Effect of upstream priming on transient downstream platelet-substrate interactions.

Upstream exposure of platelets to activating proteins 'primes' platelets for increased downstream adhesion, though the mechanics of platelet translocation before permanently arresting are not well understood. To investigate platelet translocation on platelet-binding proteins, primed platelets' transient contacts with immobilized proteins were recorded and analyzed. Using a microfluidic channel, representative of a vascular graft, platelet-activating proteins were covalently attached to the upstream priming, center, and downstream capture positions.

Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation.

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region. This flow-based system was used to assay how effectively antiplatelet agents suppress shear-induced platelet adhesion and activation downstream of the stenotic region. Microcontact printing was used to covalently attach one of three platelet binding proteins [fibrinogen, collagen, or von Willebrand factor (vWf)] to the surface of the downstream capture region.

Effects of elapsed time on downstream platelet adhesion following transient exposure to elevated upstream shear forces.

Transient exposure to elevated shear forces is known to prime platelets for enhanced downstream adhesion, but how far downstream these priming effects persist is not known. In the present study, the platelet capture regions, prepared by immobilizing fibrinogen, collagen, or von Willebrand factor, were placed at three different distances from the upstream stenotic region to vary the elapsed time of circulating platelets downstream. Platelet adhesion increased with the increase of upstream wall shear rates from 1620 s to 11,560 s for all three downstream proteins, but only the adhesion to fibrinogen increased significantly with the distance between the upstream stenotic region and the downstream capture region.

Downstream platelet adhesion and activation under highly elevated upstream shear forces.

Elevated shear force caused by an anastomotic stenosis is a common complication at the blood vessel-vascular implant interface. Although elevated shear forces were found to cause platelet aggregation around a stenotic region, transient platelet exposure to elevated shear forces and subsequent downstream events occurring under lower shear force were not extensively studied. We hypothesize that effects of elevated shear forces on pre-activation of platelets for downstream adhesion and activation are relevant in understanding the increased thrombotic risk associated with blood-contacting devices.

Silica Nanoparticle-Endothelial Interaction: Uptake and Effect on Platelet Adhesion under Flow Conditions.

Silica nanoparticles are extensively used in biomedical applications and consumer products. Little is known about the interaction of these NPs with the endothelium and effect on platelet adhesion under flow conditions in circulation. In this study, we investigated the effect of silica nanoparticles on the endothelium and its inflammation, and subsequent adhesion of flowing platelets in vitro.

Effects of upstream shear forces on priming of platelets for downstream adhesion and activation.

Unlabelled: Platelets in flowing blood are sometimes exposed to elevated shear forces caused by anastomotic stenosis at the blood vessel-vascular implant interface. The objective of this study was to determine how effective upstream shear forces are in priming platelets for downstream adhesion and activation. Flow chambers with upstream stenotic regions (shear rates of 400-1000 s) were manufactured by relief molding of polydimethylsiloxane.

Astrocyte spreading and migration on aggrecan-laminin dot gradients.

The surface concentration gradient of two extracellular matrix (ECM) macromolecules was developed to study the migratory and morphological responses of astrocytes to molecular cues typically found in the central nervous system injury environment. The gradient, prepared using microcontact printing, was composed of randomly positioned micrometer-sized dots of aggrecan (AGG) printed on a substrate uniformly coated with laminin (LN). AGG dots were printed in an increasing number along the 1000 μm long and 50 μm wide gradient area which had on each end either a full surface coverage of AGG or LN.

Binary agonist surface patterns prime platelets for downstream adhesion in flowing whole blood.

As platelets encounter damaged vessels or biomaterials, they interact with a complex milieu of surface-bound agonists, from exposed subendothelium to adsorbed plasma proteins. It has been shown that an upstream, surface-immobilized agonist is capable of priming platelets for enhanced adhesion downstream. In this study, binary agonists were integrated into the upstream position of flow cells and the platelet priming response was measured by downstream adhesion in flowing whole blood.

