Publications

Cartilage oligomeric matrix protein (COMP) is a 524,000-Da protein that is expressed at high levels in the territorial matrix of chondrocytes. The sequences of rat and bovine COMP indicate that it is a member of the thrombospondin gene family. In this study, we have cloned and sequenced human COMP. Phylogenetic analysis using progressive sequence alignment and two parsimony-based algorithms indicates that the COMP gene and a precursor of the thrombospondin-3 and -4 genes were produced by a gene duplication that occurred 750 million years ago. An interspecific backcross mapping panel has been used to map the murine COMP gene to the central region of mouse chromosome 8. Southern blot analysis of a somatic cell hybrid DNA panel and in situ hybridization to human metaphase chromosomes indicate that the human COMP gene is located on chromosome 19 in band p13.1. These data confirm and extend the known regions of homology between human and mouse chromosomes and establish that COMP, like thrombospondin-1, -2, -3, and -4, is present in the human and mouse genomes.

Thrombospondin-1 is a component of the extracellular matrix which is thought to play important roles in cell migration and proliferation, during embryogenesis and wound repair. To understand the basis for these activities, we are mapping the regions of the molecule with cell adhesive activity. Here, we use antagonists of specific cell binding sites, adhesion-perturbing thrombospondin monoclonal antibodies and proteolytic fragments of platelet thrombospondin, to investigate the adhesive mechanisms used by G361 melanoma cells, human intestinal smooth muscle cells (HISM), epidermal keratinocytes and MG-63 osteosarcoma cells. When attached to the same preparations of platelet thrombospondin, HISM and MG-63 cells underwent spreading, whereas G361 cells and keratinocytes did not. Attachment of all four cell types involved the carboxyterminal domain. The type 1 repeats and the amino-terminal heparin binding domain were important for stable attachment of G361, HISM and MG-63 cells, but were not involved in keratinocyte attachment. GRGDSP peptide caused near complete inhibition of HISM and MG-63 cell attachment, partially inhibited G361 attachment, but did not inhibit keratinocyte attachment. Attachment of HISM and MG-63 cells involved the alpha v beta 3 integrin. The integrity of the thrombospondin molecule was important for its adhesivity towards G361, HISM, and MG-63 cells, whereas keratinocytes attached to the 140 kDa tryptic fragment as effectively as they did to the intact molecule. These results show that cell attachment to platelet thrombospondin typically involves multiple binding interactions, but the exact profile of interactions is cell type specific. Usage of particular cell-binding sites does not predict whether cells will undergo spreading or not. These data may, in part, explain some of the current controversies surrounding the mechanisms of cell attachment to thrombospondin.

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.

Thrombospondin-1 is an adhesive glycoprotein that is involved in cellular attachment, spreading, migration, and proliferation. To date, four genes have been identified that encode for the members of the thrombospondin gene family. These four genes are homologous to each other in the EGF-like (type 2) repeats, the calcium-binding (type 3) motifs, and the COOH-terminal. The latter has been reported to be a cell-binding domain in thrombospondin-1. Phylogenetic trees have been constructed from the multisequence alignment of thrombospondin sequences from human, mouse, chicken, and frog. Two different algorithms generate comparable results in terms of the topology and the branch lengths. The analysis indicates that an early form of the thrombospondin gene duplicated about 925 million years ago. The gene duplication that produced the thrombospondin-1 and -2 branches of the family is predicted to have occurred 583 million years ago, whereas the gene duplication that produced the thrombospondin-3 and -4 branches of the family is predicted to have occurred 644 million years ago. These results indicate that the members of the thrombospondin gene family have existed throughout the evolution of the animal kingdom and thus probably participate in functions that are common to most of its members.

