Evidence for calcium-sensitive structure in platelet thrombospondin. Isolation and partial characterization of thrombospondin in the presence of calcium.

Self Cite

562

341

Abstract. Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cellto-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repots constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-glyasp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

Section: Discussionmentioning

confidence: 70%

mentioning

confidence: 99%

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

Lawler¹,

Hynes²

1986

Self Cite

562

341

“…Similar results were obtained with human and bovine platelet TS. Purification of TS in the presence ofCa" is believed to preserve a more native structure of the protein (23). However, no differences were observed between the binding of human platelet TS purified in the presence ofeither 50 g.M Caz+ or EDTA (unpublished experiments).…”

Section: Resultsmentioning

confidence: 95%

Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen.

Mumby¹,

Raugi²,

Börnstein³

1984

199

105

Thrombospondin (TS), a protein first described in platelets, was recently shown to be synthesized and secreted by endothelial cells, fibroblasts, and smooth muscle cells. The presence of TS in the extracellular matrix of cultured cells has prompted us to examine the associations of this protein with matrix macromolecules . Interactions of TS with both matrix and serum proteins were tested using an enzyme-linked immunosorbent assay . With this assay we assessed the binding of TS in solution to proteins adsorbed to polystyrene microtiter plates . Among collagens, platelet TS bound to type V but not to types I, III, or IV. This selective interaction was confirmed in experiments using proteins linked to cyanogen bromide-activated Sepharose . TS released from platelets in response to thrombin activation, as well as that secreted by endothelial cells in culture, bound to type V but not to type I collagen-Seph arose. No binding was observed to denatured type V collagen-Sepharose . The binding region for type V collagen was located in a chymotrypsin-produced fragment of TS with chains of M, = 70,000, after reduction . Interactions of TS with a number of other proteins, including fibronectin, fibrinogen, and laminin, could be demonstrated using the enzyme-linked immunosorbent assay technique but the interpretation of these findings is difficult since comparable binding to protein-Sepharose was not always observed. Our findings suggest that both the extravascular distribution and function of TS in vivo may involve an interaction with type V Collagen.Thrombospondin (TS)' is a glycoprotein consisting of three, possibly identical, disulfide-bonded chains of -140,000 mol wt (25, 31). The protein was initially described in platelets and was shown to be released from storage in a-granules by the action of thrombin (2, 18). A fraction of the secreted TS binds to the platelet surface (15, 39) . Subsequently TS, or a protein very similar to it, was shown to be secreted by endothelial cells (9,34,36,42,45) and by various other cells in culture, including fibroblasts (21, 40), smooth muscle cells (40), and granular pneumocytes (43). The observation by immunofluorescence of TS in a fibrillar extracellular meshwork in cultured cells (21,40) suggested that TS may function as a normal component of the extracellular matrix in vivo.It has been proposed that TS acts as an endogenous lectin in platelet aggregation by binding to fibrinogen associated 'Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; TS, thrombospondin . 646with the activated platelet surface (20). Support for this proposal has come from the observation that fibrinogen in solution can form a complex with TS adsorbed to a plastic surface (26). In addition, Lahav et al. (22) have provided evidence for the interaction ofTS with fibronectin during platelet adhesion and aggregation. Using a radioactive cross-linking agent, an interaction was observed between TS released from activated platelets and fibronectin or collagen adsorbed to a glass surf...

“…MG-63 human osteosarcoma cells (Billiau et al, 1977) were grown in DME with 10% FCS. Human platelets were isolated from citrated platelet-rich plasma as described by Lawler et al (1982). Human peripheral blood monocytes were isolated as described by Wright and Silverstein (1982) and were kindly provided by L. Van De Water (Beth Israel Hospital, Boston, MA).…”

Section: Cel/smentioning

confidence: 99%

Antibodies to the conserved cytoplasmic domain of the integrin beta 1 subunit react with proteins in vertebrates, invertebrates, and fungi.

Marcantonio

Hynes

1988

311

171

Abstract. The integrin family of cell surface receptors can be divided into three groups on the basis of their homologous 13 subunits: 13t, 132, and 133. We have raised an antibody against a synthetic peptide corresponding to the COOH-terminal domain of the chicken integrin 13~ subunit that reacts with 13 subunits from a variety of vertebrates, invertebrates, and fungi, demonstrating strong evolutionary conservation of sequences in this domain. In Drosophila cells, the antibody recognizes integrin ct13 complexes that appear to be identical with position-specific antigens. Cross-reactive proteins are also detected in Caenorhabditis elegans and Candida albicans. The antiserum is specific for 13~ subunits and does not recognize other integrin 13 subunits in humans. In immunofluorescence analyses of cultured cells, the antibody reacts only with permeabilized cells confirming that this highly conserved COOH-terminal segment is a cytoplasmic domain.