1425 Introduction: 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. Methods: 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). Results: 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. Conclusion: 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. Disclosures: Schwartz: Octapharma: Employment. Fuchs:Octapharma: Employment. Kannicht:Octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment.
4354 Objective: The characteristic multimeric pattern of plasmatic VWF results from asymmetric cleavage by ADAMTS13. Regulation of VWF multimer distribution is critical for its physiological function. In human plasma, VWF multimer gels reveal species of various multimeric sizes with flanking satellite bands (triplets). The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Defects in VWF secretion, impaired assembly of multimers, or increased proteolysis can cause von Willebrand Disease (VWD). Distribution of VWF triplet bands is significantly altered in some plasma-derived VWF concentrates. The impact of triplet structure on VWF function has not been investigated so far. Methods: Four commercially available VWF concentrates were analyzed for ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by fluorescence resonance energy transfer (FRET) assay. Samples composed of different VWF triplet distribution but comparable multimers were obtained by heparin affinity chromatography. Platelet adhesion under flow was determined using a flow-chamber model. Results: VWF concentrates markedly differed with respect to their content of ADAMTS13 antigen and activity. A higher content of ADAMTS13 correlated with an altered triplet structure reflected by an increased presence of the faster migrating triplet band, indicating VWF proteolysis. VWF-mediated platelet adhesion under flow over time was increased using a VWF fraction predominantly containing the slower migrating triplet band. Conclusion: These findings suggest that an intact triplet structure has an impact on platelet adhesion at physiological high arterial shear rate conditions. The relevance of VWF N-terminal domains for platelet binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated. Disclosures: Fuchs: octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment. Kannicht:Octapharma: Employment. Schwartz:Octapharma: Employment.
3356 Objective: Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer respectively have one shorter or longer N-terminal peptide sequence. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Distribution of VWF triplet bands is significantly altered in some types of VWD, however, the impact of triplet structure on VWF function has not been investigated so far. Methods: Platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition obtained by size exclusion in addition to heparin affinity chromatography were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, while preparation B predominantly contained larger triplet bands. Collagen- and GPIb-binding was determined by surface plasmon resonance (SPR). Platelet adhesion under flow was determined using flow-chamber models. Results: Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb, but an unchanged affinity for collagen type III, when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Conclusion: Thus, VWF forms lacking larger size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow. Disclosures: Schwartz: Octapharma: Employment. Kannicht:Octapharma: Employment. Fuchs:octapharma: Employment. Kröning:octapharma: Employment. Solecka:Octapharma: Employment.
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