Many extracellular matrix glycoproteins–including laminin, fibronectin, thrombospondin, type I collagen, and other collagens–bind the glycosaminoglycan heparin, yet little is known about the functional significance of these interactions. It is also not known if heparin-binding extracellular matrix proteins recognize distinct structural elements in heparin, nor whether all extracellular matrix proteins recognize the same or different aspects of heparin structure. If extracellular matrix proteins each recognize distinct features of heparin, such specificity could be of importance in vivo, where structurally distinct heparan sulfate species occur. To investigate specificity in the binding between extracellular matrix proteins and heparin, the method of affinity coelectrophoresis (ACE) was used [Lee, M. K., & Lander, A. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2768-2772]. Low M(r) (approximately 6 kDa) 125I-heparin was fractionated by electrophoresis through agarose gel lanes containing extracellular matrix proteins at various concentrations; from heparin migration patterns, binding affinities were calculated. The results indicate that fibronectin, type I collagen, and laminin–but not thrombospondin–each fractionate heparin into subpopulations that differ substantially in binding affinity. From ACE gels containing either fibronectin, type I collagen, or laminin, fractions of heparin were isolated that represent the 25% of molecules most strongly bound and the 25% least strongly bound by each of these proteins. Subsequent ACE analysis of these six fractions showed that (1) for each of fibronectin, type I collagen, and laminin, strongly- and weakly-binding heparin subfractions differ approximately 5-30-fold in Kd; (2) heparin that binds strongly to any one of fibronectin, type I collagen, or laminin also binds strongly to the other two; (3) heparin that binds weakly to any one of fibronectin, type I collagen, or laminin, also binds weakly to the other two; (4) heparin subfractions that differ greatly in affinity for fibronectin, type I collagen, and laminin show little difference in Kd for thrombospondin or for the heparin-binding growth factor basic fibroblast growth factor (bFGF); (5) neither heterogeneity in molecular charge [as measured by diethylaminoethyl (DEAE) chromatography] nor size nor the presence or absence of antithrombin III recognition sequences can account for the selective binding of heparin subpopulations to fibronectin, type I collagen, and laminin. These results suggest that structural elements within heparin can confer preferential binding to extracellular matrix proteins. Sensitivity of some, but not all, extracellular matrix proteins to these structural features suggests that similar features, if present in heparan sulfates or other glycosaminoglycans, may be physiologically relevant in vivo.

The biosynthetic profile of endothelial cells responding to hyperthermia is altered by extracellular matrix components. The extracellular matrix components influence the quantitative expression of members of the HSP70 family and HSP90. The expression of several HSP70 mRNA species, which are strictly stress inducible, are modulated by extracellular matrix components. Both laminin and collagen type IV decrease the amount of HSP70 protein and mRNA expressed by endothelial cells exposed to hyperthermia relative to control cultures attached to virgin plastic. In contrast, both laminin and collagen type IV increased the amount of HSP90 mRNA constitutively expressed by endothelial cells at 37 degrees C. When endothelial cells were exposed to elevated temperatures, these two extracellular matrix proteins decrease the amount of HSP90 mRNA relative to control cultures attached to virgin plastic. Our observations are consistent with the proposal that the extracellular matrix components regulate gene expression and cell behavior in regard to thermotolerance.

Thrombospondin is a 420,000-dalton adhesive glycoprotein that is composed of three subunits of equivalent molecular weight. When the cDNA for the complete coding region of the human endothelial cell thrombospondin subunit is expressed in mouse NIH 3T3 cells, a 420,000-dalton protein is synthesized and secreted. The expressed protein comigrates with human platelet thrombospondin both in the presence and in the absence of a reducing agent. The expressed protein binds to a monoclonal anti-thrombospondin antibody, heparin, and calcium. In addition to the 420,000-dalton protein, the transfected cell lines also express a variable amount of a 140,000-dalton polypeptide. When the culture supernatants that are produced by cells that are expressing thrombospondin are applied to heparin-Sepharose, the 420,000-dalton and the 140,000-dalton proteins are bound to the column and are eluted with buffer containing 0.55 and 0.3 M NaCl, respectively. The 140,000-dalton protein only binds to heparin-Sepharose in the presence of calcium. Deletion of the region of homology with procollagen results in defective assembly of the trimer. Deletion of the type 1 or type 2 repeats results in decreased stability of the subunit with the predominant polypeptides that are expressed having molecular weights of 127,000 and 130,000, respectively. These polypeptides retain low-affinity heparin-binding activity. High-affinity heparin binding is markedly diminished by mutations in either of two sequence motifs that include clusters of lysines and arginines.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.

Thrombospondin is an adhesive glycoprotein that is thought to play a role in tissue genesis and repair. We have used a monoclonal anti-thrombospondin antibody, designated 5G11, to localize thrombospondin in paraformaldehyde fixed, paraffin-embedded sections of developing mouse embryos. Thrombospondin expression is observed in uterine smooth muscle, endometrial glands, the decidua, and trophoblastic giant cells during the initial phase of post-implantation development in the embryo. Cardiac myocytes and neuroepithelial cells show positive staining for thrombospondin at day 8.5 of gestation, and this expression continues throughout the development of the myocardium and central nervous system. Strong staining for thrombospondin is seen in developing bone and in the liver. Thrombospondin is also observed in developing smooth muscle and skeletal muscle, as well as in a variety of epithelia, including the epidermis, small intestinal epithelium, lens epithelium, renal tubular epithelium, and the epithelium of the developing tooth. Comparison of thrombospondin staining with that of two known cell surface receptors for thrombospondin, syndecan and the vitronectin receptor, reveals remarkable colocalization of thrombospondin and syndecan in all tissues, but almost no coexpression with the vitronectin receptor. Coexpression of thrombospondin and syndecan may play an important role in cell-cell or cell-matrix interactions during development.