Regulation of High Shear Rate Induced von Willebrand Factor Mediated Platelet Thrombi by ADAMTS-13 .
Authors: Donadelli Roberta , Orje Jennifer N. , Galbusera Miriam , Remuzzi Giuseppe , Ruggeri Zaverio M. 
Published: April 2020

Abstract
ADAMTS-13 is a metalloproteinase that cleaves von Willebrand factor (VWF) at the peptide bond Tyr842-Met843 within the A2 domain of the mature subunit, thus contributing to the regulation of multimers size in the circulation. Cleavage is effective on newly released VWF bound to the surface of endothelial cells, but the extent to which the protease acts on circulating VWF or limits the adhesive properties of multimers during thrombus formation remains unclear. To begin to address these questions, we have established a real-time videomicroscopy technique to visualize the formation of platelet aggregates mediated by the binding of VWF A1 domain to the platelet membrane glycoprotein (GP) Ibα. In this process, single platelets first adhere to surface-immobilized VWF, and then bind soluble VWF from plasma and aggregate through a mechanism dependent on shear rate above a critical threshold. Platelet aggregates formed promptly when whole human blood containing the thrombin inhibitor D-phenyl alanyl-L-prolyl-L-arginine chloromethyl ketone dihydrochloride as an anticoagulant (80 μM) was perfused over immobilized VWF at wall shear rates above 10,000 s−1. The size of aggregates increased during the first 5 min of perfusion, then started to decrease and was less than 30% of maximum after 10 min of perfusion. When the metal ion chelator, EDTA (5mM), was added to the blood before perfusion, the size of aggregates was larger than in control blood and showed no decrease over a 10 min period. A similar result was obtained when plasma was removed and washed blood cells were suspended in a buffer containing 20 μg/ml purified VWF multimers, suggesting that a metal ion dependent plasma protease was responsible for the time-dependent reduction of platelet aggregate size. To evaluate this hypothesis, recombinant human ADAMTS-13 purified from the culture medium of stably transfected D. melanogaster cells was added to washed blood cells resuspended in buffer/VWF, and the suspension was perfused over immobilized VWF at shear rates above 10,000 s−1. In this case, VWF-mediated thrombi started to form but began to dissipate within 3 min. After 6 min, the size of thrombi was 30% or less of that seen in the absence of ADAMTS-13. When the latter was added to washed blood cells resuspended in buffer/VWF after 5 min of perfusion, when platelet aggregates had reached their maximum dimensions, a reduction in size to 30% or less of maximum occurred within 5 min. Pre-incubation of ADAMTS-13 with the blood cell/VWF suspension before perfusion did not accelerate or enhance the reduction of platelet aggregate size, indicating that the enzyme likely acts only under flow conditions and/or after VWF multimers are bound to platelet GP Ibα and exposed to shear stress. ADAMTS-13 also had no significant effect on the size of platelet thrombi formed onto collagen type I fibrils at shear rates as high as 6,000 s−1. These findings suggest that ADAMTS-13 provides a selective mechanism to regulate the size of platelet thrombi, but the effect may be limited to conditions under which the cohesion between platelets depends mainly on VWF binding induced by pathologically elevated shear stress.