title: Role of chloride ions in modulation of the interaction between von Willebrand factor and ADAMTS-13.
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.
authors.affiliation: Hemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy. rdecristofaro@rm.unicatt.it
authors.forename: Raimondo
authors.initials: R
authors.lastname: De Cristofaro
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
authors.forename: Roberta
authors.initials: R
authors.lastname: Palla
authors.forename: Silvia
authors.initials: S
authors.lastname: Lavoretano
authors.forename: Rossana
authors.initials: R
authors.lastname: Lombardi
authors.forename: Giuliana
authors.initials: G
authors.lastname: Merati
authors.forename: Federica
authors.initials: F
authors.lastname: Romitelli
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Stasio
authors.forename: Pier Mannuccio
authors.initials: PM
authors.lastname: Mannucci
mesh_terms: D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012310:Ristocetin / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002712:Chlorides; D007477:Ions; D011994:Recombinant Proteins; D012492:Salts; D014841:von Willebrand Factor; D012310:Ristocetin; D014443:Tyrosine; D012965:Sodium Chloride; D014508:Urea; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human