 138% B 10% 11% 16% 104% C 10% 32% 36% 63% ADAMTS-13-Antigen in VWF concentrates given as ng ADAMTS-13 Antigen per I.E. Units VWF:RCo VWF concentrate ADAMTS-13 Antigen [ng/I.E. U VWF:RCo] A not detectable B 6.7 C 0.8

The loss in VWF:RCo in the samples containing either Imidazole buffer or congenital TTP plasma compared to the loss in the samples containing acquired TTP plasma hints at auto-proteolysis due to specific action of ADAMTS-13. All concentrates seem to contain some ADAMTS-13 activity, but ADAMTS-13 activity in the solutions of 100 I.E. U/ml VWF:RCo used here is always lower than the activity in normal plasma (which contain per definition 1U/ml ADAMTS-13 activity). The ratio of ADAMTS-13 activity/VWF:RCo is thus always lower than 1:100 in all the investigated concentrates. However, concentrate B seems to show the highest ADAMTS-13 activity as well as the largest amount of ADAMTS-13 antigen. Concentrate B is followed by concentrate C. Concentrate A only shows traces of ADAMTS-13 activity and non detectable levels of ADAMTS-13 antigen. The results demonstrate that concentrates B and C should not be used as VWF substrates in ADAMTS-13 activity assays. The ADAMTS-13 content in the various VWF concentrates may influence stability, recovery and half-lives of the concentrates.