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Thrombospondin promotes platelet aggregation

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Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP-promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP-potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.
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... A number of studies have demonstrated that TSP promotes cell-substratum adhesion of a variety of cells including fibroblasts [5], endothelial cells [5,6], epithelial cells [5] and various tumour cell lines such as melanoma cells5678, small-cell carcinoma cells [8] , and osteosarcoma cells [9]. Recent studies have also postulated a role for TSP in cell proliferation [10] and angiogenesis [11] The first is GPIIb-III., the platelet fibrinogen receptor, which reversibly binds TSP in vitro [15] and antibodies against which inhibit TSP-promoted platelet aggregation and adhesion [13,15]. The second is GPIa-IIa, a platelet collagen receptor [16]. ...
... The second is GPIa-IIa, a platelet collagen receptor [16]. (GPIa-IIa) monoclonal antibodies (mAbs) completely inhibited the TSP-dependent adhesion of normal and thrombasthenic platelets [13]. This inhibition was dose-dependent and complete inhibition could be achieved with as little as 2 ,ug/ml of antibody, while no more than 50 % inhibition of adhesion could be achieved with anti-(GPIIb-IIIa) mAb at concentrations in excess of 50 ,ug/ ml. ...
... (6F1) to block binding. mAb 6F1 has previously been shown by us to quantitatively block TSP-dependent platelet adhesion [13]. We found that 6F1 blocked the binding of GPI.-IIa to TSP. ...
Article
The interaction of human thrombospondin (TSP) with GPIa-IIa and GPIIb-IIIa was studied. The binding for both proteins became time-independent after 60 min. A 7-fold excess concentration of unlabelled GPIa-IIa added either initially, or after time-dependent binding, resulted in a 50% inhibition of GPIa-IIa bound to TSP. GPIa-IIa and GPIIb-IIIa specifically bound TSP since: (a) the binding of GPIIb-IIIa to TSP was dependent on the presence of 1 mM MgCl2 and 1 mM CaCl2, whereas binding of GPIa-IIa was ion-independent. (b) The binding was saturable, with dissociation constants of 0.69 +/- 0.17 microM and 3.77 +/- 1.02 microM for GPIa-IIa and GPIIb-IIIa respectively. (c) GPIIb-IIIa and GPIa-IIa did not significantly bind to BSA. (d) GPIIb-IIIa bound fibrinogen ion-specifically, whereas little or no binding of GPIa-IIa was detectable. (e) Both GPIIb-IIIa and GPIa-IIa bound collagen in an ion-independent manner. (f) GPIIb-IIIa did not compete with GPIa-IIa for binding to TSP. (g) Binding of GPIa-IIa to TSP was inhibited with anti-(GPIa-IIa) (6F1), whereas mouse IgG and anti-(GPIIb-IIIa) (AP-2) had no effect. (h) The interaction of GPIa-IIa with TSP is 5.5-fold more favourable than that of GPIIb-IIIa suggesting that GPIa-IIa may be a preferred binding protein for TSP-mediated platelet adhesion.
... The effect is blocked by Fab fragments of anti-TSP-1 antibodies (Kao et al., 1986). TSP-1 also potentiates aggregation of platelets activated by higher concentrations of thrombin and facilitates the aggregation of unstimulated platelets (Wolff et al., 1986;Tuszynski et al., 1988). The inhibitory effects of antibodies to the Nterminal heparin binding domain and C-terminal domain, or heparin binding peptides from the amino-terminal domain suggest that the N and C-terminal regions are involved in platelet aggregation (Gartner et al., 1984;Dixit et al., 1985;Kosfeld & Frazier, 1993: Legrand etal., 1994Chung et al., 1997). ...
... TSP-1 potentiated aggregation of platelets is specific and can be inhibited by anti-aiibP;, antibodies in activated platelets. The same integrin has been demonstrated to be involved in TSP-1 binding to the membranes of activated platelets (Wolff et al., 1986;Tuszynski et al., 1988;McGregor et al., 1989). TSP-1 promotion of neurite outgrowth is mediated by RGD dependent interactions with integrins (O'Shea et al., 1991;Neugebauer et al., 1991). ...
