Abstract
Background: Thrombotic thrombocytopenic purpura (TTP), a potentially life-threatening blood clotting disorder, is resulted from severe deficiency of ADAMTS13 activity. Plasma exchange is the only effective initial therapy for acquired TTP, which replenishes ADAMTS13 while removing autoantibodies. However, the in-hospital mortality rate is still high, suggesting that another more effective therapy is urgently needed.
Objectives: The present study aims to develop a novel therapy using platelets as carriers to deliver recombinant ADAMTS13 (rADAMTS13) to the sites of vascular injury, where platelets are activated and de-granulated to release rADAMTS13 from the storage granules. Our specific aims are: 1) determine the in vitro uptake of rADAMTS13 by platelets; 2) determine the efficacy of platelet-delivered rADAMTS13 in anti-arterial thrombosis under flow and in vivo.
Methods: Freshly isolated human platelets from whole blood were incubated with an increasing concentration of rADAMTS13 (0, 5, 20, 50, and 100 µg/ml) at 25°C and 37°C for hours. Platelets were then washed and lysed with 1% Triton X-100 to assess the rADAMTS13 activity via a FRETS-VWF73 assay. Western blotting and confocal microscopy determined the amounts of rADAMTS13 protein and localization in platelets, respectively. The anti-thrombotic activity of the released rADAMTS13 from activated platelets was determined by a microfluidic system (BioFlux) and by a ferric chloride induced thrombus formation in mesenteric arterioles/venules in Adamts13-/- mice.
Results: Human washed platelets were able to uptake rADAMTS13 in a temperature- and concentration-dependent manner. The rADAMTS13 protein packed inside human platelets remained intact and active towards the FRET-VWF73 substrate. Confocal microscopic analysis demonstrated its localization of uptaken rADAMTS13 in the alpha granules, partially overlapped with VWF. The rADAMTS13 released from activated human platelets under shear (100 dyne/cm2) was capable of cleaving VWF and inhibiting platelet agglutination and aggregation on a collagen-coated surface in a concentration-dependent manner. Transfusion of platelets expressing rADAMTS13 into Adamts13-/- mice dramatically reduced the rate of thrombus formation in the mesenteric arterioles/venules after ferric chloride injury.
Conclusions: Our results demonstrate that human platelets can be reengineered in vitro to contain sufficient amounts of rADAMTS13 in the alpha granules. The rADAMTS13 inside platelets is releasable and proteolytically functional under shear stress or in vivo under flow. Our findings suggest that the transfusion of platelets loaded with rADAMTS13 may represent a novel therapeutic strategy for acquired TTP with inhibitors.
Zheng: Alexion: Speakers Bureau; Ablynx: Consultancy.
Author notes
Asterisk with author names denotes non-ASH members.
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