Synergies of phosphatidylserine and protein disulfide isomerase in tissue factor activation

Abstract

Tissue factor (TF), the cellular receptor and cofactor for factor VII/VIIa, initiates haemostasis and thrombosis. Initial tissue distribution studies suggested that TF was sequestered from the circulation and only present at perivascular sites. However, there is now clear evidence that TF also exists as a blood-borne form with critical contributions not only to arterial thrombosis following plaque rupture and to venous thrombosis following endothelial perturbation, but also to various other clotting abnormalities associated with trauma, infection, or cancer. Because thrombin generation, fibrin deposition, and platelet aggregation in the contexts of haemostasis, thrombosis, and pathogen defence frequently occur without TF de novo synthesis, considerable efforts are still directed to understanding the molecular events underlying the conversion of predominantly non-coagulant or cryptic TF on the surface of haematopoietic cells to a highly procoagulant molecule following cellular injury or stimulation. This article will review some of the still controversial mechanisms implicated in cellular TF activation or decryption with particular focus on the coordinated effects of outer leaflet phosphatidylserine exposure and thiol-disulfide exchange pathways involving protein disulfide isomerase (PDI). In this regard, our recent findings of ATP-triggered stimulation of the purinergic P2X7 receptor on myeloid and smooth muscle cells resulting in potent TF activation and shedding of procoagulant microparticles as well as of rapid monocyte TF decryption following antithymocyte globulin-dependent membrane complement fixation have delineated specific PDI-dependent pathways of cellular TF activation and thus illustrated additional and novel links in the coupling of inflammation and coagulation.

Keywords: Tissue factor; phosphatidylserine; protein disulfide isomerase; thiol-disulfide exchange; thrombosis.

Figure 1. Saturation with FVIIa does not interfere with ATG-mediated cellular TF activation

Monocytic THP1 cells were preincubated with buffer (control) or 100 nM recombinant human activated FVII (rFVIIa) for 30 min at 37°C. Following loading with rabbit IgG or ATG, cell-associated PCA was measured by single-stage clotting assay as described in Langer et al. [68]. A representative experiment is shown.

Figure 2. TF activation on THP1 cells by HgCl₂ or ATG is poorly correlated with extracellular PS

Activation of monocytic THP1 cells by ATG (100 μg/ml) or HgCl₂ (100 μM) was carried out using the conditions described in Langer et al. [68]. (A) PCA was measured by single-stage clotting assay and (B) PS exposure by flow cytometry using fluorescently labelled annexin V. Representative experiments are shown.

Figure 3. Saturating concentrations of annexin V only partially inhibit TF-dependent FXa generation on ATG-activated THP1 cells

Effect of annexin V on (A) PCA and (B) TF-dependent FXa generation on ATG-activated THP1 cells (means ± SD, n=3). Inhibitory TF antibody (αTF) was used for comparison.

Figure 4. Mechanisms of thiol-disulfide exchange-dependent cellular TF activation involving cell surface PDI

On intact cells, TF is maintained in a predominantly non-coagulant or cryptic state by sequestration of PS to the inner membrane leaflet and by a reduced or broken membrane-proximal Cys¹⁸⁶-Cys²⁰⁹ allosteric disulfide bond. Experiments provided evidence for modification of this bond by reduction, S-nitrosylation/-glutathionylation of the cysteine at position 209, or by the formation of mixed TF disulfides with other thiol-regulated proteins such as thioredoxin (Trx), integrins, or PDI. ATP-triggered activation of the purinergic P2X7 receptor or complement activation with subsequent C5b-7 membrane insertion results in PDI-dependent thiol-disulfide exchange with optimal TF folding and oxidation in the context of simultaneously exposed PS.