Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.     

Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties

Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin.     

Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A

Recent studies indicate that angiogenesis depends, in part, on ligation of integrin alpha(5)beta(1) by fibronectin. Evidence is now provided that integrin alpha(5)beta(1) regulates the function of integrin alpha(v)beta(3) on endothelial cells during their migration in vitro or angiogenesis in vivo. Secretion of fibronectin by endothelial cells leads to the ligation of integrin alpha(5)beta(1), which potentiates alpha(v)beta(3)-mediated migration on vitronectin without influencing alpha(v)beta(3)-mediated cell adhesion. Endothelial cell attachment to vitronectin suppresses protein kinase A (PKA) activity, while addition of soluble anti-alpha(5)beta(1) restores this activity. Moreover, agents that activate intracellular PKA, such as forskolin, dibutyryl cAMP or alpha(5)beta(1) antagonists, suppress endothelial cell migration on vitronectin in vitro or angiogenesis in vivo. In contrast, inhibitors of PKA reverse the anti-migratory or anti-angiogenic effects mediated by alpha(5)beta(1) antagonists. Therefore, alpha(v)beta(3)-mediated endothelial cell migration and angiogenesis can be regulated by PKA activity, which depends on the ligation state of integrin alpha(5)beta(1).     

Endostatin inhibits adhesion of endothelial cells to collagen I via alpha(2)beta(1) integrin, a possible cause of prevention of chondrosarcoma growth

Endostatin derived from collagen XVIII is a potent endogenous anti-angiogenic factor that induces regression of various tumors of epithelial origin. Endostatin has been shown to inhibit endothelial cell functions, however, its effect remains controversial. We first attempted here to apply the inhibitory effect of recombinant human endostatin on chondrosarcomas, which originate from the mesenchyme, in nude mice. Endostatin induced reduction of chondrosarcoma growth and tumor angiogenesis in vivo. However, endostatin showed no effect on the proliferation and migration of chondrosarcoma cells in vitro. Next, we investigated the interactions between endostatin and endothelial cells in detail. Endostatin inhibited the migration on and attachment to collagen I but did not affect the proliferation of endothelial cells. Although the migration of endothelial cells was stimulated by angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor, endostatin showed similar inhibitory effects on it in the presence and absence of the stimulants. Moreover, the inhibitory effect against endothelial cell attachment to collagen I was attenuated or modulated in the presence of neutralizing antibodies of alpha(2), alpha(5)beta(1), and alpha(V)beta(3) integrins but not that of alpha(1) integrin. Our results suggest that endostatin might suppress the alpha(2)beta(1) integrin function of endothelial cells via alpha(5)beta(1) or alpha(V)beta(3) integrin. We propose here that endostatin might be effective for anti-angiogenic therapy for human chondrosarcomas through the suppression of alpha(2)beta(1) integrin functions in endothelial cells.     

Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking alphavbeta5 integrin

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.     

Lebectin, a Macrovipera lebetina venom-derived C-type lectin, inhibits angiogenesis both in vitro and in vivo

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.     

Integrins and angiogenesis: a sticky business

From an evolutionary point of view, the development of a cardiovascular system allowed vertebrates to nourish the several organs that compose their wider multicellular organism and to survive. Acquisition of new genes encoding for extracellular matrix (ECM) proteins and their cognate integrin receptors as well as secreted pro- and anti-angiogenic factors proved to be essential for the development of vascular networks in the vertebrate embryo. Postnatal tissue neo-vascularization plays a key role during wound healing and pathological angiogenesis as well. There is now clear evidence that building blood vessels in the embryo and in the adult organism relies upon different endothelial integrins and ECM ligands. A successful vascular development depends on fibronectin and its major receptor alpha5beta1 integrin, but not on alphavbeta3, alphavbeta5, and alpha6beta4 integrins that are instead central regulators of postnatal tumor angiogenesis. Here, endothelial alphavbeta3 elicits anti- or pro-angiogenic signals depending respectively on whether it is occupied by a soluble (e.g. type IV collagen derived tumstatin) or an insoluble (vitronectin) ECM ligand. The laminin-5 receptor alpha6beta4 integrin, expressed only by endothelial cells of mature blood vessels, controls the invasive phase of tumor angiogenesis in the adult organism. Finally, regulation of vascular morphogenesis relies upon the fine modulation of integrin activation by chemoattractant and chemorepulsive cues, such as angiogenic growth factors and semaphorins.     

Integrin alpha2beta1 is the required receptor for endorepellin angiostatic activity

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.     

Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth

Angiogenesis, the formation of new blood vessels, is required for physiological development of vertebrates and repair of damaged tissue, but in the pathological setting contributes to progression of cancer. During tumor growth, angiogenesis is supported by up-regulation of angiogenic stimulators (pro-angiogenic) and down-regulation of angiogenic inhibitors (anti-angiogenic). The switch to the angiogenic phenotype (angiogenic switch) allows the tumors to grow and facilitate metastasis. The bioactive NC1 domain of type IV collagen alpha3 chain, called tumstatin, imparts anti-tumor activity by inducing apoptosis of proliferating endothelial cells. Tumstatin binds to alphaVbeta3 integrin via a mechanism independent of the RGD-sequence recognition and inhibits cap-dependent protein synthesis in the proliferating endothelial cells. The physiological level of tumstatin is controlled by matrix metalloproteinase-9, which most effectively cleaves it from the basement membrane and its physiological concentration in the circulation keeps pathological angiogenesis and tumor growth in check. These findings suggest that tumstatin functions as an endogenous inhibitor of pathological angiogenesis and functions as a novel suppressor of proliferating endothelial cells and growth of tumors.     

Inhibition effect of pcDNA-tum-5 on the growth of S180 tumor

Tumor growth and metastasis depend on vessel formation, and inhibition of angiogenesis of tumor by production of anti-angiogenic drugs should be a promising approach for cancer therapy. Tumstatin is an angiogenesis inhibitor. The anti-angiogenic activity of tumstatin is localized to the 54–132 amino acids. The gene fragment encoding amino acids 45–132 of tumstatin (tum-5) was subcloned into pcDNA3.1 (pcDNA-tum5). Tum-5 protein could be expressed and secreted in CHO cells after transfection. The conditioned medium (containing tum-5 protein) from the transfectant has an anti-angiogenic effect on HUVEC cells in vitro. The anti-tumor effect of pcDNA-tum5 on mice bearing S180 tumors was evaluated. The results showed that pcDNA-tum-5 has a significant inhibition activity in the growth of the tumors. This study suggests that the gene delivery of tum-5 may be an effective strategy for cancer therapy.     

Integrin regulation by vascular endothelial growth factor in human brain microvascular endothelial cells: role of alpha6beta1 integrin in angiogenesis

The precise role of vascular endothelial growth factor (VEGF) in regulating integrins in brain microvascular endothelial cells is unknown. Here, we analyzed VEGF effects on integrin expression and activation in human brain microvascular endothelial cells (HBMECs). Using human cDNA arrays and ribonuclease (RNase) protection assays, we observed that VEGF up-regulated the mRNA expression of alpha(6) integrin in HBMECs. VEGF significantly increased alpha(6)beta(1) integrin expression, but not alpha(6)beta(4) integrin expression in these cells. Specific down-regulation of alpha(6) integrin expression by small interfering RNA (siRNA) oligonucleotides inhibited both the capillary morphogenesis of HBMECs and their adhesion and migration. Additionally, VEGF treatment resulted in activation of alpha(6)beta(1) integrins in HBMECs. Functional blocking of alpha(6) integrin with its specific antibody inhibited the VEGF-induced adhesion and migration as well as in vivo angiogenesis, and markedly suppressed tumor angiogenesis and breast carcinoma growth in vivo. Thus, VEGF can modulate angiogenesis via increased expression and activation of alpha(6)beta(1) integrins, which may promote VEGF-driven tumor angiogenesis in vivo.     

Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion

The NC1 domains of human type IV collagen, in particular alpha3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the alpha3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-alpha3NC1 domain.     

Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect

betaig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether betaig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only betaig-h3 itself but also all four fas-1 domains of betaig-h3 mediate endothelial cell adhesion and migration through interaction with the alphavbeta3 integrin. We found that the alphavbeta3 integrin-interacting motif of the four fas-1 domains of betaig-h3 is the same YH motif that we reported previously to interact with alphavbeta5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that betaig-h3 bears alphavbeta3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.     

Integrin alpha3beta1, a novel receptor for alpha3(IV) noncollagenous domain and a trans-dominant Inhibitor for integrin alphavbeta3

Exogenous soluble human alpha3 noncollagenous (NC1) domain of collagen IV inhibits angiogenesis and tumor growth. These biological functions are attributed to the binding of alpha3NC1 to integrin alphavbeta3. However, in some tumor cells that express integrin alphavbeta3, the alpha3NC1 domain does not inhibit proliferation, suggesting that integrin alphavbeta3 expression is not sufficient to mediate the anti-tumorigenic activity of this domain. Therefore, in the present study, we searched for novel binding receptors for the soluble alpha3NC1 domain in cells lacking alphavbeta3 integrin. In these cells, soluble alpha3NC1 bound integrin alpha3beta1; however, unlike alphavbeta3, alpha3beta1 integrin did not mediate cell adhesion to immobilized alpha3NC1 domain. Interestingly, in cells lacking integrin alpha3beta1, adhesion to the alpha3NC1 domain was enhanced due to activation of integrin alphavbeta3. These findings indicate that integrin alpha3beta1 is a receptor for the alpha3NC1 domain and transdominantly inhibits integrin alphavbeta3 activation. Thus integrin alpha3beta1, in conjunction with integrin alphavbeta3, modulates cellular responses to the alpha3NC1 domain, which may be pivotal in the mechanism underpinning its anti-angiogenic and anti-tumorigenic activities.     

Overexpressed alpha3beta1 and constitutively activated extracellular signal-regulated kinase modulate the angiogenic properties of ECV304 cells.

