High Shear Stress and Immobilization of Von Willebrand Factor Promotes the VWF-Cleaving Protease Activity as Assessed by the Cone and Plate(let) Analyzer.

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) that is released from endothelial cells as a large multimeric protein. However, the specific physiological conditions required for the function of this von Willebrand factor-cleaving protease (VWF-CP) are not yet established. In this study we determined the optimal conditions for the VWF-CP activity using the Cone and Plate(let) Analyzer (CPA). Proteolysis of a soluble recombinant VWF by a recombinant VWF-CP in the absence or the presence of BaCl₂ (for induction of VWF-CP) was performed under static and flow (2050 s⁻¹) conditions. The resulted fragments of the soluble VWF were immobilized on a polystyrene surface and non-adhering sites were blocked with 1% BSA. In parallel, polystyrene surface immobilized VWF was similarly treated by VWF-CP. The immobilized cleaved VWF fragments by the above protocols served as a substrate for citrated whole blood platelet adhesion under flow (2050 s⁻¹). Reduction of platelet adhesion (surface coverage, SC) in BaCl₂ treated compared to untrerated sample reflects the degree of VWF-CP activity.

Platelet adhesion (SC, %) to VWF proteolytic products generated under the indicated conditions:

Maximal VWF-CP activity as reflected by maximal reduction (85%) of platelet adhesion was observed when immobilized VWF was treated by VWF-CP under flow. Minimal effect of VWF-CP activity was observed when soluble VWF was treated under static condition (26% reduction). Intermediate effect was observed with soluble VWF under flow (41%) and with immobilized VWF under static condition (37%). It should be noted that in the case of immobilized VWF application of flow alone (without BaCl₂) was sufficient to induce a remarkable decrease of platelet adhesion (66%). In conclusion, both immobilization of VWF and high shear stress are important conditions for VWF-CP function, suggesting that stretching of immobilized VWF under flow exposes the VWF-cleavage site and thereby may serve as a control mechanism of platelet adhesion at the site of thrombus formation.