Carboxyl Terminus of ADAMTS13 Directly Inhibits Platelet Aggregation and Ultra Large von Willebrand Factor String Formation Under Flow in a Free-Thiol–Dependent Manner

Abstract

Summary Objective ADAMTS13 cleaves von Willebrand factor (VWF), thereby inhibiting thrombus formation. Proteolytic cleavage relies on the amino-terminal (MDTCS) domains, but the role of the more distal carboxyl-terminal domains of ADAMTS13 is not fully understood. A previous study demonstrated the presence of multiple surface- exposed free sulfhydryls on ADAMTS13 that appeared to interact with those on VWF under shear. Here, we determined the physiological relevance of such an interaction in antithrombotic responses under flow. Approaches and Results A microfluidic assay demonstrated that a carboxyl-terminal fragment of ADAMTS13, comprising either 2-8 thrombospondin type 1 (TSP1) repeats and CUB domains (T2C) or 5-8 TSP1 repeats and CUB domains (T5C), directly inhibited platelet adhesion/aggregation on a collagen surface under arterial shear. In addition, an intravital microscopic imaging analysis showed that the carboxyl-terminal fragment of ADAMTS13 (T2C or T5C) was capable of inhibiting the formation and elongation of platelet-decorated ultra large (UL) VWF strings and the adhesion of platelets/leukocytes on endothelium in mesenteric venules after oxidative injury. The inhibitory activity of T2C and T5C on platelet aggregation and ULVWF string formation was dependent on the presence of their surface free thiols; pretreatment of T2C and T5C or full-length ADAMTS13 with N-ethylmaleimide that reacts with free sulfhydryls abolished or significantly reduced its antithrombotic activity. Conclusion Our results demonstrate for the first time that the carboxyl-terminus of ADAMTS13 has direct antithrombotic activity in a free-thiol dependent manner. The free thiols in the carboxyl-terminal domains of ADAMTS13 may also contribute to the overall antithrombotic function of ADAMTS13 under pathophysiological conditions.

High-resolution epitope mapping by HX MS reveals the pathogenic mechanism and a possible therapy for autoimmune TTP syndrome

“…The MDTCS construct comprises five domains (Metalloprotease, Disintegrin, TSP1, Cys-rich, and Spacer). It is fully functional in catalyzing proteolysis of VWF under various conditions (27)(28)(29)(30)(31)(32). Therefore, to reduce the complexity of data analysis, the MDTCS fragment, expressed in Drosophila Schneider 2 (S2) cells (SI Materials and Methods), was used for the epitopemapping study.…”

The D173G mutation in ADAMTS-13 causes a severe form of congenital thrombotic thrombocytopenic purpura

“…ADAMTS-13 is a zinc metalloprotease present in plasma at a concentration of about 1 µg/ml (4,5). The enzyme consists, from the N-terminus, of a relatively short propeptide, the catalytic site, the disintegrin-like (DLD), a first thrombospondin-1 (TSP1) repeat, the Cys-rich domain, the spacer domain, seven TSP1 repeats and 2 CUB (complement components C1r/C1s, urinary Epidermal Growth factor and bone morphogenetic protein-1) domains at the C-terminus (6), whose free thiols have also direct antithrombotic effects (7). When there is a deficiency of this enzyme, uncleaved, ultra large VWF multimers accumulate in microcirculation, causing increased platelet adhesion and aggregation, resulting in the formation of VWF-and platelet-rich thrombi (1,8,9).…”

Utility of microfluidic devices to study the platelet–endothelium interface

“…[65] PDMS-based platforms have shown promise for use as clinical diagnostic devices in assessing the thrombotic potential of patients on antiplatelet therapy[66] or to perform mechanistic studies to define the ADAMTS13 domains required for VWF cleavage. [67] These studies may assist in development of targeted therapies for the treatment of patients with debilitating diseases such as chronic TTP by allowing high throughput screening of known ADAMTS13 mutations. [68]…”