In vitro modeling of the microvascular occlusion and thrombosis that occur in hematologic diseases using microfluidic technology.

In hematologic diseases, such as sickle cell disease (SCD) and hemolytic uremic syndrome (HUS), pathological biophysical interactions among blood cells, endothelial cells, and soluble factors lead to microvascular occlusion and thrombosis. Here, we report an in vitro "endothelialized" microfluidic microvasculature model that recapitulates and integrates this ensemble of pathophysiological processes. Under controlled flow conditions, the model enabled quantitative investigation of how biophysical alterations in hematologic disease collectively lead to microvascular occlusion and thrombosis. Using blood samples from patients with SCD, we investigated how the drug hydroxyurea quantitatively affects microvascular obstruction in SCD, an unresolved issue pivotal to understanding its clinical efficacy in such patients. In addition, we demonstrated that our microsystem can function as an in vitro model of HUS and showed that shear stress influences microvascular thrombosis/obstruction and the efficacy of the drug eptifibatide, which decreases platelet aggregation, in the context of HUS. These experiments establish the versatility and clinical relevance of our microvasculature-on-a-chip model as a biophysical assay of hematologic pathophysiology as well as a drug discovery platform.

Biophysical investigations of engineered polyproteins: implications for force data.

Dynamic force spectroscopy is rapidly becoming a standard biophysical technique. Significant advances in the methods of analysis of force data have resulted in ever more complex systems being studied. The use of cloning systems to produce homologous tandem repeats rather than the use of endogenous multidomain proteins has facilitated these developments. What is poorly addressed are the physical properties of these constructed polyproteins. Are the properties of the individual domains in the construct independent of one another or attenuated by adjacent domains? We present data for a construct of eight fibronectin type III domains from the human form of tenascin that exhibits approximately 1 kcal mol(-1) increase in stability compared to the monomer. This effect is salt and pH dependent, suggesting that the stabilization results from electrostatic interactions, possibly involving charged residues at the interfaces of the domains. Kinetic analysis shows that this stabilization reflects a slower unfolding rate. Clearly, if domain-domain interactions affect the unfolding force, this will have implications for the comparison of absolute forces between types of domains. Mutants of the tenascin 8-mer construct exhibit the same change in stability as that observed for the corresponding mutation in the monomer. And when Phi-values are calculated for the 8-mer construct, the pattern is similar to that observed for the monomer. Therefore, mutational analyses to resolve mechanical unfolding pathways appear valid. Importantly, we show that interactions between the domains may be masked by changes in experimental conditions.