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PHYSIOLOGY OF
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THROMBOSPONDIN Deane F. Mosher, M.D.
Departments of Medicine and Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706 KEY WORDS:
hemostasis, cell growth, platelet-derived growth factor, platelet IX-granule proteins, malaria.
ABSTRACT
Thrombospondin is a large, multifunctional glycoprotein released from activated platelets and secreted by growing cells. It binds to components of the cell surface and extracellular milieu. Thrombospondin probably modulates a number of processes, including aggregation of platelets, for mation and lysis of fibrin, adhesion and migration of cells, and progression of cells through the growth cycle. Studies relating thrombospondin to disease processes are just beginning. INTRODUCTION
Thrombospondin was first identified as "thrombin-sensitive protein", i.e. a major protein of unstimulated platelets but not of platelets stimulated with thrombin ( 1, 2). Subsequent studies revealed that loss of throm bospondin was due to release from a-granules upon stimulation rather than degradation of a platelet surface protein by thrombin (3, 4). Purified thrombospondin was found to be a disulfide-bonded trimer of large (approximately 150 kilodaltons, kd) identical subunits (5). Interest in thrombospondin increased with the findings that it is a major synthetic product of cells in culture (6, 7) and that it accounts for the "endogenous lectin" activity of stimulated platelets (8). There have been many studies of thrombospondin and several comprehensive reviews (9-12). The present review focuses on items that the author thinks are particularly interesting. 85 0066-4219/90/0401-0085$02.00
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STRUCTURE OF THROMBOSPONDIN Gene Structure
Analyses of the gene for thrombospondin are in progress. The recently reported sequence of the 5' flanking region, first exon, and first intron contains interesting regulatory motifs that are also found in the growth regulated c-fos gene ( 13).
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Structure of mRNA
Human thrombospondin cDNA has been cloned from libraries of endo thelial cells and fibroblasts (14, 15). The sequence of 5802 basepairs (bp) includes a coding region of 3510 bp (specifying a protein of 1 170 amino acids) and a long 3' untranslated region. The 3' region is rich in adenine (A) and thymidine (T) and contains TATT and ATTT sequences that may mediate the stability of mRNAs for cytokines, lymphokines, and oncogenes ( 15). Some cells also contain a smaller mRNA that lacks the 3' AfT-rich sequences and may represent a more stable form of the message ( 15). Structure of Protein
Electron microscopy, proteolytic fragmentation, and inspection of the amino acid sequence predicted from the cDNA have yielded a consistent model of the structure of thrombospondin ( 1 1, 14). Eight types of sequence occur in the following order from the amino-terminus to the carboxy terminus: 1. A signal sequence, which is lost during intracellular processing. 2. A globular heparin-binding domain. 3. A pair of cysteinyl residues to participate in the intersubunit disulfide bonds that hold the trimer together. 4. A cysteine-rich sequence homologous to the N-propeptide of collagen. 5. Three homologous repeats of a cysteine-rich sequence also found in malarial cell surface proteins and complement proteins (16, 17). 6. Three homologous repeats of cysteine-rich sequence for which epi dermal growth factor is the paradigm. 7. Approximately eight homologous repeats of a unique cysteine- and aspartate-rich sequence predicted to bind divalent cations because of the arrangement of aspartate residues. A potential cell adhesion sequence, arginine-glycine-aspartate, is found in this region. 8. A globular region containing a free cysteinyl residue. The trivalent, multifunctional thrombospondin protamer has been likened to a "bola" (18), with cysteine-rich repeating sequences of the
PHYSIOLOGY OF THROMBOSPONDIN
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three subunits acting as "thongs" to connect clustered amino-terminal globu lar domains to the three individual carboxy-terminal globular domains. A special feature of thrombospondin is cooperative binding of calcium ions, probably to the aspartate-rich sequences ( 19). Such binding is associated with a major conformational change. FUNCTIONS OF THROMBOSPONDIN
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Role in Formation of the Hemostatic Plug
Thrombospondin is stored in oc-granules of platelets alongside other mul tifunctional glycoproteins (fibronectin, fibrinogen, von Willebrand's factor), heparin-binding proteins (platelet factor 4, fJ-thromboglobulin), and growth factors (platelet-derived growth factor, transforming growth factor-fJ) (20; Figure I). Thrombospondin, unlike the other multifunctional glycoproteins, is present at only low « 250 ng/ml) concentration in plasma. Alpha-granule proteins are secreted upon platelet activation, and
many, including thrombospondin, bind to the surface of the activated platelet (9-12, 20-22). Candidates for thrombospondin binding sites on platelet surfaces include (a) the 88-kd glycoprotein (GP) known as CD36, GPIV, or GPIIIb (1 0, 1 2, 23, 24); (b) fibrinogen already bound to the GPIIb/lIIa complex ( 10, 12, 23, 24); (c ) the complex of GPlIIa with the oc-chain of the vitronectin receptor (25, 26); and (d) sulfated glycolipids (27) and proteoglycans (27, 28). Antibodies to thrombospondin, especially the carboxy-terminal globu lar domain, inhibit platelet aggregation (9-12, 23). One theory proposes that thrombospondin binds to both fibrinogen and CD36 and thus strengthens the interaction of fibrinogen with the platelet surface (1 0, 1 2). Thrombo spondin's free cysteine and at least one intrachain disulfide bond are protected by calcium ion; when these groups are unprotected, inter molecular thiol-disulfide exchange and aggregate formation occur (29). Aggregated thrombospondin has the potential to agglutinate cells (30). Such agglutination is probably mediated by the amino-terminal globular domains ( 1 1, 27, 30, 31). These domains are clustered and probably func tion as a single unit in protameric thrombospondin. Aggregation and multimerization of thrombospondin would create a multivalent molecule, which like IgM, is active in agglutination. The relationships among throm bospondin binding to platelets, thrombospondin-induced agglutination of platelets, and platelet aggregation are complex, given the many possible interactions of thrombospondin with molecules at or near the platelet surface (31, 32).