Thesis
Muskelin is a novel intracellular protein involved in cell responses to the adhesion modulating matrix component, Thrombospondin-1 (TSP-1). The muskelin polypeptide sequence contains six motifs with homology to the tandem kelch motifs, initially identified in the Drosophila Kelch protein, a component of ring canals. The focus of the thesis project has been to identify the molecular interactions of muskelin by use of a number of approaches including the yeast dihybrid system, copprecipitation, pharmacological agents and gel overlays. The two hybrid approach did not prove to be appropriate, because expression of muskelin had toxic effects on yeast cells. Treatment of cell extracts with a panel of pharmacological agents, to target components of cell signalling pathways, had no effect on the distribution of muskelin between detergent soluble (cytosol and membranes) and insoluble (cytoskeleton) fractions. Using a panel of cell lines including skeletal myoblasts, a set of proteins migrating with apparent molecular weights between 45-57 kDa were specifically detected upon muskelin overlays of SDS-polyacrylamide gels. These proteins did not correspond to the most abundant proteins within cell extract. Interaction of muskelin with these proteins was detectable throughout myoblast fusion into myotubes. Fascin, tubulin and actin were investigated as potential candidates for the proteins detected by muskelin gel overlay. A combination of muskelin and fascin overlays of cell extracts demonstrated that muskelin did not directly bind fascin. Muskelin did not interact with purified tubulin and actin in gel overlay assays and cosedimentation studies revealed that muskelin does not interact direct with microtubules or microfilaments. The most prominant interaction of muskelin in a gel overlay was with a 45 kDa protein of pI 6.0. This interaction was enhanced during myoblast fusion. Apparent postranslational modification of the 45 kDa protein was detected by muskelin overlay of extracts resolved on 2-Dimensional IEF/SDS-PAGE gels. A two step approach was developed to isolate the 45 kDa protein by using a strong anion exchange column to enrich for the 45 kDa protein, followed by 2D gel anaKsis. Identifying muskelin's binding partners will enhance understanding of its functional role.
... Les TSP sont présentes dans les granules α plaquettaires (Wencel-Drake et al., 1985), dans la paroi vasculaire notamment au niveau de l'intima et de la média (Stenina et al., 2003) (Jurk et al., 2003;Nergiz-Unal et al., 2011). Les TSP sont impliquées dans l'agrégation plaquettaire (Boukerche and McGregor, 1988;Isenberg et al., 2008;Tuszynski et al., 1988) et jouent également un rôle en thrombose en recrutant les plaquettes et en stabilisant le thrombus (Bonnefoy et al., 2006). ...
Thesis
L’adhérence, l’activation et l’agrégation des plaquettes assurent l’hémostase, mais sont également à l’origine de la thrombose artérielle responsable de pathologies ischémiques graves. Ces pathologies sont traitées par des antiplaquettaires ayant prouvé leur efficacité, mais qui augmentent le risque de saignement. L’objectif de ce travail a été de mieux comprendre les mécanismes impliquant les plaquettes en hémostase, en thrombose artérielle et dans l’arrêt des saignements en conditions inflammatoires afin de réduire le risque hémorragique lié à ces traitements. L’identification d’une différence du rôle de la GPVI dans la formation du thrombus entre l’Homme et la souris indique que les agents anti-GPVI en développement pourraient être plus efficaces chez les patients que ce que laissait prévoir les études précliniques. L’utilisation de souris déficientes pour l’intégrine α5β1 plaquettaire n’a pas mis en évidence un rôle majeur de ce récepteur en thrombose artérielle, malgré une réduction de l’agrégation plaquettaire sur une surface de fibronectine en flux, suggérant que ce récepteur n’est pas une cible antithrombotique intéressante. Enfin, l’identification d’un rôle des intégrines β1 et β3 plaquettaires dans l’arrêt des saignements en conditions inflammatoires confirme le risque de saignement lié au ciblage des intégrines β3 plaquettaires
... Furthermore, Plt-EVs are known to have heterogeneous and highly diverse cargo, and studies have identified the presence of both prothrombin and thrombospondin, a dominant procoagulant α granule protein, in Plt-EVs. 28,37,38 Given this, it may be that the Plt-EVs potentiation of the Plt aggregation response to thrombin can be explained by differential contents of thrombin pathway-specific procoagulant proteins compared with the other Plt aggregation pathways tested in this study. In addition, proteomic studies have demonstrated differences in the content of Plt MVs by size distribution, with smaller MVs enriched for increased procoagulant content than larger MVs. ...
