title: The hyperglycemic byproduct methylglyoxal impairs anticoagulant activity through covalent adduction of antithrombin III.
abstract: BACKGROUND
  The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition.
  
  
  METHODS
  MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition.
  
  
  CONCLUSIONS
  MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof.
authors.affiliation: Vanderbilt University Medical Center, Department of Orthopaedics, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Center for Bone Biology, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Pharmacology, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Richard
authors.initials: R
authors.lastname: Jacobson
authors.affiliation: Vanderbilt University Medical Center, Department of Orthopaedics, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Pathology, Microbiology and Immunology, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Mignemi
authors.affiliation: Vanderbilt University, Mass Spectrometry Research Center, Proteomics Laboratory, Nashville, TN 37232 United States.
authors.forename: Kristie
authors.initials: K
authors.lastname: Rose
authors.affiliation: Vanderbilt University Medical Center, Department of Orthopaedics, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Lynda
authors.initials: L
authors.lastname: O'Rear
authors.affiliation: Vanderbilt University Medical Center, Department of Pathology, Microbiology and Immunology, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Suryakala
authors.initials: S
authors.lastname: Sarilla
authors.affiliation: Vanderbilt University Medical Center, Department of Pharmacology, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
authors.affiliation: Vanderbilt University Medical Center, Department of Pharmacology, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Joey V
authors.initials: JV
authors.lastname: Barnett
authors.affiliation: Vanderbilt University Medical Center, Department of Pathology, Microbiology and Immunology, 2200 Children's Way, Nashville, TN 37232-9565 United States.
authors.forename: Ingrid M
authors.initials: IM
authors.lastname: Verhamme
authors.affiliation: Vanderbilt University Medical Center, Department of Orthopaedics, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Center for Bone Biology, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Pathology, Microbiology and Immunology, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Pharmacology, 2200 Children's Way, Nashville, TN 37232-9565 United States; Vanderbilt University Medical Center, Department of Pediatrics, 2200 Children's Way, Nashville, TN 37232-9565 United States. Electronic address: jon.schoenecker@vanderbilt.edu.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Schoenecker
mesh_terms: D000925:Anticoagulants / Q000008:administration & dosage / Q000097:blood / Q000737:chemistry; D000990:Antithrombin III / Q000037:antagonists & inhibitors*; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D006493:Heparin / Q000097:blood / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D006943:Hyperglycemia / Q000097:blood*; D011485:Protein Binding; D011765:Pyruvaldehyde / Q000097:blood* / Q000737:chemistry*
chemical_list: D000925:Anticoagulants; D011765:Pyruvaldehyde; D000990:Antithrombin III; D006493:Heparin
keywords: Antithrombin III; Diabetes; Hypercoagulability; Methylglyoxal