ADAMTS13 is a highly specific multidomain plasma metalloprotease that regulates the multimeric size and function of von Willebrand factor (VWF) through cleavage at a single site in the VWF A2 domain. The precise role that the ADAMTS13 disintegrin-like domain plays in its function remains uncertain. Truncated ADAMTS13 variants suggested the importance of the disintegrin-like domain for both enzyme activity and specificity. Targeted mutagenesis of nonconserved regions (among ADAMTS family members) in the disintegrin-like domain identified 3 of 8 ADAMTS13 mutants (R349A, L350G, V352G) with reduced proteolytic activity. Kinetic analyses revealed a 5-to 20-fold reduction in catalytic efficiency of VWF115 (VWF residues 1554-1668) proteolysis by these mutants. These residues form a predicted exposed exosite on the surface of the disintegrin-like domain that lies approximately 26 Å from the active site. Kinetic analysis of VWF115 carrying the D1614A mutation suggested that Arg349 in the ADAMTS13 disintegrin-like domain interacts directly with Asp1614 in VWF A2. We hypothesize that this interaction assists in positioning the scissile bond within the active site of ADAMTS13 and therefore plays a major role in determining cleavage parameters (K m and k cat ), as opposed to binding affinity (K d ) of ADAMTS13 for VWF, the latter being primarily determined by the spacer domain. IntroductionVon Willebrand factor (VWF) is a large multidomain plasma glycoprotein. It circulates as covalently associated multimers that range from 2 to 40 VWF units. 1 VWF has 2 hemostatic roles: (1) to mediate platelet tethering at sites of vascular injury, and (2) it acts as a carrier protein for factor VIII. 1,2 Normally, plasma VWF circulates in a globular form that does not readily interact with circulating platelets. However, after vessel damage, VWF binds subendothelial collagen via its A3 domain. 3,4 Once bound, the shear forces exerted by the flowing blood induce VWF unfolding that reveals further binding sites. 5 The exposed VWF A1 domain can now bind to the GPIb-IX-V receptor complex on the surface of circulating platelets. 6 This results in platelet tethering, and ultimately the formation of a primary platelet plug. Large VWF multimers are more hemostatically active than smaller forms. This is because they contain more platelet and collagen binding sites, and also because they unravel more readily in response to shear. VWF multimers are synthesized intracellularly by dimerization in the endoplasmic reticulum and then by multimerization in the Golgi apparatus. 7 After their secretion from endothelial cells, VWF multimers can be converted to smaller less adhesive forms by the plasma metalloprotease, ADAMTS13. [8][9][10][11][12][13] ADAMTS13 is expressed predominantly in the liver. 14 It has also been shown to be expressed in hepatic stellate cells, 15 in platelets, 16 by cultured endothelial cells, 17,18 and by glomerular podocytes. 19 It is secreted into the blood as an active enzyme 20 and circulates at a plasma concentration of approxi...
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