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Background: Platelet (Plt) derived-extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular endothelial cell permeability, similar to fresh Plts. To investigate this hypothesis we utilized in vitro and in vivo models of vascular endothelial compromise and bleeding. Methods: In vitro: Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function was assessed by trans - endothelial electrical resistance (TEER) measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts were assessed by multiple electrode Plt aggregometry. In vivo: The effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in NOD-SCID mice by an established Miles Assay of vascular permeability and a tail snip bleeding assay. Results: In vitro: Plt-EVs displayed exosomal size distribution and expressed Plt specific surface markers. Plts and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced Thrombin Receptor Activating Peptide (TRAP) mediated aggregation of whole blood, whereas Plts enhanced TRAP, Arachidonic Acid (ASPI), Collagen, and Adenosine Diphosphate (ADP) mediated aggregation. In vivo: Plt-EVs are equivalent to Plts in attenuating VEGF-A induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model. Conclusion: Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability and mitigate the endotheliopathy of trauma (EOT). Study type: Original Article LEVEL OF EVIDENCE: This is a pre-clinical study so it does not confirm to the level of evidence table for all clinical studies and case reports.
... Earlier studies reported contradictory evidence regarding the role of TSP1 in platelet activation (135,280), and the first evaluation of platelet function in Thbs1 -/mice reported normal hemostatic function as assessed by tail bleeding and thrombin activation of washed platelets (127). Therefore, TSP1 was concluded to not be required for normal platelet function. ...
Article
Significance: In contrast to structural elements of the extracellular matrix, matricellular proteins appear transiently during development and injury responses, but their sustained expression can contribute to chronic disease. Through interactions with other matrix components and specific cell surface receptors matricellular proteins regulate multiple signaling pathways, including those mediated by reactive oxygen and nitrogen species and H2S. Dysregulation of matricellular proteins contributes to the pathogenesis of vascular diseases and cancer. Defining the molecular mechanisms and receptors involved is revealing new therapeutic opportunities. Recent Advances: Thrombospondin-1 regulates NO, H2S, and superoxide production and signaling in several cell types. The thrombospondin-1 receptor CD47 plays a central role in inhibition of NO signaling, but other thrombospondin-1 receptors also modulate redox signaling. The matricellular protein CCN1 engages some of the same receptors to regulate redox signaling, and ADAMTS1 regulates NO signaling in Marfan syndrome. In addition to mediating matricellular protein signaling, redox signaling is emerging as an important pathway that controls the expression of several matricellular proteins. Critical issues: Redox signaling remains unexplored for many matricellular proteins. Their interactions with multiple cellular receptors remains an obstacle to defining signaling mechanisms, but improved transgenic models could overcome this barrier. Future directions: Therapeutics targeting the TSP1 receptor CD47 may have beneficial effects for treating cardiovascular disease and cancer and have recently entered clinical trials. Biomarkers are needed to assess their effects on redox signaling in patients and to evaluate how these contribute to their therapeutic efficacy and potential side effects.
