Thrombospondins: Purification of human platelet thrombospondin-1.

Thrombospondins are a family of five secreted proteins that have diverse roles in modulating cellular function. Thrombospondins-1 and 2 were identified as matricellular proteins based on their functional roles combined with their transient appearance or accumulation in extracellular matrix at specific times during development and in response to injury or stress in mature tissues. Thrombospondin-1 is a major component of platelet α-granules, which provides a convenient source for purification of the protein. Methods are described to prepare thrombospondin-1 from human platelets in a biologically active form with minimal degradation or contamination with other platelet proteins. A nondenaturing method is described for removing bound transforming growth factor-ß1.

Thrombospondin-1 (TSP-1), a new bone morphogenetic protein-2 and -4 (BMP-2/4) antagonist identified in pituitary cells.

Bone morphogenetic proteins (BMPs) regulate diverse cellular responses during embryogenesis and in adulthood including cell differentiation, proliferation, and death in various tissues. In the adult pituitary, BMPs participate in the control of hormone secretion and cell proliferation, suggesting a potential endocrine/paracrine role for BMPs, but some of the mechanisms are unclear. Here, using a bioactivity test based on embryonic cells (C3H10T1/2) transfected with a BMP-responsive element, we sought to determine whether pituitary cells secrete BMPs or BMP antagonists. Interestingly, we found that pituitary-conditioned medium contains a factor that inhibits action of BMP-2 and -4. Combining surface plasmon resonance and high-resolution mass spectrometry helped pinpoint this factor as thrombospondin-1 (TSP-1). Surface plasmon resonance and co-immunoprecipitation confirmed that recombinant human TSP-1 can bind BMP-2 and -4 and antagonize their effects on C3H10T1/2 cells. Moreover, TSP-1 inhibited the action of serum BMPs. We also report that the von Willebrand type C domain of TSP-1 is likely responsible for this BMP-2/4-binding activity, an assertion based on sequence similarity that TSP-1 shares with the von Willebrand type C domain of Crossveinless 2 (CV-2), a BMP antagonist and member of the chordin family. In summary, we identified for the first time TSP-1 as a BMP-2/-4 antagonist and presented a structural basis for the physical interaction between TSP-1 and BMP-4. We propose that TSP-1 could regulate bioavailability of BMPs, either produced locally or reaching the pituitary via blood circulation. In conclusion, our findings provide new insights into the involvement of TSP-1 in the BMP-2/-4 mechanisms of action.

Expression, purification and structural characterization of functionally replete thrombospondin-1 type 1 repeats in a bacterial expression system.

The matrix glycoprotein thrombospondin-1 (TSP-1) is a prominent regulator of endothelial cells and angiogenesis. The anti-angiogenic and anti-tumorigenic properties of TSP-1 are in part mediated by the TSP-1 type 1 repeat domains 2 and 3, TSR(2,3). Here, we describe the expression and purification of human TSR(2,3) in milligram quantities from an Escherichia coli expression system. Microvascular endothelial cell migration assays and binding assays with a canonical TSP-1 ligand, histidine-rich glycoprotein (HRGP), indicate that recombinant TSR(2,3) exhibits anti-chemotactic and ligand binding properties similar to full length TSP-1. Furthermore, we determined the structure of E. coli expressed TSR(2,3) by X-ray crystallography at 2.4Å and found the structure to be identical to the existing TSR(2,3) crystal structure determined from a Drosophila expression system. The TSR(2,3) expression and purification protocol developed in this study allows for facile expression of TSR(2,3) for biochemical and biophysical studies, and will aid in the elucidation of the molecular mechanisms of TSP-1 anti-angiogenic and anti-tumorigenic activities.

Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation.

During cutaneous wound repair, platelets, dermal fibroblasts (DF) and endothelial cells all cooperate. We have presently investigated the regulation of endothelial cell tubulogenesis by human platelet thrombospondin-1 (TSP-1), in comparison to transforming growth factor-beta1 (TGF-beta1) and total platelet lysates (PL), in a fibrin matrix cell culture system incorporating DF. TSP-1, TGF-beta1 and PL all stimulated VEGF expression in DF dose dependently at mRNA and protein level. TSP-1- and PL-treated DF supernatants significantly stimulated capillary-like structure formation (tubulogenesis) by dermal microvascular endothelial cells (HMEC-1 and HDMEC), in part via VEGF, as confirmed with neutralizing anti-VEGF antibodies. In contrast, TGF-beta1-treated DF supernatants did not induce tubulogenesis. This apparent discrepancy could be explained by the differential expression regulation in HMEC-1 of fibrinolysis and metalloproteinase mediators by TSP-1 and TGF-beta1. TSP-1 potently reduced the expression of plasminogen activator inhibitor-1 (PAI-1) (mRNA and protein), whereas TGF-beta1 enhanced it. The crucial role of PAI-1 in tubulogenesis was confirmed via the addition of active recombinant PAI-1, which abrogated tubulogenesis. In contrast, neutralizing PAI-1 antibodies enhanced tubulogenesis. Our results suggest that platelet TSP-1 released in a wound stimulates endothelial cell tubulogenesis through an upregulation of DF VEGF expression and a downregulation of endothelial cell PAI-1 expression.

