Frank A. Meyer

Macromolecular basis of globular protein exclusion and of swelling pressure in loose connective tissue (umbilical cord).

The macromolecular basis of tissue swelling pressure and of the ability of tissue to exclude globular proteins, according to size, have been investigated using human umbilical cord. Exclusion data of tissue, and tissue from which the polysaccharides had been removed by hyaluronidase were compared. Exclusion of globular proteins by the polysaccharides, obtained by difference from the two sets of data, was similar to that reported for isolated polysaccharides in solution. It can be described by a sphere/cylinder geometric exclusion model. The exclusion behavior of the polysaccharide-free tissue was accounted for in terms of the component collagen fibrils, glycoprotein microfibrils and cells. Average pore diameters of 18 and 110 nm, respectively, for the intact tissue and for the polysaccharide-free tissue were estimated. Swelling pressure measurements were performed on intact, on hyaluronidase-treated and on hyaluronidase and then Pronase-treated tissues to obtain the contributions of the polysaccharides, of collagen and of microfibrils. Close to the in vivo volume of tissue, the swelling pressure is given almost entirely by the polysaccharides and is consistent with the osmotic pressure expected from the relative amounts of hyaluronic acid and proteoglycan present and their distribution in the extrafibrillar, extracellular space. Upon swelling or deswelling a small net contribution of the fibrillar system to the swelling pressure is evident.

Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton's jelly

Whartons jelly of human umbilical cord is known to contain hyaluronic acid and sulphated glycosaminoglycans (probably as proteoglycans) immobilized in an insoluble collagen fibril network. A secondary, independent, insoluble network based on glycoprotein microfibrils of 13 nm diameter and interpenetrated with the collagen network has now been found in amounts corresponding to 9% of the weight of collagen. Elastin, however, is absent. Tissue slices placed in physiological buffer swell to two-fold their in vivo volume. This is due to the influence of the polysaccharides since treatment with either testicular hyaluronidase, Streptomyces hyaluronidase or chondroitinase ABC, causes their quantitative removal and abolishes the swelling tendency of tissue. Tissue so treated remains close to its in vivo volume indicating that for this state the fibrillar network, overall, is in its relaxed unstressed configuration. Subsequent treatment with a protease causes the degradation of the glycoprotein microfibril network and a two-fold increase in tissue volume while treatment with bacterial collagenase, resulting in the solubilization of 46% of the collagen, causes only a slight deswelling. These results suggest that the unstressed configuration of the network system at the in vivo volume of tissue is due to the collagen network being held in compression by the microfibril network. With intact tissue protease digestion with trypsin, in addition, causes a preferential release of sulphated glycosaminoglycans. Hyaluronic acid, however, remains largely immobilized.

Structure and Function of Mucus

A glycoprotein building block is common to mammalian mucins. This structure is composed of several protein chains having the same sequence. The carbohydrate side chains, which constitute over three-quarters of the weight, coat only some two-thirds of the backbone chain. The bare protein chains are linked by disulphide bridges and can be digested away with trypsin. Either procedure rapidly solubilizes mucus and results in a structural unit of about 500 000 molecular weight. Mucus solubilizes spontaneously. The first size unit which reaches solution is about 15 X 10(6) molecular weight but continues to break down further. Mechanical agitation considerably speeds up this process. The gel-like character which is an essential feature of mucus--which cannot otherwise act as transport coupler--is thus a transient phenomenon. The problem of how such a structure can arise from the building blocks known to be available is discussed.

On the role of sialic acid in the rheolgical properties of mucus

The importance of sialic acid in the rheological properties of mucus has been investigated. Both bovine cervical mucus, which is a gel, and the structural glycoprotein derived from it were studied before and after treatment with neuraminidase which selectively cleaves terminal sialic acid residues. The storage modulus, viscosity and circular dichroism spectrum were all essentially changed after removal of the sialic acid. These results would indicate that removal of sialic acid does not affect the physical structure of the glycoprotein and it is concluded that sialic acid has no significant role in the rheological properties of cervical mucus.

Tissue structure and macromolecular diffusion in umbilical cord. Immobilization of endogenous hyaluronic acid.

Diffusion of endogenous hyaluronic acid and 125I-labelled albumin, monitored by desorption from umbilical cord (Whartons jelly) slices, was studied in relation to tissue structure. Diffusion of hyaluronic acid was Fickian and some two orders of magnitude slower than that in free solution. After treatment of tissue with trypsin which removes proteoglycan(s) and degrades glycoprotein microfibrils, hyaluronic acid mobility through the collagen fibril network that remains is increased by an order of magnitude. These findings indicate that the mobility of hyaluronic acid in tissue is reduced both by the collagen network and by the presence of proteoglycan(s) and/or microfibrils. Estimates of the reduction in mobility due to physical entanglements with the fibrillar networks show that these play a major role. The mobility of hyaluronic acid found for intact tissue is sufficient for it to permeate the extracellular space within its metabolic turnover time. Labelled albumin diffusion is intact tissue, on the other hand, is reduced by only some 30% relative to free solution. This is consistent with the approximate 10% reduction found for the polysaccharide-free tissue (given by the excluded volume fraction) and the approximate 20% reduction expected for the polysaccharides in the interstitial fluid. Similar effects appear to be involved in the mobility of endogenous diffusible proteins in tissue.

Comparison of structural glycoproteins from mucus of different sources.

