Carlo R Bartoli1, Jooeun Kang2, David J Restle2, David M Zhang2, Cameron Shabahang2, Michael A Acker2, Pavan Atluri2. 1. Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address: carlo.bartoli@uphs.upenn.edu. 2. Division of Cardiovascular Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania.
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/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.
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/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 humanADAMTS-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.