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization.

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.

Apoptotic cell thrombospondin-1 and heparin-binding domain lead to dendritic-cell phagocytic and tolerizing states.

Apoptotic cells were shown to induce dendritic cell immune tolerance. We applied a proteomic approach to identify molecules that are secreted from apoptotic monocytes, and thus may mediate engulfment and immune suppression. Supernatants of monocytes undergoing apoptosis were collected and compared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and differentially expressed proteins were identified using tandem mass spectrometry. Thrombospondin-1 (TSP-1) and its cleaved 26-kDa heparin-binding domain (HBD) were identified. We show that TSP-1 is expressed upon induction of monocyte apoptosis in a caspase-dependent pattern and the HBD is cleaved by chymotrypsin-like serine protease. We further show that CD29, CD36, CD47, CD51, and CD91 simultaneously participate in engulfment induction and generation of an immature dendritic cell (iDC) tolerogenic and phagocytic state. We conclude that apoptotic cell TSP-1, and notably its HBD, creates a signalosome in iDCs to improve engulfment and to tolerate engulfed material prior to the interaction with apoptotic cells.

Specific interaction of oxidized low-density lipoprotein with thrombospondin-1 inhibits transforming growth factor-beta from its activation.

Oxidized LDL (Ox-LDL) plays atherogenic roles, whereas thrombospondin-1 (TSP-1) is thought to be anti-atherogenic through activation of TGF-beta that contributes to plaque stabilization. Ox-LDL was prepared by incubating of human LDL with CuSO4. Effect of Ox-LDL on TSP-1-induced TGF-beta activation was examined in the present study. Incubation of Ox-LDL with mouse peritoneal macrophages for 3 days resulted in reduction in amounts of active TGF-beta in the culture medium by 70-78% when compared with that of parallel incubation without Ox-LDL. TSP-1 could enhance conversion of latent TGF-beta1 into active TGF-beta1 in a cell-free system. This TSP-1-mediated latent TGF-beta1 activation was inhibited by 30% by Ox-LDL, suggesting the possible interaction of Ox-LDL with TSP-1. Incubation of TSP-1 with [125I]Ox-LDL or [125I]LDL, followed by immunoprecipitation with an anti-TSP-1 antibody demonstrated that a significant amount of [125I]Ox-LDL was co-precipitated with TSP-1 while precipitation of [125I]LDL was negligible. Furthermore, upon TSP-1-conjugated Sepharose 4B affinity chromatography, both [125I]Ox-LDL and [125I]latent TGF-beta1 bound to the affinity gel were eluted by unlabeled Ox-LDL. These findings indicate that Ox-LDL interacts with TSP-1 and suppresses subsequent TSP-1-dependent TGF-beta activation, revealing a novel atherogenic function of Ox-LDL.

Platelet-derived thrombospondin-1 is necessary for the vitamin D-binding protein (Gc-globulin) to function as a chemotactic cofactor for C5a.

The chemotactic activity of C5a and C5a des Arg can be enhanced significantly by the vitamin D-binding protein (DBP), also known as Gc-globulin. DBP is a multifunctional 56-kDa plasma protein that binds and transports several diverse ligands. The objective of this study was to investigate the mechanisms by which DBP functions as a chemotactic cofactor for C5a using neutrophils and U937 cells transfected with the C5aR (U937-C5aR cells). The results demonstrate that U937-C5aR cells show C5a chemotactic enhancement only to DBP in serum, but, unlike mature neutrophils, this cell line cannot respond to DBP in plasma or to purified DBP. Analysis by SDS-PAGE and isoelectric focusing revealed no structural difference between DBP in serum compared with DBP in plasma. However, plasma supplemented with either serum, DBP-depleted serum, or activated platelet releasate provides a required factor and permits DBP to function as a chemotactic cofactor for C5a. Fractionation of activated platelet releasate revealed that the additional factor possessed the properties of thrombospondin-1 (TSP-1). Finally, purified TSP-1 alone could reproduce the effect of serum or platelet releasate, whereas Abs to TSP-1 could block these effects. These results provide clear evidence that TSP-1 is needed for DBP to function as a chemotactic cofactor for C5a.