... T HROMBOSPONDIN (TSP) 1 is a large multi-domain protein that was originally purified from platelets (15) but has since been found in many tissues including bone (23), muscle (39), skin (38,39), and brain (20). A major physiological function of this protein appears to involve cell-extracellular matrix interaction because purified preparations of TSP promote cell-substratum interaction of a variety of cell types (34), including platelets (30,33). Support for a role of TSP in matrix-cell interaction was demonstrated in a recent study by Arbeille et al. (3) who showed that TSP was localized in microfibrils at the junction between basement membrane and connective tissue in sections of human placenta, porcine arteries and skin. ...
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We have previously shown that peptides derived from the thrombospondin sequence, CSVTCG, promoted tumor cell adhesion. To further investigate this observation, the CSVTCG-tumor cell adhesion receptor from A549 human lung adenocarcinoma cells was isolated and characterized. A single protein peak was isolated by CSVTCG affinity chromatography which also analyzed as a single peak by anion exchange chromatography. The purified protein had a pI of 4.7 and analyzed on SDS-gels as a single band of M(r) = 50,000 under nonreducing conditions and as two protein bands of M(r) = 50,000, and 60,000 under reducing conditions. Purified CSVTCG binding protein (CBP) bound either CSVTCG- or TSP-Sepharose but showed little interaction with either VCTGSC- or BSA-Sepharose. CBP was cell surface exposed. CSVTCG derivatized with [125I] Bolton-Hunter reagent was taken up by cells in a dose-dependent manner and the cell association was inhibited with a monospecific polyclonal anti-CBP antibody. Examination of the cell proteins crosslinked to labeled CSVTCG by SDS-gel electrophoresis revealed one band that comigrated with purified CPB. Using an in vitro binding assay, purified CBP bound mannose, galactose, and glucosamine-specific lectins. CBP bound TSP saturably and reversibly. The binding was Ca+2/Mg+2 ion dependent and inhibited with fluid phase TSP and anti-CBP. Little or no binding was observed on BSA, fibronectin, GRGES, and GRGDS. Heparin, but not lactose, inhibited binding. Anti-CBP IgG and anti-CSVTCG peptide IgG inhibited A549 cell spreading and adhesion on TSP but not on fibronectin and laminin. These results indicate that CBP and the CSVTCG peptide domain of TSP can mediate TSP-promoted tumor cell adhesion.
Thesis
Platelet activation results in two major changes at the platelet surface: a change in GPIIb- Illa, exposing the previously cryptic fibrinogen binding site and fusion of platelet and granule membranes, leading to the expression of the two known granule membrane proteins, GMP-140 (α-granule) and GP53 (lysosome). Anti-GPIIb-IIIa monoclonal antibodies (MAbs) were raised and characterised in functional and flow cytometric assays. Competitive binding studies, with these and a panel of anti-GPIIb-IIIa MAbs, identified distinct epitopes on the complex important in influencing fibrinogen receptor exposure. Platelet aggregation was totally inhibited by MAbs binding to two conformation-dependent sites, one on GPIIb-IIIa and one on GPIIIa. A second group of antibodies, heterogeneous in their inhibitory effects on platelet aggregation, bound to overlapping sites on GPIIb-IIIa, GPIIb and GPIIIa. Three conformation-specific sites were identified, which induced platelet aggregation in the absence of any other agonist. All inhibitory MAbs totally inhibited fibrinogen binding to ADP-stimulated platelets and evidence is presented that fibrinogen and von Willebrand Factor bind to GPIIb-IIIa in a comparable and competitive way. Anti-GMP-140and anti-GP53 MAbs, raised against activated platelets, were characterised in Western blotting and phenotypic studies. Anti-GP53 MAbs were heterogeneous in their reactivity with a number of cell types; in particular, one MAb, RFAC-4, failed to bind to platelets from albino patients: a finding that may reflect the homology of GP53 with the melanoma-associated antigen, ME491. Flow cytometric, antibody binding studies showed that whilst the expression of both GMP-140 and GP53 was up-regulated over very similar thrombin concentrations, their expression ADP-stimulated platelets differed: GMP-140 was not detected on these cells but a partial expression of GP53 was seen. Using a whole blood, flow cytometric method, which minimises platelet activation in vitro. GP53 was shown to be a useful marker of platelet activation in the ex vivo analysis of platelets in a several clinical conditions.