The Biomechanical Effects of Resuscitation Colloids on the Compromised Lung Endothelial Glycocalyx.

Background: The endothelial glycocalyx is an important component of the vascular permeability barrier, forming a scaffold that allows serum proteins to create a gel-like layer on the endothelial surface and transmitting mechanosensing and mechanotransduction information that influences permeability. During acute inflammation, the glycocalyx is degraded, changing how it interacts with serum proteins and colloids used during resuscitation and altering its barrier properties and biomechanical characteristics. We quantified changes in the biomechanical properties of lung endothelial glycocalyx during control conditions and after degradation by hyaluronidase using biophysical techniques that can probe mechanics at (1) the aqueous/glycocalyx interface and (2) inside the glycocalyx.

Functional assay of antiplatelet drugs based on margination of platelets in flowing blood.

A novel functional assay of antiplatelet drug efficacy was designed by utilizing the phenomena of platelet margination in flowing blood and transient platelet contacts with surface-immobilized platelet agonists. Flow margination enhances transient contacts of platelets with the walls of flow chambers covered with surface-immobilized proteins. Depending on the type and the surface density of the immobilized agonists, such transient interactions could "prime" the marginated platelet subpopulation for enhanced activation and adhesion downstream.

Two surface gradients of polyethylene glycol for a reduction in protein adsorption.

Polyethylene glycol (PEG) coatings have been commonly used in reducing protein adsorption with the intent of improving a biomaterial's biocompatibility. To elucidate the role of PEG surface density in reducing protein adsorption, two types of grafted PEG surface density gradients were evaluated for the adsorption and desorption of albumin and fibrinogen, two blood proteins. PEG density gradients were characterized using contact angle measurements and X-ray photoelectron spectroscopy.

Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone.

Cell substrate patterning with glycosaminoglycans to study their biological roles in the central nervous system.

Microcontact printing (μCP) based techniques have been developed for creating cell culture substrates with discrete placement of CNS-expressed molecules. These substrates can be used to study various components of the complex molecular environment in the central nervous system (CNS) and related cellular responses. Macromolecules such as glycosaminoglycans (GAGs), proteoglycans (PGs), or proteins are amenable to printing.

Exploiting differential surface display of chondroitin sulfate variants for directing neuronal outgrowth.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are known to be primary inhibitors of neuronal regeneration at scar sites. However, a variety of CSPGs are also involved in neuronal growth and guidance during other physiological stages. Sulfation patterns of CS chains influence their interactions with various growth factors in the central nervous system (CNS), thus influencing neuronal growth, inhibition, and pathfinding.

Sugar glues for broken neurons.

Proteoglycans (PGs) regulate diverse functions in the central nervous system (CNS) by interacting with a number of growth factors, matrix proteins, and cell surface molecules. Heparan sulfate (HS) and chondroitin sulfate (CS) are two major glycosaminoglycans present in the PGs of the CNS. The functionality of these PGs is to a large extent dictated by the fine sulfation patterns present on their glycosaminoglycan (GAG) chains.

Astrocytes specifically remove surface-adsorbed fibrinogen and locally express chondroitin sulfate proteoglycans.

Surface-adsorbed fibrinogen (FBG) was recognized by adhering astrocytes, and was removed from the substrates in vitro by a two-phase removal process. The cells removed adsorbed FBG from binary proteins' surface patterns (FBG+laminin, or FBG+albumin) while leaving the other protein behind. Astrocytes preferentially expressed chondroitin sulfate proteoglycan (CSPG) at the loci of fibrinogen stimuli; however, no differences in overall CSPG production as a function of FBG surface coverage were identified.

Use of reflectance interference contrast microscopy to characterize the endothelial glycocalyx stiffness.

Reflectance interference contrast microscopy (RICM) was used to study the mechanics of the endothelial glycocalyx. This technique tracks the vertical position of a glass microsphere probe that applies very light fluctuating loads to the outermost layer of the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx. Fluctuations in probe vertical position are used to estimate the effective stiffness of the underlying layer.

