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At least 19 recordsLinked to original sources
PubMed1986

Structural and immunological comparison of human thrombospondins isolated from platelets and from culture supernatants of endothelial cells and fibroblasts. Evidence for a thrombospondin polymorphism.

Thrombospondin is a 450-kDa glycoprotein secreted by a variety of cells including endothelial cells, fibroblasts and platelets. The aim of this study was to compare the structural and immunological properties of human endothelial, fibroblast and platelet thrombospondins. All three thrombospondins were purified, digested with thermolysin, and the subsequent thermolysin-generated fragments isolated on a Superose 12 gel-permeation column using non-denaturating conditions. Each isolated proteolytic fragment of thrombospondins was then detected using either a radioimmunoassay with a polyclonal antibody or an enzyme-linked immunosorbent assay with three monoclonal antibodies (P10, MA-I, MA-II) directed against different epitopes of whole platelet thrombospondin. The fragmentation pattern of human endothelial thrombospondin consists of six major thermolysin-generated fragments (135-110, 98-82, 54-47, 25-20, 18-15 and 10 kDa) having molecular masses very similar to those observed with human fibroblast thrombospondin (115-100, 92-80, 54-49, 27-21, 17-13 and 12-10 kDa). Treatment of platelet thrombospondin with thermolysin only generated four proteolytic fragments having molecular masses of 110, 50, 25 and 12/10 kDa respectively. All these proteolytic fragments of endothelial, fibroblast and platelet thrombospondins were recognized by a polyclonal antibody. Monoclonal antibodies MA-I and P10 essentially recognized two proteolytic fragments (135-110, 98-82 kDa) of endothelial and fibroblast (115-100, 92-80 kDa) thrombospondins, and the 110-kDa fragment of platelet thrombospondin. Monoclonal antibody MA-II recognized three proteolytic fragments (54-47, 25-20, 18-15 kDa) of endothelial and fibroblast (54-49, 27-21, 17-13 kDa) thrombospondins, and two fragments (50, 25 kDa) of platelet thrombospondin, different from those detected by P10 an MA-I. The results clearly demonstrate that, under non-denaturating conditions, endothelial and fibroblast thrombospondins are structurally different from platelet thrombospondin since two fragments of endothelial thrombospondin (98-82, 18-15 kDa), equivalent to those of fibroblast thrombospondin (92-80, 17-13 kDa), are not released from platelet thrombospondin after thermolysin treatment. These three forms of thrombospondin are, however, immunologically indistinguishable. To investigate further the structural differences observed between platelet and the two other forms of thrombospondin, their degree of polymerization was compared. Prior to thermolysin treatment, the three forms of thrombospondin were separated into several oligomers ranging from 450 kDa to 3300 kDa when injected onto a Superose 6 gel-permeation column.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed2005

Biophysical characterization of the signature domains of thrombospondin-4 and thrombospondin-2.

The signature domain of thrombospondins consists of tandem epidermal growth factor-like modules, 13 calcium-binding repeats, and a lectin-like module. Although very similar, the signature domains of thrombospondin-1 and -2 differ in several potentially important ways from the domains of thrombospondin-3, -4, and -5. We have compared matching recombinant segments representing the signature domains of thrombospondin-2 and -4. In the presence of 2 mM CaCl2, the far UV circular dichroism spectra of thrombospondin-2 and -4 constructs contain a strong negative band at 202 nm, but only the thrombospondin-2 construct has a band at 216 nm. Chelation of calcium shifted the negative bands to lower magnitudes. Titrations of the spectra demonstrated lower cooperativity and affinity for binding of calcium to thrombospondin-4 compared with thrombospondin-2. Atomic absorption spectroscopy demonstrated that the thrombospondin-4 constructs bind seven less calcium than the thrombospondin-2 construct at 0.6 mM CaCl2. In 2 mM CaCl2, the near UV circular dichroism spectra of thrombospondin-2, but not thrombospondin-4, contain a positive band at 292 nm that disappears upon calcium chelation. Intrinsic fluorescence spectra for both proteins were also sensitive to calcium, but the changes were simpler and more marked for thrombospondin-2 than for thrombospondin-4. In differential scanning calorimetry, the thrombospondin-2 construct melted in two distinct transitions at 53.5 and 81.8 degrees C, whereas the first transition for thrombospondin-4 constructs was observed at 63.5 degrees C. Thus, the studies revealed significant differences between the signature domains of thrombospondin-2 and thrombospondin-4 in calcium binding, fine structure, and inter-modular interactions.

