A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis

Dongying Ma; Xueqing Xu; Su An; Huan Liu; Xuening Yang; John F. Andersen; Yipeng Wang; Fuyuki Tokumasu; José M. C. Ribeiro; Ivo M. B. Francischetti; Ren Lai

doi:10.1160/TH11-01-0029

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2011; 105(06): 1032-1045
DOI: 10.1160/TH11-01-0029

Platelets and Blood Cells

Schattauer GmbH

A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbβ3 or αVβ3 and inhibits platelet aggregation and angiogenesis

Dongying Ma^*

¹Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China

,

Xueqing Xu^*

²Vector Biology Section Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

Su An^*

¹Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China

⁴School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China

,

Huan Liu

¹Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China

,

Xuening Yang

³Malaria Genetics Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

John F. Andersen

²Vector Biology Section Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

Yipeng Wang

¹Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China

,

Fuyuki Tokumasu

³Malaria Genetics Section, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

José M. C. Ribeiro

²Vector Biology Section Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

Ivo M. B. Francischetti

²Vector Biology Section Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

,

Ren Lai

¹Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, China

› Author Affiliations

Abstract

Summary

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet α_IIbβ₃ and endothelial cell α_Vβ₃ integrins, but not for α₅β₁ or α₂β₁. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC₅₀ ~1 nM) and marginally to fibronectin (IC₅₀ ~1 μM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that α_IIbβ₃ binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin α_IIbβ₃ monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block α_IIbβ₃, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Keywords

