Peptide 4N1K Research Articles

The Pathological Significance and Prognostic Roles of Thrombospondin-1, and -2, and 4N1K-peptide in Bladder Cancer.

Thrombospondins (TSPs) play a role as inhibitors of angiogenesis under various pathological conditions. The aim of the study was to evaluate the pathological significance and prognostic role of the 4N1K-peptide (KRFYVVMWKK), which is derived from TSP-1 and -2, in bladder cancer. Two-hundred and six bladder cancer tissues were examined for expression of TSP-1, TSP-2, and 4N1K-peptide by immunohistochemistry. Cancer cell proliferation, apoptosis, angiogenesis and matrix metalloproteinase (MMP)-9 immunoreactivity were also examined. Expression of TSP-2 and 4N1K-peptide was negatively associated with T stage, metastasis, and grade. TSP-2 expression was negatively associated with cancer cell proliferation and MMP-9 expression, whereas 4N1K-peptide was significantly associated with apoptosis, angiogenesis, and MMP-9 expression. Multivariate analysis showed that 4N1K-peptide expression was a significant predictor of metastasis (hazard ratio=3.90, p=0.002). TSP-2 and 4N1K peptide played important roles in malignant aggressiveness and progression of bladder cancer via complex mechanisms involving cell proliferation, apoptosis, angiogenesis, and MMP-9.

Single-molecule-force spectroscopy study of the mechanism of interactions between TSP-1 and CD47

The 4N1K peptide, which is derived from the C-terminal domain of thrombospondin-1 (TSP-1), is usually used as a functional mimic peptide for TSP-1. Knowledge about the interaction force of 4N1K/CD47 is important in explaining how TSP-1 affects the biological effect of CD47. Here we used a single-molecule force spectroscopy (SMFS) technique to explore the interaction of 4N1K/CD47 on both normal and oxidative human red blood cells (hRBCs) at single-molecule level. There was no interaction force between 4N1K and CD47 on normal hRBCs; however, we did find 4N1K-bound CD47 on oxidative hRBCs. We also detected interaction forces for 4N1K/CD47ex (extracellular domain of human CD47), and 4N1K/oxidative CD47ex. The interaction forces of 4N1K/CD47ex were almost consistent with those of 4N1K/oxidative CD47ex at the same loading rate. These results suggest that the conformational change of CD47 is critical for 4N1K-CD47 interaction on oxidative hRBCs.

CD47 does not mediate amyloid-β(1–42) protofibril-stimulated microglial cytokine release

Neuroinflammation triggered by accumulation of amyloid-β protein (Aβ) is a significant component of the Alzheimer’s disease (AD) brain. Senile plaques composed of Aβ attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aβ activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aβ recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aβ species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aβ(1–42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) secretion stimulated by Aβ(1–42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aβ protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aβ(1–42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47−/− microglia and 4N1K still potently inhibited the Aβ protofibril response even in the CD47−/− microglia. The overall findings demonstrated that the microglial proinflammatory response to Aβ(1–42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity.

Deficits in cerebellar granule cell development and social interactions in CD47 knockout mice.

CD47 is involved in neurite differentiation in cultured neurons, but the function of CD47 in brain development is largely unknown. We determined that CD47 mRNA was robustly expressed in the developing cerebellum, especially in granule cells. CD47 protein was mainly expressed in the inner layer of the external granule layer (EGL), molecular layer, and internal granule layer (IGL), where granule cells individually become postmitotic and migrate, leading to neurite fasciculation. At postnatal day 8 (P8), CD47 knockout mice exhibited an increased number of proliferating granule cells in the EGL, whereas the CD47 agonist peptide 4N1K increased the number of postmitotic cells in primary granule cells. Knocking out the CD47 gene and anti-CD47 antibody impaired the radial migration of granule cells from the EGL to the IGL individually in mice and slice cultures. In primary granule cells, knocking out CD47 reduced the number of axonal collaterals and dendritic branches; by contrast, overexpressing CD47 or 4N1K treatment increased the axonal length and numbers of axonal collaterals and dendritic branches. Furthermore, the length of the fissure between Lobules VI and VII was decreased in CD47 knockout mice at P21 and at 14 wk after birth. Lastly, CD47 knockout mice exhibited increased social interaction at P21 and depressive-like behaviors at 10 wk after birth. Our study revealed that the cell adhesion molecule CD47 participates in multiple phases of granule cell development, including proliferation, migration, and neurite differentiation implying that aberrations of CD47 are risk factors that cause abnormalities in cerebellar development and atypical behaviors.

CD47-independent effects mediated by the TSP-derived 4N1K peptide.

