Human Platelet Thrombospondin Research Articles

Heparin-binding peptides from the type I repeats of thrombospondin. Structural requirements for heparin binding and promotion of melanoma cell adhesion and chemotaxis.

Synthetic peptides derived from the type I repeats of human platelet thrombospondin containing a consensus sequence Trp-Ser-Xaa-Trp bind to heparin, promote cell adhesion, and inhibit heparin-dependent interactions of melanoma cells with extracellular matrix components (Guo, N. H., Krutzsch, H. C., Nègre, E., Vogel, T., Blake, D. A., and Roberts, D. D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3040-3044). In the present study, we further examined the structural requirements for activity of these peptides. The minimal active sequence for heparin or sulfatide binding based on inhibition studies is Trp-Ser-Pro-Trp, although an octapeptide is required for optimal activity. The 2 Trp residues and the Ser residue are essential. Peptides with more than 2 residues between the Trp residues are inactive. The Pro residue is essential for activity of the pentapeptide Trp-Ser-Pro-Trp-Ser, but some larger peptides with substitutions for the Pro residue are active. For direct high affinity binding to heparin, both the consensus sequence and a flanking sequence of basic amino acids are essential. Peptides containing the consensus sequence promote cell adhesion and act cooperatively with the adjacent basic amino acid sequence to promote cell spreading. Chemical modification of the Trp residues in the peptides with amino-terminal basic amino acids abolished both cell adhesion and heparin-binding. Peptides containing the consensus sequence and basic amino acids are chemotactic for A2058 human melanoma cells. The functional importance of this novel heparin and sulfatide-binding motif is suggested by its conservation in other members of the thrombospondin gene family, complement components, and in many members of the cytokine receptor and transforming growth factor beta superfamilies.

Molecular Requirements for the Interaction of Thrombospondin With Thrombin-Activated Human Platelets: Modulation of Platelet Aggregation

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.

Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation

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.

Expression and mutagenesis of thrombospondin

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)

Thrombospondin: a component of microfibrils in various tissues.

We used antisera directed against human platelet thrombospondin (TSP) and microfibril-associated GP 128 to localize the presence of these glycoproteins in fixed sections of human placenta or porcine arteries and skin by immunogold labeling, using electron microscopy. These two antibodies reacted with both human and porcine tissues and always recognized the same structures. In all three tissues the antibodies were associated with the basement membranes and, more precisely, with the microfibrillar structures present at the junction between the basement membrane and the adjacent connective tissue. This localization indicates that GP 128 and TSP are associated with the microfibrils, and suggests their possible role in the attachment of basement membrane to the connective tissue meshwork. Their presence in microfibrils associated with the subendothelial basement membrane in arteries may be important in regard to the thrombogenicity of the subendothelium since, after an endothelial lesion, they may be directly accessible to blood platelets.

Models of human platelet thrombospondin in solution. A dynamic light-scattering study

The translational diffusion coefficient (D20,w) of human platelet thrombospondin was measured by dynamic light-scattering. D20,w, measured in 20 mM-Hepes buffer, pH 7.4, containing 350 mM-NaCl and 2 mM-CaCl2, was 1.73(+/- 0.02) x 10(-7) cm2.s-1. After removal of bound Ca2+ by addition of EDTA, D20,w decreased to 1.56(+/- 0.04) x 10(-7) cm2.s-1; this was not a consequence of aggregation. D20,w showed little sensitivity to NaCl concentration between 130 and 550 mM. Through hydrodynamic analysis combining D20,w and other parameters taken from the literature, two major types of models for thrombospondin can be proposed: either classic compact models (i.e. low degree of hydration) such as prolate or oblate ellipsoids with a high axial ratio (greater than 20) or models of low axial ratio made of multiple subunits with significant cavities (i.e. high degree of hydration).

Immunoelectron-microscopic studies of human platelet thrombospondin, Von Willebrand factor, and fibrinogen redistribution during clot formation

The relative distributions of the human platelet alpha-granule proteins fibrinogen, thrombospondin, and von Willebrand factor were mapped by immunoelectron microscopy in thin cryosections of activated platelets, platelet aggregates, and clots during the first 24 h of in vitro clot formation. In early activated platelets, the results suggest that the canalicular system constitutes a significant component of the external platelet surface, and may act as a compartment for biochemical reactions occurring during granule release. Further, detection of coagulation proteins by various non-morphological procedures may reflect protein contained within canalicular elements. Later in the release process, von Willebrand factor was detected as a major antigen on the platelet canalicular and plasma membranes; thrombospondin, on the other hand, showed minimal binding to platelets and only limited binding to the extensive fibrin network. Comparison of radioimmunoassays of supernatants of thrombin-stimulated platelets in plasma, clotted whole blood, and Triton X-100 platelet releasates indicated that virtually all of the platelet thrombospondin appears in serum. These data confirm the immunocytochemical results indicating that very little platelet thrombospondin binds to the platelet surface, compared with von Willebrand factor, studied here under the same conditions, which binds extensively to the platelet membrane following release and clot formation.

