Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis

Daniella M. Mizurini; Ivo M. B. Francischetti; John F. Andersen; Robson Q. Monteiro

doi:10.1160/TH10-03-0186

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2010; 104(06): 1116-1123
DOI: 10.1160/TH10-03-0186

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis^[*]

Authors

Daniella M. Mizurini

¹Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Ivo M. B. Francischetti

²Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
John F. Andersen

²Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
Robson Q. Monteiro

¹Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Abstract

Summary

Nitrophorin 2 (NP2) is a 20 kDa lipocalin identified in the salivary gland of the blood sucking insect, Rhodnius prolixus. It functions as a potent inhibitor of the intrinsic pathway of coagulation upon binding to factor IX (FIX) or FIXa. Herein we have investigated the in vivo antithrombotic properties of NP2. Surface plasmon resonance assays demonstrated that NP2 binds to rat FIX and FIXa with high affinities (K_D = 43 and 47 nM, respectively), and prolongs the aPTT without affecting the PT. In order to evaluate NP2 antithrombotic effects in vivo two distinct models of thrombosis in rats were carried out. In the rose Bengal/laser induced injury model of arterial thrombosis, NP2 increased the carotid artery occlusion time by ≈35 and ≈155%, at doses of 8 and 80 μg/kg, respectively. NP2 also inhibited thrombus formation in an arterio-venous shunt model, showing ≈60% reduction at 400 μg/kg (i.v. administration). The antithrombotic effect lasted for up to 48 hours after a single i.v. dose. Notably, effective doses of NP2 did not increase the blood loss as evaluated by tail-transection model. In conclusion, NP2 is a potent and long-lasting inhibitor of arterial thrombosis with minor effects on haemostasis. It might be regarded as a potential agent for the treatment of human cardiovascular diseases.

Keywords

Animal models - arterial thrombosis - coagulation inhibitors - haemostasis - nitrophorin