Thrombospondin-1 promotes fibroblast-mediated collagen gel contraction caused by activation of latent transforming growth factor beta-1.

BACKGROUND: Grafting of cultured epithelium has become a useful technique for the treatment of epithelial defects, since grafted epithelial cells secrete factors promoting wound healing. We identified one such factor produced by cultured oral epithelial cells as thrombospondin-1 (TSP-1). Recently, TSP-1 was reported to act as an activator of transforming growth factor-beta1 (TGF-beta1). OBJECTIVE: The role of TSP-1 in wound healing and its mechanism were investigated in vitro and in vivo. METHODS: The cultured oral epithelial cell-conditioned medium was harvested and applied to Heparin-Sepharose affinity chromatography. Proteins were analyzed by N-Terminal sequencer. TSP-1 and the other factors were applied to fibroblasts-mediated collagen gel contraction assay. The amount of TGF-beta1 (latent TGF-beta1 (LTGF) and active TGF-beta1) in collagen gels was quantified by ELISA and Western blotting analysis. Collagen sponges were soaked with TSP-1 and implanted subcutaneously into rats. RESULTS: A 38 kDa protein secreted from cultured oral epithelial cells was found to be human TSP-1. TSP-1 promoted collagen gel contraction activity, and anti-human TSP-1 and TGF-beta1 antibody inhibited the activity. The diameters of the gels treated with LTGF and TSP-1 were reduced to a greater extent than those of gels treated with either factor alone. Although there were no significant differences in the amounts of total TGF-beta1, which include LTGF, the amount of 25 kDa TGF-beta1 was 3.30-fold greater in TSP-1-treated samples than controls. In vivo, 7 days after implantation, increased numbers of fibroblasts were observed in the sponges treated with TSP-1. CONCLUSION: These findings suggested that TSP-1 causes collagen gel contraction by activation of LTGF. TSP-1 is expected to be especially suitable for regulating wound healing.

Purification and analysis of thrombospondin-1.

Thromboapondin 1 (TSP-1) is a trimeric matricellular protein that is expressed by many cells. It contains several different domains that allow it to participate in cell adhesion, cell migration, and cell signaling. Recently TSP-1 has been shown to activate transforming growth factor beta (TGF-beta) and to inhibit both angiogenesis and tumor growth. This unit contains protocols for the purification of TSP-1 from platelet-rich plasma and the purification of TSP-1 proteolytic fragments.

Thrombospondin-1 binds to polyhistidine with high affinity and specificity.

Thrombospondin-1 (TSP1) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a (125)I solid-phase binding assay to demonstrate that TSP1 binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of (125)I-labelled TSP1 to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by TSP1. Heparin was a potent inhibitor of binding with a K(i) of 50 nM, suggesting that the heparin-binding domain of TSP1 may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of TSP1 to directly compete for TSP1 binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that TSP1 in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that TSP1 contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of TSP1 to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with TSP1 warrants further study.

Tumor-host interactions in the gallbladder suppress distal angiogenesis and tumor growth: involvement of transforming growth factor beta1.

Angiogenesis inhibitors produced by a primary tumor can create a systemic anti-angiogenic environment and maintain metastatic tumor cells in a state of dormancy. We show here that the gallbladder microenvironment modulates the production of transforming growth factor (TGF)-beta1, a multifunctional cytokine that functions as an endogenous anti-angiogenic and anti-tumor factor in a cranial window preparation. We found that a wide variety of human gallbladder tumors express TGF-beta1 irrespective of histologic type. We implanted a gel impregnated with basic fibroblast growth factor or Mz-ChA-2 tumor in the cranial windows of mice without tumors or mice with subcutaneous or gallbladder tumors to study angiogenesis and tumor growth at a secondary site. Angiogenesis, leukocyte-endothelial interaction in vessels and tumor growth in the cranial window were substantially inhibited in mice with gallbladder tumors. The concentration of TGF-beta1 in the plasma of mice with gallbladder tumors was 300% higher than that in the plasma of mice without tumors or with subcutaneous tumors. In contrast, there was no difference in the plasma levels of other anti- and pro-angiogenic factors. Treatment with neutralizing antibody against TGF-beta1 reversed both angiogenesis suppression and inhibition of leukocyte rolling induced by gallbladder tumors. TGF-beta1 also inhibited Mz-ChA-2 tumor cell proliferation. Our results indicate that the production of anti-angiogenesis/proliferation factors is regulated by tumor-host interactions.

The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.