Objective-Platelet-and fibrin-dependent thrombus formation is regulated by blood flow and exposure of collagen and tissue factor. However, interactions between these blood-borne and vascular components are not well understood. Approach and Results-Here, we developed a method to assess whole-blood thrombus formation on microspots with defined amounts of collagen and tissue factor, allowing determination of the mechanical properties and intrathrombus composition. Confining the collagen content resulted in diminished platelet deposition and fibrin formation at high shear flow conditions, but this effect was compensated by a larger thrombus size and increased accumulation of fibrin in the luminal regions of the thrombi at the expense of the base regions. These thrombi were more dependent on tissue factortriggered thrombin generation. Microforce nanoindentation analysis revealed a significantly increased microelasticity of thrombi with luminal-oriented fibrin. At a low shear rate, fibrin fibers tended to luminally cover the thrombi, again resulting in a higher microelasticity. Studies with blood from patients with distinct hemostatic insufficiencies indicated an impairment in the formation of a platelet-fibrin thrombus in the cases of dilutional coagulopathy, thrombocytopenia, Scott syndrome, and hemophilia B. Conclusions-Taken together, our data indicate that (1) thrombin increases the platelet thrombus volume; (2) tissue factor drives the formation of fibrin outside of the platelet thrombus; (3) limitation of platelet adhesion redirects fibrin from bottom to top of the thrombus; (4) a lower shear rate promotes thrombus coverage with fibrin; (5) the fibrin distribution pattern determines thrombus microelasticity; and (6) the thrombus-forming process is reduced in patients with diverse hemostatic defects. Swieringa et al Platelet Control of Fibrin Distribution in Thrombus Formation 693Together, these in vivo findings indicate that the relative availability of platelet-adhesive (like collagen) and coagulation-triggering (like TF) substances determines the buildup of a thrombus and, by implication, the roles of thrombin and fibrin herein. In support of this idea, different patterns of fibrin deposition have been detected in injury models, such as fibrin structures around a thrombus, 16 and thrombin/fibrin-rich areas in the thrombus core region. 17,18 How the platelet and coagulant activities of the thrombogenic surface, for example, the relative abundance of collagen and TF, influence the buildup and composition of a platelet-fibrin thrombus has not been examined so far.Viscoelasticity is considered to be an important hemostatic property of a fibrin-containing thrombus. For instance, in patients with severe factor XI deficiency, a low-density fibrin network is associated with impaired hemostasis. 19,20 However, the conditions that favor high elasticity of a platelet-fibrin thrombus or clot are largely unknown. The limited evidence available suggests that a local, high concentration of thrombin enforces the fibrin net...

Nano-indentation is widely used for probing the micromechanical properties of materials. Based on the indentation of surfaces using probes with a well-defined geometry, the elastic and viscoelastic constants of materials can be determined by relating indenter geometry and measured load and displacement to parameters which represent stress and deformation. Here we describe a method to derive the viscoelastic properties of soft hydrated materials at the micro-scale using constant strain rates and stress-free initial conditions. Using a new self-consistent definition of indentation stress and strain and corresponding unique depth-independent expression for indentation strain rate, the epsilon dot method, which is suitable for bulk compression testing, is transformed to nano-indentation. We demonstrate how two materials can be tested with a displacement controlled commercial nano-indentor using the nano-espilon dot method (nano-ε̇M) to give values of instantaneous and equilibrium elastic moduli and time constants with high precision. As samples are tested in stress-free initial conditions, the nano-ε̇M could be useful for characterising the micro-mechanical behaviour of soft materials such as hydrogels and biological tissues at cell length scales.

AimsA single isolated cardiomyocyte is the smallest functional unit of the heart. Yet, all single isolated cardiomyocyte experiments have been limited by the lack of proper methods that could reproduce a physiological cardiac cycle. We aimed to investigate the contractile properties of a single cardiomyocyte that correctly mimic the cardiac cycle.Methods and resultsBy adjusting the parameters of the feedback loop, using a suitably engineered feedback system and recording the developed force and the length of a single rat cardiomyocyte during contraction and relaxation, we were able to construct force–length (FL) relations analogous to the pressure–volume (PV) relations at the whole heart level. From the cardiac loop graphs, we obtained, for the first time, the power generated by one single cardiomyocyte.ConclusionHere, we introduce a new approach that by combining mechanics, electronics, and a new type optical force transducer can measure the FL relationship of a single isolated cardiomyocyte undergoing a mechanical loop that mimics the PV cycle of a beating heart.

Micropipette aspiration (MPA) is an essential tool in mechanobiology; however, its potential is far from fully exploited. The traditional MPA technique has limited temporal and spatial resolution and requires extensive post processing to obtain the mechanical fingerprints of samples. Here, we develop a MPA system that measures pressure and displacement in real time with sub-nanometer resolution thanks to an interferometric readout. This highly sensitive MPA system enables studying the nanoscale behavior of soft biomaterials under tension and their frequency-dependent viscoelastic response.