The main structural glycoprotein derived from three mammalian mucociliary epithelial secretions (bovine cervical, human lung and human middle ear mucus) were compared. The glycoprotein was purified on a cesium chloride density gradient after prior cleavage of disulphide bonds. Marked similarities were seen for the glycoproteins in their sugar and amino acid composition, in electrophoretic mobility, in sedimentation rate and in their banding densities in a cesium chloride density gradient. The molecular weight of these materials was approximately 0.6-10(6). The similarity noted for these materials occurred despite two of them (lung and middle ear) being derived from pathological sources. The glycoprotein derived from an amphibian mucociliary epithelial secretion (frog palatal mucus) was different; it banded at a lower buoyant density and had a lower sugar content. These findings are discussed in terms of the rheological properties and physiological transport role of mucus on ciliated epithelia. It is suggested that reported differences in the properties of mammalian secretions from different sources is due to differences in the extent of crosslinking between glycoprotein units rather than to chemical or structural differences. In the case of the frog its very different composition may be in keeping with its rather different rheological properties.

Adhesion of platelets to collagen. The nature of the binding site from competitive inhibition studies

Abstract Platelet adhesion to collagen has been studied at 4° using a model system of washed platelets suspended in Tyrode buffer and a glass surface to which soluble collagen has been adsorbed. Platelets could be shown to adhere to this surface, though in fewer numbers than to collagen fibers and without leading to aggregation. Adhesion of platelets to collagen is thus an independent event which does not necessarily produce aggregation. Preincubation of platelets with soluble collagen, denatured soluble collagen, pepsin- and periodate-treated soluble collagen and cyanogen bromide peptides all decreased the ability of platelets to bind to the soluble collagen coated surface. Similar effects arose, moreover, with the synthetic copolypeptides (Gly-Pro-Ala-Gly-Pro-Pro) n , (Gly-Pro-Pro) n and (Pro 2 -Gly) n . In fact the homopolymers polyproline and polyhydroxyproline (but not proline or hydroxyproline) as well gave effects similar to those of the collagen materials. The effect was rather specific since preincubation with plasma proteins and synthetic polypeptides based on other major amino acids present in collagen, viz. polyalanine, polyglutamic acid, polyaspartic acid, polyarginine and polylysine, did not inhibit the binding of platelets. It would therefore appear that proline and hydroxyproline are the important, if perhaps not the only, determinants involved in platelet recognition of collagen. The choice of the more stable model test system of soluble collagen coated surfaces at 4° could be justified since similar effects of the test materials could be demonstrated also at 30°.

Function and properties of epithelial mucus.

In the case of ciliated epithelia over which transport of particulate matter takes place, mucus has been shown to fulfill the role of mechanical coupler (Sade, et al., 1970). Whereas ciliary beat provides the source of motion, the fresh secretion of mucus is essential for converting this motion into the movement of particles over the epithelial surface. For example, on the palate of the frog, the mucociliary system provides the means of transporting food particles from the mouth to the gullet. When such a palate is excised it continues to transport particles, but repeated loading of the palate, say by little steel balls, gradually exhausts it of its mucus. Particle velocity drops and eventually reaches zero even though ciliary beat continues. The palate epithelium, locally has become depleted of its mucus which is found accumulated along the cut edge. When some of this mucus is returned to the epithelium, transport of the steel balls is resumed at the original velocity. Such a biological preparation can be used, therefore, to test whether other systems can be applied to the epithelium and substitute for mucus. It can also answer the question of what properties they must possess to provide this function.

Characteristics of the major platelet membrane site used in binding to collagen.

Abstract Meyer and Weisman (1) showed that the adhesion of washed rabbit platelets to soluble collagen adsorbed to glass is inhibited by the presence of soluble proline/hydroxyproline-rich materials in the platelet suspension suggesting that there are sites on the platelet that recognize the high content of these two amino acids on the surface of collagen in its triple helical state. It is now found that similar inhibition can be obtained on a collagen fiber surface using human as well as rabbit platelets in platelet-rich plasma (PRP) or in various washed platelet suspensions. Using polyhydroxyproline (PHP) and rabbit platelets it could be shown that the action of PHP is on the platelet membrane since inhibition occurs almost immediately and is also seen when fixed platelets are used. Inhibition is still seen when free PHP is removed from the platelet suspension. The inhibitory effect of PHP is specific to collagen and was not seen for platelet adhesion to albumin- or globulin-coated surface. We have shown earlier that non-aggregating concentrations of ADP inhibit platelet adhesion as a result of binding to the platelet membrane but independent of the induced shape change (2). PHP action, however, differs from that of ADP since no time lag occurs and it is not blocked by AMP. Furthermore, PHP does not induce a shape change. Therefore, ADP release cannot have occurred. A maximum of inhibition of adhesion by PHP of ∼60±10% was similar to that given by ADP. However, addition of both agents together did not produce greater inhibition than either alone. This suggests that the same site is affected but that ADP action on the platelet surface is not direct but operates through a delayed lodal interaction mechanism. This indirect action of ADP probably also affects binding sites to other surfaces since adhesion to albumin- and globulin-coated surfaces is also reduced by ADP.

Aging and the interetitial content of loose connective tissue. A brief note

The concentration of hyaluronic acid which is the fixed loose connective tissue component determining water and other potentials in the interstitium remains constant with age. Since the collagen fiber network in the native tissue state is unstressed, changes in collagen fiber morphology and in collagen content with age have no influence.