Domain Of Thrombospondin-1 Research Articles

Antiangiogenic Treatment with Three Thrombospondin-1 Type 1 Repeats versus Gemcitabine in an Orthotopic Human Pancreatic Cancer Model

In this study, we investigated the antitumor efficacy of thrombospondin-1 three type 1 repeats (3TSR), the antiangiogenic domain of thrombospondin-1, in comparison and in combination with gemcitabine, in an orthotopic pancreatic cancer model. Human pancreatic cancer cells were injected into the pancreas of severe combined immunodeficient mice. The animals were treated with 3TSR, gemcitabine, 3TSR plus gemcitabine, or vehicle for 3 weeks. Subsequently, the effects of 3TSR and/or gemcitabine on tumor growth, tumor necrosis, microvessel density, cancer cell proliferation, apoptosis, and endothelial cell apoptosis were analyzed. After 3 weeks of treatment, 3TSR reduced tumor volume by 65%, and gemcitabine by 84%. Tumor volume was not statistically different between gemcitabine group and combinatorial treatment group. Extensive necrotic areas were observed in tumors from 3TSR-treated mice, whereas tumors from gemcitabine and combinatorially treated mice were less necrotic than control tumors. 3TSR reduced tumor microvessel density and increased tumor blood vessel endothelial cell apoptosis. In contrast, gemcitabine induced apoptosis and inhibited proliferation of cancer cells. 3TSR, the antiangiogenic domain of thrombospondin-1, showed comparable antitumor efficacy to gemcitabine in a human pancreatic cancer orthotopic mouse model. No synergistic effect was found when the two drugs were combined and possible reasons are discussed in detail. A delicate balance between normalization and excessive regression of tumor vasculature is important when initiating alternative combinatorial regimens for treatment of patients with pancreatic cancer.

Type 3 repeat/C-terminal domain of thrombospondin-1 triggers caspase-independent cell death through CD47/αvβ3 in promyelocytic leukemia NB4 cells

AbstractBy means of its antiangiogenic activity, thrombospondin-1 (TSP-1) exerts indirect antitumoral action on solid tumors. Here, we investigated potential antitumor action in an in vitro cell model for promyelocytic leukemia (NB4-LR1), resistant to retinoid maturation. Purified soluble TSP-1 added to cultures induced a strong dose-dependent growth inhibition and a slowly developing maturation-independent cell death. Recombinant fragments of TSP-1 allowed mapping of these activities to its type 3 repeat/C-terminal domain, features that are distinct from those of TSP-1 action on solid tumors, previously ascribed to the type 1 repeat domain. Cell death in leukemia was characterized as a caspase-independent mechanism, without DNA fragmentation, but phosphatidylserine externalization followed by membrane permeabilization. Mitochondria membrane depolarization was inherent to TSP-1 action but did not produce release of death-promoting proteins (eg, noncaspase apoptosis regulators, apoptosis-induced factor [AIF], endonuclease G, or Omi/HtrA2 or the caspase regulators, cytochrome c or second mitochondrial activator of caspase/direct inhibitor of apoptosis protein-binding protein with low isoelectric point [Smac/DIABLO]). Although detected, reactive oxygen species (ROS) production was likely not involved in the death process. Finally, receptor agonist RFYVVM and RGD peptides indicated that TSP-1 death effects are mediated by membrane receptors CD47 and αvβ3. These results demonstrated a new domain-specific antitumoral activity of TSP-1 on a leukemia cell line, which extends TSP-1 therapeutic potential outside the area of vascularized solid tumors. (Blood. 2005;106:658-667)

Thrombospondin-1 up-regulates expression of cell adhesion molecules and promotes monocyte binding to endothelium.

