The N-terminus of thrombospondin: the domain stands apart

Thrombospondin 1 (TSP1) was first recognized as a thrombin-sensitive protein associated with platelet membranes. It is secreted by numerous cell types and its expression is predominant in areas of active tissue remodeling. Thrombospondins 1 and 2 are large, trimeric, matricellular proteins, composed of multiple structural motifs which interact with a diverse array of receptors and molecules. Thrombospondin's capacity to bind multiple receptors renders it multifunctional. The functions of its isolated domains can be overlapping or contradictory. In this review, we focus on the N-terminus of the molecule, first recognized for its strong heparin binding properties and characterized by its susceptibility to proteolytic cleavage from the stalk region of thrombospondin. The N-terminus, called the heparin binding domain (HBD), interacts with a variety of macromolecules including heparan sulfate proteoglycans at the membrane and in the matrix, LDL receptor-related protein (LRP), sulfated glycolipids, calreticulin, and integrins. The HBD mediates endocytosis of thrombospondin. It functions both as a soluble and an insoluble modulator of cell adhesion and motility. In contrast to thrombospondin, the HBD has pro-angiogenic activity. We propose that the HBD of thrombospondins 1 and 2 are found primarily in the cellular microenvironment in conditions of cellular injury, stress and tissue remodeling and that the HBD conveys multiple signals involved in cellular adaptation to injury.     

Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment

The platelet protein thrombospondin (TSP) which is secreted from alpha-granules upon platelet activation agglutinates trypsinized, glutaraldehyde-fixed human erythrocytes. Optimal conditions for the hemagglutinating activity require that both Ca2+ and Mg2+ be present in final concentrations of 2 mM. In the presence of dithiothreitol (i.e., reduction of disulfide bonds), the lectin-like activity decreases in a manner proportional to the extent of reduction of the molecule from its native trimeric configuration into its Mr 180 000 subunits. Proteolysis of purified TSP with thermolysin, which produces discrete domains with the capacity to bind fibrinogen and heparin, also diminishes, but does not abolish, the hemagglutinating activity. Fibrinogen was without effect on hemagglutinating activity while heparin was found to be a potent inhibitor. Other proteoglycans such as hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate had no effect. That portion of the TSP molecule apparently responsible for the hemagglutinating activity was identified by incubating a thermolytic digest of TSP with red blood cells and then determining which fragment was bound to the cell surface. The binding site resides within a peptide fragment of 140 000 daltons but is absent from an Mr 120 000 fragment derived from the Mr 140 000 fragment. Under the conditions for optimal expression of hemagglutinating activity (i.e., 2 mM MgCl2 and 2 mM CaCl2), this Mr 140 000 fragment was also shown to have heparin binding activity.     

Localization of two binding domains for thrombospondin within fibronectin.

Thrombospondin is a major glycoprotein of the platelet alpha-granule and is secreted during platelet activation. Several protease-resistant domains of thrombospondin mediate its interactions with components of the extracellular matrix including fibronectin, collagen, heparin, laminin, and fibrinogen. Thrombospondin, as well as fibronectin, is composed of several discretely located biologically active domains. We have characterized the thrombospondin binding domains of plasma fibronectin and determined the binding affinities of the purified domains; fibronectin has at least two binding sites for thrombospondin. Thrombospondin bound specifically to the 29-kDa amino-terminal heparin binding domain of fibronectin as well as to the 31-kDa non-heparin binding domain located within the larger 40-kDa carboxy-terminal fibronectin domain generated by chymotrypsin proteolysis. Platelet thrombospondin interacted with plasma fibronectin in a specific and saturable manner in blot binding as well as solid-phase binding assays. These interactions were independent of divalent cations. Thrombospondin bound to the 29-kDa fibronectin heparin binding domain with a Kd of 1.35 x 10(-9) M. The Kd for the 31-kDa domain of fibronectin was 2.28 x 10(-8) M. The 40-kDa carboxy-terminal fragment bound with a Kd of 1.65 x 10(-8) M. Heparin, which binds to both proteins, inhibited thrombospondin binding to the amino-terminal domain of fibronectin by more than 70%. The heparin effect was less pronounced with the non-heparin binding carboxy-terminal domain of fibronectin. By contrast, the binding affinity of the thrombospondin 150-kDa domain, which itself lacked heparin binding, was not affected by the presence of heparin. Based on these data, we conclude that thrombospondin binds with different affinities to two distinct domains in the fibronectin molecule.     

Multiple domains are involved in the interaction of endothelial cell thrombospondin with fibronectin

Thrombospondin is a large multifunctional glycoprotein synthesized, secreted and incorporated into the extracellular matrix by several cell types in culture. It is also present in the blood platelet and is secreted following platelet activation. We have previously shown that thrombospondin co-distributes with fibronectin in the extracellular matrix and that it can bind directly to purified fibronectin. In order to elucidate the chemical aspects of thrombospondin incorporation into the extracellular matrix, we studied the interaction of endothelial cell thrombospondin and fibronectin. We find that endothelial cell thrombospondin has two distinct binding domains for fibronectin. One domain is on the 70-kDa core fragment, probably similar to that of platelet thrombospondin. The other domain is on the 27-kDa N-terminal fragment and is unique to endothelial cell thrombospondin. The dissociation constant of the intact endothelial-cell-derived molecule is 0.7 +/- 0.2 x 10(-7) M. Following fragmentation, the separate domains bind with somewhat lower affinity: the core domain binds with a Kd of 3.4 +/- 1.5 x 10(-7) M and the N-terminal domain binds with a Kd of 8.8 +/- 1.8 x 10(-7) M. Binding of the intact molecule is Ca2+-independent. By contrast, following tryptic fragmentation, binding of the 70-kDa fragment is practically lost. It can be restored, however, by removal of Ca2+, indicating that the binding site on this domain is either sequestered or becomes so following fragmentation. Heparin, which also binds to both fragments, competed with fibronectin binding to the 27-kDa fragment but not to the 70-kDa domain. The fact that heparin also competitively inhibits fibronectin binding of the intact molecule further supports sequestration of the fibronectin-binding domain on the 70-kDa core fragment. Our data suggest that endothelial-cell thrombospondin possesses two distinct binding sites for fibronectin, a low-affinity constitutively available one and a high-affinity one, possibly sequestered on the intact unbound molecule.     

Activation of human neutrophils increases thrombospondin receptor expression.

Thrombospondin (TSP), a 450-kDa extracellular matrix protein secreted by platelets may be instrumental in triggering polymorphonuclear leukocyte (PMN) activation and mediating PMN-endothelial cell interactions. TSP alone had no effect on O-2 generation but caused a significant increase in the chemoattractant FMLP-mediated O-2 generation. Purified HBD, but not the 140-kDa COOH-terminal fragment of TSP, retained the priming activity indicating that the priming effect was mediated through the HBD of TSP. The priming of FMLP-mediated O-2 generation by TSP, and our recent studies demonstrating that TSP stimulates PMN adhesion and motility suggested the presence of specific receptors for TSP on PMN. Binding studies on unactivated PMN, using 125I-TSP and competition with excess unlabeled TSP, demonstrated 2.4 x 10(3) binding sites/cell with an apparent Kd of 7 nM. Heparin did not compete for binding as effectively as unlabeled TSP. There were 1.5 x 10(3) heparin-inhibitable binding sites/cell with an apparent Kd of 8 nM that represented approximately 60% of the TSP-specific sites. Therefore, two distinct TSP receptors appeared to exist on unactivated PMN; one interacting with the heparin-binding domain of TSP and one interacting with a different site. Treating PMN with cytochalasin B followed by FMLP caused a 30-fold increase in TSP receptor expression. Binding studies on activated PMNs revealed 7.6 x 10(4) sites/cell; 60% of which were heparin inhibitable. The majority (5.3 x 10(4) sites/cell) of receptors expressed had an affinity of approximately 20 nM. About 50% of these sites were heparin inhibitable. In addition, there were 2.3 x 10(4) higher affinity sites/cell with an apparent Kd of 6 nM. Heparin-inhibitable sites comprised 70% of the higher affinity sites. The existence of a subset of TSP receptors that were heparin-inhibitable on PMN suggests that binding of TSP may trigger functionally independent responses. Increased receptor expression and expression of two high affinity binding sites following PMN activation may modulate PMN-endothelium or PMN-basement membrane interactions localized at the blood vessel wall.     

Thrombospondin stimulates motility of human neutrophils

Polymorphonuclear leukocytes (PMNs) migrate to sites of inflammation or injury in response to chemoattractants released at those sites. The presence of extracellular matrix (ECM) proteins at these sites may influence PMN accumulation at blood vessel walls and enhance their ability to move through tissue. Thrombospondin (TSP), a 450-kD ECM protein whose major proteolytic fragments are a COOH-terminal 140-kD fragment and an NH2-terminal heparin-binding domain (HBD), is secreted by platelets, endothelial cells, and smooth muscle cells. TSP binds specifically to PMN surface receptors and has been shown, in other cell types, to promote directed movement. TSP in solution at low concentrations (30-50 nM) "primed" PMNs for f-Met-Leu-Phe (fMLP)- mediated chemotaxis, increasing the response two- to fourfold. A monoclonal antibody against the HBD of TSP totally abolished this priming effect suggesting that the priming activity resides in the HBD of TSP. Purified HBD retains the priming activity of TSP thereby corroborating the antibody data. TSP alone, in solution at high concentrations (0.5-3.0 microM), stimulated chemotaxis of PMNs and required both the HBD and the 140-kD fragment of TSP. In contrast to TSP in solution, TSP bound to nitrocellulose filters in the range of 20- 70 pmol stimulated random locomotion of PMNs. The number of PMNs migrating in response to bound TSP was approximately two orders of magnitude greater than the number of cells that exhibited chemotaxis in response to soluble TSP or fMLP. Monoclonal antibody C6.7, which recognizes an epitope near the carboxyl terminus of TSP, blocked migration stimulated by bound TSP, suggesting that the activity resides in this domain. Using proteolytic fragments, we demonstrated that bound 140-kD fragment, but not HBD, promoted migration of PMNs. Therefore, TSP released at injury sites, alone or in synergy with chemotactic peptides like fMLP, could play a role in directing PMN movement.     

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of endothelial cell proliferation,adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin

Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence -Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependet and reversible. Thus, the antiproliferative activities of the thrombospondin peptides and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.     

Interactions of thrombospondin with endothelial cells: receptor- mediated binding and degradation

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.     

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.     

Effects of anti-thrombospondin monoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin

A monoclonal antibody (Mab) has been raised against native thrombospondin (TSP), the endogenous lectin of human platelets, that inhibits the hemagglutination of trypsinized, glutaraldehyde-fixed human erythrocytes by purified TSP. This Mab, designated A2.5, also inhibits the agglutination of fixed, activated platelets by TSP. Mab A2.5 immunoprecipitates a 25-kilodalton (kDa) peptide from chymotryptic digests of TSP that is not disulfide bonded to any other region of the TSP molecule. This fragment represents the previously characterized heparin binding domain of TSP [Dixit, V.M., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1984) J. Biol. Chem. 259, 10100-10105]. In agreement with this assignment, heparin inhibits the binding of Mab A2.5 to TSP. Another Mab, designated C6.7, also blocks TSP-mediated hemagglutination, yet has no effect on the agglutination of fixed, activated platelets by TSP. This Mab has been shown to inhibit the thrombin-stimulated aggregation of live platelets and to immunoprecipitate an 18-kDa fragment from chymotryptic digests, which is distinct from the heparin binding domain [Dixit, V.M., Haverstick, D.M., O'Rourke, K.M., Hennessy, S.W., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3472-3476].     

人vWF-A1多肽的融合表达及生物学活性鉴定

血管性血友病因子 (vWF)通过与血小板膜糖蛋白结合介导血小板的粘附和聚集 ,在血栓形成过程中发挥重要作用 .通过阻断血小板与vWF的结合可抑制血栓形成 .应用RT PCR方法从人脐带内皮细胞中克隆vWF A1区基因并在原核细胞内进行表达 ,经过纯化、复性 ,获得重组蛋白(rvWF A1) .用流式细胞术检测rvWF A1与转染了糖蛋白Ib(GPⅠb)的CHO K1细胞和血小板GPⅠb的结合能力 ,血小板聚集仪测定rvWF A1对瑞斯托霉素 (ristocetin)诱导的血小板聚集作用的影响 .重组表达载体pET 2 0b(+ ) vWF A1在大肠杆菌BL2 1(DE3)plus中得到有效表达 ,表达的重组蛋白量占菌体总蛋白 30 % .次氮基三乙酸镍琼脂糖 (Ni NTAagarose)柱纯化后 ,其纯度为 95 % .经复性的rvWF A1蛋白具有良好的生物学活性 ,它可与转染了GPⅠb的CHO K1细胞和血小板结合 ,阳性率分别为 96 90 %与 78 6 0 % ,且可以抑制ristocetin诱导的血小板聚集 ,其抑制效应呈剂量依赖性 .IC50 的rvWF A1浓度为 0 5 6 μmol L ,当浓度为 1 4 μmol L时抑制率最高达 84 70 % .结果表明 ,在原核细胞中表达人rvWF A1区蛋白可抑制血浆中野生型vWF与血小板的结合 ,具有抗血栓形成的潜在应用前景     

Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells

Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenocortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10⁻⁷ M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by ten-fold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not. © 1996 Wiley-Liss, Inc.     

Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains

A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.     

Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin

Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu-Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity.     

The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction

Human platelet thrombospondin (TSP) was purified to homogeneity by chromatography on fibrinogen coupled to cyanogen bromide-activated Sepharose. The yield of TSP was 1.3 mg or approximately 22% of that present in platelet-rich plasma as determined by radioimmunoassay. It analyzed on discontinuous sodium dodecyl sulfate gels as a single band having apparent molecular weights of 180,000 and greater than 400,000 under reducing and nonreducing conditions, respectively. Amino acid analysis gave results similar to previously published values. Antibodies raised in rabbits were monospecific as evaluated by radioimmunoassay. In double immunodiffusion tests, these antibodies gave one line of identity against TSP purified by this procedure and TSP purified by published procedures, confirming the identity of the material isolated. The protein possesses no lectin-like activity. The specificity of the TSP-fibrinogen interaction was investigated. TSP binding to fibrinogen-Sepharose occurred in the presence of EDTA, indicating that calcium and magnesium ions are not required for interaction of TSP with fibrinogen. The binding of TSP to fibrinogen-Sepharose was quantitatively blocked by pretreatment with an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen. Antibodies against the D and E domains of fibrinogen had no effect on the binding. Excess fibrinogen (30 mg/ml) added to platelet extract quantitatively inhibited binding of TSP to fibrinogen-Sepharose. TSP preferentially bound to uncross-linked fibrin, suggesting that the TSP-fibrinogen binding site is unavailable in cross-linked fibrin. These results indicate that TSP binds specifically to immobilized fibrinogen or uncross-linked fibrin through determinants present in the carboxyl-terminal portion of the alpha chain and that these interactions do not require calcium or magnesium ions.     

Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.     

Thrombospondin modulates focal adhesions in endothelial cells

We examined the effects of thrombospondin (TSP) in the substrate adhesion of bovine aortic endothelial cells. The protein was tested both as a substrate for cell adhesion and as a modulator of the later stages of the cell adhesive process. TSP substrates supported the attachment of some BAE cells, but not cell spreading or the formation of focal adhesion plaques. In contrast, cells seeded on fibrinogen or fibronectin substrates were able to complete the adhesive process, as indicated by the formation of focal adhesion plaques. Incubation of cells in suspension with soluble TSP before or at the time of seeding onto fibronectin substrates resulted in an inhibition of focal adhesion formation. Furthermore, the addition of TSP to fully adherent cells in situ or prespread on fibronectin substrates caused a reduction in the number of cells, which were positive for focal adhesions, although there was no significant effect on cell spreading. In a dose-dependent manner, TSP reduced the number of cells with adhesion plaques to approximately 60% of control levels. The distribution of remaining adhesion plaques in TSP-treated cells was also altered: plaques were primarily limited to the periphery of cells and were not present in the central cell body, as in control cells treated with BSA. The observed effects were specific for TSP and were not observed with platelet factor 4, beta-thromboglobulin, or fibronectin. The TSP-mediated loss of adhesion plaques was neutralized by the addition of heparin, fucoidan, other heparin-binding proteins, and by a monoclonal antibody to the heparin binding domain of TSP, but not by antibodies to the core or carboxy-terminal regions of TSP. The interaction of the heparin- binding domain of TSP with cell-associated heparan sulfate appears to be an important mechanistic component for this activity of TSP. These data indicate that TSP may have a role in destabilizing cell adhesion through prevention of focal adhesion formation and by loss of preformed focal adhesions.     

Characterization of the platelet agglutinating activity of thrombospondin

Thrombospondin (TSP) is a glycoprotein secreted from the alpha-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets. Platelets fixed with formaldehyde following activation by thrombin are agglutinated by exogenously added TSP. Fixed, nonactivated platelets are not agglutinated. The platelet agglutinating activity of TSP is optimally expressed in the presence of 2 mM each of Mg2+ and Ca2+. Reduction of the disulfide bonds within the TSP molecule inhibits its platelet agglutinating activity. TSP bound to the surface of fixed, activated platelets can be eluted by the addition of disodium ethylenediaminetetraacetate. This approach was exploited to identify the region of the TSP molecule containing the platelet binding site. The binding site resides within a thermolytic fragment of TSP with Mr 140 000 but is not present in the Mr 120 000 fragment derived from the polypeptide of Mr 140 000. Since both the Mr 140 000 and 120 000 fragments contain fibrinogen binding sites, this finding suggests that the binding of TSP to the platelet surface requires interaction with other platelet surface components in addition to fibrinogen. The observation that fibrinogen only partially inhibits the TSP-mediated agglutination of fixed, activated platelets is consistent with this interpretation.     

B型肉毒毒素受体结合区C片段在大肠杆菌中的表达及免疫原性研究

目的:在大肠杆菌中表达、纯化B型肉毒毒素受体结合区C片段(BHc-C),研究其免疫原性。方法:将BHc-C基因克隆到原核表达载体pGEX-4T-1中,转化大肠杆菌BL21(DE3),经IPTG诱导表达GST-BHc-C融合蛋白并通过亲和纯化;以纯化的融合蛋白免疫BALB/c小鼠制备免疫血清,采用ELISA检测免疫血清的效价并测定其抗B型肉毒毒素中和活性。结果:在大肠杆菌中表达了GST-BHc-C融合蛋白;以该融合蛋白免疫小鼠获得高效价免疫血清,且该免疫血清具有中和活性。结论:获得了GST-BHc-C融合蛋白,并证实其具有免疫原性。