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J. Biol. Chem., Vol. 275, Issue 25, 18615-18618, June 23, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/25/18615    most recent C000234200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yeh, C.-H. Articles by Huang, T.-F. Search for Related Content PubMed PubMed Citation Articles by Yeh, C.-H. Articles by Huang, T.-F. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Agkistin, a Snake Venom-derived Glycoprotein Ib Antagonist, Disrupts von Willebrand Factor-Endothelial Cell Interaction and Inhibits Angiogenesis^*

Chia-Hsin Yeh, Wen-Cheng Wang, Tsang-Tang Hsieh^§, and Tur-Fu Huang^¶

From the  Department of Pharmacology, College of Medicine, National Taiwan University and ^§ Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei 100, Taiwan

Received for publication, April 7, 2000, and in revised form, April 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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 _v3, ₂₁, and ₅₁ did not affect this binding reaction. However, neither agkistin (2 µg/ml) nor AP1 (40 µg/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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Angiogenesis, the development of new capillaries from preexisting blood vessels, plays a critical role in a variety of physiological processes and pathological conditions, including embryonic development, wound healing, tumor growth, metastasis, and various inflammatory disorders (1). Interactions between endothelial cells and extracellular matrices (ECMs)¹ including fibrinogen, vitronectin, collagen, laminin, and vWF, through cell surface adhesion receptors, are involved in the multiprocesses of neovascularization (2). For example, integrin _v3 is not readily detectable in quiescent vessels but becomes highly expressed in angiogenic vessels (3). The dependence of angiogenesis on vascular cell adhesion events in vivo is evidenced by the observation that antibody and snake venom proteins antagonizing integrin _v3 blocked angiogenesis in the chick chorioallantoic membrane (CAM) model (4, 5).

GPIb is an adhesive receptor expressed both on platelets and endothelial cells. Binding of plasma vWF to platelet GPIb plays a critical role in the earliest phase of primary hemostasis, which consists of the anchoring of platelets to injury vessel walls (6). In contrast to the platelets, the functions of endothelial GPIb for vWF are not well understood. Several studies have demonstrated the presence of GPIb complex expressed on the endothelial cell surface and up-regulation of endothelial GPIb by cytokines, i.e. tumor necrosis factor and interferon  (7-10). It has been shown that endothelial GPIb participates in HUVEC binding to sickle cell erythrocytes (11) and also promotes platelet bridging to endothelial cells (12). Recently, Lian et al. (13) assumed that endothelial GPIb may contribute to mediate the process of endothelial cell migration during wound repair in vivo.

Snake venoms contain many unique components that affect cell-matrix interaction. Disintegrins represent a family of low molecular weight, cysteine-rich polypeptides that bind specifically to integrins _IIb3, ₅₁, and _v3 expressed on platelets and other cells including vascular endothelial cells and some tumor cells, leading to inhibition of platelet aggregation, inhibition of cell adhesion, migration, and angiogenesis (14). On the other hand, some GPIb-binding venom proteins modulate vWF-GPIb interaction, leading to platelet activation, inhibition of platelet aggregation (15), or mediating HUVEC adhesion (16). Agkistin (also named agkicetin (17)), belonging to C-type lectin polypeptide derived from the viper venom of Agkistrodon acutus, specifically inhibited human platelet aggregation and agglutination triggered by ristocetin in the presence of vWF in vitro by acting as the platelet GPIb antagonist (18). Furthermore, agkistin is a potent anti-thrombotic agent because it pronouncedly blocked platelet plug formation in an experimental model in vivo.² In this study, we showed that agkistin specifically inhibited HUVEC adhesion to immobilized vWF and it was shown to bind endothelial GPIb in a saturable manner. The role of endothelial GPIb in angiogenesis in vivo was thus evaluated.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Materials-- Agkistin was purified from crude venom of Formosan Agkistrodon acutus by means of gel filtration and ion exchanger as described previously (18). The homogeneity of agkistin was judged by both sodium dodecyl sulfate-polyacrylamide electrophoresis and N-terminal amino acid sequencing. Rhodostomin was isolated from Agkistrodon rhodostoma venom as described previously (19). Human plasma purified vWF was purchased from Calbiochem, and vitronectin was from Life Technologies, Inc. The mAbs AP1 raised against GPIb (20) and 7E3 raised against integrin _IIb3 and _v3 (21) were kindly donated by Dr. Robert Montgomery (Southern Wisconsin Milwaukee Blood Center, Milwaukee, WI) and Dr. Barry Coller (Mount Sinai Hospital, New York, NY), respectively. Fluorescein isothiocyanate (FITC) and 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM) were purchased from Molecular Probes. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), collagen type I, and fibronectin were from Sigma.