Amino Acid Motifs↗
PubMed1991

Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.

Ammonium Chloride↗
PubMed2003

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin.

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.

Amino Acid Sequence↗
PubMed1993

Thrombospondin 1 is a tight-binding competitive inhibitor of neutrophil cathepsin G. Determination of the kinetic mechanism of inhibition and localization of cathepsin G binding to the thrombospondin 1 type 3 repeats.

Thrombospondin 1 was recently shown to bind to and inhibit the activity of neutrophil elastase (Hogg, P. J., Owensby, D. A., Mosher, D. F., Misenheimer, T. M., and Chesterman, C. N. (1993) J. Biol. Chem. 268, 7139-7146). This finding led us to question whether thrombospondin 1 also binds and inhibits the other major serine proteinase of neutrophils, cathepsin G. In a competitive binding assay, cathepsin G bound to thrombospondin 1 reversibly and saturably with a dissociation constant in the low nanomolar range. The kinetic mechanism of inhibition of cathepsin G activity by thrombospondin 1 was determined using the synthetic cathepsin G substrate, Suc-Ala-Ala-Pro-Phe-p-nitroanilide, and is consistent with hyperbolic tight-binding inhibition in which thrombospondin 1 binds cathepsin G and the Michaelis cathepsin G-substrate complex and weakens, but does not abolish, the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide. In the presence of 2 mM calcium ions, 2.9 +/- 0.4 mol of cathepsin G interacted with 1 mol of thrombospondin 1 trimer with a site-binding constant of 7.0 +/- 3.5 nM, which reduced the efficiency of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-nitroanilide 8.5 +/- 1.4-fold. A lower limit for the on rate constant of 5 x 10(6) M-1 S-1 was established. The affinity of binding and stoichiometry for the interaction between cathepsin G and thrombospondin 1 was enhanced in the absence of calcium ions. In the presence of EDTA, 5.3 +/- 0.5 mol of cathepsin G interacted with 1 mol of thrombospondin 1 with a site-binding constant of 2.1 +/- 1.6 nM, implying the existence of two binding sites for cathepsin G on each subunit of thrombospondin 1, one or both of which is variably exposed and sensitive to calcium ions. Thrombospondin 1 protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation. In summary, the binding of cathepsin G to thrombospondin 1 is tight, reversible, and close enough to the active site of cathepsin G to perturb the interactions of a small synthetic substrate and exclude a macromolecular protein substrate and platelets. Using defined proteolytic fragments and different conformers of thrombospondin 1, the binding sites for cathepsin G have been localized to the thrombospondin 1 type 3 repeats.

Amides↗
PubMed1985

Radioimmunoassay of human platelet thrombospondin: different patterns of thrombospondin and beta-thromboglobulin antigen secretion and clearance from the circulation.

