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.

Microenvironmental geometry guides platelet adhesion and spreading: a quantitative analysis at the single cell level.

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 µm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 µm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.

Spectrin domains lose cooperativity in forced unfolding.

Spectrin is a multidomain cytoskeletal protein, the component three-helix bundle domains are expected to experience mechanical force in vivo. In thermodynamic and kinetic studies, neighboring domains of chicken brain alpha-spectrin R16 and R17 have been shown to behave cooperatively. Is this cooperativity maintained under force? The effect of force on these spectrin domains was investigated using atomic force microscopy. The response of the individual domains to force was compared to that of the tandem repeat R1617. Importantly, nonhelical linkers (all-beta immunoglobulin domains) were used to avoid formation of nonnative helical linkers. We show that, in contrast to previous studies on spectrin repeats, only 3% of R1617 unfolding events gave an increase in contour length consistent with cooperative two-domain unfolding events. Furthermore, the unfolding forces for R1617 were the same as those for the unfolding of R16 or R17 alone. This is a strong indication that the cooperative unfolding behavior observed in the stopped-flow studies is absent between these spectrin domains when force is acting as a denaturant. Our evidence suggests that the rare double unfolding events result from misfolding between adjacent repeats. We suggest that this switch from cooperative to independent behavior allows multidomain proteins to maintain integrity under applied force.

Mechanical unfolding of TNfn3: the unfolding pathway of a fnIII domain probed by protein engineering, AFM and MD simulation.

Protein engineering Phi-value analysis combined with single molecule atomic force microscopy (AFM) was used to probe the molecular basis for the mechanical stability of TNfn3, the third fibronectin type III domain from human tenascin. This approach has been adopted previously to solve the forced unfolding pathway of a titin immunoglobulin domain, TI I27. TNfn3 and TI I27 are members of different protein superfamilies and have no sequence identity but they have the same beta-sandwich structure consisting of two antiparallel beta-sheets. TNfn3, however, unfolds at significantly lower forces than TI I27. We compare the response of these proteins to mechanical force. Mutational analysis shows that, as is the case with TI I27, TNfn3 unfolds via a force-stabilised intermediate. The key event in forced unfolding in TI I27 is largely the breaking of hydrogen bonds and hydrophobic interactions between the A' and G-strands. The mechanical Phi-value analysis and molecular dynamics simulations reported here reveal that significantly more of the TNfn3 molecule contributes to its resistance to force. Both AFM experimental data and molecular dynamics simulations suggest that the rate-limiting step of TNfn3 forced unfolding reflects a transition from the extended early intermediate to an aligned intermediate state. As well as losses of interactions of the A and G-strands and associated loops there are rearrangements throughout the core. As was the case for TI I27, the forced unfolding pathway of TNfn3 is different from that observed in denaturant studies in the absence of force.

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.

Atomic force microscopy: mechanical unfolding of proteins.

Mechanical unfolding of proteins using atomic force microscopy is becoming a routine biophysical technique. Mechanistic investigations in this rapidly evolving field are beginning to resolve the factors that contribute to the behaviour of biological macromolecules under force. Here we describe the force-mode apparatus, the experimental set-up, tractable systems of study, and the analysis of the resultant force data. Finally we summarise some of the recent achievements and limitations of this technique.