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.

We have used a simple process to fabricate small rectangular cantilevers out of silicon nitride. They have lengths of 9–50 μm, widths of 3–5 μm, and thicknesses of 86 and 102 nm. We have added metallic reflector pads to some of the cantilever ends to maximize reflectivity while minimizing sensitivity to temperature changes. We have characterized small cantilevers through their thermal spectra and show that they can measure smaller forces than larger cantilevers with the same spring constant because they have lower coefficients of viscous damping. Finally, we show that small cantilevers can be used for experiments requiring large measurement bandwidths, and have used them to unfold single titin molecules over an order of magnitude faster than previously reported with conventional cantilevers.

An atomic force microscope (AFM) design providing a focused spot of order 7 μm in diameter was used to analyze the motion of vibrating cantilevers in liquid. Picking an operating frequency for tapping mode AFM operation in liquid is complex because there is typically a large number of sharp peaks in the response spectrum of cantilever slope amplitude versus drive frequency. The response spectrum was found to be a product of the cantilever’s broad thermal noise spectrum and an underlying fluid drive spectrum containing the sharp peaks. The geometrical shape of transverse cantilever motion was qualitatively independent of the fluid drive spectrum and could be approximately reproduced by a simple theoretical model. The measurements performed give new insights into the behavior of cantilevers during tapping mode AFM operation in liquid.

Small cantilevers allow for faster imaging and faster force spectroscopy of single biopolymers than previously possible because they have higher resonant frequencies and lower coefficients of viscous damping. We have used a new prototype atomic force microscope with small cantilevers to produce stable tapping-mode images (1 μm×1 μm) in liquid of DNA adsorbed onto mica in as little as 1.7 s per image. We have also used these cantilevers to observe the forced unfolding of individual titin molecules on a time scale an order of magnitude faster than previously reported. These experiments demonstrate that a new generation of atomic force microscopes using small cantilevers will enable us to study biological processes with greater time resolution. Furthermore, these instruments allow us to narrow the gap in time between results from force spectroscopy experiments and molecular dynamics calculations.