Expression of cell adhesion molecules (CAM) responsible for leukocyte-endothelium interactions plays a crucial role in inflammation and atherogenesis. Up-regulation of vascular CAM-1 (VCAM-1), intracellular CAM-1 (ICAM-1), and E-selectin expression promotes monocyte recruitment to sites of injury and is considered to be a critical step in atherosclerotic plaque development. Factors that trigger this initial response are not well understood. As platelet activation not only promotes thrombosis but also early stages of atherogenesis, we considered the role of thrombospondin-1 (TSP-1), a matricellular protein released in abundance from activated platelets and accumulated in sites of vascular injury, as a regulator of CAM expression. TSP-1 induced expression of VCAM-1 and ICAM-1 on endothelium of various origins, which in turn, resulted in a significant increase of monocyte attachment. This effect could be mimicked by a peptide derived from the C-terminal domain of TSP-1 and known to interact with CD47 on the cell surface. The essential role of CD47 in the cellular responses to TSP-1 was demonstrated further using inhibitory antibodies and knockdown of CD47 with small interfering RNA. Furthermore, we demonstrated that secretion of endogenous TSP-1 and its interaction with CD47 on the cell surface mediates endothelial response to the major proinflammatory agent, tumor necrosis factor alpha (TNF-alpha). Taken together, this study identifies a novel mechanism regulating CAM expression and subsequent monocyte binding to endothelium, which might influence the development of anti-atherosclerosis therapeutic strategies.

Endogenous thrombospondin-1 is not necessary for proliferation but is permissive for vascular smooth muscle cell responses to platelet-derived growth factor

We have reexamined the role of endogenous thrombospondin-1 (TSP1) in growth and motility of vascular smooth muscle cells (SMCs). Based on the ability of aortic-derived SMCs isolated from TSP1 null mice and grown in the absence of exogenous TSP1 to grow at comparable rates and to a slightly higher density than equivalent cells from wild-type mice, TSP1 is not necessary for their growth. Low concentrations of exogenous TSP1 stimulate growth of TSP1 null SMCs, but higher doses of TSP1 or its C-terminal domain are inhibitory. However, SMCs from TSP1 null mice are selectively deficient in chemotactic and proliferative responses to platelet-derived growth factor and in outgrowth in three-dimensional cultures. Recombinant portions of the N- and C-terminal domains of TSP1 stimulate SMC chemotaxis through different integrin receptors. Based on these data, the relative deficiency in SMC outgrowth during an ex vivo angiogenic response of muscle tissue from TSP1 null mice is probably due to restriction of platelet-derived growth factor dependent SMC migration and/or proliferation.

Autocrine Regulation of T Cell Motility by Calreticulin-Thrombospondin-1 Interaction

The mechanisms regulating T lymphocyte migration within the extracellular matrix are not understood. We show in this study that the thrombospondin-1 binding site of calreticulin, spanning aa 19-32, is a major triggering factor for T cell motility and migration within a three-dimensional collagen type 1 matrix, and that exogenous motogenic factors such as chemokines can stimulate migration via a calreticulin-thrombospondin-1 pathway. Endogenous calreticulin binding to the N-terminal domain of endogenous thrombospondin-1 elicited a motogenic signal to the T cells through the C-terminal domain of thrombospondin-1 and its cell surface receptor integrin-associated protein (CD47). Our data further revealed that thrombospondin-1 was expressed on the cell surface with a high turnover, and that PI3K and the Janus family of tyrosine kinases were required for T cell motility mediated through calreticulin, thrombospondin-1, and CD47. These results unveil an autocrine mechanism of calreticulin-thrombospondin-1-CD47 interaction for the control of T cell motility and migration within three-dimensional extracellular matrix substrata.

Collagenases and aggrecanases: understanding the role of non-catalytic domains in cartilage matrix breakdown