Specific inhibition of endothelial cell proliferation by thrombospondin

Angiogenesis is a multi-step event involving endothelial cell migration, attachment, and proliferation. A thrombospondin (TSP)-like protein has recently been described as a naturally-occurring inhibitor of angiogenesis (1). We now report that human platelet TSP inhibits the in vitro proliferation of endothelial cells from the rabbit corpus luteum, bovine adrenal cortex and pulmonary artery, and human umbilical vein. The antiproliferative effect of TSP was neutralized by monoclonal antibodies against TSP. The growth arrest seen with TSP was specific for endothelial cells since TSP actually stimulated the growth of vascular smooth muscle cells and human foreskin fibroblasts. These results imply that the angiogenesis-inhibiting effect of TSP is mediated through an inhibition of endothelial cell proliferation. Elucidation of the mechanism of action of TSP on endothelial cell proliferation may lead to potential therapeutic approaches for the control of neovascular diseases.

Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region

We investigated Plasmodium falciparum parasitized erythrocyte binding to proteolytic fragments of thrombospondin and the effects of anti-thrombospondin monoclonal antibodies on this binding. Purified human platelet thrombospondin was cleaved by trypsin, chymotrypsin or thrombin. Fragments were separated by heparin-agarose affinity chromatography, removing the amino-terminal heparin-binding region. Trypsin at 5.0 μg ml −1 of thrombospondin cleaved thrombospondin to reduced 140 and 120 kDa fragments plus a reduced 25-kDa heparin-binding fragment. Infected erythrocytes bound to intact thrombospondin (3420 ± 460 infected erythrocytes mm −2) and the carboxy-terminal fragment, yielding 120–140-kDa fragments on sulfhydryl reduction, but not to the 25-kDa fragment (144 ± 104 infected erythrocytes mm −2 (mean ± s.d., N = 4). Similar results were obtained with chymotrypsin and thrombin cleavage. When the anti-thrombospondin monoclonal antibody MA-I was added to immobilized thrombospondin prior to infected erythrocytes, adherence was inhibited by 99%. At the same concentration, MA-I inhibited adherence to C32 melanoma cells by only 35%. MA-I binds to a calcium-dependent structure at the C-terminal globular region of thrombospondin. Monoclonal antibody MA-II inhibited adherence to thrombospondin by 46%, while MA-III had no effect. These antibodies bind to the N-terminal globular region which includes the heparin-binding site and the segment connecting the two globular regions, respectively. The site(s) for infected erythrocyte binding on thrombospondin reside in the large, 140- or 120-kDa, proteolytic cleavage fragments, and not in the N-terminal heparin-binding region.

The interaction of thrombospondin with platelet glycoprotein GPIIb-IIIa

The interaction of human platelet thrombospondin (TSP) with human platelet glycoproteins GPIIb-IIIa was studied using a solid-phase binding assay. Polystyrene test tubes were coated with TSP, and 125I-labeled GPIIb-IIIa was added, allowed to bind, and the bound radioactivity was measured. After 90 min, the binding became time independent, and in most experiments, more than 10% of the exogenously added radioactivity was bound to the tube. Analysis of the bound radioactivity by polyacrylamide gel electrophoresis and autoradiography indicated that it was from labeled GPIIb-IIIa. Several lines of evidence indicate that the binding of GPIIb-IIIa to TSP was specific. (a) TSP immobilized on plastic or Sepharose bound 3-10-fold more GPIIb-IIIa than immobilized bovine serum albumin. (b) Addition of unlabeled excess GPIIb-IIIa reversed the binding of 125I-labeled GPIIb-IIIa to immobilized TSP. (c) Addition of EDTA inhibited the binding of GPIIb-IIIa to TSP by more than 90%, whereas addition of 1 mM CaCl2 and 1 mM MgCl2 potentiated the binding by more than 100%. (d) Monoclonal antibodies against TSP and GPIIb-IIIa inhibited the binding by 30-70% as compared with control and polyclonal anti-fibrinogen anti-serum. (e) A plot of GPIIb-IIIa bound versus GPIIb-IIIa added was best described as a rectangular hyperbola by regression analysis with half-saturation at 60 ng/ml GPIIb-IIIa. Similar results were obtained when labeled TSP was added to tubes coated with GPIIb-IIIa. These results show that TSP and GPIIb-IIIa can specifically interact in vitro and suggest that GPIIb-IIIa may function as a platelet TSP receptor during platelet aggregation.

