Influence of the platelet-derived DAMP HMGB1 on thrombosis and inflammation
Final Report Abstract
High-mobility group box 1 (HMGB1) is a nuclear protein expressed by mammalian cells, which acts as a "damage associated molecular pattern" molecule (DAMP) and plays a critical role in sterile inflammation when released into extracellular space. Despite lacking a nucleus, platelets express HMGB1. The purpose of this project was to investigate the role of platelet-derived HMGB1 in platelet function and induction of inflammation. In collaboration with University of Pittsburgh, USA we generated and investigated transgenic mice with platelet-specific ablation of HMGB1 (PF4-Cre, HMGB1-flox/flox; termed HMGB1 PF4 mice). In mice whose platelets were lacking HMGB1, tail vein bleeding time was significantly prolonged and platelet aggregation was significantly decreased, whereas thrombin time, prothrombin time, and activated partial thromboplastin time were not significantly altered. We have identified platelet-derived HMGB1 as a critical mediator of thrombosis. In a trauma/hemorrhagic shock mouse model consisting of soft tissue injury, bone pseudofracture, hemorrhage, and liver crush and in trauma patients, we have shown that platelet-specific HMGB1 plays a critical role in initiating thrombo-inflammatory events such as microvascular thrombosis and the formation of neutrophil extracellular traps (NETs) in organs. Imaging of live platelets at the singlecell level using scanning ion conductance microscopy (SICM) revealed that platelet adhesion and spreading on collagen or vWF was increased by HMGB1. HMGB1 exerted its effect on platelets via a hitherto unrecognized non-canonical HMGB1/TLR4-driven signaling pathway in platelets that involves complex formation between MyD88 and the guanylate cyclase (GC) and regulates enzymatic activity of GC in platelets. We have also investigated the effect of platelet-derived HMGB1 on monocytes and found that HMGB1 derived from platelets promotes recruitment of monocytes and prevents monocytes from undergoing apoptosis via activation of RAGE/TLR4. During experimental trauma and hemorrhagic shock, infiltrated monocytes in the lung and liver were significantly attenuated in mice lacking HMGB1 in platelets. In an experimental myocardial infarction model, the recruitment of Mac-3- positive macrophages was markedly reduced upon intravenous injection of a neutralizing anti-HMGB1 antibody prior to transient ligation of the left anterior descending artery. In a prospective patient cohort, however, HMGB1 expression on the surface of circulating platelets was not significantly altered in patients with acute coronary syndrome at the time of percutaneous coronary intervention and no significant effect of platelet HMGB1 expression levels on outcome (composite of all-cause death and/or MI as well as single events) and cumulative event-free survival of patients was detected. We have also performed studies on the platelet NLRP3 inflammasome, an intracellular pattern recognition receptor involved in the regulation of inflammatory processes. Platelet NLRP3 regulated platelet activation, aggregation, and thrombus formation and was targetable via inhibition of Bruton Tyrosine Kinase (BTK). The platelet NLRP3 inflammasome was upregulated in an orthotopic pancreatic ductal adenocarcinoma mouse model and promoted tumor progression and affected survival of tumorbearing mice. Thus, platelet HMGB1 and NLRP3 are critical regulators of platelet function in platelet relevant diseases that are potentially targetable via MyD88/GC complex formation and BTK, respectively. Further studies will be needed to evaluate the role of platelet HMGB1 and NLRP3 signaling in disease states associated with abnormal coagulation and inflammation.
Publications
- (2015) Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factormediated tropism of mesenchymal stem cells for apoptotic cell death. Cell Death Differ, 22, 1219-1230
Vogel, S., Borger, V., Peters, C., Forster, M., Liebfried, P., Metzger, K., Meisel, R., Daubener, W., Trapp, T., Fischer, J.C., Gawaz, M. & Sorg, R.V.
(See online at https://doi.org/10.1038/cdd.2014.225) - (2015) Platelet-derived HMGB1 is a critical mediator of thrombosis. J Clin Invest, 125, 4638-4654
Vogel, S., Bodenstein, R., Chen, Q., Feil, S., Feil, R., Rheinlaender, J., Schaffer, T.E., Bohn, E., Frick, J.S., Borst, O., Munzer, P., Walker, B., Markel, J., Csanyi, G., Pagano, P.J., Loughran, P., Jessup, M.E., Watkins, S.C., Bullock, G.C., Sperry, J.L., Zuckerbraun, B.S., Billiar, T.R., Lotze, M.T., Gawaz, M. & Neal, M.D.
(See online at https://doi.org/10.1172/jci81660) - (2016) Platelet-derived high-mobility group box 1 promotes recruitment and suppresses apoptosis of monocytes. Biochem Biophys Res Commun, 478, 143-148
Vogel, S., Rath, D., Borst, O., Mack, A., Loughran, P., Lotze, M.T., Neal, M.D., Billiar, T.R. & Gawaz, M.
(See online at https://doi.org/10.1016/j.bbrc.2016.07.078) - (2017) HMGB1 Expression Level in Circulating Platelets is not Significantly Associated with Outcomes in Symptomatic Coronary Artery Disease. Cell Physiol Biochem, 43, 1627-1633
Rath, D., Geisler, T., Gawaz, M. & Vogel, S.
(See online at https://doi.org/10.1159/000482026) - (2017) Human NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase. J Allergy Clin Immunol.
Liu, X., Pichulik, T., Wolz, O.O., Dang, T.M., Stutz, A., Dillen, C., Delmiro Garcia, M., Kraus, H., Dickhofer, S., Daiber, E., Munzenmayer, L., Wahl, S., Rieber, N., Kummerle-Deschner, J., Yazdi, A., Franz-Wachtel, M., Macek, B., Radsak, M., Vogel, S., Schulte, B., Walz, J.S., Hartl, D., Latz, E., Stilgenbauer, S., Grimbacher, B., Miller, L., Brunner, C., Wolz, C. & Weber, A.N.
(See online at https://doi.org/10.1016/j.jaci.2017.01.017) - (2017) The NLRP3 inflammasome and bruton's tyrosine kinase in platelets co-regulate platelet activation, aggregation, and in vitro thrombus formation. Biochem Biophys Res Commun, 483, 230-236
Murthy, P., Durco, F., Miller-Ocuin, J.L., Takedai, T., Shankar, S., Liang, X., Liu, X., Cui, X., Sachdev, U., Rath, D., Lotze, M.T., Zeh, H.J., 3rd, Gawaz, M., Weber, A.N. & Vogel, S.
(See online at https://doi.org/10.1016/j.bbrc.2016.12.161) - (2019) The platelet NLRP3 inflammasome is upregulated in a murine model of pancreatic cancer and promotes platelet aggregation and tumor growth. Ann Hematol.
Boone, B.A., Murthy, P., Miller-Ocuin, J.L., Liang, X., Russell, K.L., Loughran, P., Gawaz, M., Lotze, M.T., Zeh, H.J., 3rd & Vogel, S.
(See online at https://doi.org/10.1007/s00277-019-03692-0)
