Abstract

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: <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: <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 concentrateNormal plasmaImidazole bufferCongenital TTPAcquired TTP
7% 106% 111% 138% 
10% 11% 16% 104% 
10% 32% 36% 63% 
VWF concentrateNormal plasmaImidazole bufferCongenital TTPAcquired TTP
7% 106% 111% 138% 
10% 11% 16% 104% 
10% 32% 36% 63% 

ADAMTS-13-Antigen in VWF concentrates given as ng ADAMTS-13 Antigen per I.E. Units VWF:RCo

VWF concentrateADAMTS-13 Antigen [ng/I.E. U VWF:RCo]
not detectable 
6.7 
0.8 
VWF concentrateADAMTS-13 Antigen [ng/I.E. U VWF:RCo]
not detectable 
6.7 
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.

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