DEMONSTRATION OF TSP RECEPTOR ON THE CELL‐SURFACE OF HUMAN ERYTHROLEUKAEMIA CELL LINE (HEL)

British Journal of HaematologyVolume 73, Issue 3 p. 430-431 Free Access DEMONSTRATION OF TSP RECEPTOR ON THE CELL-SURFACE OF HUMAN ERYTHROLEUKAEMIA CELL LINE (HEL) Nobutaka Imamura, Nobutaka Imamura Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this authorTominari Inada, Tominari Inada Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this authorDeodatus M. Mtasiwa, Deodatus M. Mtasiwa Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this authorAtsushi Kuramoto, Atsushi Kuramoto Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this author Nobutaka Imamura, Nobutaka Imamura Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this authorTominari Inada, Tominari Inada Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this authorDeodatus M. Mtasiwa, Deodatus M. Mtasiwa Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this authorAtsushi Kuramoto, Atsushi Kuramoto Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology. Hiroshima University, 1–2–3, Kasumi, Minami-Ku, Hiroshima 734, JapanSearch for more papers by this author First published: November 1989 https://doi.org/10.1111/j.1365-2141.1989.tb07773.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES Asch, A.S., Barnwell, J., Silverstein, R.L. & Nachman, R.L. (1987) Isolation of the thrombospondin membrane receptor. Journal of Clinical Investigation, 79, 1054– 1061. Imamura, N., Inada, T., Mtasiwa, D.M. & Kuramoto, A. (1989) Demonstration of TSP receptor on the cell-surface of acute megakaryoblastic leukemia. American Journal of Hematology, 31, 142– 143. Imamura, N., Kajihara, H. & Kuramoto, A. (1988) Flow cytometric analysis of peroxidase negative acute leukemias by monoclonal antibodies. II. Acute megakaryoblastic and acute pro-megakaryocytic leukemia. Leukemia Research, 12, 279– 289. Lawler, J., Derick, L.H., Connolly, J.E., Chen, J.H. & Chao, F.C. (1985) The structure of human platelet thrombospondin. Journal of Biological Chemistry, 260, 3762– 3772. Talle, M.A., Rao, P.E., Westberg, E., Allegan, N., Makowski, M. Mittler, R.S. & Goldstein, G. (1983) Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies. Cellular Immunology, 78, 83– 87. Citing Literature Volume73, Issue3November 1989Pages 430-431 ReferencesRelatedInformation

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.

Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.

Chinese hamster ovary cell adhesion to human platelet thrombospondin is dependent on cell surface heparan sulfate proteoglycan.

Thrombospondin is a 420-kD platelet alpha-granule glycoprotein that binds specifically to heparin. We examined adhesion to thrombospondin of CHO K1 cells and three mutant CHO lines with varying deficiencies in glycosaminoglycan (GAG) synthesis. In an experiment in which the parent line (K1) had 78% adherence to thrombospondin adsorbed to tissue culture plastic, CHO S745 cells, with less than 6% normal GAG synthesis had 11% adherence. CHO S677 cells, with decreased heparan sulfate proteoglycan but increased chondroitin sulfate proteoglycan, had 42% adherence. CHO S803 cells, with decreased heparan sulfate proteoglycan and normal chondroitin sulfate proteoglycan, had 31% adherence. Heparin inhibited K1 cell adhesion to thrombospondin, but not fibronectin, in a concentration-dependent manner. Dermatan sulfate but not chondroitin sulfate was also inhibitory. There was markedly decreased K1 cell adhesion to a thrombospondin core fragment that lacked the heparin binding NH2-terminal domain. Purified heparin binding domain, although poorly adhesive when adsorbed to substratum, inhibited cell adhesion to intact thrombospondin. Adhesion was better for all cell lines tested, including three human tumor cell lines, when thrombospondin was adsorbed at pH 4.0 compared with pH 7.4. When adsorption of thrombospondin was done at pH 7.4, cell adhesion was better when thrombospondin was adsorbed in the presence of greater than or equal to 0.6 mM calcium, compared to 0.1 mM calcium or EDTA. These findings suggest that thrombospondin can adsorb to plastic with varying degrees of exposure of a cell adhesion domain. We conclude that the thrombospondin cell adhesion receptor on CHO cells is a heparan sulfate proteoglycan, and that cell adhesion to thrombospondin depends on conformation of adsorbed thrombospondin.

