Transcripts coding the VWF cleaving protease are decreased under proinflammatory conditions, which is reversed by co-incubation with activated protein C and selenate

Introduction

In sepsis, the severity-dependent decrease of the VWF-cleaving protease ADAMTS13 is a common phenomenon, which may contribute to aggregation of platelets/platelet consumption and the development of sepsis-associated thrombotic microangiopathy (TMA) and organ failure. Up to now, hepatic stellate cells (HSC) are considered to function as the primary source of ADAMTS13 protein. The underlying mechanisms of the decrease in sepsis remain unclear.

Methods

We present data obtained in in vitro experiments using cultured human HSC (LX2-line) and microvascular endothelial cells (HMEC) stimulated under proinflammatory conditions. Monolayers were exposed to cytokines known to be plasma abundant/relevant during systemic inflammation (TNF, IL1β, IFNγ), to bacterial endotoxin (100 ng/ml), to a mixture of cytokines/endotoxin, or to freshly prepared serum obtained from patients (n = 12) with severe sepsis/septic shock.

Results

Both cell lines expressed ADAMTS13 mRNA as quantitated using quantitative PCR normalized to a set of unvaried genes. Overall, incubation with cytokines resulted in a decrease of ADAMTS13 mRNA to different extents ranging between 40 and 80% of the basal transcription rate in between 24 hours. Furthermore, in endotoxin-treated cells, ADAMTS13 declined to 60% (HSC) or 65% of basal levels. This effect was more pronounced by the mixture of cytokines/endotoxin to levels of 55% (HSC) or 40% (HMEC). In monolayers treated with serum from patients with sepsis, only 10% (HSC) or 49% (HMEC) of the basal level was determined. Both the trace element selenium and activated protein C, which are used in the supportive therapy of patients with sepsis, ameliorates the decrease in serum-treated HSC and increased the level of ADAMTS13 transcript in endothelial cells. Continuous infusion adapted to body weight also abolished the decrease of ADAMTS13 expression in hepatic tissues during the course of polymicrobial sepsis in mice.

Conclusions

We found that mRNA coding ADAMTS13 protein is also present in endothelial cells. Also we observed a marked decrease in both cell lines undergoing proinflammatory stimulation. This mechanism may contribute to the decline of proteolytic activity of ADAMTS13 in patients with sepsis and sepsis-associated TMA. Furthermore, the amelioration of this effect by selenate and APC may function as mechanisms resulting in the more favorable outcome observed in a number of clinical studies.