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Biomedical subjects

L W Cunningham

Publications and source records attributed to L W Cunningham.

At least 37 records · Page 2Linked to original sources
PubMed1981

Platelet-collagen adhesion-membrane fluidity and the development of high affinity adhesion through multiple interacting sites.

Multiple, linked interactions between the platelet surface and collagen fibers have been implicated in the initiation of platelet secretion and subsequent aggregation. The formation of such multiple simultaneous interactions could give rise to high affinity adhesion of platelets to collagen even though the affinity of the individual interactions may be much weaker. This concept has been tested by measuring the adhesion of platelets to collagen under conditions which could effect the formation of multiple interactions. Adhesion is markedly diminished at 4 degrees C but not at 23 or 37 degrees C. Metabolic inhibitors such as 2-deoxyglucose and Antimycin A do not inhibit adhesion although they virtually abolish subsequent aggregation. Brief formaldehyde fixation of platelets greatly reduces adhesion. These results are consistent with the concept that the formation of multiple linked interactions between the platelet surface and collagen are important in platelet-collagen adhesion and that mobility of platelet membrane components is required for the clustering of these interactions in focussed regions on the platelet surface.

Antimycin A↗
PubMed1980

Collagen-mediated platelet aggregation: the role of multiple interactions between the platelet surface and collagen.

Although the requirement for collagen fibrils to initiate platelet aggregation is well established, there has been no satisfactory explanation for this requirement. One possibility is that multiple simultaneous and linked interactions between collagen and the platelet surface must occur to initiate the release reaction and subsequent aggregation. Direct evidence in support of this proposal was obtained by examination of the ability of collagen crosslinked in a random manner with glutaraldehyde to initiate platelet aggregation. Collagen crosslinked with 0.25% glutaraldehyde is only a slightly less effective aggregating agent than native fibrillar collagen. Further studies revealed that whereas native triple helical cross-linked collagen is an effective aggregating agent, denatured crosslinked collagen is ineffective. It thus appears that crosslinking of platelet receptor sites by multiple simultaneous and linked interactions with a rigid collagen matrix is required to initiate platelet aggregation. The precise steric relationship of the collagen sites does not appear to be of great importance.

Blood Platelets↗
PubMed1980

Comparison of collagen and glycosaminoglycan synthesis in attaching control and diabetic human skin fibroblasts.

Cultured fibroblasts derived from normal subjects and juvenile diabetics attach in the absence of serum to plastic culture dishes and secrete macromolecules, including collagenous components, hyaluronic acid, and proteoglycans into the medium and onto the plastic surface where they form a microexudate carpet. Most diabetic fibroblasts examined did not spread as well as normal cells during a 4-hr interval after the initial attachment. There were no significant differences between normal and diabetic cells with respect to proline and lysine incorporation and lysine hydroxylation. The percentage glycosylation of hydroxylysine was marginally higher in the media proteins of diabetic cells, but glycosylation in both normal and diabetic cells was elevated over that typically observed in human skin collagen. Collagenous components were estimated to constitute approximately 15-20% of the microexudate carpet fraction in both normal and diabetic cell strains. Diabetic fibroblasts exhibited a marginally lower ratio of heparan sulfate to chondroitin sulfate in the cell surface to matrix microexudate carpet fraction (trypsinate) than did normal fibroblasts. The hyaluronate and chondroitin sulfate contents of this fraction of diabetic cells were not significantly different from those of normal cells.

Cell Adhesion↗
PubMed1979

Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum.

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.

Adenosine Triphosphatases↗
PubMed1979

Fibronectin and the multiple interaction model for platelet-collagen adhesion.

A rapid, sensitive, and reproducible assay to determine the adhesion of platelets to collagen has been developed. Collagen fibers and adherent platelets are retained on polycarbonate membrane filters. Chemical modification of collagen by acetylation and of platelets by treatment with chymotrypsin markedly reduces adhesion. The role of fibronectin in the collagen-platelet interaction has been examined. Treatment of platelets with purified antibody or Fab' fragments to fibronectin only slightly reduces adhesion. Preincubation of platelets with high concentrations of gelatin reduces adhesion by only 22% but fails to inhibit aggregation. Thus, fibronectin has only a limited role in the adhesion of platelets to collagen and is either not involved in the adhesion that leads to aggregation or is only one of several adhesion mechanisms, any of which alone can initiate aggregation.

