Abstract
The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.
Highlights
The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease, which cleaves VWF at the Tyr1605-Met1606 peptide bond
In the past few years, several studies have clearly shown the involvement of ultralarge VWF and ADAMTS-13 in the occurrence of the syndrome referred to as thrombotic thrombocytopenic purpura, which is characterized by thrombocytopenia, microangiopathic hemolytic anemia, and neurologic signs and other symptoms related to widespread formation of platelet thrombi in the microcirculation
Ultra-large VWFs are often present in the plasma of patients with thrombotic thrombocytopenic purpura, due to the deficiency of ADAMTS-13 activity caused by an acquired autoantibody against this metalloprotease [3,4,5] or by its inherited deficiency [6]
Summary
The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr1605-Met1606 peptide bond. Early studies by Furlan et al [7] on the specificity of the ADAMTS-13/VWF interaction showed that physiological concentrations of NaCl inhibit hydrolysis of VWF Based on this observation, current functional assays that measure ADAMTS-13 activity are carried out either in the absence of NaCl or at very low NaCl concentrations [7,8,9]. Current functional assays that measure ADAMTS-13 activity are carried out either in the absence of NaCl or at very low NaCl concentrations [7,8,9] Taken together, these observations prompted us to further investigate the effect of different ions on ADAMTS-13 interactions with VWF, in order to elucidate the mechanisms of control of ADAMTS-13 enzymatic activity under physiological conditions
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