Abstract
ADAMTS13 is a plasma metalloprotease that is essential for the normal proteolytic processing of von Willebrand factor (VWF). Dysfunctional ADAMTS13 may lead to thrombotic thrombocytopenic purpura, as uncleaved and unusually large VWF multimers accumulate in the blood and cause intravascular platelet aggregation. Many studies indicate that proteolysis of multimeric VWF involves conformational changes in the VWF A2 domain that expose the Y1605-M1606 scissile bond and also allow substrate binding to multiple exosites on ADAMTS13. For example, VWF is resistant to proteolysis by ADAMTS13 unless the VWF is subjected to fluid shear stress, mild denaturation with guanidine or urea, or adsorption onto a surface. However, the functional interactions between shear stress, various ADAMTS13 binding sites and VWF cleavage are not understood. Therefore, we investigated the effect of fluid shear stress and ADAMTS13 structure on ADAMTS13-VWF binding and VWF cleavage. Upon mixing recombinant VWF (rVWF) and ADAMTS13 in a physiological buffer (50 mM HEPES, 5 mM CaCl2, 1 μM ZnCl2, 150 mM NaCl, pH 7.4), we found that immunoprecipitation with anti-VWF also pulled down substantial amounts of ADAMTS13. Although less striking, a similar result was obtained with purified plasma VWF. Therefore, ADAMTS13 can bind VWF without gaining access to the cleavage site in VWF domain A2. When fluid shear stress was applied for 2 min with a bench-top vortexer, ADAMTS13 binding increased 3-fold and VWF was also cleaved. Lowering the ionic strength markedly increased the rate of VWF cleavage but did not affect ADAMTS13 binding, which suggests that cleavage and binding depend on distinct VWF-ADAMTS13 interactions. Shear-induced binding was reversible slowly upon removal of unbound ADAMTS13 or rapidly by addition of SDS. ADAMTS13-VWF binding was stable for at least 24 h after cessation of shear stress, indicating that the structural change in VWF that promotes binding was not readily reversible. Using a catalytically inactive ADAMTS13 variant to simplify the analysis of binding assays, 30 nM ADAMTS13(E231Q) bound to 30 μg/ml rVWF (120 nM subunits) with a stoichiometry of 0.012 ± 0.004 under static conditions and 0.098 ± 0.023 after shearing (mean ± SD, n = 3, P = 0.019). With 120 nM ADAMTS13(E231Q) the stoichiometry increased to 0.086 ± 0.036 under static conditions and 0.469 ± 0.033 after shearing for 2 min. Recombinant ADAMTS13 truncated after TSP-1 repeat 8 (lacking the C-terminal CUB domains, delCUB), or truncated after the Spacer domain (consisting of domains MDTCS), did not bind rVWF under static conditions, implicating the CUB domains in binding to VWF. In contrast, full-length ADAMTS13, delCUB and MDTCS bound similarly to rVWF after shearing. In a previous study, delCUB and MDTCS did not cleave VWF subjected to fluid shear stress (Zhang et al, Blood2007; 110: 1887–1894). However, under the conditions employed in these experiments, MDTCS and delCUB displayed significant proteolytic activity, cleaving VWF at a rate comparable to that of full length ADAMTS13 when shear stress was applied over a time course of 0–160 sec. We conclude that ADAMTS13 CUB domains contribute to binding a few sites on multimeric VWF under static conditions, whereas ADAMTS13 MDTCS domains are sufficient to bind many sites in an altered conformation of VWF that is induced by fluid shear stress. Binding of ADAMTS13 to unsheared VWF multimers may facilitate the cleavage of VWF within a growing thrombus.
Biomechanical forces enhance directed migration and activation of bone marrow-derived dendritic cells
