OC 29.2 - Neutrophil Extracellular Traps: shaping fibrinolysis and fibrin structure in vivo and ex vivo

Background: The fibrin matrix of venous and arterial thrombi is intertwined with neutrophil extracellular traps (NETs) composed of DNA, histones and proteins expelled by neutrophils. Certain routes of NET formation require peptidyl-arginine deiminase-4 (PAD4) activity, whereas the leukocyte elastase inhibitor SERPINB1 suppresses NET-formation. While the in vitro effects of individual NET components on fibrin structure and lysis have been extensively explored, our understanding of their combined impact is still limited.

Aims: Assessing the effect of NETs on fibrin structure and fibrinolysis in vivo and ex vivo.

Methods: Deep vein thrombosis was modelled in murine strains with different capacity to form NETs (wild-type (WT), PAD4-/-, Serpinb1-/-)applying inferior vena cava stenosis. Thrombi were extracted after 1 to 4 days, weighed, and fibrin structure was examined with scanning electron microscopy (SEM). Lysis of whole-blood clots was measured in a viscoelastic assay (ClotPro). Histone 3 (H3) was citrullinated with PAD4 and its binding to plasminogen measured in an immunosorbent assay. White blood cells (WBCs) for in vitro assays were isolated from bone marrow.

Results: Odds for thrombus formation was 61% lower in PAD4-/-mice compared to WT (OR 0.39;95% CI 0.17-0.94), but no significant difference was observed between Serpinb1-/- and WT mice. Thrombus weight peaked on day-3 in WT and PAD4-/-mice, on day-4 clots lysed. PAD4-/-thrombi weighed significantly less on day-3 and day-4 than WT thrombi. Treatment with N-formylmethionine-leucyl-proline retarded ClotPro lysis of WT and Serpinb1-/-clots(34.2±8.0min to 56.4±18.8min and 36.7±11.1min to 55.5±17.1min,n=6), but not PAD4-/-clots. Plasminogen activation was accelerated by PAD4-/-WBC, but not by cells from other mice. H3 citrullination enhanced the plasminogen-H3 binding. SEM measurements on day-3 revealed that significantly thinner fibrin fibers in PAD4-/-mice than WT and Serpinb1-/-mice, on day-4 this trend was reversed in PAD4-/-mice.

Conclusion(s): Suppressed histone citrullination in WBC enhances plasminogen activation ex vivo and restricts thrombus growth in murine model of venous thrombosis.