title: Inhibition of ADAMTS-13 by Doxycycline Reduces von Willebrand Factor Degradation During Supraphysiological Shear Stress: Therapeutic Implications for Left Ventricular Assist Device-Associated Bleeding.
abstract: OBJECTIVE
  The aim of this study was to investigate a potential therapy for left ventricular assist device (LVAD)-associated bleeding.
  
  
  BACKGROUND
  Nonsurgical bleeding is the most frequent complication of LVAD support. Recent evidence has demonstrated that supraphysiological shear stress from continuous-flow LVADs accelerates von Willebrand factor (vWF) metabolism by the action of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) (the vWF protease). An acquired vWF deficiency causes bleeding. This suggests that ADAMTS-13 is a clinical target to reduce vWF degradation. We tested the hypothesis that inhibition of ADAMTS-13 with doxycycline, an inexpensive, clinically approved drug, reduces vWF degradation during shear stress.
  
  
  METHODS
  Whole blood was collected from human donors (n = 15), and purified, recombinant ADAMTS-13 protein was obtained. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the dose relationship between doxycycline and ADAMTS-13 activity prior to shear stress (n = 10). To determine the effect of shear stress, plasma and recombinant ADAMTS-13 were exposed to LVAD-like supraphysiological shear stress (approximately 175 dyne/cm(2)). vWF multimers and degradation fragments were characterized with electrophoresis and immunoblotting (n = 10). Förster resonance energy transfer was used to quantify plasma ADAMTS-13 activity (n = 10). An ELISA was used to quantify vWF:collagen binding activity. Platelet aggregometry was performed with adenosine 5'-diphosphate, collagen, and ristocetin (vWF-platelet pathway) agonism (n = 10).
  
  
  RESULTS
  Doxycycline significantly decreased plasma ADAMTS-13 activity (p = 0.01) and the activity of recombinant human ADAMTS-13 protein by 21%. After plasma was exposed to shear stress, the same pattern of vWF degradation was observed as previously reported for LVAD patients, and vWF:collagen binding activity decreased significantly (p = 0.002). Doxycycline significantly decreased ADAMTS-13 activity (p = 0.04) and the activity of recombinant ADAMTS-13 by 18%, protected large vWF multimers from degradation, and significantly decreased the levels of the 5 smallest vWF fragments by 12 ± 2% (p < 0.05). As a result, vWF:collagen binding activity was significantly restored (p = 0.004). ADAMTS-13 inhibition with doxycycline did not hyperactivate platelets.
  
  
  CONCLUSIONS
  Inhibition of ADAMTS-13 by doxycycline decreased vWF degradation and improved vWF function during supraphysiological shear stress without hyperactivating platelets. ADAMTS-13 is a clinical target to reduce vWF degradation, improve vWF function, and potentially reduce bleeding during LVAD support.
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address: carlo.bartoli@uphs.upenn.edu.
authors.forename: Carlo R
authors.initials: CR
authors.lastname: Bartoli
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Jooeun
authors.initials: J
authors.lastname: Kang
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: David J
authors.initials: DJ
authors.lastname: Restle
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: David M
authors.initials: DM
authors.lastname: Zhang
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Cameron
authors.initials: C
authors.lastname: Shabahang
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Acker
authors.affiliation: Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
authors.forename: Pavan
authors.initials: P
authors.lastname: Atluri
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000071120:ADAMTS13 Protein; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000627:therapeutic use*; D004318:Doxycycline / Q000378:metabolism / Q000627:therapeutic use*; D006333:Heart Failure / Q000628:therapy; D006353:Heart-Assist Devices / Q000009:adverse effects*; D006470:Hemorrhage / Q000209:etiology* / Q000517:prevention & control*; D006801:Humans; D011474:Prosthesis Design; D016896:Treatment Outcome; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D004318:Doxycycline
keywords: ADAMTS-13; bleeding; doxycycline; heart failure; left ventricular assist device (LVAD); mechanical circulatory support; shear stress; von Willebrand factor