Thrombospondin is found in the fibrin meshwork of whole blood clots (33) and excised wounds (34). In studies of plasma or purified proteins,
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PLATELET
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}.�-:-",,;raGRANULE
Figure 1
Postulated flow of thrombospondin and related molecules in a wound area.
Platelets release alpha-granule contents: thrombospondin (TSP), fHhromboglobulin ({i-TG), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), and transforming growth factor-fJ (TGF-fJ). Thrombospondin binds to thc surfacc of a nucleated cell via heparan sulfate proteoglycan (HSPG), an integrin receptor (rxp), and/or CD36/glycoprotein IV (IV) and, like PDGF-receptor complex, is taken up by endocytosis and degraded. As a conse quence of binding at the cell surface, information is transmitted
(dotted lines) to the nucleus
to prepare the cell for DNA replication. Important to this preparation is transcription of the genes for thrombospondin and small inducible
gene (SIG) products. Secreted SIG products
and SIG homologs, platelet factor 4 and fJ-thromboglobulin, modulate the binding of thrombospondin to heparan sulfate proteoglycan.
thrombospondin interacts strongly with fibrin as well as fibrinogen and is incorporated into the fibrin clot network (35). The three subunits of thrombospondin may interact with polymerizing fibrin intermediates and serve as a trifunctional branching unit, thus inducing the formation of more numerous growing fibers and hastening fibrin polymerization (35). A structure with thinner fibers is 'produced. Inasmuch as the concentration ofthrombospondin near an activated platelet is a function of the distance from the platelet surface, thrombospondin may cause considerable hetero-
PHYSIOLOGY OF THROMBOSPONDIN
89
geneity of fibrin structure within a platelet plug. Such heterogeneity may influence the subsequent lysis of the clot (36). Importance to Cellular Function
Thrombospondin has a striking and unique distribution in the developing embryo (37). During the neurulation stage, it is present in ectoderm, neuroepithelium, epicardium and epimy ocardium, and developing somites. During the organogenesis stage, it is present in developing glands, muscles, and cartilage. During the fetal period and later, it is present in basement membranes of a number of organs. Although the distribution and intensity of immunostaining for thrombospondin are less in adult tissues than in embryonic tissues (34, 37-39), there is positive staining of basement membrane regions beneath glandular epithelium in skin and lung and intense staining at the dermal epidermal junction (38). Thrombospondin can be extracted from articular cartilage (40), decalcified bone (41), and umbilical arteries (42). Megakary ocytes contain thrombospondin (43, 44). Early wounds stain intensely for thrombospondin whereas healed wounds hardly stain at all (34). This finding predicts a correlation between the needs for cells to proliferate and migrate and the expression of throm bospondin. Cell culture experiments bear out this prediction. Proliferating cultures of endothelial cells, smooth muscle cells, and fibroblasts make more thrombospondin than do nondividing cells (45). Thrombospondin synthesis is induced by molecules released from platelet a-granules (Figure 1). Platelet-derived growth factor induces rapid (within one hour) and transient increases in synthesis of thrombospondin and its mRNA when administered to growth-arrested smooth muscle cells (46-48). Thrombo spondin mRNA is "super induced" by cycloheximide in a manner similar to that of c-myc, c-fos, and other growth-regulatory gene products (48). Transforming growth factor-p induces a more sustained increase in thrombospondin mRNA (49). Platelets induce monocytes to synthesize thrombospondin, an event that requires platelet-monocyte contact rather than soluble products released from stimulated platelets (50). Such contact may be mediated by thrombospondin itself (51).
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EXPRESSION OF Tf[ROMBOSPONDIN
EFFECTS ON CELL GROWTH Thrombospondin has modest growth-pro moting activity when added to serum-starved smooth muscle cells; this effect is blocked by heparin (52). Thrombospondin also causes increased turnover of phosphoinositides and activity of S6 kinase (53). Addition of antithrombospondin antibodies to actively growing cells cultured in serum containing medium inhibits growth (54). Such results indicate that throm bospondin is an autocrine growth factor, i.e. secreted thrombospondin binds to the cell surface and induces growth (Figure 1).