Animals↗
PubMed1977

Circular dichroism of glycopeptide fractions from alpha1-acid glycoprotein, thyroglobulin, and ovalbumin.

Using sequential pronase digestions, glycopeptide fractions were prepared from human alpha1-acid glycoprotein, hen egg ovalbumin, and bovine thyroglobulin, two types of glycopeptides being obtained from the latter. The fractions were characterized on the basis of hexose, hexosamine, sialic acid, and peptide content. The glycopeptide fraction from alpha1-acid glycoprotein is complex (i.e., the carbohydrate moiety contains mannose, galactose, N-acetylglucosamine, and sailic acid), as is one of the glycopeptide fractions from thyroglobulin (type I). The glycopeptide fraction from ovalbumin and the type II glycopeptide fractions from thyroglobulin are simple (i.e., the carbohydrate moiety contains only mannose and N-acetylglucosamine). The circular dichroic spectra of the two complex glycopeptide fractions and the ovalbumin glycopeptide fraction were similar and were characterized by a negative extremum between 207.5 nm and 211 nm with magnitudes in the range of --6400 deg-cm2-dmol-1 to --7200 deg-cm2-dmol-1 (referred to the molar concentration of N-acetylated sugars). The thyroglubulin type II glycopeptide fraction exhibited a circular dichroic spectrum with an extremum of --29 200 deg-cm2-dmol-1 at 205 nm. Removal of sialic acid from the complex glycopeptide fractions greatly increased the (negative) magnitude of ellipticity at the extremum. The circular dichroic spectra of the complex of glycopeptide fractions were reasonably additive using the spectra of monomeric sialic acid and the asialo-derivatives. This demonstrates that the contributions of sialic acid to the circular dichroic spectrum are nearly additive. The implications of this observation are that covalent attachment of these terminal residues to the oligosaccharides does not lead to strong interactions with other chromophores nor to positioning in particularly asymmetric environment. In contrast, the magnitudes of the observed ellipticity extrema in the circular dichroic spectra of the asialo-derivatives, in which N-acetylglucosamine is the major chromophore, are much greater than can be accounted for on the basis of monomeric contributions (i.e., free N-acetylglucosamine). This finding shows that the optical activity of N-acetyl-glucosamine is greatly influenced by the formation of the carbohydrate core in glycoproteins and suggests the possible formation of secondary structure in the carbohydrate moiety.

Amino Acids↗
PubMed1977

The synthesis of macromolecular 3-hydroxyproline by attaching and confluent cultures of human fibroblasts.

The synthesis of collagen has been studied during the attachment of freshly trypsinized human fibroblasts to culture vessels by measurement of the incorporation of radioactive proline into macromolecular hydroxyproline. Collagenous protein(s) was found to be a component of a substrate-attached material ('microexudate carpet') synthesized rapidly during cell attachment in the absence of serum. The ratio of 3-hydroxyproline/4-hydroxyproline in the collagenous proteins synthesized during cell attachment was found to be 4-5 fold higher than that of normal type I collagen. The synthesis of 3-hydroxyproline by confluent cultures was diminished by serum deprivation, and was shown to require higher concentrations of ascorbate than the synthesis of the 4-hydroxy isomer.

Ascorbic Acid↗
PubMed1977

Collagen-mediated platelet aggregation. Evidence for multivalent interactions of intermediate specificity between collagen and platelets.

We have shown previously that periodate oxidation of collagen carbohydrate does not affect its ability to aggregate platelets. We now describe an additional characterization of periodate-modified collagen which demonstrates that collagen devoid of intact carbohydrate is fully capable of fibril formation, and we confirm its capacity to initiate platelet aggregation. Furthermore, we demonstrate that the platelet aggregating abilities of Types I, II, and III fibrillar collagen are quite similar despite differences in carbohydrate content and amino acid sequence. We also demonstrate that monomeric, pepsin-solubilized Type I human collagen is ineffective inhibiting aggregation by performed fibrils derived from the same molecule, thus establishing that the affinity of platelets for collagen depends upon prior polymerization of collagen. We interpret these and other findings to demonstrate that the hydroxylysyl glycoside regions of collagen are not highly specific sites involved in platelet-collagen interactions leading to "physiological" aggregation, and that the possibility must be considered that multiple interactions involving collagen sites of comparatively low structural specificity may be the initiating events in release of platelet ADP and the ensuing aggregation.

Amino Acids↗