Antithrombotic activity of TNF-alpha

Abstract

Basic and clinical observations suggest that thrombosis and inflammation are closely related. Here we addressed the role played by TNF-alpha in thrombus formation and growth in an in vivo mouse model. Using intravital microscopy, we show that systemic administration of TNF-alpha at doses found in sepsis transiently inhibits thrombus formation and delays arterial occlusion upon vascular injury. These results were reflected in a prolonged bleeding time. Platelets isolated from the TNF-alpha-treated mice showed a marked decrease in fibrinogen binding and P-selectin expression as well as reduced platelet aggregation in response to various agonists. In contrast, in vitro treatment of platelets with TNF-alpha did not affect their function. TNF receptor 1- and 2-deficient mice exhibited normal thrombogenesis in the presence of TNF-alpha. Additionally, the inhibitory effect of TNF-alpha was lost either after treatment with NG-monomethyl-l-arginine, an inhibitor of NO production, or in mice deficient for iNOS. These results indicate that under inflammatory conditions, when leukocytes need free passage to transmigrate into tissues, TNF-alpha decreases platelet activation and inhibits thrombi formation. This effect is not exerted directly on platelets but mediated through the rapid generation of NO in the vessel wall.

Figure 1

Effect of TNF-α on thrombus formation in vivo. Thrombus formation in response to vascular injury was visualized in arterioles of control (upper) or TNF-α–treated mice (lower). The different lengths of time after ferric chloride application are indicated. No significant difference in initial platelet adhesion to the injured vessel was observed between the two groups (3 minutes). The appearance of thrombi was delayed by TNF-α infusion (8 minutes), however. In addition, thrombi in treated mice were unstable and often did not grow to occlusive size, leaving, after 14 minutes, a still patent vessel, whereas the control arterioles occluded. n = 12–15 mice.

Figure 2

Quantitative analysis of arterial thrombogenesis in WT, TNF-R1/2^–/–, L-NMMA–treated, and iNOS^–/– mice. Two minutes after the injury, the number of platelet-vessel wall interactions in each group of mice was comparable between the TNF-α–treated and untreated animals (a–d). In contrast to the antithrombotic effect measured in WT mice (a), TNF-α did not significantly affect thrombus formation and vessel occlusion in TNF-R1/2^–/– (b), L –NMMA–treated (c), and iNOS^–/– mice (d). Compared with WT mice, iNOS^–/– mice exhibited a significant increase in platelet deposition (P < 0.02) and a slightly shorter time in thrombus formation (P < 0.05) and in vessel occlusion (P < 0.03). n = 7–10 mice per group. *P < 0.05; **P < 0.005 versus untreated WT.

Figure 3

Tail bleeding time and plasma clotting time in TNF-α–treated mice. Mice 12–16 weeks old were infused with PBS or TNF-α. (a) The bleeding time was measured after amputation of a 3-mm portion of the tail. The mice injected with TNF-α (filled circles) exhibited a fourfold prolonged bleeding time compared with control mice (open circles) (P < 0.003). (b) Plasma clotting time was measured in an aggregometer after adding CaCl₂ solution. The clotting time was significantly shorter in mice treated with 1 ng/ml of TNF-α (black bar) than in control mice (white bar). P < 0.001, n = 8 mice per group.

Figure 4

Effect of in vivo and in vitro treatments with TNF-α on platelet function. PBS (white bars) or TNF-α (black bars) were administered for 30 minutes either to whole blood samples (a, c, and e) or by intravenous injection into mice (b, d, and f). PRP was prepared, and platelets were tested for aggregation in response to 2 μM ADP (e and f). To study platelet activation by flow cytometry, platelets were washed, activated for 5 minutes with thrombin (0.1 U/ml) or CRP (2 μg/ml), and incubated for 10 minutes at 37°C with FITC-conjugated Ab’s against human fibrinogen (a and b) or a FITC-conjugated mAb against P-selectin (c and d). (g and h) Shown are representative results of TNF-α systemic administration on fibrinogen binding and platelet aggregation. n = 6 mice per group. *P < 0.05; **P < 0.005. MFI, mean fluorescence intensity.