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METABOLISM OF THROMBOSPONDIN Thrombospondin binds to cell layers of cultured fibroblasts to two sites and in two ways, nonsaturably to extracellular matrix and saturably to the cell surface (55). Throm bospondin bound to the cell surface has a punctate distribution and is subject to receptor-mediated endocytosis and degradation in lysosomes (55). There are a number of molecules in extracellular matrix to which thrombospondin may bind, including proteoglycans, fibronectin, type V and other collagens, and osteonectin (9, 10, 55-57). Matrix-bound thrombospondin is resistant to rather harsh extraction conditions, but nevertheless is rapidly taken up by the cells and degraded (55). The major cellular mediator of thrombospondin binding and degra dation is a membrane form of heparan sulfate proteoglycan (39, 55, 5860; Figure 1). Binding and degradation are inhibited by heparin and other sulfated glycosaminoglycans (presumably because of interaction with the amino-terminal domains of thrombospondin) and by platelet factor 4 and fl-thromboglobulin (presumably because of interaction with the sidechains of the proteoglycan) (58). Thus, the three heparin-binding proteins released from platelets compete with one another for binding sites on nucleated cells (Figure 1). Interestingly, genes for homologs of platelet factor 4 and fl-thromboglobulin have been identified because, like the gene for thrombospondin, their transcription is induced by platelet-derived growth factor (see, for example 61, 62). These have been called "small inducible genes" (SIG; 61). The products of these genes are secreted (62) and may compete for thrombospondin binding sites (Figure 1). CD36 and the integrin receptor for vitronectin also interact with throm bospondin (12, 23-25) and may serve as additional mediators of throm bospondin binding to cell surfaces (Figure 1). It remains to be determined whether thrombospondin or its aggregates can cluster various cell surface receptors together. Such a scenario would explain why five different anti
thrombospondin monoclonal antibodies to widely separated epitopes ean
block cell growth (54). The literature on thrombo spondin and cell adhesion is confusing at present, with some papers focusing on pro-adhesive effects (see for example 25,63) and others on anti adhesive effects (e.g. 39,64). Among the pro-adhesion papers, reasonable arguments have been proposed that adhesion to thrombospondin is mediated by an arginine-glycine-aspartate-sensitive integrin receptor (25, 65), heparan sulfate proteoglycan (63, 66), a receptor specific for the car boxyl-terminal domain of thrombospondin (66), or some combination thereof (66). Thrombospondin is active in migration assays (67). The carboxyl-terminal domains are important for haptotaxis (directional EFFECTS ON CELL ADHESION AND MOVEMENT
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movement up a gradient of substratum-bound thrombospondin) whereas the aminoterminal domains are important for chemotaxis (movement toward increasing concentrations of soluble thrombospondin). Throm bospondin causes endothelial cells to lose focal adhesion plaques (68). This effect is neutralized by molecules that bind to the amino-terminal domain of thrombospondin. Undifferentiated keratinocytes attach and spread on a thrombospondin-coated surface, but differentiated keratinocytes do not (69). This effect requires an intact carboxyl-terminal domain and is con sistent with the localization of thromobospondin at the dermal-epidermal junction (38). Thus, thrombospondin probably does interact in important ways with the adhesive machinery of cells, possibly destabilizing cell matrix contacts so that cells can migrate or round up and pass through mitosis. CHANGES IN DISEASE STATES A ltered Platelet Thrombospondin
Thrombospondin, along with other a-granule components, is largely miss ing from platelets of patients with Gray platelet syndrome (70). The deficiency in such patients may be in the targeting of endogenously syn thesized secretory proteins to developing a-granules in megakaryocytes (71). Thrombospondin has been found to be deficient, along with GPIb, in platelets of patients with various malignancies and abnormal "platelet antithrombin tests" (72) and, along with GPIa, in platelets of an unusual patient whose defective collagen-induced platelet aggregation and hemorrhagic diathesis disappeared with menopause (73). A degraded form of thrombospondin is found in patients with primary thrombocytosis and other myeloproliferative diseases (70). Elucidation of the pathogenetic mechanisms underlying these observations and their medical significance will require a more complete understanding of glycoprotein metabolism in the megakaryocyte and of the complex roles played by platelets in blood coagulation. Levels of Plasma and Serum Thrombospondin
The normal plasma concentration of thrombospondin is 40 to 250 ngjml (70). Similar levels are found in plasma of patients with thrombocytopenia due to recent chemotherapy, which indicates that the measured protein is not released artifactually from platelets during sample processing and that the dominant sources for thrombospondin in normal plasma are various tissues (74). The half-life of injected thrombospondin is extremely short, 10 to 75 min (75, 76). Thus, the quantity of thrombospondin being released into plasma could be substantial despite the low plasma level. There is
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little correlation between the concentrations of plasma thrombospondin and fj-thromboglobulin (74). Statistically significant increases in plasma thrombospondin are found in patients with recent total hip replacement, chronic renal failure, chronic liver failure, remote splenectomy, and recent (within 24 hours) acute myocardial infarction (70). The concentration of thrombospondin in serum is proportional to the platelet count (74). At present, thrombospondin is not a clinically significant analyte. Levels have not been quantified in a number of disease states, however, and this may change in the future. Thrombospondin in Tissues
There have been few studies of thrombospondin in diseased tissues. Cyto sols of malignant breast tissue contain more thrombospondin than cytosols of normal breast, perhaps because of activation of endothelium (77). Breast cyst fluids can be very rich in thrombospondin (up to 55,000 ng/ml) (78). Systematic studies of thrombospondin in various diseased tissues could be quite informative. Tumor cells in culture regularly synthesize and secrete thrombospondin (79; Table 1). Synthesis in neoplastically transformed cells may not be as regulated as in normal cells (45). Neoplastic lesions,
therefore, especially need to be studied, with care taken to distinguish synthetic pools of thrombospondin in the tumor cells and reacting cells from thrombospondin deposited in extracellular matrix. Table 1
Synthesis and secretion of thrombospondin by cultured human tumor cells' Production of thrombospondin Cells
Normal fibroblasts
(�g/ 106 cells/24 hr) 32
SV-40 transformed fibroblasts
8.2
Fibrosarcoma
4.9
Rhabdomyosarcoma Normal glial cells
5.3 12
Glioblastoma
7.1
Wilm's tumor
0.8
Neuroblastoma
1.0
Teratocarcinoma
1.0
Choriocarcinoma
7.8
Lung carcinoma
2.8
Melanoma
9.3
Unknown primary
17
"A competitive enzyme-linked immunoassay for human thrombospondin (80) was used to measure thrombospondin in conditioned media provided to the author by Prof. Antti Vaheri, University of Helsinki. The media contained rabbit serum (which did not interfere with the assay) and was placed over just-confluent cells
for 24 hr.