A method for radioimmunoassay of human thrombospondin was developed. Monospecific precipitating anti-human thrombospondin antibody was raised in rabbits after injection of thrombospondin purified by fibrinogen-agarose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linear portion of the thrombospondin radioimmunoassay standard curve was 0.5 to 20 ng/ml. Normal platelets and platelet-poor plasma contained 28,900 +/- 14,500 ng thrombospondin per 10(9) platelets and 60.6 +/- 10.7 ng/ml (mean +/- SD), respectively. Using radioimmunoassays for beta-thromboglobulin and thrombospondin antigens, we compared platelet location and secretion of these proteins. Both antigens shared similar distributions in platelet subcellular fractions with the largest amount localized to platelet alpha-granules. With thrombin (0.25 U/ml) as a platelet agonist, 62.4% and 19.5% of total beta-thromboglobulin and thrombospondin, respectively, were secreted from suspensions of washed human platelets. Because only 20% of the total platelet thrombospondin was secreted, further studies were initiated to determine whether the remaining thrombospondin became localized on the activated platelets membrane. 125I-Fab antithrombospondin specifically bound to activated platelets but not to unstimulated platelets. In contrast, 125I-Fab anti-beta-thromboglobulin did not bind to activated platelets. Plasma clearance of human beta-thromboglobulin (half-life fast 7.6 minutes, slow 56.6 minutes) and of human thrombospondin (half-life fast 29.9 minutes, slow 190 minutes) followed a biphasic exponential curve. In conclusion, both beta-thromboglobulin and thrombospondin are located in platelet alpha-granules, but they show a different pattern of secretion and expression on the platelet membrane and plasma clearance.

Animals↗
PubMed1993

Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family.

A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.

Amino Acid Sequence↗
PubMed1984

Effect of anti-thrombospondin antibodies on the hemagglutination activities of the endogenous platelet lectin and thrombospondin.

The proposal that thrombospondin is the endogenous platelet lectin was evaluated using antisera and monoclonal antibodies to thrombospondin. The platelet-bound hemagglutinin activity of human platelets stimulated with A23187 was inhibited by rabbit anti-thrombospondin sera and by a monoclonal anti-thrombospondin IgG. A second monoclonal IgG did not inhibit platelet-bound agglutinin activity. Preparations of purified platelet thrombospondin differed in their hemagglutination activities. The hemagglutination activity of an active preparation of thrombospondin was inhibited by the monoclonal antibody that inhibited platelet-bound lectin activity. The hemagglutination activity of an almost inactive preparation of thrombospondin was enhanced by the anti-thrombospondin monoclonal antibody that did not block platelet-bound lectin activity. The results demonstrate that expression of the platelet-bound form of the endogenous lectin is thrombospondin-dependent and suggest that thrombospondin must become part of a larger complex, either by binding to the platelet surface or by becoming aggregated in solution, before hemagglutination activity can be expressed.

Antibodies, Monoclonal↗
PubMed1983

A radioimmunoassay for thrombospondin, used in a comparative study of thrombospondin, beta-thromboglobulin and platelet factor 4 in healthy volunteers.

A radioimmunoassay was developed for the platelet alpha-granule protein thrombospondin; concentrations of thrombospondin as low as 3 ng ml-1 could be measured. There was no interference from other components of human biological fluids and no crossreactivity with beta-thromboglobulin (beta-TG) or platelet factor 4 (PF4). Plasma samples were stable when stored at -20 degrees C. Normal human plasma contained 105.0 +/- 31.0 ng thrombospondin ml-1 compared with beta-TG concentrations of 37.2 +/- 10.9 ng ml-1 and PF4 concentrations of 14.7 +/- 10.1 ng ml-1 when samples were carefully taken into a platelet inhibitor cocktail and processed at 0-4 degrees C. Release of thrombospondin during clotting of blood occurred at the same time as that of beta-TG and PF4 and resulted in a serum concentration of 17.5 +/- 5.5 micrograms ml-1. Assay of whole blood gave a platelet thrombospondin content of 89.1 +/- 28.3 ng/10(6) platelets. The concentration in normal urine fluctuated widely from 3 to 22.5 ng ml-1, and was unrelated to urine flow. The half-life of thrombospondin in vivo was about 9 h, much longer than that of either beta-TG or PF4. Unlike PF4, it was not released into the blood following an intravenous heparin injection. Bovine, ovine, canine and porcine sera contained thrombospondin which crossreacted immunologically with the human molecule; these species would be suitable animal models for the study of thrombospondin and its value as a platelet release marker.

Adult↗
PubMed1992

Thrombospondin II: partial cDNA sequence, chromosome location, and expression of a second member of the thrombospondin gene family in humans.