4N1K is a peptide fragment derived from the C-terminal, globular domain of thrombospondin which has been shown to mediate integrin-dependent cell adhesion and promote integrin activation acting via the cell-surface receptor, CD47. However, some studies found that 4N1K could act independently of CD47, putting in question the specificity of 4N1K for CD47. This led us to characterize the cellular and non-cellular effects of 4N1K. We found that 4N1K stimulated a potent increase in binding of a variety of non-specific IgG antibodies to cells in suspension. We also found that these same antibodies, as well as CD47-deficient cells, could bind substrate-immobilized 4N1K significantly better than a control peptide, 4NGG. Furthermore, we found that cells treated with 4N1K at higher concentrations inhibited, while lower concentrations promoted cell adhesion to immobilized fibronectin as an integrin substrate. Importantly, both the stimulatory and the inhibitory activity of 4N1K occurred as efficiently in the CD47-deficient JinB8 cells, as it did in the CD47-expressing parental or in JinB8 cells reconstituted with CD47 expression. Given these results, we suggest that 4N1K interacts non-specifically with epitopes commonly found on the cell surface, and conclude that it is not a suitable peptide for use to study the consequences of CD47 receptor ligation.

Signal transduction pathways in erythrocyte nitric oxide metabolism under high fibrinogen levels

Previous studies show that the fibrinogen molecule modulates the metabolism of nitric oxide (NO) in erythrocyte. The in vitro induced hiperfibrinogenemia interferes in the metabolism of the NO in the erythrocyte in dependence of the phosphorylation degree of the band 3. The soluble form of fibrinogen binds into CD47 protein present in the erythrocyte membrane. The soluble thrombomodulin is an inflammatory marker that binds to the erythrocyte CD47 in a site with a sequence peptide known as 4N1K. A study done in vitro shows that when hiperfibrinogenemia was induced in the presence of the peptide 4N1K agonist of CD47 it were observed variations in the efflux of NO from erythrocyte and an increase in the concentrations of GSNO, peroxinitrite, nitrite and nitrate of the erythrocytes. The aim of this work was to study the influence of the peptide 4N1K, on the metabolism of NO in the erythrocyte under high fibrinogen concentration and in the presence of inhibitors of the status of phosphorylation of protein band 3. In this in vitro study, whole blood samples were harvested from healthy subjects and NO, peroxynitrite, nitrite, nitrate and S-nitro-glutathione (GSNO) were determined in presence of 4N1K, calpeptine, Syk inhibitor and under high fibrinogen concentrations. The results obtained in erythrocytes under high fibrinogen levels when 4N1K is present with the Syk inhibitor or with calpeptine, showed in relation to the control samples increased significant concentrations of efflux of NO and of peroxynitrite, nitrite, nitrate and GSNO. In conclusion it was verified that in the in vitro model of hiperfibrinogenemia the peptide 4N1K, agonist of CD47, induces mobilization of NO in the erythrocyte in dependence of the status of phosphorylation of protein band 3.

Thrombospondin-1-derived 4N1K peptide expression is negatively associated with malignant aggressiveness and prognosis in urothelial carcinoma of the upper urinary tract

BackgroundThrombospondin (TSP) is a multi-functional protein that appears to have dual roles in cancer, that is, either as a promoter or a suppressor. 4N1K is a TSP-derived peptide that has been reported to be associated with neovascularity, cell survival, and invasion. There is a little information regarding its pathological roles in human cancer tissues. Our aim was to clarify clinical significance and prognostic value of 4N1K expression in patients with urothelial carcinoma of the upper urinary tract (UC-UUT).MethodsWe investigated 4N1K expression in 97 surgically excised, non-metastasized UC-UUT specimens and five normal tissues via immunohistochemistry. Microvessel density (MVD), lymph vessel density (LVD), cancer cell proliferation (PI), apoptotic index (AI), and matrix metalloproteinase (MMP)-9 expression was also determined. The relationships 4N1K expression and pT stage, grade, and prognosis were analysed. In addition, correlations with these cancer-related and TSP-related factors were also investigated.ResultsStrong and moderate 4N1K expression was found in normal urothelial tissues. Of the 97 specimens, 45 patients were positive for 4N1K expression, which was primarily located in the interstitial areas of the cancer tissue. 4N1K expression was negatively associated with pT stage (p = 0.003) and grade (p = 0.002). Survival analyses revealed that 4N1K is a predictor of metastasis-free (p = 0.036) and cause-specific survival (p = 0.009). 4N1K expression was closely associated with malignant behaviour, specifically MVD (p = 0.001), AI (p = 0.013), and MMP-9 expression (p = 0.036), but not PI and LVD, as determined via multivariate analysis models.Conclusions4N1K expression appears to be associated with cancer cell progression and survival in UC-UUT patients via the regulation of angiogenesis, apoptosis, and MMP-9 expression. There is a possibility that the 4N1K-peptide may be a useful marker and novel therapeutic target in patients with UC-UUT.