ECV304, a spontaneously transformed cell line derived from the human umbilical vein endothelial cell (HUVEC) (Takahashi et al., 1990), has been developed as an in vitro angiogenesis model. In the present study, we further characterized the angiogenic properties of this cell line. Compared to HUVEC, ECV304 cells showed distinct features including a higher activity of cellular adhesion, slower but reproducible progression of angiogenesis on Matrigel, and resistance to apoptosis. Thus, the expression of integrin and activation of extracellular-signal regulated kinase 1/2 (Erk1/2), a downstream effector of the integrin pathway, were examined. Flow cytometry revealed that alpha3beta1 integrin was markedly upregulated in ECV304 cells, while alpha(v)beta1 and alpha5beta1 integrins were slightly downregulated. Consistent with this, the binding activity to collagen type IV and laminin, major extracellular matrices of Matrigel, was increased 1.4- and 1.9-fold in ECV304 cells, respectively. This tight binding may retard the initial stage of sprouting and migration in the angiogenesis of ECV304 cells. It has been further demonstrated that Erk1/2 is constitutively active in ECV304 cells, rendering them resistent to the inhibitory effect of PD98059 on proliferation. However, migration of both HUVEC and ECV304 cells was inhibited to a similar extent by PD98059 in a dose-dependent manner. Up to 50 microM of PD98059, no significant changes in cell binding and tubulogenesis on Matrigel was observed in ECV304 cells. In contrast, the tubulogenesis of HUVEC was severely impaired by PD98059. Elevated Erk1/2 activity in ECV304 cells was suppressed by dominant negative H-Ras, but not by cytochalasin D. These results suggest that the overexpression of alpha3beta1 integrin and the constitutive activation of Erk1/2 play a key role in the alteration of the angiogenic properties of ECV304 cells.     

Construction, expression, and characterization of a new targeted bifunctional fusion protein: tumstatin45-132-TNF

The anti-angiogenic activity of tumstatin45-132 is mediated by binding to alphaVbeta3 on endothelial cells and tumor vascular endothelium showing increased expression of alphaVbeta3. Tumor necrosis factor alpha (TNF-alpha) is known to not only possess direct cytotoxicity against tumor cells, but also induces tumor vessel disruption, however, clinical use of TNF-alpha as an anticancer drug is hampered by severe systemic toxicity. In this study, we explore the possibility of fusing tumstatin45-132 with human TNF-alpha in the hope of generating a targeting, bi-functional protein in tumor treatment. Tumstatin45-132-TNF was constructed and expressed in E. coli. The recombinant fusion protein was shown to be insoluble and in an inclusion body form. An effective strategy for refolding and purification of tumstatin45-132-TNF resulted in final purified yields of 3 mg purified fusion protein recovered from 1 liter of E. coli culture. The refolded tumstatin45-132-TNF with a purity of 98% assessed by denaturing SDS - PAGE showed a single band on gels. Endothelial cell proliferation assay and standard cytolytic assays against L929 indicated that the fusion protein maintains tumstatin45-132 and TNF-alpha activity. More importantly, tumstatin45-132-TNF inhibits endothelial cell proliferation more than tumstatin45-132 alone. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the tumstatin45-132 moiety specifically interacts with alphaVbeta3 integrin. These results lay the solid foundation for further investigation of antitumor activity of tumstatin45-132-TNF in vivo.     

Function of the Tetraspanin CD151–α6β1 Integrin Complex during Cellular Morphogenesis

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.     

Agkistin, a snake venom-derived glycoprotein Ib antagonist, disrupts von Willebrand factor-endothelial cell interaction and inhibits angiogenesis

Glycoprotein (GP) Ib, an adhesion receptor expressed on both platelets and endothelial cells, mediates the binding of von Willebrand factor (vWF). Platelet GPIb plays an important role in platelet adhesion and activation, whereas the interaction of vWF and endothelial GPIb is not fully understood. We report here that agkistin, a snake venom protein, selectively blocks the interaction of vWF with human endothelial GPIb and inhibits angiogenesis in vivo. Agkistin specifically blocked human umbilical vein endothelial cell (HUVEC) adhesion to immobilized vWF in a concentration-dependent manner. Fluorescein isothiocyanate (FITC)-conjugated agkistin bound to HUVECs in a saturable manner. AP1, a monoclonal antibody (mAb) raised against GPIb, specifically inhibited the binding of FITC-conjugated agkistin to HUVECs in a dose-dependent manner, but other anti-integrin mAbs raised against alpha(v)beta(3), alpha(2)beta(1), and alpha(5)beta(1) did not affect this binding reaction. However, neither agkistin (2 microgram/ml) nor AP1 (40 microgram/ml) apparently reduced HUVEC viability. Both agkistin and AP1 exhibited a profound anti-angiogenic effect in vivo when assayed by using the 10-day-old embryo chick chorioallantoic membrane model. These results suggest endothelial GPIb plays a role in spontaneous angiogenesis in vivo, and the anti-angiogenic effect of agkistin may be because of disruption of the interaction of endogenous vWF with endothelial GPIb.