Haematophagy - blood-sucking - disintegrin - thrombosis - sialogenins

Full Text

References

References
1 Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer 2008; 8: 604-617.
2 Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer 2002; 2: 91-100.
3 Zannetti A, Del Vecchio S, Iommelli F. et al. Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5). Clin Cancer Res 2009; 15: 5224-5233.
4 Huang TF. What have snakes taught us about integrins?. Cell Mol Life Sci 1998; 54: 527-540.
5 Markland FS. Snake venoms and the hemostatic system. Toxicon 1998; 36: 1749-1800.
6 Calvete JJ. Structure-function correlations of snake venom disintegrins. Curr Pharm Des 2005; 11: 829-835.
7 Rahman S, Flynn G, Aitken A. et al. Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin alphaIIbbeta3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery. Biochem J 2000; 345: 701-709.
8 Lu X, Lu D, Scully MF. et al. Structure-activity relationship studies on ADAM protein-integrin interactions. Cardiovasc Hematol Agents Med Chem 2007; 5: 29-42.
9 Sanz L, Bazaa A, Marrakchi N. et al. Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system. Biochem J 2006; 395: 385-392.
10 Fox JW, Serrano SM. Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. FEBS J 2008; 275: 3016-3030.
11 Kini RM, Evans HJ. Structural domains in venom proteins: evidence that metal-loproteinases and nonenzymatic platelet aggregation inhibitors (disintegrins) from snake venoms are derived by proteolysis from a common precursor. Toxicon 1992; 30: 265-293.
12 Watson SP, Auger JM, McCarty OJ. et al. GPVI and integrin alphaIIb beta3 signaling in platelets. J Thromb Haemost 2005; 3: 1752-1762.
13 Ludwig IS, Geijtenbeek TB, van Kooyk Y. Two way communication between neutrophils and dendritic cells. Curr Opin Pharmacol 2006; 6: 408-413.
14 Silva R, D’Amico G, Hodivala-Dilke KM. et al. Integrins: the keys to unlocking angiogenesis. Arterioscler Thromb Vasc Biol 2008; 28: 1703-1713.
15 Jackson SP, Schoenwaelder SM. Antiplatelet therapy: in search of the ‘magic bullet‘. Nat Rev Drug Discov 2003; 2: 775-789.
16 McLane MA, Marcinkiewicz C, Vijay-Kumar S. et al. Viper venom disintegrins and related molecules. Proc Soc Exp Biol Med 1998; 219: 109-119.
17 Swenson S, Ramu S, Markland FS. Anti-angiogenesis and RGD-containing snake venom disintegrins. Curr Pharm Des 2007; 13: 2860-2871.
18 Francischetti IM. Platelet aggregation inhibitors from hematophagous animals. Toxicon 2010; 56: 1130-1144.
19 Ribeiro JM, Francischetti IM. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol 2003; 48: 73-88.
20 Francischetti IM, Sa-Nunes A, Mans BJ. et al. The role of saliva in tick feeding. Front Biosci 2009; 14: 2051-2088.
21 Mans BJ, Louw AI, Neitz AW. Savignygrin, a platelet aggregation inhibitor from the soft tick Ornithodoros savignyi, presents the RGD integrin recognition motif on the Kunitz-BPTI fold. J Biol Chem 2002; 277: 21371-21378.
22 Wang X, Coons LB, Taylor DB. et al. Variabilin, a novel RGD-containing antagonist of glycoprotein IIb-IIIa and platelet aggregation inhibitor from the hard tick Dermacentor variabilis. J Biol Chem 1996; 271: 17785-17790.
23 Mazur P, Henzel WJ, Seymour JL. et al. Ornatins: potent glycoprotein IIb-IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella ornata. Eur J Biochem 1991; 202: 1073-1082.
24 McLane MA, Gabbeta J, Rao AK. et al. A comparison of the effect of decorsin and two disintegrins, albolabrin and eristostatin, on platelet function. Thromb Haemost 1995; 74: 1316-1322.
25 Xu X, Yang H, Ma D. et al. Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands. Mol Cell Proteomics 2008; 7: 582-590.
26 Ma D, Wang Y, Yang H. et al. Anti-thrombosis repertoire of blood-feeding horsefly salivary glands. Mol Cell Proteomics 2009; 8: 2071-2079.
27 Francischetti IM, Saliou B, Leduc M. et al. Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets. Toxicon 1997; 35: 1217-1228.
28 Calvo E, Tokumasu F, Marinotti O. et al. Aegyptin, a novel mosquito salivary gland protein, specifically binds to collagen and prevents its interaction with platelet glycoprotein VI, integrin alpha2beta1, and von Willebrand factor. J Biol Chem 2007; 282: 26928-26938.
29 Quinn M, Deering A, Stewart M. et al. Quantifying GPIIb/IIIa receptor binding using 2 monoclonal antibodies: discriminating abciximab and small molecular weight antagonists. Circulation 1999; 99: 2231-2238.
30 Chang SJ, Chang CN, Chen CW. Occupancy of glycoprotein IIb/IIIa by B-6 vitamers inhibits human platelet aggregation. J Nutr 2002; 132: 3603-3606.
31 Francischetti IM, Mather TN, Ribeiro JM. Tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis. Thromb Haemost 2005; 94: 167-174.
32 Assumpcao TC, Alvarenga PH, Ribeiro JM. et al. Dipetalodipin, a novel multi-functional salivary lipocalin that inhibits platelet aggregation, vasoconstriction, and angiogenesis through unique binding specificity for TXA2, PGF2alpha, and 15(S)-HETE. J Biol Chem 2010; 285: 39001-39012.
33 Sperzel M, Huetter J. Evaluation of aprotinin and tranexamic acid in different in vitro and in vivo models of fibrinolysis, coagulation and thrombus formation. J Thromb Haemost 2007; 5: 2113-2118.
34 Dejana E, Villa S, de Gaetano G. Bleeding time in rats: a comparison of different experimental conditions. Thromb Haemost 1982; 48: 108-111.
35 Subramaniam M, Frenette PS, Saffaripour S. et al. Defects in hemostasis in P-selectin-deficient mice. Blood 1996; 87: 1238-1242.
36 Seymour JL, Henzel WJ, Nevins B. et al. Decorsin. A potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora. J Biol Chem 1990; 265: 10143-10147.
37 Gould RJ, Polokoff MA, Friedman PA. et al. Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc Soc Exp Biol Med 1990; 195: 168-171.
38 Blobel CP, White JM. Structure, function and evolutionary relationship of proteins containing a disintegrin domain. Curr Opin Cell Biol 1992; 4: 760-765.
39 Scarborough RM, Rose JW, Naughton MA. et al. Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J Biol Chem 1993; 268: 1058-1065.
40 Assumpcao TC, Charneau S, Santiago PB. et al. Insight into the Salivary Transcriptome and Proteome of Dipetalogaster maxima. J Proteome Res 2011; 10: 1669-1679.
41 Faili A, Randon J, Francischetti IM. et al. Convulxin-induced platelet aggregation is accompanied by a powerful activation of the phospholipase C pathway. Biochem J 1994; 298: 87-91.
42 Clemetson KJ, Kini RM. Introduction. Toxins and Hemostasis. From bench to bedside. Springer; 2010. pp. 1-9.
43 Cheresh DA, Stupack DG. Regulation of angiogenesis: apoptotic cues from the ECM. Oncogene 2008; 27: 6285-6298.
44 Shattil SJ, Kim C, Ginsberg MH. The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 2010; 11: 288-300.
45 Griffioen AW, Vyth-Dreese FA. Angiostasis as a way to improve immunotherapy. Thromb Haemost 2009; 101: 1025-1031.
46 Marcinkiewicz C. Functional characteristic of snake venom disintegrins: potential therapeutic implication. Curr Pharm Des 2005; 11: 815-827.
47 Munnix IC, Cosemans JM, Auger JM. et al. Platelet response heterogeneity in thrombus formation. Thromb Haemost 2009; 102: 1149-1156.
48 Watson SP. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis. Curr Pharm Des 2009; 15: 1358-1372.
49 Jennings LK. Mechanisms of platelet activation: need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 2009; 102: 248-257.
50 Francischetti IM, Seydel KB, Monteiro RQ. Blood coagulation, inflammation, and malaria. Microcirculation 2008; 15: 81-107.