Cell Culture-- HUVECs were isolated from umbilical cord veins and maintained in Medium 199 containing 20% fetal bovine serum, 30 µg/ml endothelial cell growth supplement, 4 mM L-glutamine, and antibiotics at 37 °C with 5% CO₂ incubation. The cells were identified as positively immunofluorescent staining for human vWF antigen (DAKO) and used between the second and fourth passages.

Adhesion Assays-- Synchronized HUVECs were harvested and labeled with BCECF/AM (10 µg/ml) for 30 min at 37 °C. After washing, cells were incubated with the indicated concentrations of agkistin or PBS for another 30 min. Control or the pretreated HUVECs were applied onto ECM-precoated plates at a density of 5 × 10⁴ cells/well and incubated for 90 min at 37 °C allowing adhesion. After incubation, the non-adherent cells were removed by aspiration, and plates were subjected to a CytoFluor 2300 fluorescence plate reader (Minipore). The percent adherent HUVECs toward fibronectin (30 µg/ml), vitronectin (10 µg/ml), and vWF (10 µg/ml) was calculated to be 49.2 ± 2.2, 24.0 ± 0.7, and 40.9 ± 1.5%, respectively, based on the cell count by hemacytometer (Hausser Scientific) after trypsin deattachment. The amount of nonspecific adhesion was performed by using wells precoated with 1% BSA and estimated to be 3.26 ± 0.8%.

HUVEC Viability-- Cells were seeded at a density of 2 × 10⁴ cells/well in 24-well plates (Costar) followed by incubation with agkistin, AP1, rhodostomin, or vehicle for 48 h in a humidified atmosphere (i.e. 37 °C, 5% CO₂). For MTT assay, cells were incubated with MTT at a final concentration of 0.5 mg/ml for 4 h. After incubation, the medium was aspirated, and the cells were dissolved in dimethyl sulfoxide and then the developed color absorbance at 550 nm was measured.

Flow Cytometry-- HUVECs (1 × 10⁶) were fixed with 1% paraformaldehyde and then incubated with FITC-conjugated agkistin or FITC-conjugated BSA for 30 min. After incubation, cells were washed twice and resuspended in PBS and analyzed immediately by fluorescence-activated cell sorter(Calibur, Becton Dickinson) using excitation and emission wavelength of 488 and 525 nm, respectively. In inhibitory studies, mAb 7E3 or AP1 was added to fixed HUVEC for 30 min prior to the incubation of FITC-conjugated proteins.

Chick CAM Angiogenesis Assay-- Eggs of 10-day-old chick embryo were opened with a 1.0-cm square window that allowed direct access to underlying CAM by the method described previously (22). A filter paper disc (1.3 cm, Minipore) saturated with test compounds or an equal aliquot of PBS (final volume in 20 µl) was applied to the top of CAM. The window was covered with sterile cellophane tape, and the embryos were incubated for a further 48 h at 37 °C with 60% humidity to develop spontaneous angiogenesis.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Inhibition of HUVEC Adhesion to Immobilized vWF by Agkistin-- Multiple adhesive receptors expressed on endothelial cells mediate adhesion to extracellular matrices. First, we examined the effect of agkistin on HUVEC adhesion to a number of immobilized ECMs including collagen type I (80 µg/ml), fibronectin (30 µg/ml), vitronectin (10 µg/ml), and vWF (10 µg/ml). As shown in Fig. 1, agkistin specifically and dose dependently inhibited HUVEC adhesion to immobilized vWF (48.98 ± 4.12% inhibition at 0.066 µM) but apparently little affected those adhesive events toward other matrices used (less than 10% inhibition). Agkistin at a high concentration of 0.2 µM did not exhibit a further inhibition on HUVEC adhesion to immobilized vWF (about 52% inhibition, data not shown). These results indicate that agkistin does not interfere with the adhesion of integrins _v3, ₂₁, and ₅₁ and their respective ligands, vitronectin, collagen, and fibronectin. The interaction of vWF with HUVECs is mediated by endothelial integrin _v3 and by GPIb complex (16). Therefore, agkistin may process a rather specific binding and high affinity toward GPIb on HUVEC and functionally block vWF-endothelial GPIb interaction. A similar result (40-50%) was reported with echicetin, another GPIb antagonist (23).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Effect of agkistin on HUVEC adhesion to immobilized ECMs. HUVECs (5 × 104 cells/well) were added to 96-well plates, which were precoated with collagen type I (80 µg/ml), vitronectin (10 µg/ml), fibronectin (30 µg/ml), or vWF (10 µg/ml) in the absence or presence of various concentrations of agkistin. Results are expressed as percentage inhibition of adhesion compared with control cells in the absence of agkistin. All experiments were conducted in quadruplicate and repeated at least three times. Data are presented as mean ± S.E. (n = 3-6).