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Possible Role of Thrombospondin in Falciparum Malaria There has been considerable interest in the role that thrombospondin may play in the sequestration of Plasmodiumfalciparum. Knobs on the surface of infected erythrocytes are the points of attachment to endothelium. Such infected erythrocytes also bind specifically to thrombospondin, and antithrombospondin antibodies block adherence of infected erythrocytes to some melanoma cell lines (81). Cytoadherence is also blocked by anti body to CD36 (82). As described above, CD36 is a putative thrombospondin
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receptor, which raises the possibility that thrombospondin forms a bridge between erythrocyte knobs and endothelial CD36. Adherence of infected erythrocytes to melanoma cell lines, however, does not correlate with expression of thrombospondin (83), and expression of a CD36 cDNA clone in an otherwise nonexpressing cell line results in cytoadherence of parasitized erythrocytes but not increased binding of thrombospondin (84). Because there are no sequence homologies between thrombospondin and CD36, it seems most likely that there are two distinct adhesion mech anisms, one allowing adhesion of parasitized erythrocytes to thrombo spondin and the other adhesion to CD36 (84). ACKNOWLEDGMENTS
The experimental work described herein was supported by National Insti tutes of Health Grant HL29586.
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and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth mus cle cells. J. Cell Bioi. 101: 1059-70 47. Kobayashi, S., Eden-McCutchan, F., Framson, P., Bornstein, P. 1986. Partial amino acid sequence of human throm bospondin as determined by analysis of cDNA clones: Homology to malarial circumsporozoite proteins. Biochemistry 25: 8418-25 48. Majack, R. A., Milbrandt, J., Dixit, V. M. 1987. Induction of thrombospondin messenger RNA levels occurs as an immediate primary response to platelet derived growth factor. J. BioI. Chem. 262: 8821-25 49. Penttinen, R. P., Kobayashi, S., Bornstein, P. 1988. Transfonning growth factor fJ increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stab ility. Proc. Natl. A cad. Sci. USA 85: 1105-8
50. Schwartz, B. S. 1989. Monocyte syn thesis of thrombospondin: The role of platelets. J. Bioi. Chem. 264: 7512-17 51. Silverstein, R. L., Nachman, R. L. 1987. Thrombospondin binds to monocytes macrophages and mediates platelet monocyte adhesion. J. Clin. Invest. 79: 867-74 52. Majack, R. A., Cook, S. C., Bornstein, P. 1986. Control of smooth muscle growth by components of the extra cellular matrix: Autocrinc role for thrombospondin. Proc. Natl. Acad. Sci. USA 83: 9050-54 53. Burden, T. S., Resink, T. J., Baur, U., Burgin, M., Biihler, F. R. 1988. Acti vation of S6 kinase in cultured vascular smooth muscle cells by submitogenic levels of thrombospondin. Biochem. Biophys. Res. Commun. 150: 278-86 54. Majack, R. A., Goodman, L. V. , V. M. 1988. Cell surface thrombo spondin is functionally essential for vas cular smooth muscle cell prolifera tion. J. Cell Bioi. 106: 415-22 55. McKeown-Longo, P . J., Hanning, R., Mosher, D. F. 1984. Binding and degra dation of platelet thrombospondin by cultured fibroblasts. J. Cell Bioi. 98: 2228 56. Clezardin, P., Malaval, L., Ehren sperger, A. S., Delmas, P. D., Dechav anne, M., et al. 1988. Complex for mation of human thrombospondin with osteonectin. Eur. J. Biochem. 175: 27584 57. Dreyfus, M., Lahav, J. 1988. The build up of the thrombospondin extracellular matrix. An apparent dependence on syn.
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thesis and on preformed fibrillar matrix. Eur. J. Cell Bioi. 47: 257-82 58. Murphy-Ullrich, J. E., Mosher, D. F. 1987. Interactions of t hrombo spon din with endothelial cells: Receptor mediated binding and de gr adati on . J. Cell Bio!. 105: 1603-11 59. Murphy-Ullrich, J. E., Westrick, L. G., Esko, J. D.,Mosher,D. F. 1988. Altered m etabolism of thrombospondin by Chinese hamster ovary cells defective in gl ycosaminoglycan synthesis. J. Bioi. Chern. 263: 640(H) 60. Vischer, P., Vol ke r W., Schmi dt A., Sinclair, N. 1988. Association of throm bospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks. Eur. J. Cell BioI. 47: 36--46 61. Kawahara, R. S. , Deuel, T. F. 1989. Pla telet-derived growth factor-inducible gene JEis a member of a family of small inducible genes related to platelet factor 4. J. Bioi. Chern. 264: 679-82 62. Oquendo, P. , Alberta, J., Wen, D. , Graycar, J. L., De rynck R., et al. 1989. The platelet-derived growth factor inducible KC gene encodes a secretory protein related to platelet IX-granule proteins. J. BioI. Chern. 264: 413337 63. Kaesberg, P. R., Ershler, W. B., Es ko , J. D., Mosher, D. F. 1989. Chinese ham ster ovary cell adhesion to human plate let thrombospondin is dependent on cell surface heparin sulfate proteoglycan. J. Clin. Invest. 83: 994--1001 64. Lahav, J. 1988. Thrombospondin inhibits adhesion of endothelial cells. Exp. Cell Res. 177: 199-204 65. Tuszynski, G. P., Karczewski, T., Smith, L., Murphy, A., Rothman, V., et a1. 1989. The G PIIb/IIIa -li ke complex may function as a human melanoma cell adhesion receptor for thrombospondin. Exp. Cell Res. 182: 473-81 66. Roberts, D. D . , Sherwood, J. A., Gins burg, V. 1987. Platelet thrombospondin mediates attachment and spreading of hum an melanoma cells. J. Cell Bioi. 104: 131-39 67. Taraboletti, G., Roberts, D. D., Liotta, L. A. 1987. Thrombospondin-induced tumor cell migration: Haptotaxis and chemotaxis are mediated by different molecular domains. J. Cell Bioi. 105: 2409-15 68. Murphy-Ullrich, J. E. , Hook, M. 1989. Thrombospondin modulates focal ad hesions in endothelial cells. J. Cell Bio i. 109: 1309 19 69. Varani, J. , Nickoloff, B. J., Riser, B. L., Mitra, R. S., O Ro ur ke , K., et a1. 1988. ,
