Rapid Activation of Unstimulated Endothelial Cells Containing Tissue Factor Following Laser-Induced Injury as Monitored Via Calcium Mobilization.

Abstract

The induction of thrombus formation in vivo through a laser-induced injury to the vessel wall in the living mouse has demonstrated the rapid expression of tissue factor antigen, thrombin generation and fibrin formation during thrombus formation. This thrombosis model is dominated by a tissue factor-dependent mechanism of platelet activation and fibrin formation. Specifically, this model does not lead to exposure of detectable sub-endothelial collagen, and the growth of the resulting thrombus is not dependent upon collagen-induced signaling. In light of prior in vivo results suggesting the rapid expression of active tissue factor on the vessel wall, we have determined if endothelial cells are activated in response to the injury induced by a pulsed laser. Laser-induced activation of individual cultured endothelial cells and cell populations were monitored through elevation of intracellular calcium and fluorescence microscopy. Cultured human umbilical vein endothelial cells (HUVECs) were loaded with the calcium-sensitive dye Fluo-4 and subjected to a single pulse of the nitrogen dye-tuned laser. HUVEC activation, was characterized by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. Comparable calcium elevation could be achieved by exogenous addition of either ADP (10 μM) or thrombin (1 U/ml). Although reduced, significant laser-induced elevation of intracellular calcium remained when cells were bathed in calcium-free media, thus suggesting that both calcium influx and calcium mobilization play a part in the total calcium elevation observed. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation of the targeted cell and was followed by a wave of calcium elevation in surrounding cells. These results imply either a release of secondary mediators or cell-cell communication. Using both isolated and confluent cultured HUVECs, we performed widefield immunofluorescence and differential interference contrast microscopy to detect tissue factor (TF) in unstimulated HUVECs using an anti-TF antibody of high affinity and specificity. Tissue factor was not detected in intact endothelial cells but was localized to abundant small granules within the cytoplasm in Triton X-100-permeabilized cultured HUVECs. The J82 bladder carcinoma cell line, which constitutively expresses TF on the plasma membrane, was used as a positive control for surface expression of TF and an isotype-matched non-immune antibody used as a negative control. These results indicate the presence of a preformed intracellular pool of TF within HUVECs that under resting conditions is not detectable on the cell surface. Given the rapid time course of TF expression and platelet accumulation in vivo following laser-induced injury of the endothelium, any TF expression on the vessel wall must be preformed and not derived via endothelial protein synthesis. Although to date, we have not been able to detect TF antigen on the plasma membrane of laser-activated HUVECs it is possible that the amount of TF antigen is below the limits of sensitivity of immunodetection. Alternatively, TF, like P-selectin, may be rapidly recycled from the plasma membrane to the cell interior. Nonetheless, we suspect that tissue factor is translocated to the endothelial cell surface following cell activation and that preformed endothelial cell tissue factor, following laser-induced injury of the endothelium, plays a critical role in thrombin generation and fibrin formation in this thrombosis model.