Chapter
Gefäßerkrankungen und damit verbundene Akutkomplikationen stellen unverändert die Haupttodesursache des Diabetikers dar. Die Entwicklung der letztlich lebensterminierenden thrombotischen Akutereignisse an endothelialen Läsionsstellen des Gefäßsystems ist verstärkt. Morbidität und Mortalität des Diabetikers hängen damit von den gefäßvermittelten Komplikationen schicksalhaft ab. Die Integrität des Blutflusses als Voraussetzung einer bedarfsgerechten Organperfusion resultiert aus Vasomotion, Plasmazusammensetzung, Eigenschaften zellulärer Blutelemente, der Gefäßstruktur sowie v. a. aus der ungestörten Interaktion dieser Komponenten an der endothelialen Grenzfläche. Die funktionelle Thromboresistenz des Endothels ist beim Diabetiker vermindert. Neben gesteigerter intravasaler Thrombinbildung und verminderter re-parativer Fibrinolyse führen v. a. primär funktionsgesteigerte Thrombozyten zu einem präthrombotischen Zustand. Im Gegensatz zu hämorrheologischen Mechanismen kann eine thrombotische Diathese zur akuten Strombahnobstruktion führen. Aktivierte Thrombozyten sind dabei dreifach schädigend: 1) primäre Mikroembolisierung der Kapillarstrombahn, 2) lokale Progression von Gefäßwandläsionen durch Sekretion vasokonstriktiver, mitogener und oxydativ wirksamer Substanzen, 3) Auslösung einer arteriellen Akutthrombose. Aus diesen Gründen liegt eine ergänzende thrombozytenfunktionshemmende Präventionsmedikation bei Diabetikern nahe. Voraussetzung für eine weitere Verbesserung der Nutzen-Risiko-Abwägung solcher Therapieansätze ist die Analyse des individuellen Risikos eines aktivierten zellulären Hämostasesystems z. B. mit der durchflußzytometrischen Aktivierungsmarkeranalyse nach dem Düsseldorf-III-Protokoll („thrombotic risk assessment“).
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Human platelet thrombospondin adsorbed on plastic promotes attachment and spreading of human G361 melanoma cells. Attachment is rapid, and spreading is maximal by 90 min with 60-90% of the attached cells spread. In contrast, thrombospondin promotes attachment but not spreading of human C32 melanoma cells, which attach and spread only on laminin substrates. The specificity of these interactions and the regions of the thrombospondin molecule involved in attachment and spreading were examined using proteolytic fragments of thrombospondin and by inhibition studies. The sulfated fucan, fucoidan, and monoclonal antibody A2.5, which is directed against the heparin-binding domain of thrombospondin, selectively inhibit spreading but only weakly inhibit attachment. Monoclonal antibodies against some other domains of thrombospondin, however, are potent inhibitors of attachment. The amino-terminal heparin-binding domain of thrombospondin does not promote attachment. Large fragments lacking the heparin-binding domain support attachment but not spreading of G361 cells. Attachment activity is lost following removal of the 18-kD carboxyl-terminal domain. These results suggest that at least two melanoma ligands are involved in cell attachment and spreading on thrombospondin. The carboxyl-terminal region and perhaps other regions of the molecule bind to receptor(s) on the melanoma surface that promote initial attachment but not cell spreading. Interaction of the heparin-binding domain with sulfated glycoconjugates on melanoma surface proteoglycans and/or sulfated glycolipids mediates spreading. Monoclonal antibodies A2.5 and C6.7 also reverse spreading of G361 cells growing on glass culture substrates, suggesting that binding to thrombospondin mediates attachment of these melanoma cells in culture.
