Characterization of the Threshold Response of Initiation of Blood Clotting to Stimulus Patch Size

This article demonstrates that the threshold response of initiation of blood clotting to the size of a patch of stimulus is a robust phenomenon under a wide range of conditions and follows a simple scaling relationship based on the Damkohler number. Human blood and plasma were exposed to surfaces patterned with patches presenting clotting stimuli using microfluidics. Perturbations of the complex network of hemostasis, including temperature, variations in the concentration of stimulus (tissue factor), and the absence or inhibition of individual components of the network (factor IIa, factor V, factor VIII, and thrombomodulin), did not affect the existence of this response. A scaling relationship between the threshold patch size and the timescale of reaction for clotting was supported in numerical simulations, a simple chemical model system, and experiments with human blood plasma. These results may be useful for understanding the spatiotemporal dynamics of other autocatalytic systems and emphasize the relevance of clustering of proteins and lipids in the regulation of signaling processes

Using chemistry and microfluidics to understand the spatial dynamics of complex biological networks

Understanding the spatial dynamics of biochemical networks is both fundamentally important for understanding life at the systems level and also has practical implications for medicine, engineering, biology, and chemistry. Studies at the level of individual reactions provide essential information about the function, interactions, and localization of individual molecular species and reactions in a network. However, analyzing the spatial dynamics of complex biochemical networks at this level is difficult. Biochemical networks are non-equilibrium systems containing dozens to hundreds of reactions with nonlinear and time-dependent interactions, and these interactions are influenced by diffusion, flow, and the relative values of state-dependent kinetic parameters. To achieve an overall understanding of the spatial dynamics of a network and the global mechanisms that drive its function, networks must be analyzed as a whole, where all of the components and influential parameters of a network are simultaneously considered. Here, we describe chemical concepts and microfluidic tools developed for network-level investigations of the spatial dynamics of these networks. Modular approaches can be used to simplify these networks by separating them into modules, and simple experimental or computational models can be created by replacing each module with a single reaction. Microfluidics can be used to implement these models as well as to analyze and perturb the complex network itself with spatial control on the micrometer scale. We also describe the application of these network-level approaches to elucidate the mechanisms governing the spatial dynamics of two networks-hemostasis (blood clotting) and early patterning of the Drosophila embryo. To investigate the dynamics of the complex network of hemostasis, we simplified the network by using a modular mechanism and created a chemical model based on this mechanism by using microfluidics. Then, we used the mechanism and the model to predict the dynamics of initiation and propagation of blood clotting and tested these predictions with human blood plasma by using microfluidics. We discovered that both initiation and propagation of clotting are regulated by a threshold response to the concentration of activators of clotting, and that clotting is sensitive to the spatial localization of stimuli. To understand the dynamics of patterning of the Drosophila embryo, we used microfluidics to perturb the environment around a developing embryo and observe the effects of this perturbation on the expression of Hunchback, a protein whose localization is essential to proper development. We found that the mechanism that is responsible for Hunchback positioning is asymmetric, time-dependent, and more complex than previously proposed by studies of individual reactions. Overall, these approaches provide strategies for simplifying, modeling, and probing complex networks without sacrificing the functionality of the network. Such network-level strategies may be most useful for understanding systems with non-linear interactions where spatial dynamics is essential for function. In addition, microfluidics provides an opportunity to investigate the mechanisms responsible for robust functioning of complex networks. By creating nonideal, stressful, and perturbed environments, microfluidic experiments could reveal the function of pathways thought to be nonessential under ideal conditions

Effects of shear rate on propagation of blood clotting determined using microfluidics and numerical simulations

This paper describes microfluidic experiments with human blood plasma and numerical simulations to determine the role of fluid flow in the regulation of propagation of blood clotting. We demonstrate that propagation of clotting can be regulated by different mechanisms depending on the volume-to-surface ratio of a channel. In small channels, propagation of clotting can be prevented by surface-bound inhibitors of clotting present on vessel walls. In large channels, where surface-bound inhibitors are ineffective, propagation of clotting can be prevented by a shear rate above a threshold value, in agreement with predictions of a simple reaction-diffusion mechanism. We also demonstrate that propagation of clotting in a channel with a large volume-to-surface ratio and a shear rate below a threshold shear rate can be slowed by decreasing the production of thrombin, an activator of clotting. These in vitro results make two predictions, which should be experimentally tested in vivo. First, propagation of clotting from superficial veins to deep veins may be regulated by shear rate, which might explain the correlation between superficial thrombosis and the development of deep vein thrombosis (DVT). Second, nontoxic thrombin inhibitors with high binding affinities could be locally administered to prevent recurrent thrombosis after a clot has been removed. In addition, these results demonstrate the utility of simplified mechanisms and microfluidics for generating and testing predictions about the dynamics of complex biochemical networks

Propagation of blood clotting in the complex biochemical network of hemostasis is described by a simple mechanism

Hemostasis is the complex biochemical network that controls blood clotting. We previously described a chemical model that mimicked the dynamics of hemostasis based on a simple regulatory mechanisma threshold response due to the competition between production and removal of activators. Here, we used human blood plasma in phospholipid-coated microfluidic channels to test predictions based on this mechanism. We demonstrated that, for a given geometry of channels, clot propagation from an obstructed channel into a channel with flowing blood plasma is dependent on the shear rate in the channel with flowing blood plasma. If confirmed in vivo, these results may explain clot propagation from a small vessel to a larger, clinically relevant vessel. In addition, these results would further validate the use of modular mechanisms, simplified chemical models, and microfluidics to study complex biochemical networks