Inhibition of FITC-conjugated Agkistin Binding to HUVEC by AP1-- To further explore the binding receptor of agkistin on HUVECs, we conjugated agkistin and BSA with FITC by a previously described method (24) and then examined the binding reaction of FITC-conjugated agkistin toward HUVEC by flow cytometry. Nonspecific binding was performed using FITC-BSA as a probe. After incubation of FITC-conjugated agkistin with HUVECs, the increment of relative fluorescence intensity of FITC-agkistin bound cells was concentration-dependent and reached a saturated binding at a concentration of 1 µM (Fig. 2). The different concentration range of agkistin used in this binding assay and in the cell adhesive assay (Fig. 1) is attributable to the different number of cells used in the two independent systems (i.e. 1 × 10⁶ cells for agkistin binding and 5 × 10⁴ cells for cell adhesion). Thus, the concentration of agkistin in reaching binding saturation is about 14-fold higher than the maximal effective concentration used in adhesion study (1.0 versus 0.07 µM). AP1, a mAb raised against the N-terminal 45-kDa domain of GPIb (25), exerted a dose-dependent inhibition on the binding of FITC-agkistin to HUVECs. However, all of anti-_v3 mAb 7E3 and anti-₂₁, ₃₁, ₄₁, and ₅₁ integrin mAbs (50 µ g/ml, Chemicon) did not affect this binding (Fig. 3 and data not shown). These data indicate that agkistin and AP1 might bind to a common epitope on endothelial GPIb whereas agkistin did not bind to other endothelial integrins including _v3, ₂₁, and ₅₁, consistent with the inference obtained from the above described adhesive experiment.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Binding isotherm of FITC-conjugated agkistin to HUVECs. HUVECs (1 × 106 cells) were incubated with various concentrations of FITC-conjugated agkistin or FITC-conjugated BSA for 30 min and then analyzed by flow cytometry. Specific binding () is calculated by subtracting the nonspecific binding (, as probed by FITC-BSA) from total binding (, as probed by FITC-agkistin). This is a representative one of three similar results.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effect of mAbs on the binding of FITC-conjugated agkistin to HUVECs. HUVECs (1 × 106 cells) pretreated with 7E3 (A, thick line, 50 µg/ml), various concentrations of AP1 (B-D, thick lines, 10, 20, or 50 µg/ml, respectively), or nonimmune IgG (A-D, gray areas) were incubated with FITC-conjugated agkistin (0.5 µM) and analyzed by flow cytometry. Nonspecific binding was carried out by incubating cells with FITC-conjugated BSA (A-D, thin lines, 0.5 µM). Similar results were obtained in at least four separate experiments. E, quantitative analyses of the binding of FITC-agkistin and FITC-BSA in the presence of mAbs were presented as mean fluorescence intensity. Data are presented as mean ± S.E. (n = 4).

Neither Agkistin nor AP1 Affected HUVEC Viability-- Primary endothelial cells are anchorage-dependent, and disruption of integrin-ECM interaction induces HUVEC death (26, 27). In addition, _v3 receptor blockade resulted in unscheduled programmed cell death (apoptosis) of proliferating vascular cells (4). Recently, Feng et al. (28) indicated that GPIb complex expressed on Chinese hamster ovary cells regulates cell proliferation. Thus, it is interesting to investigate if the blockade of endothelial GPIb reduces the viability of primary endothelial cells. HUVEC viability assay was performed by determining the cell metabolic activity with MTT. As shown in Fig. 4, neither agkistin (at a high concentration of 2 µg/ml, i.e. 0.066 µM), which exerted a maximal effect in blocking vWF-HUVEC adhesion (Fig. 1), nor AP1 (40 µg/ml) affected HUVEC viability. However, rhodostomin (2 µg/ml), a member of disintegrins, significantly reduced HUVEC viability. These data suggest that blockade of the vWF-endothelial GPIb interaction is not sufficient to induce cell death and endothelial GPIb receptor is not essential for cell survival.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Effect of agkistin, AP1, and rhodostomin on HUVEC viability. HUVECs (4 × 104/ml) were seeded on 24-well plates. After attachment, cells were treated with the indicated concentration of test compounds for 48 h and followed by MTT assay. Results are expressed as percentage inhibition of viability compared with control cells in the absence of agkistin (PBS). Data are presented as mean ± S.E. (n = 4).

