Thrombospondin: synthesis and secretion by cells in culture.

Abstract

Thrombospondin, a high molecular weight glycoprotein secreted by platelets in response to activation by thrombin, has been identified by immunofluorescence in bovine aortic endothelial cells, human foreskin fibroblasts, and human aortic smooth muscle cells. Immunofluorescence patterns were found to be similar using antisera raised to thrombospondins purified either from bovine aortic endothelial cells or from human platelets. Radioimmune precipitation of pulse-labeled cellular proteins confirmed the presence of thrombospondin in positively stained cells. A sensitive quantitative enzyme-linked immunosorbent assay (ELISA) was developed and used to determine that the accumulation of secreted thrombospondin was similar for endothelial cells and fibroblasts but was higher for smooth muscle cells. The presence of thrombospondin in a variety of cells suggests that its function may not be limited to an involvement in platelet interactions.Thrombospondin (TS), a high molecular weight glycoprotein, is released from a-granules after activation of platelets by thrombin (1, 2). After release, the protein binds to the activated platelet surface in a calcium-dependent fashion (3) and may participate in platelet-platelet interactions (4). Earlier work in this laboratory (5) identified a high molecular weight glycoprotein secreted by endothelial cells in culture that represented a substantial portion of the noncollagenous protein synthesized and secreted by these cells. This glycoprotein was subsequently shown to be indistinguishable from TS by a variety of criteria, including co-purification, molecular weight, amino acid composition, immunological cross-reactivity, and peptide maps (6).Although TS has been shown to have lectinlike activity in platelet-platelet interactions (4), its function in endothelial cells is not known. This led us to examine other cells in culture for the synthesis and secretion of TS. We have now identified TS in a variety of mesenchymal cells. Since TS is secreted and deposited in the cell layer of these cells in culture, we postulate that it may function as a matrix protein in vivo. MATERIALS AND METHODS Cell CultureBovine aortic endothelial (BAE) ceils were provided by Dr. S. Schwartz (University of Washington). They were maintained in Waymouth's medium supplemented with 10% fetal calf serum (FCS) (Reheis Chemical Company, Phoenix, AZ), penicillin (100 U/nil), and streptomycin (100 gg/ml). Human dermal fibroblasts were isolated from foreskin explants and maintained in Dulbecco's modified Eagle's medium (DME) supplemented with 10% FCS and antibiotics. Smooth muscle ceils were obtained from human aortae and were provided by Dr. Russell Ross (University of Washington). They were maintained in DME supplemented with 20% FCS and antibiotics. Ceils were grown at 37°C in an atmosphere of 5% CO2. Purification of Thrombospondins BAE cell TS was isolated according to the method of McPherson el al. (6).Human platelet TS was isolated from l-d-old blood bank platelets according to the procedure of Lawler a...

Thrombin Modulates the Expression of a Set of Genes Including Thrombospondin-1 in Human Microvascular Endothelial Cells

“…Although the precise mechanism that governs its secretion has not been fully elucidated, a direct correlation between THBS1 transcription and secretion into culture media has long been observed (42,43). To confirm that the increase in THBS1 mRNA correlated with an increase protein expression and to quantify the amount of THBS1 released into the culture medium, a competitive ELISA approach was employed.…”

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

“…Following incubation, the samples were centrifuged for 1 min at 10000 x g and the supernatants were removed and kcpt for electrophoretic analysis. The Affi-gel beads were washed with Tris/NaCI con- (1) and absence (2) of Ca"; lanes 3 and 4, thrombospondin bound to ovalbumin-Affi-gel in the presence (3) and absence (4) of Ca" taining either CaCI2 or EDTA for samples that contained digests of CaZ ' -thrombospondin or EDTA-throinbospondin, respectively. The beads were then transferred to new tubes to avoid contamination of fibronectin-bound material with that bound to the tube walls.…”

“…Several of the thrombospondin-synthesizing cells, among them endothelial cells [7], fibroblasts and smooth muscle cells [2], incorporate it into their extracellular matrices. In vivo, thrombospondin was shown to be an endogenous component of extracellular matrices of a variety of human tissues [XI. Thrombospondin is a multifunctional protein capable of interacting with numerous macromolecules, e. g. fibronectin [9, 101, heparin [ll], collagen 19, 121 and sulfated glycolipids [13].…”

Phosphatidylinositol 3-Kinase and Focal Adhesion Kinase Are Early Signals in the Growth Factor–Like Responses to Thrombospondin-1 Seen in Human Vascular Smooth Muscle

“…3,4 TSP-1 was originally described as a product of platelet ␣-granules 5 but has since been shown to be synthesized by a number of different cell types, including vascular endothelial and smooth muscle cells. 6,7 TSP-1 is present in negligible amounts in normal human blood vessels, but the expression of this protein is markedly upregulated in injured and diseased vasculature, 8 -10 suggesting a possible role for TSP-1 in the abnormal cellular response to vascular injury.…”