Abstract
Background: ADAMTS13 is an anti-thrombotic metalloenzyme that cleaves ultra large von Willebrand factor (VWF) released from endothelium, which prevents platelet accumulation at the sites of vascular injury. Deficiency of plasma ADAMTS13 activity resulting from either ADAMTS13 mutations or acquired autoantibodies against ADAMTS13 leads to disseminated occlusive microvascular thromboses, a hallmark of pathology in a potentially fatal blood disorder, thrombotic thrombocytopenic purpura (TTP). However, it is still not known if ADAMTS13 has other biological functions besides cleaving VWF and inhibiting thrombosis.
Objectives: The present study aims to determine the effects of ADAMTS13 on an agonist-induced platelet activation and aggregation and its underlying molecular mechanisms.
Methods: Platelet aggregation, activation, and apoptosis were determined by a light transmission assay, flow cytometry, and Western blotting, respectively.
Results: We found that a recombinant full-length ADAMTS13 (rADAMTS13) but not a C-terminal or N-terminal truncated form of ADAMTS13 (e.g., MDTCS or TSP2-CUB) was able to enhance the aggregation of isolated human and murine platelets induced by a fibrillar collagen. This enhancing activity was rADAMTS13 concentration- and conformation-dependent, but did not require its proteolytic activity. While heat-inactivated rADAMTS13 showed no enhancing effect, the EDTA-treated rADAMTS13 could still enhance the collagen-induced aggregation of human platelets. Of notes, rADAMTS13 at the same concentrations did not enhance AYPGKF, a peptide that acts as an agonist for the protease-activated receptor 4 (PAR4) on platelets, or 2MesADP-induced murine (Adamts13-/-) platelet aggregation. Flow cytometric analysis demonstrated that an addition of rADAMTS13 to the fibrillar collagen resulted in significantly more activation and apoptosis of washed murine platelets than the collagen alone, apparently via an increase of AKT phosphorylation inside platelets. Moreover, an addition of anti-CD47 IgG that binds platelet surface CD47, a glycoprotein that binds to signal-regulatory protein α on macrophages and regulates phagocytosis, further enhanced the activation and aggregation of Adamts13-/- murine platelets induced by a combination of rADAMTS13 and fibrillar collagen.
Conclusions: Our findings revealed a novel but paradoxical function of rADAMTS13 promoting hemostasis while inhibiting thrombosis at the same time, like thrombin that has both prothrombotic and antithrombotic activities.