The role of well-defined patterned substrata on the regeneration of DRG neuron pathfinding and integrin expression dynamics using chondroitin sulfate proteoglycans.

Injured neurons intrinsically adapt to and partially overcome inhibitory proteoglycan expression in the central nervous system by upregulating integrin expression. It remains unclear however, to what extent varying proteoglycan concentrations influence the strength of this response, how rapidly neurons adapt to proteoglycans, and how pathfinding dynamics are altered over time as integrin expression is modulated in response to proteoglycan signals. To investigate these quandaries, we created well-defined substrata in which postnatal DRG neuron pathfinding dynamics and growth cone integrin expression were interrogated as a function of proteoglycan substrata density.

Competitive Adsorption of Three Human Plasma Proteins onto Sulfhydryl-to-sulfonate Gradient Surfaces.

Competitive adsorption of three human plasma proteins: albumin (HSA), fibrinogen (Fgn), and immunoglobulin G (IgG) from their ternary solution mixtures onto a sulfhydryl-to-sulfonate gradient surface was investigated using spatially-resolved total internal reflection fluorescence (TIRF) and autoradiography. The concentration of each protein in the ternary solution mixture was kept at an equivalent of 1/100 of its physiological concentration in blood plasma. The three proteins displayed different adsorption and desorption characteristics.

Multiprotein microcontact printing with micrometer resolution.

Depositing multiple proteins on the same substrate in positions similar to the natural cellular environment is essential to tissue engineering and regenerative medicine. In this study, the development and verification of a multiprotein microcontact printing (μCP) technique is described. It is shown that patterns of multiple proteins can be created by the sequential printing of proteins with micrometer precision in registration using an inverted microscope.

Inducing alignment in astrocyte tissue constructs by surface ligands patterned on biomaterials.

Planar substrates with patterned ligands were used to induce astrocyte alignment whereas substrates with uniform fields of ligand were used to produce random cell orientation. DRG neurons plated on top of oriented astrocyte monolayers exhibited directional outgrowth along aligned astrocytes, demonstrating that purely biological cues provided by the oriented astrocytes were sufficient to provide guidance cues. Antibody blocking studies demonstrated that astrocyte associated FN played a major mechanistic role in directing engineered neurite extension.

The effect of upstream platelet-fibrinogen interactions on downstream adhesion and activation.

Circulating activated platelets roll and make transient contacts before ultimately adhering to a substrate. However, despite the dynamic nature of platelet adhesion, most in vitro adhesion and activation studies have focused on establishing local cause and effect relationships. Here, we determined the effect of exposing platelets to immobilized upstream human fibrinogen on downstream adhesion and activation.

Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy.

The mechanical properties of endothelial glycocalyx were studied using atomic force microscopy with a silica bead (diameter ∼18 μm) serving as an indenter. Even at indentations of several hundred nanometers, the bead exerted very low compressive pressures on the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx and allowed for an averaging of stiffness in the bead-cell contact area. The elastic modulus of BLMVEC glycocalyx was determined as a pointwise function of the indentation depth before and after enzymatic degradation of specific glycocalyx components.

Using microcontact printing of fibrinogen to control surface-induced platelet adhesion and activation.

The ability to promote or inhibit specific platelet-surface interactions in well-controlled environments is crucial to studying fundamental adhesion and activation mechanisms. Here, microcontact printing was used to immobilize human fibrinogen covalently in the form of randomly placed, micrometer-sized islands at an overall surface coverage of 20, 50, or 85%. The nonprinted background region was blocked with covalently immobilized human albumin.

Natural Underwater Adhesives.

The general topic of this review is protein-based underwater adhesives produced by aquatic organisms. The focus is on mechanisms of interfacial adhesion to native surfaces and controlled underwater solidification of natural water-borne adhesives. Four genera that exemplify the broad range of function, general mechanistic features, and unique adaptations are discussed in detail: blue mussels, acorn barnacles, sandcastle worms, and freshwater caddisfly larva.