Physical characterization of platelet thrombospondin.

559

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: Discussionsupporting

confidence: 70%

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

Lawler¹,

Hynes²

1986

559

341

“…A useful tool for characterizing the fragments of TS as well as the complexes of TS with mAbs and its protein ligands is the electron microscope (19,32). The native molecular mass of calcium-replete, trimeric TS is •450,000 D. The mass of the subunits, based on estimates from SDS gel electrophoresis under reducing conditions, was in the range of 150,000-185,000 (36,41). Recent sequencing of TS cDNA reveals that the true molecular mass of the subunit is somewhat less than these values, '~140,000 (see below).…”

Section: Modular Structure Of Thrombospondinmentioning

confidence: 99%

Thrombospondin: a modular adhesive glycoprotein of platelets and nucleated cells.

Frazier¹

1987

191

Platelet adhesion to the subendothelial matrix and the subsequent aggregation of the activated platelets is a special case of intercellular adhesion that is highly regulated and for which multiple, possibly redundant adhesive systems exist. The large "adhesive glycoproteins" such as fibrinogen, von Willebrand factor, and fibronectin and their receptors, which participate in platelet adhesion and aggregation, are not unique to platelets, and probably serve analogous functions in a variety of cell types and locations. Thrombospondin (TS)/the most abundant protein of platelet alpha-granules, has been recognized as a new member of this class of adhesive glycoproteins. Whereas other adhesive glycoproteins are contained within alpha-granules and secreted upon platelet activation, they also exist in plasma at high concentrations. Hence TS is the only member of the group that is unique to alpha-granules and whose expression in quantity at sites of platelet aggregation is absolutely dependent on platelet activation.Since its discovery in 1971, TS has been thought to have a role in platelet aggregation (3,4). This suspicion has recently been confirmed by the fnding that both monoclonal (14) and polyclonal (22, 37) antibodies against platelet TS can block the secondary or secretion-dependent phase of platelet aggregation. This result in itself, however, does not speak directly to the mechanism of TS involvement in this complex process, since antibodies against fibrinogen (62), fibronectin (13), and the platelet glycoprotein IIb/IIIa (43), the receptor on platelets for fibrinogen and fibronectin, will all block platelet aggregation. Other agents that block aggregation are short peptide sequences related either to RGDS, the cellbinding sequence of fibronectin and fibrinogen (21), or to the COOH-terminal segment of the fibrinogen gamma chain (27). Both interfere with the binding of fibrinogen, fibronectin (58), and presumably yon Willebrand factor (61) to IIb/IIIa.In addition to its localization in platelets and its presumed synthesis in megakaryocytes (42), TS has been found, using immunofluorescence localization techniques, in a variety of extracellular locations in human tissues including blood vessel, muscle, skin, kidney, and glandular epithelium (64). The biosynthesis of TS has been demonstrated in a number of normal and transformed cells in culture, including endothelial, smooth muscle, and glial cells, type II pneumocytes, fibroblasts, keratinocytes, and macrophages (1,25,26,45,46,50,57,65). Furthermore, several cell lines derived from malignancies such as HT-1080 fibrosarcoma cells and the melanoma lines C32 and G361 also make TS (54,63). This widespread expression of TS and its appearance as a component of the extracellular matrix have led to the suggestion that TS might have a role analogous to that of fibronectin and laminin in cell attachment, motility, and perhaps differentiation. TS does not appear to be an obligatory attachment factor for cultured cells such as fibroblasts, smooth muscle cells, or endothelial ...

“…Thrombospondin is an Mr 450,000 glycoprotein composed of three probably identical disulfide-bonded subunits of Mr 150,000 (1, 37,38,49). Thrombospondin was first described as a component of platelet a-granules (1) and is now known to be synthesized by endothelium, fibroblasts, granular pneumocytes, and smooth muscle cells in culture (30,51,52,65,71,72).…”

Section: -I-hrombospondinmentioning

confidence: 99%

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells.

Majack¹,

Cook²,

Börnstein³

1985

228

154

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The ind0ction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 ~,g/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 ~g/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.