Relevance of Intact Von Willebrand Factor (VWF) Triplet Structure for VWF Function.

The characteristic multimer pattern of plasmatic von Willebrand factor (VWF) results from asymmetric cleavage by the processing metalloprotease ADAMTS13 between Y1605/M1606 within the VWF A2 domain. In normal plasma, characteristic species of various multimeric sizes with flanking satellite bands (triplets) encircling the major band on VWF multimer gels are present. The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Even though the distribution of VWF satellite bands is significantly altered in some types of von Willebrand disease (VWD) and several commercial VWF concentrates, the impact of triplet structure on VWF function has not been investigated so far.

Four commercially available VWF concentrates were analyzed with respect to ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by the fluorescence resonance energy transfer (FRET) assay. VWF zymogram gels were used to test for ADAMTS13 activity. Samples composed of different VWF triplet distribution but comparable VWF multimers were obtained by fractionation of plasmatic VWF using heparin affinity chromatography. VWF affinity to collagen was measured by surface plasmon resonance (SPR).

VWF concentrates markedly differed in their content of ADAMTS13 antigen and activity. A higher ADAMTS13 content correlated with an increased portion of the proteolyzed faster migrating VWF triplet band. The degree of VWF proteolysis, i.e. lack of an additional N-terminal fragment, correlated with a decreased collagen binding level measured by SPR.

Proteolytic cleavage of N-terminal domains of VWF resulting in a higher content of faster migrating satellite bands affects the function of VWF. The impact of VWF N-terminal domains on collagen binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated.

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