Antithrombin regulates neutrophil activities through the stimulation of C-type lectin family 1A

A screening method using co-expression of AT and a candidate receptor in HEK293T cells identified CLEC1A as a novel receptor for AT.

Antithrombin regulates neutrophil cell shape and ROS production through C-type lectin family 1A.

It has been suggested that a serine proteinase inhibitor, antithrombin (AT), exerts anti-inflammatory effects on different types of cells, independent of thrombin inhibition. In the present study, we aimed to identify a specific receptor for antithrombin by a screening method using a transmembrane tethered-AT ligand expressed on HEK293T cells together with the co-expression of candidate receptors, followed by the immunoprecipitation of a complex of AT ligand with a receptor. We identified C-type lectin family 1A (CLEC1A) as a receptor for AT. We confirmed the binding of AT to the extracellular domain of CLEC1A using surface plasmon resonance. Recombinant as well as native AT concentration-dependently induced the rounding of purified human neutrophils in shape, associated with the suppression of spontaneous ROS production in vitro but argatroban did not, indicating the independence of AT effects on thrombin inhibition. Native AT maintained the passage of neutrophils through the artificial microcapillaries. Both AT also enhanced the phagocytosis of pHrodo-labeled E.coli and prolonged the viability of the neutrophils. The cellular effects of AT were similar to those of HRG which has the same CLEC1A as a receptor, and were partially inhibited by the addition of anti-CLEC1A antibody to the incubation media. These results suggested that CLEC1A is a novel receptor for AT and the stimulation of CLEC1A by AT at least in part mediates the important functional changes of human neutrophils.