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,
'
Thrombospondin-induced adhesion of human keratinocytes. J. CUn. Invest. 81: 1537--44 70. Booth, W. J., Berndt, M. C. 1987. Thrombospondin in clinical disease states. Semin. Thromb. Hemostasis 13: 298-306 71. Rosa, J.-P. , George, J. N., Bainton, D. F.,Nurden,A. T.,Caen,J. P.,et a1. 1987. Gray platelet syndrome. Demonstration of alpha granule membranes that can fuse with the cell surface. J. Clin. Invest. 80: 1138-46 72. Sioand, E. M. , Kenney, D. M. , Chao, F. c., Lawler, J., Tullis, J. L. 1987. Platelet antithrombin defect in malignancy: Pla telet protein alterations. Blood 69: 47985 73. Kehrel, B., Balleisen, L., Kokott, R., Mesters, R., Stenzinger, W., et al. 1988. Deficiency of intact thrombospondin and membrane glycoprotein Ia in pla telets with defective collagen-induced aggregation and spontaneous loss of dis order. Blood 71: 1074--7 8 74. Dawes, J., Pratt, D. A. , Dewar, M. S. , Preston, F. E. 1988. Do extra-platelet sources contribute to the plasma le vel of thrombospondin. Thromb. Haemostasis 59:273-76 75. Switalska, H. I. , Niewiarowski, S., Tu s zyns ki, G. P., R ucins ki , B., Schma ier, A. H., et a1. 1985. Radioimmuno assay of human platelet thrombospon din: Differen t patterns of thrombospon din and fi-thromboglobulin antigen secretion and clearance from the circulation. J. Lab. CUn. Med. 106: 690700 76. Perlman, S. B., Foits, J. D ., Hammes, R. J., Besozzi, M. c., Mosher, D. F. 1987. . The accumulation of a platelet protein, thrombospondin, at the site of arterial thrombus formation: Preliminary r eport. Eur. J. Nuc!. Med. 12: 492-95 77. Pratt, D A., Miller, W. R., Dawes, J. 1989. Thrombospondin in malignant and non�malignant breast tissue. Eur. J. Cancer CUn. Oneol. 25: 343-50 78. Miller, W. R., Dawes, J. 1985. Platelet associated proteins i� human breast cyst fluids. CUn. Chim. Acta 152: 37-42 79. Varani, J., Riser, B. L., Hu ghes, L. A., Carey, T. E., Fligiel, S. E. , et a1. 1989. Characterization of thrombospondin synthesis, secretion and cell surface expression by human tumor cells. Clin. Exp. Metastasis 7: 265-76 80. Jaffe, E. A., Ruggiero, J. T., Leung, L. L. K., Doyle, M. J., McKeown-Longo, P. J., et a1. 1983. Cultured human fibro blasts synthesize and secrete thrombo spondin and incoroporate it into extra-
PHYSIOLOGY OF THROMBOSPONDIN cellular matrix. Proc. Nat!. A cad. Sci. USA 80: 998�1002 81. Roberts, D. D., Sherwood, J. A., Spi talnik, S. L. , Panton, L. J., Howard , R. J. , falciparum malaria parasitized eryth rocytcs and may mediate cytoadhcrence. Nature 318: 64-66 82. Barnwell, J. W., Ockenhouse, C. F., Knowles, D. M. II. 1985. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum�
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infected
erythrocytes
to
monocytes,
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endothelial, and C32 melanoma cells. J. Immunol. 135: 3494--97 83. Sherwood , J. A., Roberts, D. D., Spi talnik , S. L. , Marsh, K., Harvey, E. B., et al. 1989. Studies of the receptors on melanoma cells for Plasmodium fal ciparum infected erythrocytes. Am. J. Trop. Aled. lfyg. 40: 119�27 84. Oquendo, P., Hundt, E., Lawler, J., Seed, B. 1989. CD36 directly mediates cytoadherence of Plasmodium fal ciparum parasitized erythrocytes. Cell 58: 95�101
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PHYSIOLOGY OF
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THROMBOSPONDIN Deane F. Mosher, M.D.
Departments of Medicine and Physiological Chemistry, University of Wisconsin, Madison, Wisconsin 53706 KEY WORDS:
hemostasis, cell growth, platelet-derived growth factor, platelet IX-granule proteins, malaria.
ABSTRACT
Thrombospondin is a large, multifunctional glycoprotein released from activated platelets and secreted by growing cells. It binds to components of the cell surface and extracellular milieu. Thrombospondin probably modulates a number of processes, including aggregation of platelets, for mation and lysis of fibrin, adhesion and migration of cells, and progression of cells through the growth cycle. Studies relating thrombospondin to disease processes are just beginning. INTRODUCTION
Thrombospondin was first identified as "thrombin-sensitive protein", i.e. a major protein of unstimulated platelets but not of platelets stimulated with thrombin ( 1, 2). Subsequent studies revealed that loss of throm bospondin was due to release from a-granules upon stimulation rather than degradation of a platelet surface protein by thrombin (3, 4). Purified thrombospondin was found to be a disulfide-bonded trimer of large (approximately 150 kilodaltons, kd) identical subunits (5). Interest in thrombospondin increased with the findings that it is a major synthetic product of cells in culture (6, 7) and that it accounts for the "endogenous lectin" activity of stimulated platelets (8). There have been many studies of thrombospondin and several comprehensive reviews (9-12). The present review focuses on items that the author thinks are particularly interesting. 85 0066-4219/90/0401-0085$02.00
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STRUCTURE OF THROMBOSPONDIN Gene Structure
Analyses of the gene for thrombospondin are in progress. The recently reported sequence of the 5' flanking region, first exon, and first intron contains interesting regulatory motifs that are also found in the growth regulated c-fos gene ( 13).
