M. R. Dyer1, Q. Chen1, A. Gutierrez1, B. Zuckerbraun1, M. Neal1 1University Of Pittsburgh,Surgery,Pittsburgh, PA, USA
Introduction: Deep vein thrombosis (DVT) is a common and highly morbid complication following trauma. The damage-associated molecular pattern molecule, high-mobility group box-1 (HMGB1) has been shown to be a key mediator of the inflammatory response following trauma. In pro-inflammatory states neutrophils can be stimulated to release their nuclear content including DNA, histones, and granule contents to form what is referred to as neutrophil extracellular traps (NETs). NETs have been shown to be pro-thrombotic and are implicated in the development of DVT. Recently platelet-derived HMGB1 was found to be a key mediator of microvascular thrombosis and formation of NETs in the lung following trauma. We hypothesized that HMGB1 released from activated platelets would promote DVT formation via NET production.
Methods: We created transgenic mice lacking HMGB1 specifically on platelets (HMGB1 PF4), and subjected them to IVC ligation to induce DVT formation, with comparison to control (HMGB1 Flox). Recombinant HMGB1 or RNase free DNase was administered via tail vein injection 30 minutes prior to IVC ligation in some experiments. Clots were harvested at 24 and 72 hours following IVC ligation, quantified by length and weight, and analyzed for NET expression by immunofluorescence (IF). Blood was obtained by cardiac puncture to determine HMGB1 circulating levels.
Results: HMGB1 levels are increased following DVT formation compared to sham (32 vs. 10.8 ng/ml) and platelets are the predominant source of HMGB1 during DVT (HMGB1 Flox 44.3 vs. HMGB1 PF4 16.3 ng/ml, p<0.05). Recombinant HMGB1 increased clot burden at 24 hours compared to control (25.3 vs 11.6 mg, p<0.05). HMGB1 PF4 mice have decreased thrombus formation at 24 hours (HMGB1 Flox 12.9 vs. HMGB1 PF4 7.4 mg, p<0.05) and 72 hours (HMGB1 Flox 32.6 vs. HMGB1 PF4 19.5 mg, p<0.05). IF staining of harvested clots for citrullinated histone H3, a marker of NETs, demonstrated significantly less NETs in HMGB1 PF4 mice (HMGB1 Flox 2291 vs. HMGB1 PF4 126, p<0.05), indicating a role for platelet HMGB1 in NET formation. Treatment with DNase, which degrades NETs, eliminated the difference in clot burden between HMGB1 PF4 and HMGB1 Flox mice (9 vs. 11 mg, p=0.18).
Conclusion: HMGB1 is increased following DVT and platelets are the significant source of circulating HMGB1. Platelet released HMGB1 increases thrombus burden via formation of NETs in a murine model of DVT. These data provide a potential critical link between inflammation and post-trauma thrombotic complications.