Introductionvon Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays an essential role in tethering platelets at the site of vascular injury. VWF is synthesized in endothelial cells, where a portion is stored in granules called Weibel-Palade bodies as "unusually large" or "ultralarge" multimers (ULVWF) and secreted upon endothelial stimulation. 1,2 Secreted ULVWF multimers bind platelets with relatively high affinity and are thought to be prothrombotic. ULVWF is cleaved into smaller and less dangerous multimers by the metalloprotease ADAMTS13, a member of the A Disintegrin And Metalloprotease with ThromboSpondin type I repeat family. [3][4][5] Inherited or acquired deficiency of ADAMTS13 causes life-threatening microvascular thrombosis that is characteristic of thrombotic thrombocytopenic purpura. 4,6,7 Conversely, mutations in von Willebrand disease type 2A cause bleeding by increasing the cleavage of VWF by ADAMTS13 and impairing platelet adhesion. [8][9][10] Therefore, normal hemostasis depends on the precise regulation of VWF proteolysis.ADAMTS13 cleaves the Tyr 1605 -Met 1606 bond in the A2 domain of VWF, but this bond is buried and relatively inaccessible until the A2 domain is unfolded, presumably by tensile force in vivo. 10,11 The shear stress required to apply this force will vary depending on whether VWF is immobilized at the vessel wall or moving with the flowing blood, and whether platelets are bound to it. The rate of VWF cleavage also can be modulated by cofactors that bind to the A1 domain, including platelet GPIb and heparin. 12 Thus, ADAMTS13 is presented with VWF multimers in plasma or on endothelial cell surfaces that vary in their susceptibility to cleavage, with or without attached platelets.The relevance of each of these potential substrates to the catabolism of VWF is unknown. Several studies suggest that VWF strings on endothelial cells must be cleaved to inhibit thrombus growth, 13,14 but the role of proteolysis in the fluid phase has not been established. Therefore, the cleavage of VWF by ADAMTS13 was assessed in a cone-plate viscometer to minimize the contribution of surface interactions. The results indicate that proteolysis of fluid phase VWF-platelet complexes is likely to determine the steady state size distribution of circulating VWF multimers in vivo.
Methods
Recombinant ADAMTS13Full-length human ADAMTS13 with a C-terminal V5 tag was expressed in TRex 293 cells (Invitrogen) as described previously 15 and partially purified by anion exchange chromatography. In brief, conditioned medium containing recombinant ADAMTS13 was supplemented with proteinase inhibitors (0.1 mol/L D-Phe-Pro-Arg-chloromethane and 144 mol/L phenylmethylsulfonyl fluoride and applied to tandem columns of HiTrap Q Sepharose (2 ϫ 5 mL; GE Healthcare, Chalfont St Giles, United Kingdom). The columns were washed with 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, and developed with a linear gradient of 0 to 50 mM CaCl 2 in 20 mM Tris-HCl, pH 8.0, and 100 mM NaCl. Fractions containing ADAMTS13 were combined,...