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Thrombospondin (TSP) is a multifunctional platelet glycoprotein synthesized by a variety of cells in culture including monocytes and macrophages. We now report that 125I-TSP binds specifically, saturably, and reversibly to mouse peritoneal macrophages and to cells of the monocyte-like human cell line U937 with dissociation constants of 6.7-14.5 X 10(-8) M and 3-4 X 10(5) binding sites per cell. TSP mediates an adhesive interaction between thrombin-stimulated platelets and both U937 cells and human blood monocytes. Using a sensitive rosetting assay, we found that monocytes were not rosetted by resting platelets whereas greater than 90% were rosetted by thrombin-stimulated platelets. Monoclonal and polyclonal anti-TSP antibodies markedly inhibited rosetting as did TSP itself. Neither control antibodies nor heparin, fibronectin, fibrinogen, nor the fibronectin adhesion tetrapeptide Arg-Gly-Asp-Ser inhibited rosetting. TSP may thus serve as a molecular bridge linking activated platelets with monocytes at sites of early vascular injury. Such interaction may be of critical importance in the regulation of thrombosis and the initiation of atherosclerosis.
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The human platelet glycoprotein thrombospondin (TSP) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). Binding of 125I-TSP to lipids from sheep and human erythrocytes and human platelets resolved on thin layer chromatograms indicates that sulfatides are the only lipids in the membrane which bind TSP. Binding to less than 2 ng of sulfatide could be detected. TSP failed to bind to other purified lipids including cholesterol 3-sulfate, phospholipids, neutral glycolipids, and gangliosides. Binding of 125I-TSP was inhibited by unlabeled TSP, by low pH, and by reduction of intersubunit disulfide bonds with dithiothreitol. A monoclonal antibody against TSP (A2.5), which inhibits hemagglutination and agglutination of fixed activated platelets by TSP, strongly inhibited TSP binding to sulfatides. A second monoclonal antibody (C6.7), which inhibits hemagglutination and aggregation of thrombin-activated live platelets, weakly inhibited sulfatide binding. Binding was inhibited by high ionic strength and by some monosaccharide sulfates including methyl-alpha-D-GlcNAc-3-sulfate. Neutral sugars did not inhibit. Fucoidan, a sulfated fucan, strongly inhibited binding with 50% inhibition at 0.3 micrograms/ml fucoidan. Other sulfated polysaccharides including heparin and dextran sulfates were good inhibitors, whereas hyaluronic acid and keratan sulfate were very weak.
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Thrombospondin and histidine-rich glycoprotein are two proteins with diverse biological activities which have been associated with human platelets and other cell systems. Using an enzyme-linked immunosorbent assay, we have demonstrated that purified human platelet thrombospondin formed a complex with purified human plasma histidine-rich glycoprotein. The formation of the thrombospondin-histidine-rich glycoprotein complex was specific, concentration dependent, and saturable. Significant binding was detected when histidine-rich glycoprotein was incubated with thrombospondin immobilized on anti-thrombospondin IgG-coated plates, indicating that the observed complex formation was not due to a thrombospondin interaction with the plastic surface. Sucrose-density-gradient ultracentrifugation of a mixture of thrombospondin and histidine-rich glycoprotein also revealed the formation of fluid-phase complexes, with an estimated stoichiometry of 1 thrombospondin: 3.5 histidine-rich glycoprotein. Fibrinogen, which has been previously shown to bind to absorbed thrombospondin, did not inhibit the formation of the thrombospondin-histidine-rich glycoprotein complex. Histidine-rich glycoprotein complexed with thrombospondin was capable of binding heparin and neutralizing the anticoagulant activity of heparin in plasma. Specific complex formation between thrombospondin and histidine-rich glycoprotein may play a significant role in influencing platelet blood vessel wall interactions as well as modulating the association of various cells with the extracellular matrix.