Regulation of Cross-linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells by Convergence of Distinct β1 and β3 Integrin Pathways

To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.

Role of oxidative stress and caspase 3 in CD47‐mediated neuronal cell death

CD47 or integrin-associated protein promotes cell death in blood and tumor cells. Recently, CD47 signaling has been identified in neurons as well. In this study, we investigated the role of CD47 in neuronal cell death. Exposure of primary mouse cortical neurons to the CD47 ligand thrombospondin-1 or the specific CD47-activating peptide 4N1K induced cell death. Activation of CD47 elevated levels of active caspase 3 and increased the generation of reactive oxygen species (ROS) in a time-dependent manner. Both ROS scavengers and caspase inhibitors attenuated cell death. But ROS scavenging did not reduce the activation of caspase 3, and combination treatments with a caspase inhibitor plus free radical scavenger did not yield additive protection. Taken together, these data suggest that parallel and redundant pathways of oxidative stress and caspase-mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase-dependent and caspase-independent pathways.

Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced alpha(v)beta(3) activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.

CD47 mediates killing of breast tumor cells via Gi-dependent inhibition of protein kinase A.

Thrombospondins (TSPs) have been implicated as antitumor and antimetastasis factors in breast cancer. Although this effect has been attributed to the antiangiogenic activity of TSPs, recent observations suggest other mechanisms may be at work. The TSP receptor CD47 (integrin-associated protein) has recently been reported to mediate a novel form of apoptosis. Here, we have studied the response of breast cancer cells to CD47 ligands TSP-1, the CD47 agonist peptide 4N1K derived from TSP-1, and the anti-CD47 monoclonal antibody 1F7. All of these ligands killed four different breast cancer cell lines. This CD47-mediated cell death did not require active caspases or Bcl-2 degradation and did not cause DNA laddering or cytochrome c release. Pertussis toxin (PTX) prevented CD47-mediated death, indicating the involvement of Gi alpha. 4N1K dramatically reduced intracellular cAMP levels, an effect reversed with PTX. Forskolin, 8-bromo cAMP, and isobutylmethylxanthine (IBMX) all prevented CD47-mediated apoptosis, indicating the involvement of cAMP. H89 and protein kinase A (PKA) inhibitor peptide prevented rescue of breast cancer cells by PTX, 8-Br-cAMP, and forskolin, suggesting that the effects of cAMP are mediated via PKA-dependent phosphorylation events. Epidermal growth factor also inhibited CD47-induced apoptosis via a PKC-dependent but ERK-independent pathway. Thus, CD47-mediated killing of breast cancer cells occurs by a novel pathway involving regulation of cAMP levels by heterotrimeric Gi with subsequent effects mediated by PKA.

Increased erythrocyte adhesion in mice and humans with hereditary spherocytosis and hereditary elliptocytosis

AbstractMice with disruptions of the red blood cell (RBC) cytoskeleton provide severe hemolytic anemia models in which to study multiorgan thrombosis and infarction. The incidence of cerebral infarction ranges from 70% to 100% in mice with α-spectrin deficiency. To determine whether mutant RBCs abnormally bind adhesive vascular components, we measured adhesion of mouse and human RBCs to immobilized human thrombospondin (TSP) and laminin (LM) under controlled flow conditions. Mutant RBCs had at least 10-fold higher adhesion to TSP compared with normal RBCs (P < .006). Mutant relative to unaffected RBC adhesion to LM was significantly (P < .01) increased as well. Treatment of RBCs with the anionic polysaccharide dextran sulfate inhibited mutant RBC adhesion to TSP (P < .001). Treatment of RBCs with antibodies to CD47 or the CD47-binding TSP peptide 4N1K did not inhibit TSP adhesion of RBCs. Previously, we have shown that infarcts in α-spectrin–deficient sph/sph mice become histologically evident beginning at 6 weeks of age. TSP adhesion of RBCs from 3- to 4- and 6- to 8-week-old sph/sph mice was significantly higher than RBCs from adult mice (> 12 weeks old; P < .005). While the mechanism of infarction in these mice is unknown, we speculate that changes in RBC adhesive characteristics contribute to this pathology.