Levosimendan may improve survival in patients requiring mechanical assist devices for post-cardiotomy heart failure

INTRODUCTION: Most case series suggest that less than half of the patients receiving a mechanical cardiac assist device as a bridge to recovery due to severe post-cardiotomy heart failure survive to hospital discharge. Levosimendan is the only inotropic substance known to improve medium term survival in patients suffering from severe heart failure. METHODS: This retrospective analysis covers our single centre experience. Between July 2000 and December 2004, 41 consecutive patients were treated for this complication. Of these, 38 patients are included in this retrospective analysis as 3 patients died in the operating room. Levosimendan was added to the treatment protocol for the last nine patients. RESULTS: Of 29 patients treated without levosimendan, 20 could be weaned off the device, 9 survived to intensive care unit discharge, 7 left hospital alive and 3 survived 180 days. All 9 patients treated with levosimendan could be weaned, 8 were discharged alive from ICU and hospital, and 7 lived 180 days after surgery (p < 0.002 for 180 day survival). Plasma lactate after explantation of the device was significantly lower (p = 0.002), as were epinephrine doses. Time spent on renal replacement therapy was significantly shorter (p = 0.023). CONCLUSION: Levosimendan seems to improve medium term survival in patients failing to wean off cardiopulmonary bypass and requiring cardiac assist devices as a bridge to recovery. This retrospective analysis justifies prospective randomised investigations of levosimendan in this group of patients

Remotely Activated Protein-Producing Nanoparticles

The development of responsive nanomaterials, nanoscale systems that actively respond to stimuli, is one general goal of nanotechnology. Here we develop nanoparticles that can be controllably triggered to synthesize proteins. The nanoparticles consist of lipid vesicles filled with the cellular machinery responsible for transcription and translation, including amino acids, ribosomes, and DNA caged with a photolabile protecting group. These particles served as nanofactories capable of producing proteins including green fluorescent protein (GFP) and enzymatically active luciferase. In vitro and in vivo, protein synthesis was spatially and temporally controllable, and could be initiated by irradiating micrometer-scale regions on the time scale of milliseconds. The ability to control protein synthesis inside nanomaterials may enable new strategies to facilitate the study of orthogonal proteins in a confined environment and for remotely activated drug delivery.National Cancer Institute (U.S.) (MIT-Harvard Center for Cancer Nanotechnology Excellence Grant U54 CA151884)Marie D. and Pierre Casimir-Lambert FundNational Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Grant EB000244

Stem cell membrane engineering for cell rolling using peptide conjugation and tuning of cell–selectin interaction kinetics

Dynamic cell–microenvironment interactions regulate many biological events and play a critical role in tissue regeneration. Cell homing to targeted tissues requires well balanced interactions between cells and adhesion molecules on blood vessel walls. However, many stem cells lack affinity with adhesion molecules. It is challenging and clinically important to engineer these stem cells to modulate their dynamic interactions with blood vessels. In this study, a new chemical strategy was developed to engineer cell–microenvironment interactions. This method allowed the conjugation of peptides onto stem cell membranes without affecting cell viability, proliferation or multipotency. Mesenchymal stem cells (MSCs) engineered in this manner showed controlled firm adhesion and rolling on E-selectin under physiological shear stresses. For the first time, these biomechanical responses were achieved by tuning the binding kinetics of the peptide-selectin interaction. Rolling of engineered MSCs on E-selectin is mediated by a Ca[superscript 2+] independent interaction, a mechanism that differs from the Ca[superscript 2+] dependent physiological process. This further illustrates the ability of this approach to manipulate cell–microenvironment interactions, in particular for the application of delivering cells to targeted tissues. It also provides a new platform to engineer cells with multiple functionalities.National Heart, Lung, and Blood Institute (Programs of Excellence in Nanotechnology Award Contract HHSN268201000045C)National Institutes of Health (U.S.) (Grant 2-P30-CA14051)Armed Forces Institute of Regenerative Medicine (Award W81XWH-08-2-0034

Angiogenesis PET Tracer Uptake (⁶⁸Ga-NODAGA-E[(cRGDyK)]₂) in Induced Myocardial Infarction and Stromal Cell Treatment in Minipigs

Angiogenesis is considered integral to the reparative process after ischemic injury. The αvβ3 integrin is a critical modulator of angiogenesis and highly expressed in activated endothelial cells. 68Ga-NODAGA-E[(cRGDyK)]2 (RGD) is a positron-emission-tomography (PET) ligand targeted towards αvβ3 integrin. The aim was to present data for the uptake of RGD and correlate it with histology and to further illustrate the differences in angiogenesis due to porcine adipose-derived mesenchymal stromal cell (pASC) or saline treatment in minipigs after induction of myocardial infarction (MI). Three minipigs were treated with direct intra-myocardial injection of pASCs and two minipigs with saline. MI was confirmed by 82Rubidium (82Rb) dipyridamole stress PET. Mean Standardized Uptake Values (SUVmean) of RGD were higher in the infarct compared to non-infarct area one week and one month after MI in both pASC-treated (SUVmean: 1.23 vs. 0.88 and 1.02 vs. 0.86, p &lt; 0.05 for both) and non-pASC-treated minipigs (SUVmean: 1.44 vs. 1.07 and 1.26 vs. 1.04, p &lt; 0.05 for both). However, there was no difference in RGD uptake, ejection fractions, coronary flow reserves or capillary density in histology between the two groups. In summary, indications of angiogenesis were present in the infarcted myocardium. However, no differences between pASC-treated and non-pASC-treated minipigs could be demonstrated