Platelet-Delivered Recombinant ADAMTS13 Inhibits Platelet Adhesion and Aggregation on Collagen Surface Under Flow in the Presence of Autoantibodies from Acquired Thrombotic Thrombocytopenic Purpura

Abstract

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal syndrome. It is primarily caused by severe deficiency of plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor (VWF), thereby reducing the platelet adhesion and aggregation under flow. Plasma exchange is the only effective initial therapy for acquired autoimmune TTP. However, in hospital mortality rate remains as high as 10-20% in addition to the concern about the complications associated with the placement of central catheter and the use of plasma products. Therefore, a better and more effective therapy is urgently needed.Objectives: To develop a more effective therapy, we determined the uptake and package of recombinant ADAMTS13 (rA13) in platelets and assessed the efficacy of platelets-delivered rA13 in inhibiting platelet adhesion and aggregation and thrombus formation under arterial shear.Methods: Human platelets were isolated from whole blood, washed, and incubated with various concentrations of rA13 (1-100 microgram/ml) at 25 and 37 degree Celsius for various times. The amount of rA13 uptake by platelets was determined in cell lysate by Western blotting and FRETS-VWF73 assays and in fixed cells by immunofluoresent microscopy and flow cytometry. The function of platelet delivered rA13 was also determined by shear-induced platelet adhesion and aggregation on collagen surface using a microfluidic system.Results: Freshly isolated and blood bank stored platelets were able to update rA13 in a temperature, concentration, and time-dependent manner. When rA13 (5 microgram/ml) was incubated with platelets, nearly all platelets were positive for rA13, assessed by immunofluoresent microscopy and flow cytometry. The rA13 inside platelets remained intact and was proteolytically active in cleaving FRETS-VWF73. Confocal imaging revealed that rA13 was partially co-localized with VWF in alpha granules of platelets. Microfluidic assay demonstrated that platelet-delivered rA13 was able to dramatically inhibit platelet adhesion and aggregation on collagen-coated surface under arterial shear (100 dyne/cm2) in the absence and presence of a human monoclonal antibody against ADAMTS13 (scFv4-20) that was isolated from a patient with acquired autoimmune TTP. These results were consistent with the inhibitory effects observed with mouse transgenic platelets expressing rA13 under the same conditions. When 1/3 of whole blood Adamts13-/- platelets were replaced with transgenic platelets (containing rA13), the coverage of all fluorescent platelets onto a VWF-collagen surface was dramatically reduced (data not shown).Conclusion: Our results demonstrate that platelets uptake and deliver rA13 to the site of thrombus formation under flow and the platelet-delivered rA13 may be efficacious for treating acquired TTP with inhibitors. DisclosuresZheng:Ablynx: Consultancy; Alexion: Research Funding.