Ligation of CD47 During Monocyte Differentiation into Dendritic Cells Results in Reduced Capacity for Interleukin‐12 Production

We have previously shown that the CD47-binding thrombospondin-1 (TSP-1)-derived peptide 4N1K induces a rapid apoptosis-like death of human monocytes and dendritic cells (DCs). However, not all cells were susceptible to the peptide-induced cell death and here, we have investigated whether surviving monocytes could differentiate into functionally normal DCs. We found that the cell-surface phenotype, the T-cell stimulatory capacity and the ability to undergo lipopolysaccharide (LPS)-induced maturation into CD83+ DCs were essentially identical in 4N1K-derived and control DCs. Interleukin-10 (IL-10) production was also normal, but a significant downregulation of the pro-inflammatory cytokines IL-12 and tumour necrosis factor-alpha (TNF-alpha) was observed in the 4N1K-derived DCs. To the contrary, simultaneous stimulation of control DCs with 4N1K and LPS + interferon-gamma did not alter IL-12 production. These results indicate that although activation of the TSP-1-binding region of CD47 on monocytes induces apoptosis in a large proportion of the cells, it does not hamper the overall capacity of the surviving cells to differentiate into DCs. Such DCs, however, have a reduced capacity for IL-12 and TNF-alpha production, and the possibility that this is linked to the uptake of apoptotic cells is discussed.

CD47 Ligation Induces a Rapid Caspase‐Independent Apoptosis‐Like Cell Death in Human Monocytes and Dendritic Cells

CD47 is a versatile cell-surface molecule expressed on nearly all haematopoietic cells. In its capacity as a thrombospondin-1 (TSP-1) receptor, CD47 has recently been shown to mediate cell death in certain cells, for example, activated but not resting T cells. Here, we have investigated the possibility that human monocytes and dendritic cells (DCs) undergo cell death, following CD47 ligation. Using the TSP-1-derived CD47-binding peptide 4N1K, we found that both freshly isolated monocytes and monocyte-derived DCs underwent a rapid, caspase-independent cell death. This was characterized by the simultaneous presence of phosphatidylserine exposure, plasma membrane permeability, reduced mitochondrial membrane potential and highly fragmented DNA. Not all cells were sensitive to 4N1K-induced apoptosis; a plateau of cell death reached at an average of 38% of the monocyte and DCs populations. The results presented here, thus, show that CD47 can mediate a rapid apoptosis-like cell death of human monocytes and DCs.

Involvement of a CD47-dependent pathway in platelet adhesion on inflamed vascular endothelium under flow

AbstractResting platelet adhesion to inflammatory vascular endothelium is thought to play a causal role in secondary thrombus formation or microcirculatory disturbance after vessel occlusion. However, though adhesion receptors involved in platelet-matrix interactions have been extensively studied, the molecular mechanisms involved in platelet-endothelium interactions are incompletely characterized and have been mainly studied under static conditions. Using human platelets or platelets from wild-type and CD47–/– mice in whole blood, we demonstrated that at low shear rate, CD47 expressed on human and mouse platelets significantly contributes to platelet adhesion on tumor necrosis factor-α (TNF-α)–stimulated vascular endothelial cells. Using the CD47 agonist peptide 4N1K and blocking monoclonal antibodies (mAbs), we showed that CD47 binds the cell-binding domain (CBD) of endothelial thrombospondin-1 (TSP-1), inducing activation of the platelet αIIbβ3 integrin that in turn becomes able to link the endothelial receptors intercellular adhesion molecule 1 (ICAM-1) and αvβ3. Platelet CD36 and GPIbα are also involved because platelet incubation with blocking mAbs directed against each of these 2 receptors significantly decreased platelet arrest. Given that anti-CD47 treatment of platelets did not further decrease the adhesion of anti-CD36–treated platelets and CD36 is a TSP-1 receptor, it appears that CD36/TSP-1 interaction could trigger the CD47-dependent pathway. Overall, CD47 antagonists may be potentially useful to inhibit platelet adhesion on inflamed endothelium.

Opposite effects of thrombospondin-1 via CD36 and CD47 on homotypic aggregation of monocytic cells.

