Background: Monocytic tissue factor (TF), an initiator of extrinsic blood coagulation, is often activated under various inยฏammatory conditions including endotoxemia. This activation could be a contributing factor to the manifestation of disseminated intravascular coagulation following septic shock.
Hypothesis: We herein determine if extracellular Ca 2 ([Ca 2 ] ex ) regulates bacterial endotoxin (LPS)-inducible monocytic TF activation.
Methods: We have employed a model monocytic cell line (THP-1) to explore the mode of action of [Ca 2 ] ex on the modulation of LPS-induced TF activation. TF activity was measured by a single stage clotting assay, while TF expression as well as LPS recognition and its receptor expression were studied in immunoยฏuorescent approaches.
Results: LPS-induced TF activation was inversely correlated to [Ca 2 ] ex . Upon exposure of THP-1 cells to LPS (1 . 5 mg ml ร1 ) for 6 h in the Hanks' medium without CaCl 2 , TF was activated by nearly 10-fold. TF activation appreciably decreased with the increasing [Ca 2 ] ex . No more than 3 . 5-fold TF activation was detected at 5 mM [Ca 2 ] ex . Consistent with the signiยฎcantly lower degree of TF activation, LPS-induced TF expression at 5 mM [Ca 2 ] ex was 60 per cent less than that without [Ca 2 ] ex . FACScan analysis showed that LPS recognition was signiยฎcantly blocked at 5 mM [Ca 2 ] ex which however had no eect on the expression of CD14 and CD11b, the proposed major LPS receptors. Moreover, LPS binding in vitro was signiยฎcantly inhibited by 5 mM CaCl 2 .
Conclusion: Our results demonstrate that [Ca 2 ] ex blocked LPS recognition without aecting its receptor expression on THP-1 monocytes. This insensitivity to LPS thereby resulted in the depressed inducible monocytic TF expression and activation.