A novel form of human thrombospondin was identified during the screening of a human fibroblast cDNA library. We report the cDNA sequence for 1.8 kb of the 3' end of the cDNA, plus an additional 937 bp of 3'-untranslated sequence. The translated sequence reveals a high degree of similarity to thrombospondin I. The homology ranges from 56 to 80% for different regions within the two proteins. The repeating segments of amino acid sequence identified in thrombospondin I were found to be conserved in thrombospondin II. The new form of thrombospondin hybridizes to a 7.5-kb message by Northern analysis. The THBS2 gene is located at the distal long arm of chromosome 6 at 6q27. The gene is transcribed in fibroblasts, smooth muscle cells, and an osteosarcoma cell line, at levels somewhat lower than that of thrombospondin I. Umbilical vein endothelial cells do not transcribe thrombospondin II under the conditions of this study. These findings suggest that previous studies of thrombospondin function need to be reassessed to identify the functions specific to each molecule.

Amino Acid Sequence↗
PubMed1989

Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells. A model to study the different effects of thrombospondin in cell adhesion.

In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450-kDa glycoprotein initially found in platelets. Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts. SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti-thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells. Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG-63, SaOs-2) were performed to assess the ability of these cells to adhere to thrombospondin and type-I collagen. Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG-63 and SaOs-2 cells, both when it was directly adsorbed to plastic and when it was bound to type-I collagen. Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion.

Animals↗
PubMed1995

Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin-1: implications for the anti-adhesive activities of thrombospondin-1.

The organisation of the actin cytoskeleton was examined in H9c2 and human intestinal smooth muscle cells adherent on fibronectin or thrombospondin-1. Whereas cells adherent on fibronectin adopted a polygonal shape and rapidly assembled prominent stress fibres and focal contacts, cells adherent on thrombospondin-1 assumed a more irregular morphology with large lamellae containing radial actin microspikes. Focal contacts were not detected in cells adherent on thrombospondin-1, as determined by indirect immunofluorescence staining for vinculin and other focal contact components. Instead, the radial microspikes stained positively for the actin-bundling protein, 55 kDa/fascin, and myosins. In cells adherent on fibronectin, 55 kDa/fascin immunoreactivity was diffuse and tended to be concentrated in the perinuclear region. In long-term adherent cells cultured in serum-containing medium, 55 kDa/fascin was detected in membrane ruffles, in stress fibres and in the perinuclear region. The microspikes formed within 40 minutes of plating cells on thrombospondin-1 and remained present when cells were treated with sodium orthovandate and hydrogen peroxide to increase intracellular phosphotyrosine levels. Indeed, although vanadate-treated cells tended to retract, the microspikes became more prominent and showed an increased intensity of staining for fascin. Under these conditions, a proportion of the microspikes did not appear to be in contact with the substratum: these spikes stained weakly for focal adhesion kinase, talin and vinculin. Cells treated with genistein also spread and formed fascin-containing microspikes which tended to be more slender than those of control cells. In contrast, cells adherent on fibronectin displayed a complex rearrangement of the actin cytoskeleton and a transient enrichment of 55 kDa/fascin-containing structures at the cell surface when treated with sodium orthovanadate and hydrogen peroxide. These observations indicate that cell interactions with fibronectin or thrombospondin-1 send distinct organisational signals to the actin cytoskeleton and may offer a mechanistic framework for further investigations of the anti-adhesive properties of thrombospondin-1.

Actins↗
PubMed1983

Cooperative binding of calcium to thrombospondin. The effect of calcium on the circular dichroism and limited tryptic digestion of thrombospondin.