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Structure of mRNA
Human thrombospondin cDNA has been cloned from libraries of endo thelial cells and fibroblasts (14, 15). The sequence of 5802 basepairs (bp) includes a coding region of 3510 bp (specifying a protein of 1 170 amino acids) and a long 3' untranslated region. The 3' region is rich in adenine (A) and thymidine (T) and contains TATT and ATTT sequences that may mediate the stability of mRNAs for cytokines, lymphokines, and oncogenes ( 15). Some cells also contain a smaller mRNA that lacks the 3' AfT-rich sequences and may represent a more stable form of the message ( 15). Structure of Protein
Electron microscopy, proteolytic fragmentation, and inspection of the amino acid sequence predicted from the cDNA have yielded a consistent model of the structure of thrombospondin ( 1 1, 14). Eight types of sequence occur in the following order from the amino-terminus to the carboxy terminus: 1. A signal sequence, which is lost during intracellular processing. 2. A globular heparin-binding domain. 3. A pair of cysteinyl residues to participate in the intersubunit disulfide bonds that hold the trimer together. 4. A cysteine-rich sequence homologous to the N-propeptide of collagen. 5. Three homologous repeats of a cysteine-rich sequence also found in malarial cell surface proteins and complement proteins (16, 17). 6. Three homologous repeats of cysteine-rich sequence for which epi dermal growth factor is the paradigm. 7. Approximately eight homologous repeats of a unique cysteine- and aspartate-rich sequence predicted to bind divalent cations because of the arrangement of aspartate residues. A potential cell adhesion sequence, arginine-glycine-aspartate, is found in this region. 8. A globular region containing a free cysteinyl residue. The trivalent, multifunctional thrombospondin protamer has been likened to a "bola" (18), with cysteine-rich repeating sequences of the
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three subunits acting as "thongs" to connect clustered amino-terminal globu lar domains to the three individual carboxy-terminal globular domains. A special feature of thrombospondin is cooperative binding of calcium ions, probably to the aspartate-rich sequences ( 19). Such binding is associated with a major conformational change. FUNCTIONS OF THROMBOSPONDIN
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Role in Formation of the Hemostatic Plug
Thrombospondin is stored in oc-granules of platelets alongside other mul tifunctional glycoproteins (fibronectin, fibrinogen, von Willebrand's factor), heparin-binding proteins (platelet factor 4, fJ-thromboglobulin), and growth factors (platelet-derived growth factor, transforming growth factor-fJ) (20; Figure I). Thrombospondin, unlike the other multifunctional glycoproteins, is present at only low « 250 ng/ml) concentration in plasma. Alpha-granule proteins are secreted upon platelet activation, and
many, including thrombospondin, bind to the surface of the activated platelet (9-12, 20-22). Candidates for thrombospondin binding sites on platelet surfaces include (a) the 88-kd glycoprotein (GP) known as CD36, GPIV, or GPIIIb (1 0, 1 2, 23, 24); (b) fibrinogen already bound to the GPIIb/lIIa complex ( 10, 12, 23, 24); (c ) the complex of GPlIIa with the oc-chain of the vitronectin receptor (25, 26); and (d) sulfated glycolipids (27) and proteoglycans (27, 28). Antibodies to thrombospondin, especially the carboxy-terminal globu lar domain, inhibit platelet aggregation (9-12, 23). One theory proposes that thrombospondin binds to both fibrinogen and CD36 and thus strengthens the interaction of fibrinogen with the platelet surface (1 0, 1 2). Thrombo spondin's free cysteine and at least one intrachain disulfide bond are protected by calcium ion; when these groups are unprotected, inter molecular thiol-disulfide exchange and aggregate formation occur (29). Aggregated thrombospondin has the potential to agglutinate cells (30). Such agglutination is probably mediated by the amino-terminal globular domains ( 1 1, 27, 30, 31). These domains are clustered and probably func tion as a single unit in protameric thrombospondin. Aggregation and multimerization of thrombospondin would create a multivalent molecule, which like IgM, is active in agglutination. The relationships among throm bospondin binding to platelets, thrombospondin-induced agglutination of platelets, and platelet aggregation are complex, given the many possible interactions of thrombospondin with molecules at or near the platelet surface (31, 32).
Thrombospondin is found in the fibrin meshwork of whole blood clots (33) and excised wounds (34). In studies of plasma or purified proteins,
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PLATELET
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}.�-:-",,;raGRANULE
Figure 1
Postulated flow of thrombospondin and related molecules in a wound area.