Degradation of cartilage matrix, which mainly consists of collagen fibrils and aggrecan, seriously impairs the function of joints. The primary cause of this process is elevated proteolytic enzymes. Collagen fibrils are degraded by a group of matrix metalloproteinases (MMPs) including collagenases (MMP-1, MMP-8 and MMP-13), gelatinase A (MMP-2) and MT1-MMP (MMP-14). Aggrecan is degraded by MMPs and 'aggrecanases' belonging to the ADAMTS family. Inhibitors of these metalloproteinases are considered as potential therapeutic agents to protect cartilage degradation, and many active site-directed inhibitors with a zinc-chelating moiety have been developed. Unfortunately, those compounds have serious side effects possibly due to lack of selectivity [1]. In the hope of developing new types of inhibitors we have been investigating the mechanisms of action of collagenases and aggrecanases. Collagenases are unique as they can degrade triple helical interstitial collagens into three-quarter and one-quarter fragments, a crucial step for collagenolysis in the tissue. Typical collagenases consist of a catalytic domain and a hemopexin domain that are connected by a linker peptide. Both domains are essential for collagenolysis. The three-dimensional structure of a prototypic collagenase, collagenase 1 (MMP-1), however, indicated that the active site of the enzyme is too narrow to accommodate triple-helical collagen. We have recently shown that collagenase locally unwinds triple helical collagen before it hydrolyses the peptide bonds [2]. Based on a series of mutagenesis we predict that the collagen binding site is created by both the catalytic and the hemopexin domains. This potential collagen binding groove is partially blocked by the prodomain in the zymogen of MMP-1 (proMMP-1), explaining its inability to bind to collagen unless it is activated. Overall proMMP-1 has a 'closed' configuration in contrast to the 'open' configuration of the active MMP-1. The exact mechanism of how collagenase unwinds collagen is under investigation. Another key collagenase is MT1-MMP. It is highly expressed in rheumatoid synovial lining cells invading into the cartilage. Overexpression of recombinant MT1-MMP in COS7 cells in vitro degrade reconstituted type I collagen films. This reaction requires dimerisation of the enzyme on the cell surface through the hemopexin domains. When dimerisation of MT1-MMP is prevented by overexpression of the membrane-anchored MT1-MMP hemopexin domain, the collagenolytic activity was blocked, but not its general photolytic activity. Among 19 ADAMTSs in humans, six have been shown to have aggrecanase activity. Their non-catalytic domains include one disintegrin, one or more thrombospondin, one cysteine-rich and one spacer domain. Full-length ADAMTS-4 (aggrecanase 1) is active on aggrecan only, but when the C-terminal spacer domain is deleted it expresses broader proteolytic activity and digests fibronectin, fibromodulin, decorin and biglycan, as well as maintaining aggrecanase activity [3]. Further deletion of the cysteine-rich and thrombospondin domains greatly reduces both activities [3]. These studies suggest that activities of collagenases and aggrecanases will be attenuated by inhibitors or antibodies that interact directly with their non-catalytic ancillary domains (exosite/allosteric inhibitors). Such molecules will be attractive for therapy as they will be highly selective because they are based on the unique mechanism of each proteinase.

Mapping Netrin Receptor Binding Reveals Domains of Unc5 Regulating Its Tyrosine Phosphorylation

Netrin and its receptors Unc5 and deleted in colorectal carcinoma (DCC) regulate axon guidance and cell migration. We defined domains involved in the interactions between netrin-1, DCC, and Unc5c. We show that Unc5 requires both Ig domains to interact with netrin. DCC binds through the fourth fibronectin type III domain, whereas netrin binds through multiple domains to both receptors. We examined the functional consequences of removing the netrin binding and nonbinding domains from Unc5 in vitro and in vivo. In human embryonic kidney 293 cells, removal of the netrin binding second Ig domain causes an increase in basal tyrosine phosphorylation, whereas removal of the netrin nonbinding thrombospondin domains decreases tyrosine phosphorylation. Moreover, experiments in Caenorhabditis elegans indicate that both netrin binding and nonbinding domains are necessary for phenotypic rescue of an unc-5 loss of function mutation.

Characterization of the Binding Interaction between ADAMTS13 and von Willebrand Factor (VWF).

