Platelet-derived HMGB1 is a critical mediator of thrombosis
Sebastian Vogel, … , Meinrad Gawaz, Matthew D. Neal
Sebastian Vogel, … , Meinrad Gawaz, Matthew D. Neal
Published November 9, 2015
Citation Information: J Clin Invest. 2015;125(12):4638-4654. https://doi.org/10.1172/JCI81660.
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Research Article Hematology

Platelet-derived HMGB1 is a critical mediator of thrombosis

Abstract

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Authors

Sebastian Vogel, Rebecca Bodenstein, Qiwei Chen, Susanne Feil, Robert Feil, Johannes Rheinlaender, Tilman E. Schäffer, Erwin Bohn, Julia-Stefanie Frick, Oliver Borst, Patrick Münzer, Britta Walker, Justin Markel, Gabor Csanyi, Patrick J. Pagano, Patricia Loughran, Morgan E. Jessup, Simon C. Watkins, Grant C. Bullock, Jason L. Sperry, Brian S. Zuckerbraun, Timothy R. Billiar, Michael T. Lotze, Meinrad Gawaz, Matthew D. Neal

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Figure 1

Platelet-derived HMGB1 promotes platelet aggregation and thrombus formation.

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Platelet-derived HMGB1 promotes platelet aggregation and thrombus format...
(A) Hmgb1 Pf4 mice have prolonged bleeding time compared with Hmgb1 Flox control mice. (B) PT, (C) aPTT, and (D) TT are not altered in Hmgb1 Pf4 mice as compared with control mice. (E and F) Reduction in collagen-induced platelet aggregation in Hmgb1 Pf4 mice. (G) Blood derived from Hmgb1 Pf4 mice is less thrombogenic in a flow chamber system. Exogenous HMGB1 increases thrombus formation in blood from both Hmgb1 Pf4 and Hmgb1 Flox mice. (H–J) FeCl3-induced thrombus formation is inhibited in Hmgb1 Pf4 mice, with (H) improved blood flow and (I) prolonged time to vessel occlusion (as measured by laser Doppler imaging, quantified in J). (K) Immunofluorescence staining of thrombi from Hmgb1 Flox control mice demonstrates large clusters of CD41-positive platelets and surrounding deposition of HMGB1 overlying a fibrinogen network. Thrombi from Hmgb1 Pf4 animals show smaller clusters without HMGB1 expression. Scale bar: 100 μm (top row), 40 μm (bottom row). (L) Ly6G-positive immune cell infiltrates and citH3-positive NETs detected in FeCl3-induced thrombi from Hmgb1 Flox mice but not Hmgb1 Pf4 mice. Scale bar: 100 μm (top row), 40 μm (bottom row). (M) Western blot of clots isolated from Hmgb1 Pf4 mice reveals almost no expression of HMGB1 as compared with control. Data show mean ± SD from at least 3 separate experiments and (B–D) n = 3, (G, H, and J) n = 4, (F) n = 5, and (A) n = 12 mice per group. (E, I, and K–M) Representative images from at least 4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test in A–D, F, H, and J; 1-way ANOVA with Tukey’s post-hoc test in G).