Expression of tissue factor procoagulant activity: regulation by cytosolic calcium.

Abstract

Intact bovine fibroblasts, pericytes, and kidney cells manifested significantly less tissue factor procoagulant activity than their disrupted counterparts. Addition of calcium ionophore A23187 rapidly and reversibly enhanced the cellsurface expression of tissue factor in intact cells up to the level achieved by disruption. Inhibitors of calmodulin blocked the ionophore-dependent enhancement of procoagulant activity. Similar kinetic parameters were obtained for factor X hydrolysis by tissue factor-factor VIla on unperturbed pericytes and phosphatidyicholine vesicles. Increase in Vmax and decrease in apparent Km for this reaction were seen after either disruption or ionophore stimulation of the pericytes. Addition of phosphatidylserine to the reconstituted phospholipid vesicles also increased the Vmax and decreased the apparent Km for factor X hydrolysis. These data agree with the hypothesis that the expression of tissue factor procoagulant activity on cell surfaces is modulated by calcium-mediated changes in the asymmetric distribution of phosphatidylserine in plasma membrane.Tissue factor (TF) is a membrane-anchored cell-surface protein that initiates coagulation when blood contacts a damaged tissue (1, 2). The expression of TF procoagulant activity is essential in the hemostatic response to vascular injury. TF may also trigger the clotting associated with heart attacks, strokes, cancer, and other thrombohemorrhagic diseases. Thus, the description of how TF activity is regulated in vivo is basic to understanding the biology and pathology of coagulation.TF-initiated coagulation begins with plasma factor VII (FVII) or factor VIla (FVIIa) binding to TF (3, 4). The TF-FVII/FVIIa complex catalyzes peptide bond cleavages in two other plasma proteins, factor IX (FIX) and factor X (FX), both zymogens of serine proteases, which are converted to active enzymes (FIXa and FXa) by limited proteolysis (5-7).The interactions between these clotting factors have been studied extensively in vitro (3-12). Nevertheless, several fundamental questions regarding the initiation of coagulation in vivo remain unresolved. For example, how the expression of TF activity on cell surfaces is modulated so that clotting occurs only when and where it is needed is unclear.One model for the biological regulation of TF-initiated coagulation is that the endothelium forms a TF-deficient barrier between FVII in the plasma and underlying cells that contain TF. Thus, coagulation may be triggered by damage to the endothelium, causing the association of plasma FVII and TF on cell surfaces outside the blood. Anatomical localization, however, may not be the only mechanism controlling TF-initiated coagulation. TF in unperturbed cells appears cryptic (13). In this study we have examined the relationship between number of TF molecules in a cell and expressed procoagulant activity to understand the mechanism of TF encryption and assess its importance as a physiological modulator of coagulation. MATERIALS AND METHODSTF was purified from bovine brain and ...

Overexpression of the 78-kDa Glucose-regulated Protein/Immunoglobulin-binding Protein (GRP78/BiP) Inhibits Tissue Factor Procoagulant Activity

“…In support of this concept, GRP78/BiP overexpression attenuates TF PCA induced by ionomycin, suggesting that alterations in ER Ca 2ϩ stores play an important role in the regulation of TF PCA by GRP78/BiP. Given that GRP78/BiP is a major Ca 2ϩ -binding protein (10,11) and that TF PCA is mediated by changes in intracellular levels of free Ca 2ϩ (47)(48)(49)(50), overexpression of GRP78/BiP could potentially inhibit TF PCA indirectly by sequestering intracellular Ca 2ϩ . Although it is not completely understood as to how alterations in intracellular Ca 2ϩ increase TF PCA, it may involve the production of ROS.…”

“…In addition, GRP78/BiP overexpression could potentially limit the accessibility of anionic phospholipids essential for TF PCA. Exposure of anionic phospholipids on the outer plasma membrane increases TF PCA (47,66,75,76) and is regulated by Ca 2ϩ levels (47,75,77). Therefore, modulation of any component/cofactors involved in this pathway by GRP78/ BiP could ultimately attenuate TF PCA.…”

“…studies have demonstrated that increases in cytosolic Ca 2ϩ levels by treatment with the Ca 2ϩ ionophores, ionomycin or A23187, increases TF PCA, at a rate independent of de novo protein synthesis (47)(48)(49)(50). Treatment of cells with hydrogen peroxide also increases TF PCA, which is independent of an increase in TF protein levels (42,46).…”

“…Infection with adenovirus, as well as other respiratory viruses, increases TF expression and procoagulant activity (PCA) (33). Growth factors (34) and cytokines (35,36), endotoxin (37,38), hypoxia (39), cell injury (40), reactive oxygen species (ROS) (41)(42)(43), oxidized LDL (44 -46), and increases in intracellular free Ca 2ϩ (47)(48)(49)(50) are also known to induce TF PCA in a variety of cell types, including vascular endothelial cells, smooth muscle cells, and circulating monocytes. Furthermore, the majority of tumor cells express TF (51)(52)(53)(54), and the prothrombotic state observed in cancer patients has been largely attributed to this expression (53).…”

Viscoelasticity and Ultrastructure in Coagulation and Inflammation: Two Diverse Techniques, One Conclusion

“…Calcium has also been shown to modulate the expression of TF pro-coagulant activity on the cell surface [ 169 ].…”