Sequence and domain arrangements influence mechanical properties of elastin-like polymeric elastomers.

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self-assembly that aligns lysine residues for crosslinking. As a result, both the full-length monomer as well as elastin-like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full-length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self-assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full-length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties.

Fibrillins, fibulins, and matrix-associated glycoprotein modulate the kinetics and morphology of in vitro self-assembly of a recombinant elastin-like polypeptide.

Elastin is the polymeric protein responsible for the properties of extensibility and elastic recoil of the extracellular matrix in a variety of tissues. Although proper assembly of the elastic matrix is crucial for its durability, the process by which this assembly takes place is not well-understood. Recent data suggest the complex interaction of tropoelastin, the monomeric form of elastin, with a number of other elastic matrix-associated proteins, including fibrillins, fibulins, and matrix-associated glycoprotein (MAGP), is important to achieve the proper architecture of the elastic matrix. At the same time, it is becoming clear that self-assembly properties intrinsic to tropoelastin itself, reflected in a temperature-induced phase separation known as coacervation, are also important in this assembly process. In this study, using a well-characterized elastin-like polypeptide that mimics the self-assembly properties of full-length tropoelastin, the process of self-assembly is deconstructed into "coacervation" and "maturation" stages that can be distinguished kinetically by different parameters. Members of the fibrillin, fibulin, and MAGP families of proteins are shown to profoundly affect both the kinetics of self-assembly and the morphology of the maturing coacervate, restricting the growth of coacervate droplets and, in some cases, causing clustering of droplets into fibrillar structures.

Investigation of recombinant human elastin polypeptides as non-thrombogenic coatings.

We investigated the use of a recombinant human elastin polypeptide as a coating on synthetic materials with a view to determining if these polypeptides could improve the blood compatibility of cardiovascular devices such as vascular conduits and arterial/venous catheters. Platelet adhesion and activation were studied in vitro using three commercially available synthetic materials: polyethylene terephthalate (Mylar), a poly(tetrafluoroethylene/ethylene) copolymer (Tefzel) and a polycarbonate polyurethane (Corethane). Coated with adsorbed polypeptide, all three synthetic materials demonstrated reduced platelet activation and adhesion in platelet rich plasma in vitro. Compared to non-coated controls, there was a significant decrease (p=0.05) in both platelet microparticle release and P-selectin expression for the polypeptide-coated surfaces. Scanning electron microscopy indicated fewer adhering platelets on coated surfaces compared to non-coated controls. In vivo, in a rabbit model, evaluations of polyurethane catheters coated with the polypeptide showed a marked increase in catheter patency and a significant decrease in fibrin accretion and embolism when compared to uncoated controls. This polypeptide shows a strong potential for use as a non-thrombogenic coating for small diameter vascular grafts. In addition, the results of this study indicate that the elastin polypeptide would be a valuable component of a tissue engineered vascular conduit.

Recombinant human elastin polypeptides self-assemble into biomaterials with elastin-like properties.

Processes involving self-assembly of monomeric units into organized polymeric arrays are currently the subject of much attention, particularly in the areas of nanotechnology and biomaterials. One biological example of a protein polymer with potential for self-organization is elastin. Elastin is the extracellular matrix protein that imparts the properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Tropoelastin, the approximately 70 kDa soluble monomeric form of elastin, is highly nonpolar in character, consisting essentially of 34 alternating hydrophobic and crosslinking domains. Crosslinking domains contain the lysine residues destined to form the covalent intermolecular crosslinks that stabilize the polymer. We and others have suggested that the hydrophobic domains are sites of interactions that contribute to juxtaposition of lysine residues in preparation for crosslink formation. Here, using recombinant polypeptides based on sequences in human elastin, we demonstrate that as few as three hydrophobic domains flanking two crosslinking domains are sufficient to support a self-assembly process that aligns lysines for zero-length crosslinking, resulting in formation of the crosslinks of native elastin. This process allows fabrication of a polymeric matrix with solubility and mechanical properties similar to those of native elastin.

Sequence and structure determinants for the self-aggregation of recombinant polypeptides modeled after human elastin.

Elastin is a polymeric structural protein that imparts the physical properties of extensibility and elastic recoil to tissues. The mechanism of assembly of the tropoelastin monomer into the elastin polymer probably involves extrinsic protein factors but is also related to an intrinsic capacity of elastin for ordered assembly through a process of hydrophobic self-aggregation or coacervation. Using a series of simple recombinant polypeptides based on elastin sequences and mimicking the unusual alternating domain structure of native elastin, we have investigated the influence of sequence motifs and domain structures on the propensity of these polypeptides for coacervation. The number of hydrophobic domains, their context in the alternating domain structure of elastin, and the specific nature of the hydrophobic domains included in the polypeptides all had major effects on self-aggregation. Surprisingly, in polypeptides with the same number of domains, propensity for coacervation was inversely related to the mean Kyte-Doolittle hydropathy of the polypeptide. Point mutations designed to increase the conformational flexibility of hydrophobic domains had the unexpected effect of suppressing coacervation and promoting formation of amyloid-like fibers. Such simple polypeptides provide a useful model system for understanding the relationship between sequence, structure, and mechanism of assembly of polymeric elastin.

Elastin as a self-organizing biomaterial: use of recombinantly expressed human elastin polypeptides as a model for investigations of structure and self-assembly of elastin.

Elastin is the major extracellular matrix protein of large arteries such as the aorta, imparting characteristics of extensibility and elastic recoil. Once laid down in tissues, polymeric elastin is not subject to turnover, but is able to sustain its mechanical resilience through thousands of millions of cycles of extension and recoil. Elastin consists of ca. 36 domains with alternating hydrophobic and cross-linking characteristics. It has been suggested that these hydrophobic domains, predominantly containing glycine, proline, leucine and valine, often occurring in tandemly repeated sequences, are responsible for the ability of elastin to align monomeric chains for covalent cross-linking. We have shown that small, recombinantly expressed polypeptides based on sequences of human elastin contain sufficient information to self-organize into fibrillar structures and promote the formation of lysine-derived cross-links. These cross-linked polypeptides can also be fabricated into membrane structures that have solubility and mechanical properties reminiscent of native insoluble elastin. Understanding the basis of the self-organizational ability of elastin-based polypeptides may provide important clues for the general design of self-assembling biomaterials.