VWF multimers are cleaved into smaller less thrombogenic fragments in plasma by ADAMTS13. Deficiency of ADAMTS13 activity leads to thombotic thrombocytopenic purpura (TTP), a disease characterized by thrombocytopenia, microangiopathic hemolytic anemia, fever, neurological decline, and renal insufficiency. ADAMTS13 is a member of the A Disintegrin and Metalloprotease with ThromboSpondin repeats family that has characteristic motifs including a signal sequence, a propeptide, a catalytic metalloprotease domain, a disintegrin domain, a thrombospondin-1 (TSP1) repeat, a cysteine-rich region, and a spacer domain. These domains are followed in ADAMTS13 by seven additional TSP1 repeats and two C-terminal CUB domains. Previous work has shown that ADAMTS13 truncated after the spacer domain retains proteolytic activity towards VWF, but little is known about the potential role of the additional C-terminal TSP1 and CUB domains in VWF binding or cleavage. We therefore developed an enzyme-linked immunosorbent assay (ELISA)-based system to study ADAMTS13-VWF binding. VWF was immobilized in microtiter wells and incubated with plasma or recombinant ADAMTS13 variants. Bound ADAMTS13 was detected directly by solubilization and Western blotting, or indirectly by ELISA. ADAMTS13 proteolytic activity towards VWF is enhanced by denaturation of VWF with urea or guanidine, but ADAMTS13 bound specifically to VWF without prior denaturation. EDTA increased the binding of ADAMTS13 to VWF and prevented proteolysis of the immobilized VWF. Binding was saturable and time-dependent with maximal binding in two hours. Binding was reversible with a half-time for dissociation of four hours. ADAMTS13 in normal human plasma but not in plasma from a patient with TTP bound immobilized VWF. The stoichiometry of VWF monomers to ADAMTS13 at saturation was approximately 2 (range 1–4) and the Kd of recombinant ADAMTS13 binding to VWF was 12 nM (range 5–26 nM). This Kd for binding is similar to the Km for VWF cleavage of 16 nM determined independently, and both are comparable to the estimated plasma ADAMTS13 concentration of 8 nM. The properties of C-terminally truncated ADAMTS13 constructs suggest a regulatory function for certain domains. Truncation after the 8th TSP1 domain decreased the Kd 2-fold. Further truncation after either the 6th or 7th TSP1 domain increased the Kd 2-fold relative to full-length ADAMTS13. Truncation after the spacer domain gave binding properties indistinguishable from full-length ADAMTS13. Truncation after the metalloprotease domain gave no detectable binding to VWF. Therefore, binding of ADAMTS13 to VWF requires sequences in the cysteine-rich or spacer domains, and is modulated by sequences in at least the 8th TSP1 repeat and the CUB domains.

Heightened Proteolysis of Plasma ADAMTS13 Antigen in Patients with Thrombotic Thrombocytopenic Purpura.

A total of 140 subjects with thrombotic thrombocytopenic purpura (TTP)/hemolytic-uremic syndrome (HUS), in which 25 were Upshaw-Schulman syndrome (USS, belonged to 20 families), 107 were acquired TTP (96 idiopathic and 11 drug-induced), and 8 were acquired O157:H7-associated HUS, were analyzed on their plasma ADAMTS13 antigen (:AGN), using 3 novel anti-ADAMTS13 murine monoclonal IgG antibodies (A10, C7, and WH2-1–11). Epitope of these antibodies resided on the disintegrin-like, 7th/8th thrombospondin-1, and 4th thrombospondin-1 domains, respectively. The WH2-11–1 alone was immunoreactive on western blot (WB) under reducing conditions. Further, in a static assay system, A10 totally and C7 partially inhibited ADAMTS13 activity (:ACT), but WH2-11–1 did not. A sandwich ELISA using C7 as the 1st antibody and biotinylated A10 as the 2nd antibody revealed a significant reduction of plasma ADAMTS13:AGN level in patients with acquired TTP, but a considerable variation in those with USS, suggesting that the quantitative ELISA has a limited diagnostic value.A quantitative and semi-qualitative WB analysis using A10 and WH2-11-1, however, demonstrated that normal plasmas had a 190/180 kD-doublet band under non-reducing conditions. A size exclusion chromatography of normal plasma was able to separate a 190 kD-protein from a 180 kD-band, and the former turned to be a single 200 kD-band on WB under reducing conditions, but in the latter such protein band was completely invisible, indicating that the 180 kD-band had undergone proteolytic modification. All the patient plasmas with USS and a roughly half of those with acquired TTP had a broad and fuzzy ~190/180 kD-band on WB before reduction, but it almost entirely disappeared after reduction. In patient plasmas with acquired HUS, however, the pattern of WB analysis was indistinguishable from that of the normal subjects.Aforementioned WB analysis was then performed on a girl of acquired TTP with high-titer inhibitors against ADAMTS13, using the sequential plasma samples obtained during the past 3 years, and confirmed complete normalization of the abnormal ADAMTS13:AGN patterns in accord to the increase of plasma ADAMTS13:ACT, together with her clinical improvements. Protease(s) responsible for this heightened cleavage is currently under investigation.

Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans.

Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family in Caenorhabditis elegans and the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacin-like metalloprotease with an epidermal growth factor (EGF)-like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoter-driven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wild-type rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.

Internalization but not binding of thrombospondin-1 to low density lipoprotein receptor-related protein-1 requires heparan sulfate proteoglycans.

The amino-terminal domain of the extracellular matrix (ECM) protein thrombospondin-1 (TSP-1) mediates binding to cell surface heparan sulfate proteoglycans (HSPG) as well as binding to the endocytic receptor, low density lipoprotein-related protein (LRP-1). We previously found that recombinant TSP-1 containing the amino-terminal residues 1-214, retained both of these interactions (Mikhailenko et al. [1997]: J Biol Chem 272:6784-6791). Here, we examined the activity of a recombinant protein containing amino-terminal residues 1-90 of TSP-1 and found that this domain did not retain high-affinity heparin-binding. The loss of heparin-binding correlated with decreased binding to the fibroblast cell surface. However, both ligand blotting and solid phase binding studies indicate that this truncated fragment of TSP-1 retained high-affinity binding to LRP-1. Consistent with this, it also retained the ability to block the uptake and degradation of (125)I-TSP-1. However, TSP-1(1-90) itself was poorly endocytosed and this truncated amino-terminal domain was considerably more effective than the full-length heparin-binding domain (HBD) of TSP-1 in blocking the catabolism of endogenously expressed TSP-1. These results indicate that TSP-1 binding to LRP-1 does not require prior or concomitant interaction with cell surface HSPG but suggest subsequent endocytosis requires high-affinity heparin-binding.

Phosphonamide-Based Metalloproteinase Inhibitors: A New Approach to the Development of Safer Metalloproteinase Inhibitors

Two closely related zinc metalloprotease (MP) families i.e. MMPs (matrix metalloproteinases) and ADAMs (a disintegrin-like and metalloproteinase-containing protein) including ADAMTSs (ADAMs with one or more thrombospondin domain) have lately become important therapeutic targets in drug discovery research. Although a vast number of MMP inhibitors have been developed in recent years, the results from clinical trials with these compounds have been insufficient for their approval as effective and safe drugs. One of the obstacles to the success MMP inhibitors in clinical development has been the adverse effects caused by undesirable inhibition of non-target MMPs and / or ADAMs. A series of phosphonamide-based compounds recently disclosed by our laboratory includes potent metalloproteinase inhibitors with increased enzyme selectivity relative to the corresponding sulfonamide-based compounds. This increased selectivity arises from the chirality of the phosphonamides core structure, which alters the inhibitor binding mode at the active site of the enzyme. In addition, these phosphonamidebased compounds may be useful for the development of safe metalloproteinase inhibitors, as antedrugs, since phosphonamide structures are known for their instability in the body. This instability may provide a solution to the problem of side effects known to conventional MMP inhibitors and allow new applications for metalloproteinase inhibitors in general.

Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion

AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

Thrombospondin-bound Integrin-associated Protein (CD47) Physically and Functionally Modifies Integrin αIIbβ3 by Its Extracellular Domain

Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, alphaIIbbeta3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to alphaIIbbeta3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the alphaIIbbeta3 with mutated beta3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type alphaIIbbeta3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of alphaIIbbeta3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to alphaIIbbeta3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified alphaIIbbeta3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with alphaIIbbeta3 and can change alphaIIbbeta3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin.