Removal of calcium from thrombospondin with EDTA results in a decrease in the intensity of the negative CD peak between 200 and 250 nm. Quantitative analysis of the CD spectrum of thrombospondin indicates that thrombospondin contains approximately 11% alpha-helix, 43% beta-sheet, and 46% random coil in the presence of calcium and that a small change in secondary structure may occur upon removal of calcium with EDTA. When the change in the CD at 220 nm is measured as a function of calcium concentration, a sigmoidal curve with a transition midpoint of 120 microM is obtained, indicating that the binding is cooperative. Analysis of Hill plots of the data revealed a Hill coefficient of 12.3. Calcium was also found to affect the peptide pattern produced by limited tryptic digestion of thrombospondin, with some portions of the molecule being resistant to trypsin in the presence of calcium. When the change in quantity of a 65,000-dalton tryptic fragment was measured as a function of calcium concentration, a sigmoidal curve was again obtained. The midpoint of this transition is achieved at a free calcium concentration of 45 microM at 0 degrees C and 103 microM at 25 degrees C. These data indicate that thrombospondin contains at least 12 binding sites for calcium and that cooperative interactions between sites are associated with a conformational change in the thrombospondin molecule.

Blood Platelets↗
PubMed2005

Thrombospondins, metallo proteases and thrombospondin receptors messenger RNA and protein expression in different tumour sublines of the Dunning prostate cancer model.

Thrombospondin is a potent inhibitor of angiogenesis and might therefore be important in controlling tumour growth. TSP interacts with a number of proteases and receptors and in this way inhibits stimulation of angiogenesis. An earlier study showed that thrombospondin is expressed in benign prostatic hyperplasia (BPH) and high-grade prostatic intraepithelial neoplasia (PIN) but is absent in prostate cancer. The present study was therefore designed to evaluate the expression of thrombospondin 1 and 2 (TSP-1, TSP-2), TSP receptors CD36 and CD47, and matrix-metalloproteases 2 and 9 (MMP-, MMP-9) in a rat prostate cancer model. By using immunohistochemistry, Western blot, and real-time PCR the expression patterns of TSP-1, TSP-2, CD36, CD47, MMP-2, and MMP-9 were investigated in normal rat prostate tissue and five malignant Dunning sublines tissue. TSP-1 mRNA levels were decreased in all tumours compared with normal prostate. However, there was no difference in expression of TSP-2 and CD36 mRNA in these samples. MMP-2 was increased with malignancy, but no expression of MMP-9 was seen. The CD47 receptor did slightly increase with malignancy except for H3327. The results showed that thrombospondin is expressed in normal prostate but not in prostate tumours in a rat model. Simultaneously, MMP-2 expression increases with malignancy.

Animals↗
PubMed2002

Trimeric assembly of the C-terminal region of thrombospondin-1 or thrombospondin-2 is necessary for cell spreading and fascin spike organisation.

Thrombospondin-1 (TSP-1) and the highly related protein thrombospondin-2 (TSP-2) are trimeric extracellular molecules that have complex roles in wound healing, angiogenesis and matrix organisation. At the cellular level, TSP-1 supports cell adhesion and migration by the organisation of fascin spike cytoskeletal structures. To define the molecular requirements for assembly of fascin spikes by thrombospondins, we developed a panel of recombinant protein units of TSP-1 and TSP-2; these were designed according to the domain boundaries and included matched monomeric and trimeric units. These proteins were tested for their effects on cell attachment and fascin spike organisation using C2C12 skeletal myoblasts and vascular smooth muscle cells. In monomeric units, cell attachment activity was localised to the type 1 repeats or type 3 repeats/C-terminal globule, and both regions need to be present in the same molecule for maximal activity. On a molar basis, cell-attachment activities with monomeric units were low compared with intact TSP-1, and no monomeric unit induced cell spreading. Trimeric versions of the type 1 repeats were more adhesive but did not induce cell spreading. Strikingly, trimers that contained the type 3 repeats/C-terminal globule of either TSP-1 or TSP-2 supported cell spreading and fascin spike organisation, producing a similar activity to intact TSP-1. We conclude that trimeric assembly of the highly conserved TSP C-terminal region is necessary for organisation of the fascin-based cytoskeletal structures that are needed for thrombospondin-induced cell motility.

Actin Cytoskeleton↗
PubMed2005

Thrombospondin 1, thrombospondin 2 and the eye.

