Altered ADAMTS-13 Expression in Human Umbilical Vein Endothelial Cells.

Abstract

von Willebrand factor (vWF), as secreted by endothelial cells (ECs), is a highly thrombogenic, ultra-large multimeric protein which promotes platelet adhesion and clot formation. This ultra-large vWF would lead to platelet aggregation even without vascular injury if not for the activity of a plasma vWF-cleaving protease, ADAMTS-13, which cleaves the ultra-large vWF multimers upon secretion into the more moderately-sized, less thrombogenic multimers commonly found circulating in normal plasma. The production of ADAMTS-13 was first described in hepatic stellate cells, but has since been found to be produced in a variety of tissues and cell types including ECs. Production of ADAMTS-13 by ECs is of interest as the newly secreted ultra-large vWF is immediately cleaved on the surface of ECs by ADAMTS-13. Quiescent ECs normally exist as a confluent non-thrombogenic monolayer lining the lumen of the vasculature. Damage to the EC monolayer results in cell activation, stimulating not only thrombosis but proliferation as the ECs attempt to prevent blood loss and repair vascular wall damage. To assess the production of ADAMTS-13 by human umbilical vein ECs, as measured by a commercially-available ELISA (American Diagnostica Inc., Stamford, CT), cellular supernatants were collected and cellular proteins extracted from either confluent ECs (EC monolayers), damaged confluent ECs (EC monolayers which had narrow strips of cells removed with a sterile pipette tip), or subconfluent ECs (50% confluence). After four hours, supernatants from subconfluent ECs contained 2.5X as much ADAMTS-13 (161.5±29.1 pg/ml/μg cellular protein; p<0.002) as compared with either confluent or damaged confluent ECs (60.2±22.5 and 56.0±19.2 pg/ml/μg cellular protein, respectively). Similarly, cell lysates from subconfluent ECs contained ∼2.5X as much ADAMTS-13 (645.1±73.1 pg/ml/μg cellular protein; p<0.002) as compared with either confluent or damaged confluent ECs (257.8±32.8 and 311.3±29.3 pg/ml/μg cellular protein, respectively). In time-course experiments, confluent, damaged confluent, and subconfluent ECs were fixed 4, 24, 48, 72, or 96 hours following injury (producing the damaged confluent ECs). ADAMTS-13 expression was subsequently detected by immunohistochemistry using a rabbit polyclonal anti-human ADAMTS-13 antibody directed against the metalloproteinase domain (Abcam Inc., Cambridge, MA) and a fluorescent secondary antibody. Subconfluent ECs expressed more intense staining for ADAMTS-13 throughout the time course when compared with the confluent ECs. By 72 hours, the damaged confluent ECs began expressing comparable intense staining, particularly in regions where the ECs were reestablishing the monolayer. Previous work has suggested that the hepatic stellate cells, as well as the other cell types which express ADAMTS-13, produce this protease constitutively and that this production is not otherwise regulated. The data presented herein demonstrate that ADAMTS-13 production, at least in ECs, is affected by the growth state of the ECs. This enhanced production of ADAMTS-13 may limit the extent of platelet thrombosis at sites of vascular injury.