Procoagulant Soluble Tissue Factor Is Released From Endothelial Cells in Response to Inflammatory Cytokines

Abstract

Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-alpha and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-alpha (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-alpha and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223+/-0.0069 versus 0.0012+/-0.0006 for control, P<0.001 and 0.0022+/-0.0004 versus 0.0012+/-0.0007, P<0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-alpha stimulation, supporting factor Xa generation after relipidation (6.55+/-1.13 U versus 2.99+/-0.59 U in control supernatant, P<0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.

Human alternatively spliced tissue factor is not secreted and does not trigger coagulation

“…We observed that MP-TF activity in cancer patients with VTE is higher than in matched cancer patients without VTE. This finding strongly supports previous observations of a relationship between elevated MP-TF activity and VTE in patients with cancer from small studies of selected cancer patients [1,[4][5][6].…”

“…We observed that MP-TF activity in cancer patients with VTE is higher than in matched cancer patients without VTE. This finding strongly supports previous observations of a relationship between elevated MP-TF activity and VTE in patients with cancer from small studies of selected cancer patients [1,[4][5][6].It is interesting that the large majority of patients who develop VTE during chemotherapy have normal MP-TF activity, suggesting that an acute thrombotic event itself is not the cause of an elevated MP-TF activity. Our data point to a contribution of cancer cells themselves as (in) direct cause of elevated MP-TF activity and ask for novel studies better designed to study prospectively the relationship between chemotherapy, elevated MP-TF activity and venous thrombosis.…”

“…With regard to secretion of asTF by primary cells, the conditioned medium of TNF-a stimulated HUVEC was shown to contain asTF, indicating secretion of asTF. Although this secreted asTF was procoagulant, its activity was < 0.1% of the corresponding TF activity [4].…”

“…At present, the coagulant properties of asTF are disputed. Although recombinant asTF shortened the clotting time of platelet poor plasma [1], asTF was enriched in human thrombi [1] and triggered activation of factor (F)X when secreted by human umbilical vein endothelial cells (HUVEC) [4], asTF expressed by human embryonic kidney (HEK) cells failed to activate FX [5]. To further explore the putative role of asTF in coagulation, we studied the procoagulant properties of asTF cloned from HUVEC in an expression model of MIA PaCa-2 cells, a human pancreatic TF-deficient cell line [2,[6][7][8].…”

“…Thereafter, two studies were published by the same investigators, who investigated the production and procoagulant properties of native asTF from HUVEC [4,10]. Although in both studies identical experimental conditions resulted in similar quantities of TF, as detected by Western blot, in the study of Szotowski et al the amounts of asTF exceeded those of TF, whereas in the study of Eisenrach et al only small amounts of asTF were found compared with TF.…”