Thrombospondin 1 and thrombospondin 2 (TSP1 and TSP2), which comprise the subgroup A thrombospondins, are matricellular proteins. As matricellular proteins, they modulate interactions between cells and the cellular environment, regulate cell adhesion and typically are expressed during tissue formative processes. In general, TSP1 and TSP2 counter angiogenesis (including tumour angiogenesis) and play important but contrasting roles during cutaneous repair. The two proteins are involved in development, including that of the eye, although evidence suggests that they have their greatest impact during tissue production in the adult. In the normal adult eye, they tend to be found at sites of ongoing matrix synthesis or cell-matrix interactions. At these sites, the two proteins possibly influence cellular differentiation and/or basement membrane deposition. TSP1 is also present in the intraocular fluids and drainage pathway, where it may function in maintaining the anti-angiogenic environment and in intraocular pressure control, respectively. TSP1 could also be involved in ocular immune privilege. Unlike in skin wounds, where TSP1 is derived from the blood and is present only in the early phases of repair, ocular tissue damage appears to lead to protacted TSP1 synthesis by local cells. This response might help suppress angiogenesis in the transparent tissues of the eye and so lessen visual axis opacification following injury. However, TSP2, which is also produced by damaged ophthalmic tissue and may be especially important in matrix organisation, seems to augment contraction in anomalous intraocular fibrosis. Elucidating the roles of TSP1 and TSP2 in ocular physiology and pathobiology may lead to improved therapies for neovascular, neoplastic, reparative and other ophthalmic diseases.

Animals↗
PubMed1999

Relative abundance of thrombospondin 2 and thrombospondin 3 mRNAs in human tissues.

The levels of thrombospondin 2 (TSP2) and thrombospondin 3 (TSP3) mRNAs in a variety of human tissues were determined by analysis of multiple-tissue mRNA dot blots. For TSP2 mRNA, aorta and fetal heart had the greatest relative abundance. High levels were also detected for muscle, fetal, endocrine, immune, and nerve tissues. The pattern of expression of TSP3 mRNA was very different: kidney, pituitary gland, trachea, uterus, and fetal kidney had the greatest abundance. In general, TSP3 mRNA was expressed at high levels in endocrine, muscle, and fetal tissues. In addition to the tissue-specific differences, a more even distribution of TSP3 mRNA among tissues was observed. The high relative abundance of the two mRNAs in a variety of tissues and the tissue-specific differences in expression could be significant for understanding the diverse roles implicated for TSP2 and TSP3.

Blotting, Northern↗
PubMed1997

Endothelial cell mitogenesis induced by LPA: inhibition by thrombospondin-1 and thrombospondin-2.

We examined the effects of thrombospondin-1 (TSP1) and thrombospondin-2 (TSP2) on the uptake of tritiated thymidine by bovine aortic endothelial (BAE) cells in response to two growth factors, basic fibroblast growth factor (bFGF) and lysophosphatidic acid (LPA). bFGF and LPA stimulate cell proliferation through distinct receptors that have convergent signaling pathways. The doses of LPA that trigger proliferation of BAE cells, which have not been reported previously, were 1 to 30 micromol/L, as opposed to the 5 to 100 micromol/L concentrations required to stimulate proliferation of human foreskin fibroblasts. Baseline mitogenic activity and activity stimulated by either bFGF or LPA on BAE cells was inhibited by human TSP1 purified from platelets or a recombinant source with a similar dose response. These results demonstrate that the anti-proliferative effect of platelet TSP1 is not caused by contaminants from the stimulated platelet. Recombinant mouse TSP2 inhibited BAE cell proliferation in response to LPA in a dose range similar to that of TSP1. Inasmuch as TSP2 does not activate latent TGFbeta1 (Schultz-Cherry et al., J Biol Chem 1995;270: 7304), these results show that inhibition of angiogenesis by TSPs is not related to control of activation of TGFbeta. Together, these studies suggest that structural motifs common to TSP1 and TSP2 inhibit endothelial cell proliferation. Furthermore, TSPs inhibit cell proliferation stimulated by two growth factor receptors that act through distinct signaling pathways.

Animals↗