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 developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. We validate FCFM on soft polyacrylamide gels. We find that the creep behavior of living cells conforms to a power-law material model. By recording short (50-60 ms) force clamp measurements in rapid succession, we generate, for the first time, two-dimensional maps of power-law exponent and modulus scaling parameter. Although these maps reveal large spatial variations of both parameters across the cell surface, we obtain robust mean values from the several hundreds of measurements performed on each cell. Measurements on mouse embryonic fibroblasts show that the mean power-law exponents and the mean modulus scaling parameters differ greatly among individual cells, but both parameters are highly correlated: stiffer cells consistently show a smaller power-law exponent. This correlation allows us to distinguish between wild-type cells and cells that lack vinculin, a dominant protein of the focal adhesion complex, even though the mean values of viscoelastic properties between wildtype and knockout cells did not differ significantly. Therefore, FCFM spatially resolves viscoelastic sample properties and can uncover subtle mechanical signatures of proteins in living cells.

Scanning ion conductance microscopy ͑SICM͒ is an emerging tool for the noncontact investigation of biological samples such as live cells. It uses an ion current through the opening of a tapered nanopipette filled with an electrolyte for topography measurements. Despite its successful application to numerous systems no systematic investigation of the image formation process has yet been performed. Here, we use finite element modeling to investigate how the scanning ion conductance microscope images small particles on a planar surface, providing a fundamental characterization of the imaging process. We find that a small particle appears with a height that is only a fraction of its actual height. This has significant consequences for the quantitative interpretation of SICM images. Furthermore, small and low particles are imaged as rings in certain cases. This can cause small, closely spaced particles to appear with a lateral orientation that is rotated by 90°. Considering both real space and spatial frequency space we find that a reasonable and useful definition of lateral resolution of SICM is the smallest distance at which two small particles can clearly be resolved from each other in an image. We find that this resolution is approximately equal to three times the inner radius of the pipette tip opening.

An altered platelet lipidome might be associated with thrombotic disposition in CAD, a mechanism potentially regulated by CXCL12-CXCR4-CXCR7 axis.

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.

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Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.

Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell's elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM.