Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.     

Characterization of METH-1/ADAMTS1 processing reveals two distinct active forms

METH-1/ADAMTS1 is a member of a newly described family of genes that contain metalloprotease, disintegrin, and thrombospondin-like motifs. We have recently shown that METH-1 protein is a potent inhibitor of angiogenesis. Here, we demonstrate that secreted human pro-METH-1 is processed in two consecutive steps to release both p87 and p65 active forms. The p87 form lacks the N-terminal prodomain and p65 results from an additional processing event in the C-terminal end. Generation of p87 was blocked with specific inhibitors of furin, and incubation of pro-METH-1 with purified furin released the p87 fragment but not p65. Generation of p65 required preformation of p87 and was suppressed by inhibitors of matrix metalloproteases. We demonstrate that matrix metalloproteases 2, 8, and 15 were able to release p65 when p87 was used as substrate. This second processing step removes two thrombospondin repeats from the carboxyl-terminal end of p87-METH-1 and alters the affinity of the protein to heparin and endothelial cultures. Furthermore, this deletion was associated with a reduced activity upon suppression of endothelial cell proliferation. We hypothesize that METH-1 processing is relevant for the modulation of the anti-angiogenic properties displayed by the protein.     

Biophysical characterization,including disulfide bond assignments,of the anti-angiogenic type 1 domains of human thrombospondin-1

Thrombospondin-1 (TSP1), a modular secreted glycoprotein, possesses anti-angiogenic activity both in vitro and in vivo. This activity has been localized to the thrombospondin type 1 repeats/domains (TSR). A TSP1 monomer contains three TSRs, each with a hydrophobic cluster with three conserved tryptophans (WxxWxxW), a basic cluster with two conserved arginines (RxR), and six conserved cysteines. Using the baculovirus system, we expressed TSRs of human TSP1 as either the three domains in tandem (P123) or the third domain alone (P3) and demonstrated that both P123 and P3 at nanomolar concentrations inhibit either basic fibroblast-growth-factor or sphingosine-1-phosphate induced endothelial cell migration. Far-UV circular dichroism (CD) indicated that P123 and P3 have a common global fold that is very similar to properdin, a protein with six TSRs. Near-UV CD and fluorescence quenching studies indicated the conserved tryptophans are in a structured, partially solvent-accessible, positively charged environment. N-terminal sequence and mass spectrometry analysis of trypsin-digested TSRs indicated that the RFK linker sequence between P1 and P2 is readily proteolyzed and the conserved arginines are solvent accessible. By a combination of proteolysis and mass spectrometry, the recombinant TSRs were determined to be fully disulfide bonded with a connectivity of 1-5, 2-6, and 3-4 (cysteines are numbered sequentially from N- to C-terminus). TSRs are found in numerous extracellular proteins. These TSRs share the hydrophobic and basic clusters of the TSP TSRs but some have quite different placement of cysteine residues. We propose a sorting of TSRs into six groups that reconciles our results with information about other TSRs.     

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.     

Protein engineering and properties of human metalloproteinase and thrombospondin 1

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.     

A second, expressed thrombospondin gene (Thbs2) exists in the mouse genome.

The diverse and occasionally conflicting properties described for the extracellular, cell surface-associated protein thrombospondin (TSP) have raised the possibility that functionally distinct forms of the protein exist in the same organism. We have isolated and characterized a partial cDNA clone for mouse TSP that is clearly homologous to, but distinct from, the coding sequence for mouse TSP deduced from a mouse genomic clone (Bornstein, P., Alfi, D., Devarayalu, L., Framson, P., and Li, P. (1990) J. Biol. Chem. 265, 16691-16698). This second TSP, which we term thrombospondin 2, is the product of a separate gene (Thbs2) and is expressed in a variety of mouse tissues in a pattern that differs from that for TSP1. Based on their translated amino acid sequences, it seems likely that TSP1 and TSP2 will be found to have both common and unique properties and that the functional consequences of TSP production will reflect the ratio of the levels of these two related proteins.     

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.     

Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.     

cDNA cloning and characterization of vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.     

Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family

A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.     

A novel human metalloprotease synthesized in the liver and secreted into the blood: possibly, the von Willebrand factor-cleaving protease?

We identified a novel metalloprotease, which could be responsible for cleaving the Tyr842-Met843 peptide bond of von Willebrand factor (vWF). This metalloprotease was purified from Cohn Fraction-I precipitate of human pooled plasma by the combination of gel filtration, DEAE chromatography, and preparative polyacrylamide gel electrophoresis in the presence of SDS. The NH2-terminal amino acid sequence of the isolated protein was: AAGGILHLELLVAVGPDVFQAHQEDTRRY. Based on this sequence, we searched human genomic and EST databases, and identified compatible nucleotide sequences. These results suggested that this protein is a novel metalloprotease, a member of the family of a disintegrin and metalloprotease with thrombospondin type-1 motifs (ADAMTS), and its genomic DNA was mapped to human chromosome 9q34. Multiple human tissue northern blotting analysis indicated that the mRNA encoding this protease spanned approximately 5 kilobases and was uniquely expressed in the liver. Furthermore, we determined the cDNA sequence encoding this protease, and found that this protease was comprised of a signal peptide, a proregion followed by the putative furin cleavage site, a reprolysin-type zinc-metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (TSP1) motif, a cysteine-rich region, a spacer domain, and COOH-terminal TSP1 motif repeats.     

Non-peptidic Thrombospondin-1 Mimics as Fibroblast Growth Factor-2 Inhibitors: AN INTEGRATED STRATEGY FOR THE DEVELOPMENT OF NEW ANTIANGIOGENIC COMPOUNDS*

Endogenous inhibitors of angiogenesis, such as thrombospondin-1 (TSP-1), are promising sources of therapeutic agents to treat angiogenesis-driven diseases, including cancer. TSP-1 regulates angiogenesis through different mechanisms, including binding and sequestration of the angiogenic factor fibroblast growth factor-2 (FGF-2), through a site located in the calcium binding type III repeats. We hypothesized that the FGF-2 binding sequence of TSP-1 might serve as a template for the development of inhibitors of angiogenesis. Using a peptide array approach followed by binding assays with synthetic peptides and recombinant proteins, we identified a FGF-2 binding sequence of TSP-1 in the 15-mer sequence DDDDDNDKIPDDRDN. Molecular dynamics simulations, taking the full flexibility of the ligand and receptor into account, and nuclear magnetic resonance identified the relevant residues and conformational determinants for the peptide-FGF interaction. This information was translated into a pharmacophore model used to screen the NCI2003 small molecule databases, leading to the identification of three small molecules that bound FGF-2 with affinity in the submicromolar range. The lead compounds inhibited FGF-2-induced endothelial cell proliferation in vitro and affected angiogenesis induced by FGF-2 in the chicken chorioallantoic membrane assay. These small molecules, therefore, represent promising leads for the development of antiangiogenic agents. Altogether, this study demonstrates that new biological insights obtained by integrated multidisciplinary approaches can be used to develop small molecule mimics of endogenous proteins as therapeutic agents.     

Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains. VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn(2+) and Ca(2+) ions are conserved in the protease domain. The Cys-rich domain contains an RGDS sequence that could mediate integrin-dependent binding to platelets or other cells. Alternative splicing gives rise to at least seven potential variants that truncate the protein at different positions after the protease domain. Alternative splicing may have functional significance, producing proteins with distinct abilities to interact with cofactors, connective tissue, platelets, and von Willebrand factor.     

Thrombospondin II: partial cDNA sequence, chromosome location, and expression of a second member of the thrombospondin gene family in humans.

A novel form of human thrombospondin was identified during the screening of a human fibroblast cDNA library. We report the cDNA sequence for 1.8 kb of the 3' end of the cDNA, plus an additional 937 bp of 3'-untranslated sequence. The translated sequence reveals a high degree of similarity to thrombospondin I. The homology ranges from 56 to 80% for different regions within the two proteins. The repeating segments of amino acid sequence identified in thrombospondin I were found to be conserved in thrombospondin II. The new form of thrombospondin hybridizes to a 7.5-kb message by Northern analysis. The THBS2 gene is located at the distal long arm of chromosome 6 at 6q27. The gene is transcribed in fibroblasts, smooth muscle cells, and an osteosarcoma cell line, at levels somewhat lower than that of thrombospondin I. Umbilical vein endothelial cells do not transcribe thrombospondin II under the conditions of this study. These findings suggest that previous studies of thrombospondin function need to be reassessed to identify the functions specific to each molecule.     

Differential Interactions of Thrombospondin-1, -2, and -4 with CD47 and Effects on cGMP Signaling and Ischemic Injury Responses

Thrombospondin-1 regulates nitric oxide (NO) signaling in vascular cells via CD47. Because CD47 binding motifs are conserved in the C-terminal signature domains of all five thrombospondins and indirect evidence has implied CD47 interactions with other family members, we compared activities of recombinant signature domains of thrombospondin-1, -2, and -4 to interact with CD47 and modulate cGMP signaling. Signature domains of thrombospondin-2 and -4 were less active than that of thrombospondin-1 for inhibiting binding of radiolabeled signature domain of thrombospondin-1 or SIRPα (signal-regulatory protein) to cells expressing CD47. Consistent with this binding selectivity, the signature domain of thrombospondin-1 was more potent than those of thrombospondin-2 or -4 for inhibiting NO-stimulated cGMP synthesis in vascular smooth muscle cells and downstream effects on cell adhesion. In contrast to thrombospondin-1- and CD47-null cells, primary vascular cells from thrombospondin-2-null mice lack enhanced basal and NO-stimulated cGMP signaling. Effects of endogenous thrombospondin-2 on NO/cGMP signaling could be detected only in thrombospondin-1-null cells. Furthermore, tissue survival of ischemic injury and acute recovery of blood flow in thrombospondin-2-nulls resembles that of wild type mice. Therefore, thrombospondin-1 is the dominant regulator of NO/cGMP signaling via CD47, and its limiting role in acute ischemic injury responses is not shared by thrombospondin-2.Nitric oxide (NO) is a major mediator of intracellular and paracellular signal transduction. NO preserves vascular health by minimizing the adhesion of inflammatory cells to the vessel wall, limiting platelet activation, and increasing blood vessel diameter and blood flow by relaxing vascular smooth muscle cells (VSMC).³ These actions of NO are mediated by activating soluble isoforms of guanylate cyclase (sGC) to increase cGMP levels, resulting in downstream activation of cGMP-dependent protein kinases and ion channels (1).Physiological NO/cGMP signaling is limited by several phosphodiesterases that degrade cGMP and by thrombospondin-1 (TSP). TSP1 is a secreted protein that is produced by vascular and inflammatory cells that regulates cellular behavior by engaging several cell surface receptors. Recently we reported that TSP1 potently blocks NO-stimulated prosurvival responses in endothelial and VSMC (2, 3). TSP1 also plays a role in promoting platelet thrombus formation and hemostasis by antagonizing the antithrombotic activity of NO (4). In all of these vascular cells, picomolar concentrations of TSP1 are sufficient to block NO-stimulated fluxes in cGMP by engaging its receptor CD47 (5). Nanomolar concentrations of TSP1 further inhibit the same signaling pathway by inhibiting CD36-mediated uptake of myristate into vascular cells (6). In vivo, mice lacking TSP1 demonstrate elevated basal tissue cGMP levels and greater increases in regional blood flow in response to a NO challenge than wild type controls (4). After an ischemic insult, the absence of TSP1 or CD47 in transgenic mice is associated with better maintenance of tissue perfusion and enhanced tissue survival. Similarly, targeting TSP1 or CD47 using function blocking antibodies enhances ischemic tissue perfusion and survival in wild type mice and pigs (7, 8).TSP1 belongs to a family of five secreted glycoproteins that share an evolutionarily conserved C-terminal signature domain (9). TSP1 and TSP2 form a distinct subfamily of trimeric proteins that exhibit similar anti-angiogenic activities for endothelial cells in vitro and activities in vivo to block tumor growth. Despite their similarities in structure, TSP1 and TSP2 have markedly different expression patterns after tissue injury, with TSP1 being immediately expressed and maximal at day 3, whereas TSP2 was not expressed until day 7 and was maximal 10 days after injury (10). In addition, large amounts of TSP1 but not TSP2 are stored in platelet α-granules and released into the wound environment. Polymorphisms in TSP1 and TSP2 have been linked to altered risk of premature myocardial infarction (11, 12). A 3′-untranslated region polymorphism in TSP2 is also associated with type 2 diabetes in men (13). The molecular basis for these associations is unclear.Less is known about the roles of the pentameric TSP3–5 in vascular cells. TSP3 and TSP5 (also known as cartilage oligomeric matrix protein) appear to serve their primary functions in bone development (14, 15). However, a polymorphism in TSP4 is associated with premature myocardial infarcts in certain populations (11, 16, 17). A proatherogenic activity for the A387P variant of TSP4 was proposed based on its differential ability to modulate proliferation of endothelial and VSMC (18). Cardiovascular functions of TSP4 may also be linked to the high expression of TSP4 in heart (19) and its altered expression in that tissue during hypertensive heart failure (20).The C-terminal domain of TSP1 is sufficient to mediate CD47-dependent inhibition of cGMP signaling (5). Of the two CD47 binding VVM motifs identified in this domain of TSP1, the first is conserved among all five TSPs, suggesting that CD47 binding could be a universal attribute of this family (21). Based on structural evidence that the VVM motifs may not be accessible (22, 23), however, conservation of VVM motifs may not be sufficient to predict CD47 binding. Uncertainty regarding the location of the CD47 binding site in the G domain of TSP1 therefore limits interpretation of the known sequence homology to predict CD47 binding to other TSP family members.Although CD47 recognition of other TSPs has not been demonstrated experimentally, a local deficiency of inflammation-associated T cell apoptosis shared by TSP1-, CD47-, and TSP2-null mice is consistent with this hypothesis (24). Furthermore, a 21-residue peptide from the C-terminal domain of TSP4 was found to decrease human umbilical vein endothelial cell proliferation similar to the CD47 binding peptides from TSP1, although it lacks the VVM motif and no interaction with CD47 was demonstrated (25).To directly address whether other TSP family members can inhibit NO responses and signaling in vascular cells, we now compare binding of recombinant signature domains of TSP1, TSP2, and TSP4 to cell surface CD47 and inhibition of NO-stimulated cell responses and cGMP signaling by these domains. We also compared acute tissue blood flow and perfusion responses to ischemic challenge in TSP1 and TSP2-null mice and cGMP responses in primary cultures of vascular cells isolated from these mice. These studies clearly demonstrate that CD47 selectively interacts with TSP1 and that the signature domains of TSP2 and TSP4 are less potent inhibitors of NO signaling in vascular cells in vitro. Furthermore, we show that the role of TSP1 to acutely limit recovery from ischemic injury in vivo is not shared by TSP2.     

