Objective: 

Inflammation and thrombosis are closely linked and elevated serum levels of the proinflammatory cytokine TNFa correlate with an increased risk for atherothrombotic diseases. It is still unclear, however, how TNFa influences arterial thrombosis in vivo and what role the TNF-receptor-subtypes play in this context. We investigated the effect of TNFa on arteriolar thrombus formation and platelet-endothelium-interaction in vivo in wildtyp- (WT) and TNFR-deficient mice.

Methods: 

We measured time to thrombotic vessel occlusion upon vessel injury by ferric chloride and analyzed rolling of fluorescently labeled platelets on the endothelium by intravital microscopy in the dorsal skinfold chamber model. In cultured human endothelial cells (HUVEC, HMEC) we investigated the influence of TNFa on O₂^- formation (cytochrom C-assay), translocation of the p65-subunit of NF-kB, the upregulation of cell surface molecules (immunofluorescence) and PAI-1 (RT-PCR). Ex vivo we performed platelet aggregation studies in platelet rich plasma (PRP).

Results: 

In WT-mice TNFa-stimulation (5ng/ml, 4h; dosage for all experiments) led to a significant decrease of thrombotic vessel occlusion time in vivo, which in TNFR1-/- animals was even shorter. Platelet-interaction with the endothelium after TNFa-stimulation was increased only in TNFR1-/- mice, resulting in a greater amount of rolling platelets than in WT-mice. These effects could not be shown in TNFR2-/- and TNFR1-/-2-/- mice. In vitro we measured a significant increase of endothelial O₂^- release after TNFa-stimulation, translocation of the p65-subunit of NFkB to the nucleus, as well as an upregulation of p-selectin and tissue factor on endothelial cell surface. TNFa also led to an increase in mRNA levels of PAI-1. ADP- (20mM) or collagen- (10mg/ml) induced platelet aggregation in PRP was not altered by TNFa.

Conclusion: 

TNFa exerts prothrombotic effects in vivo, which are not due to a direct influence on platelets but rather mediated through endothelial mechanisms. TNFR2 is required for the prothrombotic properties, while signaling through TNFR1 seems to compensate, indicating very distinctive roles for the TNF-receptor subtypes in this context.