Platelets release alpha-granule contents: thrombospondin (TSP), fHhromboglobulin ({i-TG), platelet factor 4 (PF-4), platelet-derived growth factor (PDGF), and transforming growth factor-fJ (TGF-fJ). Thrombospondin binds to thc surfacc of a nucleated cell via heparan sulfate proteoglycan (HSPG), an integrin receptor (rxp), and/or CD36/glycoprotein IV (IV) and, like PDGF-receptor complex, is taken up by endocytosis and degraded. As a conse quence of binding at the cell surface, information is transmitted
(dotted lines) to the nucleus
to prepare the cell for DNA replication. Important to this preparation is transcription of the genes for thrombospondin and small inducible
gene (SIG) products. Secreted SIG products
and SIG homologs, platelet factor 4 and fJ-thromboglobulin, modulate the binding of thrombospondin to heparan sulfate proteoglycan.
thrombospondin interacts strongly with fibrin as well as fibrinogen and is incorporated into the fibrin clot network (35). The three subunits of thrombospondin may interact with polymerizing fibrin intermediates and serve as a trifunctional branching unit, thus inducing the formation of more numerous growing fibers and hastening fibrin polymerization (35). A structure with thinner fibers is 'produced. Inasmuch as the concentration ofthrombospondin near an activated platelet is a function of the distance from the platelet surface, thrombospondin may cause considerable hetero-
PHYSIOLOGY OF THROMBOSPONDIN
89
geneity of fibrin structure within a platelet plug. Such heterogeneity may influence the subsequent lysis of the clot (36). Importance to Cellular Function
Thrombospondin has a striking and unique distribution in the developing embryo (37). During the neurulation stage, it is present in ectoderm, neuroepithelium, epicardium and epimy ocardium, and developing somites. During the organogenesis stage, it is present in developing glands, muscles, and cartilage. During the fetal period and later, it is present in basement membranes of a number of organs. Although the distribution and intensity of immunostaining for thrombospondin are less in adult tissues than in embryonic tissues (34, 37-39), there is positive staining of basement membrane regions beneath glandular epithelium in skin and lung and intense staining at the dermal epidermal junction (38). Thrombospondin can be extracted from articular cartilage (40), decalcified bone (41), and umbilical arteries (42). Megakary ocytes contain thrombospondin (43, 44). Early wounds stain intensely for thrombospondin whereas healed wounds hardly stain at all (34). This finding predicts a correlation between the needs for cells to proliferate and migrate and the expression of throm bospondin. Cell culture experiments bear out this prediction. Proliferating cultures of endothelial cells, smooth muscle cells, and fibroblasts make more thrombospondin than do nondividing cells (45). Thrombospondin synthesis is induced by molecules released from platelet a-granules (Figure 1). Platelet-derived growth factor induces rapid (within one hour) and transient increases in synthesis of thrombospondin and its mRNA when administered to growth-arrested smooth muscle cells (46-48). Thrombo spondin mRNA is "super induced" by cycloheximide in a manner similar to that of c-myc, c-fos, and other growth-regulatory gene products (48). Transforming growth factor-p induces a more sustained increase in thrombospondin mRNA (49). Platelets induce monocytes to synthesize thrombospondin, an event that requires platelet-monocyte contact rather than soluble products released from stimulated platelets (50). Such contact may be mediated by thrombospondin itself (51).
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EXPRESSION OF Tf[ROMBOSPONDIN
EFFECTS ON CELL GROWTH Thrombospondin has modest growth-pro moting activity when added to serum-starved smooth muscle cells; this effect is blocked by heparin (52). Thrombospondin also causes increased turnover of phosphoinositides and activity of S6 kinase (53). Addition of antithrombospondin antibodies to actively growing cells cultured in serum containing medium inhibits growth (54). Such results indicate that throm bospondin is an autocrine growth factor, i.e. secreted thrombospondin binds to the cell surface and induces growth (Figure 1).
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METABOLISM OF THROMBOSPONDIN Thrombospondin binds to cell layers of cultured fibroblasts to two sites and in two ways, nonsaturably to extracellular matrix and saturably to the cell surface (55). Throm bospondin bound to the cell surface has a punctate distribution and is subject to receptor-mediated endocytosis and degradation in lysosomes (55). There are a number of molecules in extracellular matrix to which thrombospondin may bind, including proteoglycans, fibronectin, type V and other collagens, and osteonectin (9, 10, 55-57). Matrix-bound thrombospondin is resistant to rather harsh extraction conditions, but nevertheless is rapidly taken up by the cells and degraded (55). The major cellular mediator of thrombospondin binding and degra dation is a membrane form of heparan sulfate proteoglycan (39, 55, 5860; Figure 1). Binding and degradation are inhibited by heparin and other sulfated glycosaminoglycans (presumably because of interaction with the amino-terminal domains of thrombospondin) and by platelet factor 4 and fl-thromboglobulin (presumably because of interaction with the sidechains of the proteoglycan) (58). Thus, the three heparin-binding proteins released from platelets compete with one another for binding sites on nucleated cells (Figure 1). Interestingly, genes for homologs of platelet factor 4 and fl-thromboglobulin have been identified because, like the gene for thrombospondin, their transcription is induced by platelet-derived growth factor (see, for example 61, 62). These have been called "small inducible genes" (SIG; 61). The products of these genes are secreted (62) and may compete for thrombospondin binding sites (Figure 1). CD36 and the integrin receptor for vitronectin also interact with throm bospondin (12, 23-25) and may serve as additional mediators of throm bospondin binding to cell surfaces (Figure 1). It remains to be determined whether thrombospondin or its aggregates can cluster various cell surface receptors together. Such a scenario would explain why five different anti
thrombospondin monoclonal antibodies to widely separated epitopes ean
block cell growth (54). The literature on thrombo spondin and cell adhesion is confusing at present, with some papers focusing on pro-adhesive effects (see for example 25,63) and others on anti adhesive effects (e.g. 39,64). Among the pro-adhesion papers, reasonable arguments have been proposed that adhesion to thrombospondin is mediated by an arginine-glycine-aspartate-sensitive integrin receptor (25, 65), heparan sulfate proteoglycan (63, 66), a receptor specific for the car boxyl-terminal domain of thrombospondin (66), or some combination thereof (66). Thrombospondin is active in migration assays (67). The carboxyl-terminal domains are important for haptotaxis (directional EFFECTS ON CELL ADHESION AND MOVEMENT