Inhibition of Spontaneous Angiogenesis by Agkistin and AP1 in CAM Model-- Several useful models have been used to study the role of cell adhesion molecule in angiogenesis. One of the most common in vivo models is the chick embryo CAM assay (29). To evaluate the role of endothelial GPIb complex in neovascularization in vivo, the effects of agkistin and anti-GPIb mAb AP1 on spontaneous angiogenesis occurring in the chick CAM model were examined. Upon dissection of the CAM of 12-day-old chick embryo, both agkistin (Fig. 5, B and C) and AP1 (Fig. 5, D and E), which were topically applied on CAM for 48 h, dose dependently inhibited the spontaneous angiogenesis in the CAM model as examined under filter disc as compared with control CAMs (Fig. 5A, PBS). These data suggest that endothelial GPIb receptor may significantly contribute to spontaneous angiogenesis in vivo. Moreover, blockade of vWF-GPIb interaction either by anti-GPIb mAb (i.e. AP1) or by polypeptide (i.e. agkistin) markedly inhibited neovascularization. On the other hand, accutin, a disintegrin derived from the same viper venom of A. acutus, inhibited angiogenesis by acting as endothelial integrin _v3 antagonist and by inducing apoptosis (5). Taken together, snake venom constituents affect cell-matrix interaction via multiple mechanisms, resulting in modification of platelet and vascular cell behaviors, an important cellular process in thrombosis, hemostasis, and angiogenesis (30). Furthermore, at a higher dose (2 µg/embryo), both agkistin and AP1 significantly disrupted preexisting blood vessels as evidenced by disappearance of large vessels (Fig. 5, C and E). This is different from the previous observation with integrin _v3 antagonists, which inhibited angiogenesis without affecting preexisting blood vessels (4, 5). Therefore, the action mechanisms of anti-angiogenic activity elicited by endothelial GPIb antagonists are quietly different from those of integrin _v3 antagonists and remain to be further investigated. To our knowledge, it is the first report describing the functional role of endothelial GPIb in spontaneous angiogenesis, and GPIb antagonists can markedly inhibit this process. It remains to be elucidated regarding whether agkistin affects the multisteps in the angiogenic process such as tissue degradation, migration, and differentiation elicited by a specific angiogenic factor (e.g. vascular endothelial growth factor or basic fibroblast growth factor).

View larger version (81K): [in this window] [in a new window]   Fig. 5.   Effect of agkistin and AP1 on spontaneous angiogenesis in CAM assay. Filter discs soaked in buffer (A, PBS), agkistin (B and C, 0.2 and 2 µg/embryo, respectively), or AP1 (D and E, 0.4 and 2 µg/embryo, respectively) in a total volume of 20 µl were applied on 10-day-old CAMs. After a 48-h incubation, CAMs were resected, fixed, and photographed. This is a representative one of three similar experiments.

In conclusion, agkistin specifically inhibited HUVEC adhesion to immobilized vWF through selective binding of endothelial GPIb, resulting in a blockade of interaction between vWF and endothelial GPIb. Agkistin binds to endothelial GPIb in a specific and saturable manner. Furthermore, both agkistin and AP1 significantly inhibited spontaneous angiogenesis in the chick CAM model in vivo but apparently did not affect HUVEC viability. In addition to their anti-thrombotic activity of agkistin and crotalin, another venom platelet GPIb antagonist (31), these GPIb antagonists may be useful tools for developing a new therapeutic strategy in angiogenesis-related diseases, such as inflammation and tumor metastasis.

	ACKNOWLEDGEMENTS

We appreciate very much the generous supply of monoclonal antibodies from Dr. R. Montgomery (AP1) and Dr. B. Coller (7E3). We also thank S. C. Huang for preparing HUVECs.

	FOOTNOTES

^* This work was supported by National Science Council of Taiwan Grant NSC 84-2331-B002-112BC and National Health Research Institute Grant NHRI-GT-EX89B920L.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Rd, Taipei 100, Taiwan.

Published, JBC Papers in Press, April 21, 2000, DOI 10.1074/jbc.C000234200

² C-H. Yeh, M-C. Chang, H-C. Peng, and T-F. Huang, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; BCECF/AM, 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester; BSA, bovine serum albumin; CAM, chorioallantoic membrane; FITC, fluorescein isothiocyanate; GP, glycoprotein; HUVEC, human umbilical vein endothelial cell; mAb, monoclonal antibody; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; vWF, von Willebrand factor.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 275/25/18615    most recent C000234200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yeh, C.-H. Articles by Huang, T.-F. Search for Related Content PubMed PubMed Citation Articles by Yeh, C.-H. Articles by Huang, T.-F. Social Bookmarking                      What's this?