Elsevier

JACC: Heart Failure

Volume 3, Issue 11, November 2015, Pages 860-869
JACC: Heart Failure

Mini-Focus Issue: Mechanical Circulatory Support: Opportunities and Challenges
Inhibition of ADAMTS-13 by Doxycycline Reduces von Willebrand Factor Degradation During Supraphysiological Shear Stress: Therapeutic Implications for Left Ventricular Assist Device-Associated Bleeding

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Abstract

Objectives

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/cm2). 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.

Key Words

ADAMTS-13
bleeding
doxycycline
heart failure
left ventricular assist device (LVAD)
mechanical circulatory support
shear stress
von Willebrand factor

Abbreviations and Acronyms

ADAMTS-13
a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13
ELISA
enzyme-linked immunosorbent assay
FRET
Förster resonance energy transfer
GST
glutathione-S-transferase
HRP
horseradish peroxidase
LVAD
left ventricular assist device
SDS
sodium dodecyl sulfate
TBS
Tris-buffered saline
TTP
thrombotic thrombocytopenia purpura
vWF
von Willebrand factor

Cited by (0)

The authors have reported that they have no relationships relevant to the contents of this paper to disclose.