Volltext

Referenzen

References
1 Mann KG. Biochemistry and physiology of blood coagulation. Thromb Haemost 1999; 82: 165-174.
2 Owens 3rd AP, Mackman N. Tissue factor and thrombosis: The clot starts here. Thromb Haemost 2010; 104: 432-439.
3 van Dieijen G, van Rijn JL, Govers-Riemslag JW. et al. Assembly of the intrinsic factor X activating complex--interactions between factor IXa, factor VIIIa and phospholipid. Thromb Haemost 1985; 53: 396-400.
4 Lozier JN, Kessler CM. Clinical aspects and therapy of hemophilia. In: Hematology: Basic Principles and Practice. 4th ed. New York: Churchill Livingstone; 2005. pp. 2047-2069.
5 Gailani D. Activation of factor IX by factor XIa. Trends Cardiovasc Med 2000; 10: 198-204.
6 Weitz JI. Emerging anticoagulants for the treatment of venous thromboembolism. Thromb Haemost 2006; 96: 274-284.
7 Gray E, Mulloy B, Barrowcliffe TW. Heparin and low-molecular-weight heparin. Thromb Haemost 2008; 99: 807-818.
8 Ahrens I, Lip GY, Peter K. New oral anticoagulant drugs in cardiovascular disease. Thromb Haemost 2010; 104: 49-60.
9 Levine MN, Raskob G, Beyth RJ. et al. Hemorrhagic complications of anticoagulant treatment: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004; 126: 287S-310S.
10 Gailani D, Renné T. The intrinsic pathway of coagulation: a target for treating thromboembolic disease?. J Thromb Haemost 2007; 5: 1106-1112.
11 Howard EL, Becker KC, Rusconi CP. et al. Factor IXa inhibitors as novel anticoagulants. Arterioscler Thromb Vasc Biol 2007; 27: 722-727.
12 Koh CY, Kini RM. Molecular diversity of anticoagulants from haematophagous animals. Thromb Haemost 2009; 102: 437-453.
13 Ribeiro JM, Francischetti IM. Role of arthropod saliva in blood feeding: sialome and post-sialome perspectives. Annu Rev Entomol 2003; 48: 73-88.
14 Andersen JF, Gudderra NP, Francischetti IM. et al. The role of salivary lipocalins in blood feeding by Rhodnius prolixus . Arch Insect Biochem Physiol 2005; 58: 97-105.
15 Hellmann K, Hawkins RI. Prolixins-S and prolixin-G; two anticoagulants from Rhodnius prolixus . Nature 1965; 207: 265-267.
16 Ribeiro JM, Schneider M, Guimarães JA. Purification and characterization of prolixin S (nitrophorin 2), the salivary anticoagulant of the blood-sucking bug Rhodnius prolixus . Biochem J 1995; 308: 243-9.
17 Sun J, Yamaguchi M, Yuda M. et al. Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus . Thromb Haemost 1996; 75: 573-577.
18 Zhang Y, Ribeiro JM, Guimarães JA. et al. Nitrophorin-2: a novel mixed-type reversible specific inhibitor of the intrinsic factor-X activating complex. Biochemistry 1998; 37: 10681-10690.
19 Isawa H, Yuda M, Yoneda K. et al. The insect salivary protein, prolixin-S, inhibits factor IXa generation and Xase complex formation in the blood coagulation pathway. J Biol Chem 2000; 275: 6636-6641.
20 Gudderra NP, Ribeiro JM, Andersen JF. Structural determinants of factor IX(a) binding in nitrophorin 2, a lipocalin inhibitor of the intrinsic coagulation pathway. J Biol Chem 2005; 280: 25022-25028.
21 Andersen JF, Ding XD, Balfour C. et al. Kinetics and equilibria in ligand binding by nitrophorins 1–4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap. Biochemistry 2000; 39: 10118-10131.
22 Morton TA, Myszka DG, Chaiken IM. Interpreting complex binding kinetics from optical biosensors: a comparison of analysis by linearization, the integrated rate equation, and numerical integration. Anal Biochem 1995; 227: 176-185.
23 Fernandes RS, Kirszberg C, Rumjanek VM. et al. On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells. J Thromb Haemost 2006; 4: 1546-1552.
24 Umetsu T, Sanai K. Effect of 1-methyl-2-mercapto-5-(3-pyridyl)-imidazole (KC-6141), an anti-aggregating compound, on experimental thrombosis in rats. Thromb Haemost 1978; 39: 74-83.
25 Matsuno H, Uematsu T, Nagashima S. et al. Photochemically induced thrombosis model in rat femoral artery and evaluation of effects of heparin and tissue-type plasminogen activator with use of this model. J Pharmacol Methods 1991; 25: 303-317.