The N-terminal module of thrombospondin-1 interacts with the link domain of TSG-6 and enhances its covalent association with the heavy chains of inter-alpha-trypsin inhibitor

We recently found that leukocytes from thrombospondin-1 (TSP1)-deficient mice exhibit significant reductions in cell surface CD44 relative to those from wild type mice. Because TSG-6 modulates CD44-mediated cellular interactions with hyaluronan, we examined the possibility that TSP1 interacts with TSG-6. We showed that recombinant full-length human TSG-6 (TSG-6Q) and the Link module of TSG-6 (Link_TSG6) bind 125I-TSP1 with comparable affinities. Trimeric recombinant constructs containing the N-modules of TSP1 or TSP2 inhibit binding of TSP1 to TSG-6Q and Link_TSG6, but other recombinant regions of TSP1 do not. Therefore, the N-modules of both TSP1 and TSP2 specifically recognize the Link module of TSG-6. Heparin, which binds to these domains of both proteins, strongly inhibits binding of TSP1 to Link_TSG6 and TSG-6Q, but hyaluronan does not. Inhibition by heparin results from its binding to TSP1, because heparin also inhibits TSP1 binding to Link_TSG6 mutants deficient in heparin binding. Removal of bound Ca2+ from TSP1 reduces its binding to full-length TSG-6. Binding of TSP1 to Link_TSG6, however, is enhanced by chelating divalent cations. In contrast, divalent cations do not influence binding of the N-terminal region of TSP1 to TSG-6Q. This implies that divalent cation dependence is due to conformational effects of calcium-binding to the C-terminal domains of TSP1. TSP1 enhances covalent modification of the inter-alpha-trypsin inhibitor by TSG-6 and transfer of its heavy chains to hyaluronan, suggesting a physiological function of TSP1 binding to TSG-6 in regulation of hyaluronan metabolism at sites of inflammation.     

Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS13

ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr1605-Met1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more C-terminal TSP1 and CUB domains are dispensable in vitro.     

Specificities of heparin-binding sites from the amino-terminus and type 1 repeats of thrombospondin-1

Interactions of heparin with intact human thrombospondin-1 (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically modified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avidity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units. The dependence on oligosaccharide size for binding to the recombinant amino-terminal heparin-binding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, suggesting that the interaction between intact TSP1 and heparin is primarily mediated by the amino-terminal domain. Based on activities of chemically modified heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation of glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl residues of the uronic acids. In contrast, all of these modifications were required for binding of heparin to the type 1 repeat peptides. Affinity purification of heparin octasaccharides on immobilized TSP1 type 1 repeat peptides revealed a preference for oligosaccharides containing the disaccharide sequence IdoA(2-OSO(3))alpha1-4-GlcNS(6-OSO(3)). Binding of these oligosaccharides to the peptide required the Trp residues. These data demonstrate that the heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and are consistent with the ability of these TSP1-derived molecules to inhibit FGF2-stimulated angiogenesis.     

Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor

Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.     

The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.