Abstract 841: Dual Modulation of Tissue-Factor and Tissue-Factor-Pathway-Inhibitor by Neutrophil Proteases
Background and Aim of the study: Growing evidence suggests inflammation as a crucial component for plaque thrombogenicity and destabilization. Neutrophils, as part of the inflammatory infiltrate, accumulate especially at rupture sites and have been linked to acute coronary syndromes by epidemiological studies. Although within the unstable plaque the lipid-rich core expresses the highest amounts of Tissue factor (TF), the main activator of the extrinsic coagulation cascade, expression of the endogen antagonist Tissue-factor-pathway-Inhibitor-1 (TFPI-1) can hardly be detected in these areas. We therefore sought to investigate if proteases released by human neutrophils can influence the TF/TFPI complex on this note.
Methods and Results: Cultured human umbilical endothelial cells (HUVEC‘s) were incubated with human elastase (HNE), cathepsin G (CG) or proteinase 3 (PR3) in vitro. HNE and CG selectively degraded TFPI on cultured endothelial cells (FACS analysis) and thereby increased their procoagulatoric activity (Factor Xa generation assay). In contrast PR3 did not degrade surface bound TFPI on endothelial cells, but induced full-length and alternatively spliced TF as well as IL-8 expression on mRNA and protein level - a effect not seen after CG or HNE stimulation. As serine proteases are known to activate proteinase-activated-receptors (PAR) we further analysed the influence of PAR-1 and PAR-2 on PR3 induced TF and IL-8 expression. Using site specific antibodies we identified cleavage of PAR-1 by PR3 suggesting PAR-1 activation. Gene silencing of PAR-1 by small interfering RNA hindered PR3 induced TF and IL-8 mRNA expression, indicating a predominant role of PAR-1 in the PR3 mediated thrombotic and inflammatory effects. All three neutrophil proteases induced the release of apoptotic, TF bearing microparticles by endothelial cells in vitro (FACS), thereby further enhancing the prothrombotic and inflammatory milieu.
Conclusion: This dual regulation of the TF/TFPI pathway by neutrophil proteases may therefore contribute to the increased thrombogenicity of the atherosclerotic plaque and might illustrate how neutrophils influence plaque progression.