26 Herbert JM, Hérault JP, Bernat A. et al. Biochemical and pharmacological properties of SANORG 32701. Comparison with the ‘synthetic pentasaccharide’ (SR 90107/ORG 31540) and standard heparin. Circ Res 1996; 79: 590-600.
27 Ribeiro JM, Hazzard JM, Nussenzveig RH. et al. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science 1993; 260: 539-541.
28 Ribeiro JM, Walker FA. High affinity histamine-binding and antihistaminic activity of the salivary nitric oxide-carrying heme protein (nitrophorin) of Rhodnius prolixus . J Exp Med 1994; 180: 2251-2257.
29 Peters RF, Lees CM, Mitchell KA. et al. The characterisation of thrombus development in an improved model of arterio-venous shunt thrombosis in the rat and the effects of recombinant desulphatohirudin (CGP 39393), heparin, and iloprost. Thromb Haemost 1991; 65: 268-274.
30 Melo FR, Mourão PA. An algal sulfated galactan has an unusual dual effect on venous thrombosis due to activation of factor XII and inhibition of the coagulation proteases. Thromb Haemost 2008; 99: 531-538.
31 Feuerstein GZ, Patel A, Toomey JR. et al. Antithrombotic efficacy of a novel murine antihuman factor IX antibody in rats. Arterioscler Thromb Vasc Biol 1999; 19: 2554-2562.
32 Himber J, Refino CJ, Burcklen L. et al. Inhibition of arterial thrombosis by a soluble tissue factor mutant and active site-blocked factors IXa and Xa in the guinea pig. Thromb Haemost 2001; 85: 475-481.
33 Choudhri TF, Hoh BL, Prestigiacomo CJ. et al. Targeted inhibition of intrinsic coagulation limits cerebral injury in stroke without increasing intracerebral hemorrhage. J Exp Med 1999; 190: 91-99.
34 Decrem Y, Rath G, Blasioli V. et al. Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis. J Exp Med 2009; 206: 2381-2395.
35 Gailani D, Renné T. Intrinsic pathway of coagulation and arterial thrombosis. Arterioscler Thromb Vasc Biol 2007; 27: 2507-2513.
36 Wang X, Cheng Q, Xu L, Feuerstein GZ. et al. Effects of factor IX or factor XI deficiency on ferric chloride-induced carotid artery occlusion in mice. J Thromb Haemost 2005; 3: 695-702.
37 Renné T, Pozgajová M, Grüner S. et al. Defective thrombus formation in mice lacking coagulation factor XII. J Exp Med 2005; 202: 271-281.
38 Gui T, Reheman A, Funkhouser WK. et al. In vivo response to vascular injury in the absence of factor IX: examination in factor IX knockout mice. Thromb Res 2007; 121: 225-234.
39 Benincosa LJ, Chow F-S, Tobia LP. et al. Pharmacokinetics and pharmacodynamics of a humanized monoclonal antibody to factor IX in cynomolgus monkeys. J Pharmacol Exp Ther 2000; 292: 810-816.
40 Nazareth RA, Tomaz LS, Ortiz-Costa S. et al. Antithrombotic properties of Ixolaris, a potent inhibitor of the extrinsic pathway of the coagulation cascade. Thromb Haemost 2006; 96: 7-13.
41 Vlasuk GP, Bradbury A, Lopez-Kinninger L. et al. Pharmacokinetics and anticoagulant properties of the factor VIIa-tissue factor inhibitor recombinant Nematode Anticoagulant Protein c2 following subcutaneous administration in man. Dependence on the stoichiometric binding to circulating factor X. Thromb Haemost 2003; 90: 803-812.
42 Francischetti IM, Valenzuela JG, Andersen JF. et al. Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex. Blood 2002; 99: 3602-3612.
43 Monteiro RQ, Rezaie AR, Ribeiro JM. et al. Ixolaris: a factor Xa heparin-binding exosite inhibitor. Biochem J 2005; 387: 871-877.
44 Monteiro RQ, Rezaie AR, Bae JS. et al. Ixolaris binding to factor X reveals a precursor state of factor Xa heparin-binding exosite. Protein Sci 2008; 17: 146-153.
45 Zingali RB, Bianconi ML, Monteiro RQ. Interaction of bothrojaracin with prothrombin. Haemostasis 2001; 31: 273-278.
46 Monteiro RQ, Zingali RB. Bothrojaracin, a proexosite I ligand, inhibits factor Va-accelerated prothrombin activation. Thromb Haemost 2002; 87: 288-293.
47 Francischetti IM, Seydel KB, Monteiro RQ. Blood coagulation, inflammation, and malaria. Microcirculation 2008; 15: 81-107.
48 Kasthuri RS, Taubman MB, Mackman N. Role of tissue factor in cancer. J Clin Oncol 2009; 27: 4834-4838.

Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis[*]

Authors

Nitrophorin 2, a factor IX(a)-directed anticoagulant, inhibits arterial thrombosis without impairing haemostasis^[*]