Formation of methionine sulfoxide by peroxynitrite at position 1606 of von Willebrand factor inhibits its cleavage by ADAMTS-13: A new prothrombotic mechanism in diseases associated with oxidative stress.
An enhanced formation of reactive oxygen species and peroxynitrite occurs in several clinical settings including diabetes, coronary artery disease, stroke, sepsis, and chronic inflammatory diseases. Peroxynitrite oxidizes methionine and tyrosine residues to methionine sulfoxide (MetSO) and 3-nitrotyrosine (NT), respectively. Notably, ADAMTS-13 cleaves von Willebrand factor (VWF) exclusively at the Tyr1605-Met1606 peptide bond in the A2 domain. We hypothesized that peroxynitrite could oxidize either or both of these amino acid residues, thus potentially affecting ADAMTS-13-mediated cleavage. We tested our hypothesis using synthetic peptide substrates based on: (1) VWF Asp1596-Ala1669 sequence (VWF74) and (2) VWF Asp1596-Ala1669 sequence containing nitrotyrosine (VWF74-NT) or methionine sulfoxide (VWF74-MetSO) at position 1605 or 1606, respectively. The peptides were treated with recombinant ADAMTS-13 and the cleavage products analyzed by RP-HPLC. VWF74 oxidized by peroxynitrite underwent a severe impairment of its hydrolysis. Likewise, VWF74-MetSO was minimally hydrolyzed, whereas VWF74-NT was hydrolyzed slightly more efficiently than VWF74. Oxidation by peroxynitrite of purified VWF multimers inhibited ADAMTS-13 hydrolysis, but did not alter their electrophoretic pattern nor their ability to induce platelet agglutination by ristocetin. Moreover, VWF purified from type 2 diabetic patients showed oxidative damage, as revealed by enhanced carbonyl, NT, and MetSO content and was partially resistant to ADAMTS-13 hydrolysis. In conclusion, peroxynitrite may contribute to prothrombotic effects, hindering the proteolytic processing by ADAMTS-13 of high-molecular-weight VWF multimers, which have the highest ability to bind and activate platelets in the microcirculation.
Lancellotti
Stefano
S
Institute of Internal Medicine and Geriatrics and Hemostasis Research Center, Catholic University School of Medicine, 00168 Rome, Italy.
De Filippis
Vincenzo
V
Pozzi
Nicola
N
Peyvandi
Flora
F
Palla
Roberta
R
Rocca
Bianca
B
Rutella
Sergio
S
Pitocco
Dario
D
Mannucci
Pier Mannuccio
PM
De Cristofaro
Raimondo
R
D051722:ADAM Proteins / Q000494:pharmacology*; D000071120:ADAMTS13 Protein; D000328:Adult; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D016022:Case-Control Studies; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D006868:Hydrolysis; D008297:Male; D008715:Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D008875:Middle Aged; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D055503:Protein Multimerization; D012310:Ristocetin / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014842:von Willebrand Diseases; D014841:von Willebrand Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D014841:von Willebrand Factor; D012310:Ristocetin; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D008715:Methionine; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; C013111:methionine sulfoxide