CD47 and the 19 kDa Interacting Protein-3 (BNIP3) in T Cell Apoptosis

CD47 is a surface receptor that induces either coactivation or apoptosis in lymphocytes, depending on the ligand(s) bound. Interestingly, the apoptotic pathway is independent of caspase activation and cytochrome c release and is accompanied by early mitochondrial dysfunction with suppression of mitochondrial membrane potential (Deltapsim). Using CD47 as bait in a yeast two-hybrid system, we identified the Bcl-2 homology 3 (BH3)-only protein 19 kDa interacting protein-3 (BNIP3), a pro-apoptotic member of the Bcl-2 family, as a novel partner. Interaction between CD47 and the BH3-only protein was confirmed by immunoprecipitation analysis, and CD47-induced apoptosis was inhibited by attenuating BNIP3 expression with antisense oligonucleotides. Finally, we showed that the C-terminal domain of thrombospondin-1 (TSP-1), but not signal-regulatory protein (SIRPalpha1), is the ligand for CD47 involved in inducing cell death. Immunofluorescence analysis of CD47 and BNIP3 revealed a partial colocalization of both molecules under basal conditions. After T cell stimulation via CD47, BNIP3 translocates to the mitochondria to induce apoptosis. These results show that the BH3-dependent apoptotic pathways, previously shown to be activated by intracellular pro-apoptotic events, can also be turned on by surface receptors. This new pathway results in a fast induction of cell death resembling necrosis, which is likely to play an important role in lymphocyte regulation at inflammatory sites and/or in the vicinity of thrombosis.

The C-terminal peptide of thrombospondin-1 stimulates distinct signaling pathways but induces an activation-independent agglutination of platelets and other cells

A peptide from the C-terminal domain of thrombospondin-1 (4N1-1) has been proposed to stimulate platelet aggregation by a novel mechanism involving both an activation-independent agglutination and an activation-dependent, glycoprotein (GP) IIb/IIIa-mediated aggregation which involves GPVI signaling but does not involve CD47. The present study demonstrates that 4N1-1 stimulated a different pattern of signal transduction pathways than the GPVI agonist convulxin. Furthermore, 4N1-1-induced platelet aggregation was activation-independent and not dependent on GPVI or GPIIb/IIIa. Interestingly, 4N1-1 also stimulated activation-independent agglutination of different megakaryocytic and non-megakaryocytic cells. 4N1-1-induced cell agglutination but not platelet signaling was inhibited by anti-CD47 antibodies.

Platelet aggregation induced by the C-terminal peptide of thrombospondin-1 requires the docking protein LAT but is largely independent of αIIb/β3

Thrombospondin-1 (TSP1) is abundantly secreted during platelet activation and plays a role in irreversible platelet aggregation. A peptide derived from the C-terminal domain of TSP1, RFYVVMWK (RFY) can activate human platelets at least in part via its binding to integrin-associated protein. Although integrin-associated protein is known to physically interact with alphaIIb/beta3, we found that this major platelet integrin had only a partial implication in RFY-mediated platelet aggregation. Accordingly, RFY induced a significant Glanzmann type I thrombasthenic platelet aggregation. The alphaIIb/beta3-dependent part of platelet aggregation induced by RFY was mainly due to secreted ADP and thromboxane A2. In the absence of alphaIIb/beta3 and fibrinogen, RFY stimulated a rapid tyrosine phosphorylation of a set of proteins, including Syk, linker for activation of T cells (LAT) and phospholipase Cgamma2. This signaling pathway was critical for RFY-mediated platelet activation as revealed by the use of pharmacological inhibitors as well as LAT-deficient mouse platelets. Phosphoinositide 3-kinase activation was also required for RFY-mediated platelet aggregation. Our results unravel a new alphaIIb/beta3 and fibrinogen-independent mechanism for platelet aggregation in response to the active peptide from the C-terminal domain of TSP1.

Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains

ADAMTS (ADisintegrin And Metalloproteinase domain, with ThromboSpondin type-1 modules) is a recently described family of zinc-dependent proteases which play important roles in a variety of normal and pathological conditions, including arthritis and cancer. In this work, we report the identification and cloning of cDNAs encoding seven new human ADAMTSs. These novel enzymes have been called ADAMTS-13, -14, -15, -16, -17, -18, and -19. All of them show a domain organization similar to that of previously characterized family members, consisting of a signal sequence, a propeptide, a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich region, and a variable number of TS-1 repeats. Expression analysis revealed that these ADAMTS genes are mainly expressed in fetal tissues, especially in lung (ADAMTS14, ADAMTS16, ADAMTS17, ADAMTS18, and ADAMTS19), kidney (ADAMTS14, ADAMTS15, and ADAMTS16), and liver (ADAMTS13, ADAMTS15 and ADAMTS18). Reverse transcriptase–polymerase chain reaction analysis also revealed the expression of some of these new ADAMTSs in different human adult tissues, such as prostate (ADAMTS13, ADAMTS17, and ADAMTS18), and brain (ADAMTS13, ADAMTS16, ADAMTS17, and ADAMTS18). High levels of ADAMTSs transcripts were also observed in some tumor biopsies and cells lines, including osteosarcomas (ADAMTS19), melanoma and colon carcinoma cells (ADAMTS13). Chromosomal location analysis indicated that the seven identified ADAMTS genes are dispersed in the human genome mapping to 9q34, 10q21, 11q25, 5p15, 15q24, 16q23, and 5q31, respectively. According to these results, together with a comparative analysis of ADAMTSs in other eukaryotic organisms, we conclude that these enzymes, with at least 18 distinct members encoded within the human genome, represent an example of a widely expanded protease family during metazoan evolution.

C-terminal peptide of thrombospondin-1 induces platelet aggregation through the Fc receptor γ-chain–associated signaling pathway and by agglutination

AbstractA peptide from the C-terminal domain of thrombospondin-1 (Arg-Phe-Tyr-Val-Val-Met-Trp-Lys; known as 4N1-1) has been reported to induce platelet aggregation and to bind to the integrin-associated protein (IAP), which is also known as CD47. In this study, it was discovered that 4N1-1 or its derivative peptide, 4N1K, induces rapid phosphorylation of the Fc receptor (FcR) γ chain, Syk, SLP-76, and phospholipase C γ2 in human platelets. A specific inhibitor of Src family kinases, 4-amino-4-(4-methylphenyl)-7-(t-butyl) pyrazola[3,4-d]pyrimidine, prevented phosphorylation of these proteins, abolished platelet secretion, and reduced aggregation by approximately 50%. A similar inhibition of aggregation to 4N1-1 was obtained in the presence of Arg-Gly-Asp-Ser in mouse platelets deficient in FcR γ chain or SLP-76 and in patients with type I Glanzmann thrombasthenia. These results show that 4N1-1 signals through a pathway similar to that used by the collagen receptor glycoprotein (GP) VI. The αIIbβ3-independent aggregation induced by 4N1-1 was also observed in fixed platelets and platelets from patients with Bernard-Soulier syndrome, which are deficient in GPIbα. Surprisingly, the ability of 4N1-1 to stimulate aggregation and tyrosine phosphorylation was not altered in platelets pretreated with anti-IAP antibodies and in IAP-deficient mice. These results show that the C-terminal peptide of thrombospondin induces platelet aggregation through the FcR γ-chain signaling pathway and through agglutination. The latter pathway is independent of signaling events and does not use GPIbα or αIIbβ3. Neither of these pathways is mediated by IAP.

A new FACIT of the collagen family: COL21A1

Interrogation of the Human Genome data for sequences related to the von Willebrand factor A-domain module identified a previously unreported 4.1 kb full-length cDNA that is predicted to encode a new member of the collagen superfamily of extracellular matrix proteins, collagen XXI. The domain organization of collagen XXI comprised an N-terminal signal sequence, followed by single von Willebrand factor A-domain and thrombospondin domains, and an interrupted collagen triple helix. The organization of these motifs predict that collagen XXI is a new member of the FACIT collagen sub-family. Expression analysis indicated that COL21A1 mRNA is present in many tissues including heart, stomach, kidney, skeletal muscle and placenta, and radiation hybrid mapping localized the COL21A1 gene to 6p11-12.