Thrombospondin-1 (TSP-1), an extracellular matrix protein, has a multimodular structure and each domain specifies a distinct biological function through interaction with a specific ligand. In this study we found that exogenously added TSP-1 inhibits phorbol myristate acetate (PMA)/LPS-induced homotypic aggregation of human monocytic U937 cells, whereas the 70-kDa fragment of TSP-1 generated by the proteolytic cleavage of the intact molecule promotes the homotypic aggregation. The aggregation was also inhibited by anti-CD47 mAb or the 4N1K peptide, of which sequence is derived from the CD47-binding site of TSP-1 and absent in the 70-kDa fragment. In contrast, the augmented cell aggregation by the 70-kDa fragment was hampered by anti-CD36 mAb or antibody against the CD36-binding site of TSP-1. The cell aggregation of U937 cells was completely blocked, even in the presence of the 70-kDa fragment, by mAb against leukocyte function associated antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1). We therefore propose that TSP-1 may regulate LFA-1/ICAM-1-mediated cell adhesion of monocytes/macrophages by either the inhibitory effect through CD47 or the promoting effect through CD36 depending on which domain/fragment is functional in a given biological setting.

CD47 ligation selectively inhibits the development of human naive T cells into Th1 effectors.

The CD47 Ag, also named integrin-associated protein, was recently reported to regulate the production of IL-12 by human monocytes and dendritic cells. The present study shows that CD47 ligation by CD47 mAb in primary cultures of cord blood mononuclear cells inhibits IL-12-driven Th1 cell development, as revealed by the cytokine secretion profile at restimulation and IFN-gamma production at the single-cell level. F(ab')(2) fragments of CD47 mAb or the synthetic peptide 4N1K, corresponding to the CD47 binding site of thrombospondin, display the same activity. CD47 engagement does not change the phenotype of IL-12-primed cells from Th1 to Th2 or affect IL-4-induced Th2 cell development. Moreover, CD47 mAb inhibits IL-12- but not IL-4-induced IL-2 production as well as IFN-gamma in primary cultures, which was correlated with a decrease of the IL-12Rbeta2 chain expression. Inclusion of exogenous IL-2 at priming corrects IL-12R expression as well as the inhibition of Th1 cell development. The data thus underline the role of IL-2 in Th1 cell development and further suggest that targeting IL-2 and IL-12 simultaneously may have some therapeutic advantage in Th1 autoimmune diseases.

Role of Thrombospondin-1-Derived Peptide, 4N1K, in FGF-2-Induced Angiogenesis

Angiogenesis involves proliferation of capillary endothelial cells and formation of lumen-containing tube-like structures. A recently established murine brain capillary endothelial cell line, IBE, can either proliferate or form tube-like structures (i.e., differentiate) in response to fibroblast growth factor-2 (FGF-2), dependent on the culture conditions. The 4N1K peptide (KRFYVVMWKK), which is derived from the C-terminal cell-binding domain of thrombospondin-1 (TSP-1), inhibited tube formation, but not proliferation of IBE cells. Polyclonal antibodies against 4N1K blocked TSP-1-induced inhibition of tube formation by IBE cells. 4N1K inhibited tyrosine phosphorylation of focal adhesion kinase and FGF-2-stimulated tyrosine phosphorylation of phospholipase C-γ in tube-forming, but not proliferating, IBE cells. The peptide also inhibited FGF-2-induced neovascularization in mouse cornea. Our results indicate that TSP-1 may exert its inhibitory effects on angiogenesis via the C-terminal cell-binding domain containing the 4N1K sequence by inhibiting tube formation by endothelial cells.

Thrombospondin-1 Acts Via IAP/CD47 to Synergize With Collagen in 2β1-Mediated Platelet Activation

Integrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of IIbβ3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin 2β1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP−/− mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of 2β1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and 2β1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.

Thrombospondin-1 Acts Via IAP/CD47 to Synergize With Collagen in α2β1-Mediated Platelet Activation

AbstractIntegrin-associated protein (IAP; or CD47) is a receptor for the cell binding domain (CBD) of thrombospondin-1 (TS1). In platelets, IAP associates with and regulates the function of αIIbβ3 integrin (Chung et al, J Biol Chem 272:14740, 1997). We test here the possibility that CD47 may also modulate the function of platelet integrin α2β1, a collagen receptor. The CD47 agonist peptide, 4N1K (KRFYVVMWKK), derived from the CBD, synergizes with soluble collagen in aggregating platelet-rich plasma. 4N1K and intact TS1 also induce the aggregation of washed, unstirred platelets on immobilized collagen with a rapid increase in tyrosine phosphorylation. The effects of TS1 and 4N1K on platelet aggregation are absolutely dependent on IAP, as shown by the use of platelets from IAP−/− mice. Prostaglandin E1 (PGE1) prevents 4N1K-dependent aggregation on immobilized collagen but does not inhibit the 4N1K peptide stimulation of α2β1-dependent platelet spreading. Finally, a detergent-stable, physical association of IAP and α2β1 integrin is detected by coimmunoprecipitation. These results imply a role for IAP and TS1 in the early activation of platelets upon adhesion to collagen.