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movement up a gradient of substratum-bound thrombospondin) whereas the aminoterminal domains are important for chemotaxis (movement toward increasing concentrations of soluble thrombospondin). Throm bospondin causes endothelial cells to lose focal adhesion plaques (68). This effect is neutralized by molecules that bind to the amino-terminal domain of thrombospondin. Undifferentiated keratinocytes attach and spread on a thrombospondin-coated surface, but differentiated keratinocytes do not (69). This effect requires an intact carboxyl-terminal domain and is con sistent with the localization of thromobospondin at the dermal-epidermal junction (38). Thus, thrombospondin probably does interact in important ways with the adhesive machinery of cells, possibly destabilizing cell matrix contacts so that cells can migrate or round up and pass through mitosis. CHANGES IN DISEASE STATES A ltered Platelet Thrombospondin
Thrombospondin, along with other a-granule components, is largely miss ing from platelets of patients with Gray platelet syndrome (70). The deficiency in such patients may be in the targeting of endogenously syn thesized secretory proteins to developing a-granules in megakaryocytes (71). Thrombospondin has been found to be deficient, along with GPIb, in platelets of patients with various malignancies and abnormal "platelet antithrombin tests" (72) and, along with GPIa, in platelets of an unusual patient whose defective collagen-induced platelet aggregation and hemorrhagic diathesis disappeared with menopause (73). A degraded form of thrombospondin is found in patients with primary thrombocytosis and other myeloproliferative diseases (70). Elucidation of the pathogenetic mechanisms underlying these observations and their medical significance will require a more complete understanding of glycoprotein metabolism in the megakaryocyte and of the complex roles played by platelets in blood coagulation. Levels of Plasma and Serum Thrombospondin
The normal plasma concentration of thrombospondin is 40 to 250 ngjml (70). Similar levels are found in plasma of patients with thrombocytopenia due to recent chemotherapy, which indicates that the measured protein is not released artifactually from platelets during sample processing and that the dominant sources for thrombospondin in normal plasma are various tissues (74). The half-life of injected thrombospondin is extremely short, 10 to 75 min (75, 76). Thus, the quantity of thrombospondin being released into plasma could be substantial despite the low plasma level. There is
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little correlation between the concentrations of plasma thrombospondin and fj-thromboglobulin (74). Statistically significant increases in plasma thrombospondin are found in patients with recent total hip replacement, chronic renal failure, chronic liver failure, remote splenectomy, and recent (within 24 hours) acute myocardial infarction (70). The concentration of thrombospondin in serum is proportional to the platelet count (74). At present, thrombospondin is not a clinically significant analyte. Levels have not been quantified in a number of disease states, however, and this may change in the future. Thrombospondin in Tissues
There have been few studies of thrombospondin in diseased tissues. Cyto sols of malignant breast tissue contain more thrombospondin than cytosols of normal breast, perhaps because of activation of endothelium (77). Breast cyst fluids can be very rich in thrombospondin (up to 55,000 ng/ml) (78). Systematic studies of thrombospondin in various diseased tissues could be quite informative. Tumor cells in culture regularly synthesize and secrete thrombospondin (79; Table 1). Synthesis in neoplastically transformed cells may not be as regulated as in normal cells (45). Neoplastic lesions,
therefore, especially need to be studied, with care taken to distinguish synthetic pools of thrombospondin in the tumor cells and reacting cells from thrombospondin deposited in extracellular matrix. Table 1
Synthesis and secretion of thrombospondin by cultured human tumor cells' Production of thrombospondin Cells
Normal fibroblasts
(�g/ 106 cells/24 hr) 32
SV-40 transformed fibroblasts
8.2
Fibrosarcoma
4.9
Rhabdomyosarcoma Normal glial cells
5.3 12
Glioblastoma
7.1
Wilm's tumor
0.8
Neuroblastoma
1.0
Teratocarcinoma
1.0
Choriocarcinoma
7.8
Lung carcinoma
2.8
Melanoma
9.3
Unknown primary
17
"A competitive enzyme-linked immunoassay for human thrombospondin (80) was used to measure thrombospondin in conditioned media provided to the author by Prof. Antti Vaheri, University of Helsinki. The media contained rabbit serum (which did not interfere with the assay) and was placed over just-confluent cells
for 24 hr.
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Possible Role of Thrombospondin in Falciparum Malaria There has been considerable interest in the role that thrombospondin may play in the sequestration of Plasmodiumfalciparum. Knobs on the surface of infected erythrocytes are the points of attachment to endothelium. Such infected erythrocytes also bind specifically to thrombospondin, and antithrombospondin antibodies block adherence of infected erythrocytes to some melanoma cell lines (81). Cytoadherence is also blocked by anti body to CD36 (82). As described above, CD36 is a putative thrombospondin
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receptor, which raises the possibility that thrombospondin forms a bridge between erythrocyte knobs and endothelial CD36. Adherence of infected erythrocytes to melanoma cell lines, however, does not correlate with expression of thrombospondin (83), and expression of a CD36 cDNA clone in an otherwise nonexpressing cell line results in cytoadherence of parasitized erythrocytes but not increased binding of thrombospondin (84). Because there are no sequence homologies between thrombospondin and CD36, it seems most likely that there are two distinct adhesion mech anisms, one allowing adhesion of parasitized erythrocytes to thrombo spondin and the other adhesion to CD36 (84). ACKNOWLEDGMENTS
The experimental work described herein was supported by National Insti tutes of Health Grant HL29586.
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l. Baenziger, N. L, Brodie, G. N., Majerus, P. W. 1971. A thrombin-sen
2.
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4.
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