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The biosynthesis of thrombospondin, a glycoprotein first described in platelets, has been studied in human endothelial cells. This glycoprotein has a molecular mass of 450 kDa. It is secreted and incorporated into the extracellular matrix of several cell types in culture. Pulse-chase experiments with [3H]leucine were performed and the synthesis and secretion of the glycoprotein was studied by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results of these experiments show that the three subunits of thrombospondin are identical in molecular mass. During synthesis there is a small but significant increase in molecular mass within 20 min after pulse labeling. The early form of thrombospondin is sensitive to endoglucosaminidase H treatment, indicating that a transformation of the oligosaccharide structures from ‘high-mannose’ to ‘complex’ structures takes place. Within 60 min after synthesis only the mature form of the glycoprotein is secreted into the medium. In the presence of tunicamycin, an inhibitor of N-glycosylation, there is a reduction in molecular mass of the subunit from 165 kDa to 155 kDa. Pulse-chase experiments in the presence of tunicamycin supported the conclusion that the carbohydrate part is processed during biosynthesis. Inhibition of glycosylation had a pronounced effect on the secretion of thrombospondin. The decreased occurrence of thrombospondin in the culture medium seemed to be due to a high intracellular degradation rate of unglycosylated thrombospondin. Characterization of the glycopeptide structures of thrombospondin metabolically labeled with [3H]mannose by Bio-Gel P-4 and concanavalin-A–Sepharose column chromatography revealed that the oligosaccharide structures of the cellular and secreted forms of thrombospondin differ in their composition.
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Thrombospondin (TS), a 450,000 molecular weight glycoprotein, is released from alpha-granules of thrombin-activated platelets and is secreted and incorporated into the extracellular matrix by several cell types in culture. We have examined the effects of cell density and transformation on the production of TS in cell culture. The levels of TS, per cell, in the culture medium of endothelial cells, smooth muscle cells, and fibroblasts were greater at lower cell densities; in fibroblasts the levels of two other extracellular matrix proteins, fibronectin and collagen, were unaffected by cell density. Our evidence indicates that the higher levels of TS in the culture medium, determined for lower-density cells, were achieved by an increased secretion of the protein rather than by a reduction in degradation or incorporation into the extracellular matrix. TS production by normal and transformed WI-38 fibroblasts was the same, although the fibronectin level in the culture medium of the transformed cells was substantially decreased. These findings suggest that the production of TS by cells in culture is regulated in a different fashion from that of fibronectin or collagen.
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Fixed platelets, bearing covalently bound fibrinogen, participate passively in aggregation of fresh platelets when the aggregation process is release related (G. Agam and A. Livne, Thromb Haemostasis 51:145-149, 1984). Inhibition of the release by aspirin abolishes the capability of the fresh platelets activated by 10 microM ADP to interact with the fixed platelets. A supernatant fraction from fresh platelets activated by 10 microM ADP (releasate) reconstitutes the interaction. Purified thrombospondin (TSP) replaces the releasate. Moreover, anti-TSP antibodies abolish the reconstituting effect of the releasate. It is concluded that TSP plays a role in the molecular mechanism of platelet-platelet recognition during release-related aggregation.
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Platelets activated by alpha-thrombin change shape from disks to spheres and aggregate. During this sequence of activation, platelets develop a membrane-bound lectin activity1,2 which is expressed after the secretion of intracellular granule contents3. This lectin activity seems to be important in mediating platelet aggregation by binding to a specific receptor on other platelets. Gartner et al.4 have recently shown that fibrinogen is the receptor for the endogenous lectin secreted by activated platelets. Thrombospondin5 (also called glycoprotein G6 and thrombin-sensitive protein7,8) is an alpha-granule constituent which has a molecular weight (MW) of 450,000 and is a disulphide-linked trimer of 160,000 MW subunits5,9,10. Secreted thrombospondin5-14 binds to platelet membranes in a calcium-dependent manner12. We show here that thrombospondin is the endogenous lectin of human platelets.