Antithrombin III inhibits nuclear factor κB activation in human monocytes and vascular endothelial cells

Abstract

The serpin antithrombin III (AT III), the most important natural inhibitor of thrombin activity, has been shown to exert marked anti-inflammatory properties and proven to be efficacious in experimental models of sepsis, septic shock, and disseminated intravascular coagulation. Moreover, clinical observations suggest a possible therapeutic role for AT III in septic disorders. The molecular mechanism, however, by which AT III attenuates inflammatory events is not yet entirely understood. We show here that AT III potently blocks the activation of nuclear factor B (NF-B), a transcription factor involved in immediate early gene activation during inflammation. AT III inhibited agonist-induced DNA binding of NF-B in cultured human monocytes and endothelial cells in a dose-dependent manner, suggesting that AT III interferes with signal transduction leading to NF-B activation. This idea was supported by demonstrating that AT III prevents the phosphorylation and proteolytic degradation of the inhibitor protein IB␣. In parallel to reducing NF-B activity, AT III inhibited the expression of interleukin-6, tumor necrosis factor-␣, and tissue factor, genes known to be under the control of NF-B. The observation that chemically modified AT III that lacks heparin-binding capacity had no effect on NF-B activation supports the current understanding that the inhibitory potency of AT III depends on the interaction of AT III with heparinlike cell surface glycosaminoglycans. This hypothesis was underscored by the finding that the AT III ␤-isoform, known to have higher affinity for glycosaminoglycans, is more effective in preventing NF-B transactivation than ␣-AT III. These data indicate that AT III can alter inflammatory processes via inhibition of NF-B activation. IntroductionAntithrombin is one of the most important endogenous regulators of coagulation, acting as the major inhibitor of thrombin and interfering with several plasma proteases such as kallikrein and factors IXa, Xa, XIa, and XIIa. 1 Apart from its role in hemostasis, there is accumulating evidence that antithrombin III (AT III) exerts anti-inflammatory properties and improves survival in animalsepsis models and disseminated intravascular coagulation (DIC). [2][3][4] There are a number of compelling reasons why AT III may also be an effective therapeutic agent in patients with sepsis. [5][6][7] AT III was shown to reduce leukocyte-endothelial interaction, 8 to prevent microvascular leakage, 9 and to ameliorate ischemia/reperfusion injury. 10 These effects, however, seem to result only in part from interference of AT III with thrombin activity because inhibition of thrombin generation alone did not prove similarly effective. 11,12 The beneficial effects of AT III rather appear mainly to result from direct, thrombin-independent effects on vascular cells. AT III was found to stimulate nitric oxide synthesis in vascular smooth muscle cells, 13 to inhibit migration and adhesion of neutrophils, 14 and to attenuate cytokine production in monocytes and endothelial cells (ECs) 15...

Antithrombin reduces ischemia/reperfusion-induced renal injury in rats by inhibiting leukocyte activation through promotion of prostacyclin production

“…11,12 In contrast to these reports, AT has been demonstrated to inhibit leukocyte activation directly. [13][14][15] These observations suggest that anti-inflammatory effects of AT may be mediated mainly by its direct inhibitory properties on leukocyte activation.…”

Antithrombin Reduces Inflammation and Microcirculatory Perfusion Failure in Closed Soft-Tissue Injury and Endotoxemia

“…Furthermore, antithrombin was capable in delaying microvascular thrombus formation in a murine model (21,22). Beside its anticoagulatory actions, antithrombin reveals anti-inflammatory activities in a variety of experimental models, which are independent of its direct influence on the coagulatory cascade (7,23). Beneficial effects of antithrombin could also be shown in patients with severe sepsis (24), which, however, have not been confirmed in the multinational KyberSept trial (25).…”

“…Antithrombin III, a plasma-derived serine protease inhibitor, controls the activity of thrombin and other proteases of the coagulation cascade (4,5). Furthermore, antithrombin has previously shown a large number of anti-inflammatory actions, independent of its anticoagulatory effects (6)(7)(8). It is supposed that antithrombin interacts with endothelial cells, thus inhibiting leukocyteendothelial cell interaction.…”

Life-Threatening Thrombosis in Mice With Targeted Arg48-to-Cys Mutation of the Heparin-Binding Domain of Antithrombin

“…Indeed, AT, via interaction with HSPG present on endothelial cells or on neutrophils, promotes the release of antiinflammatory prostacyclin and blocks the activation of the proinflammatory NF-B, thereby decreasing platelet and neutrophil activation, chemotaxis, and interaction with the endothelium, 38 -41 effects that are lost after chemically blocking the heparin-binding domain of AT. 39,40 No thrombotic phenotype was observed so far in mice with altered heparan-sulfate (HS) moieties or deficiencies in the HSPG core proteins. 3-O-sulfation of the pentasaccharide core sequence of heparin/heparan-sulfate is essential for interaction with AT.…”