Biosynthesis and secretion of thrombospondin in human melanoma cells

A polyclonal antisera specific for human platelet thrombospondin (TSP) has been utilized to study the biosynthesis and secretion of TSP in the M21 human melanoma cell line. Pulse-chase indirect immunoprecipitation analysis reveal that human melanoma cells rapidly synthesize and secrete this platelet α-granule associated glycoprotein. Topographical analysis of the melanoma cell surface by indirect immunofluorescence indicate that the TSP molecules have no obvious extra-cellular organization. The implications of thrombospondin synthesis in the metastatic process of melanoma are discussed.

Axial dependence of platelet-collagen interactions in flowing blood. Upstream thrombus growth impairs downstream platelet adhesion.

Vascular subendothelium and collagenous surfaces were exposed to flowing citrated blood. Platelet interactions with these surfaces were investigated at various axial distances from the upstream end of the exposed surfaces. A pronounced axial decrease in surface coverage with platelets and in thrombus dimensions was encountered on collagenous surfaces. This phenomenon was observed at shear rates of 200 to 2000 s-1, but was most pronounced at low shear rates (less than 650 s-1). After 5 minutes of perfusion at a shear rate of 650 s-1, 4.6 x 10(6) platelets were deposited on the most upstream 20 mm2 of the collagen surface, in contrast to 2.2 x 10(6) platelets/20 mm2 14 mm farther downstream. Depletion of von Willebrand factor and/or thrombospondin from the boundary layer of the blood flow was not responsible for this. Collagen-bound von Willebrand factor enhanced the surface coverage with platelets without affecting the axial decrement, while pretreatment of the collagen surface with thrombospondin had no effect at all. However, partial inhibition of thrombus growth by aspirin reduced the axial decrements, and less thrombogenic surfaces as human and rabbit subendothelium, which induced only a few small thrombi, produced virtually no axial differences in platelet adhesion. Raising the shear rate to 2600 s-1 also gave no axial differences in platelet-collagen adhesion; it did, however, give an axial increase in thrombus dimensions. This increase was neutralized after the addition of antibody against human platelet thrombospondin to the blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody

A rat monoclonal IgG 2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca 2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca 2+-dependent and Ca 2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of M r 88 000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca 2+ and 1 mM Mg 2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.

Thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes

Thrombospondin, a multifunctional adhesive glycoprotein originally identified in platelets, was isolated and identified from an extract of ovine articular cartilage. Immunoreactive material from a cartilage extract comigrated on gel electrophoresis with purified human platelet thrombospondin. When articular chondrocytes were cultured in the presence of 35S-methionine, metabolically labeled thrombospondin was immunoprecipitated from the culture medium and cell layer extract. These results demonstrate that thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes.

Activation of S 6 kinase in cultured vascular smooth muscle cells by submitogenic levels of thrombospondin

Purified human platelet thrombospondin was shown to activate S 6 kinase in cultured vascular smooth muscle cells in a dose- (1–9 μg/ml) and time-dependent manner. Down regulation of epidermal growth factor and somatomedin C receptors by prior treatment of cells with their respective growth factors did not reduce this effect. Kinase activation by thrombospondin was only marginally reduced in the presence of platelet-derived growth factor specific antibody at levels that totally inhibited platelet-derived growth factor (5 ng/ml) induced activation. Additionally, thrombospondin elicits a rapid dose-dependent phosphoinositide turnover response analogous to that of platelet-derived growth factor, epidermal growth factor and somatomedin C. Prior treatment of cells with phorbol ester for 48 hrs in serum-free culture medium resulted in a small enhancement of S 6 kinase activation by thrombospondin and the above mentioned growth factors but a complete loss in the ability of phorbol ester to activate this enzyme. These findings with cultured smooth muscle cells suggest a growth factor-like role for thrombospondin.

Human Keratinocytes Synthesize and Secrete the Extracellular Matrix Protein, Thrombospondin

Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.