Thromb Haemost 2018; 118(10): 1713-1728
DOI: 10.1055/s-0038-1669785
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

The First Intrinsic Tenase Complex Inhibitor with Serine Protease Structure Offers a New Perspective in Anticoagulant Therapy

Zorica Latinović
1   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
2   Jožef Stefan International Postgraduate School, Ljubljana, Slovenia
Adrijana Leonardi
1   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
Lidija Kovačič
1   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
3   Global Drug Development, Novartis Ireland Ltd, Dublin, Ireland
Cho Yeow Koh
4   Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore
5   Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
Jernej Šribar
1   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
Alenka Trampuš Bakija
6   Division of Pediatrics, University Medical Center Ljubljana, University of Ljubljana, Ljubljana, Slovenia
Divi Venkateswarlu
7   Department of Chemistry, North Carolina Agricultural and Technical State University, Greensboro, North Carolina, United States
R. Manjunatha Kini
4   Protein Science Laboratory, Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore
Igor Križaj
1   Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia
› Author Affiliations
Funding This work was supported by a grant from the Slovenian Research Agency (P1–0207), by the FP7 WeNMR (No. 261572) and H2020 West-Life (No. 675858) grants, and by a grant for the Research Cooperation of Doctoral Students Abroad in Year 2014 (Official Gazette of the Republic of Slovenia, No. 89/14).
Further Information

Publication History

17 May 2018

24 July 2018

Publication Date:
20 September 2018 (online)


Components of the intrinsic blood coagulation pathway, among them factor VIIIa (FVIIIa), have been recognized as suitable therapeutic targets to treat venous thromboembolism, pathological process behind two very serious cardiovascular diseases, deep vein thrombosis and pulmonary embolism. Here, we describe a unique glycoprotein from the nose-horned viper (Vipera ammodytes ammodytes [Vaa]) venom, Vaa serine proteinase homolog 1 (VaaSPH-1), structurally a serine protease but without an enzymatic activity and expressing potent anticoagulant action in human blood. We demonstrated that one of its targets in the blood coagulation system is FVIIIa of the intrinsic tenase complex, where it antagonizes the binding of FIXa. Anticoagulants with such characteristics are intensively sought, as they would be much safer for medical application as the contemporary drugs, which frequently induce excessive bleeding and other complications. VaaSPH-1 is unlikely to be orally available for chronic usage as it has molecular mass of 35 kDa. However, it represents a very promising template to design low molecular mass FVIIIa-directed anticoagulant substances, based on structural features of the interaction surface between VaaSPH-1 and FVIIIa. To this end, we constructed a three-dimensional model of VaaSPH-1 bound to FVIIIa. The model exposes the 157–loop and the preceding α-helix as the most appropriate structural elements of VaaSPH-1 to be considered as a guideline to synthesize small FVIIIa-binding molecules, potential new generation of anticoagulants.

Supplementary Material

  • References

  • 1 Langford Nj, Stansby G, Avital L. The management of venous thromboembolic diseases and the role of thrombophilia testing: summary of NICE Guideline CG144. Acute Med 2012; 11 (03) 138-142
  • 2 Smith ML. The expanding role of direct oral anticoagulants in the management of thromboembolic disease. Drug Top 2016; 4: 54-61
  • 3 Kim JH, Lim K-M, Gwak HS. New anticoagulants for the prevention and treatment of venous thromboembolism. Biomol Ther (Seoul) 2017; 25 (05) 461-470
  • 4 Heit JA, Spencer FA, White RH. The epidemiology of venous thromboembolism. J Thromb Thrombolysis 2016; 41 (01) 3-14
  • 5 Wolberg AS, Rosendaal FR, Weitz JI. , et al. Venous thrombosis. Nat Rev Dis Primers 2015; 1: 15006
  • 6 Kesieme E, Kesieme C, Jebbin N, Irekpita E, Dongo A. Deep vein thrombosis: a clinical review. J Blood Med 2011; 2: 59-69
  • 7 Cheng Y, Liu Z, Yao F. , et al. Current and former smoking and risk for venous thromboembolism: a systematic review and meta-analysis. In: Lowe G, ed. PLoS Med 2013; 10 (09) e1001515 . Doi:10.1371/journal.pmed.1001515
  • 8 Rosendaal FR. Venous thrombosis: the role of genes, environment, and behavior. Hematology (Am Soc Hematol Educ Program) 2005; 1: 1-12
  • 9 Wakefield TW, Caprini J, Comerota AJ. Thromboembolic diseases. Curr Probl Surg 2008; 45 (12) 844-899
  • 10 Peterson JA, Maroney SA, Mast AE. Targeting TFPI for hemophilia treatment. Thromb Res 2016; 141 (Suppl. 02) S28-S30
  • 11 Merli GJ, Groce JB. Pharmacological and clinical differences between low-molecular-weight heparins: implications for prescribing practice and therapeutic interchange. P&T 2010; 35 (02) 95-105
  • 12 Junqueira DR, Zorzela LM, Perini E. Unfractionated heparin versus low molecular weight heparins for avoiding heparin-induced thrombocytopenia in postoperative patients. Cochrane Database Syst Rev 2017; 4: CD007557
  • 13 Levy JH, Spyropoulos AC, Samama CM, Douketis J. Direct oral anticoagulants: new drugs and new concepts. JACC Cardiovasc Interv 2014; 7 (12) 1333-1351
  • 14 McRae SJ, Ginsberg JS. New anticoagulants for the prevention and treatment of venous thromboembolism. Vasc Health Risk Manag 2005; 1 (01) 41-53
  • 15 Mekaj YH, Mekaj AY, Duci SB, Miftari EI. New oral anticoagulants: their advantages and disadvantages compared with vitamin K antagonists in the prevention and treatment of patients with thromboembolic events. Ther Clin Risk Manag 2015; 11: 967-977
  • 16 Tummala R, Kavtaradze A, Gupta A, Ghosh RK. Specific antidotes against direct oral anticoagulants: a comprehensive review of clinical trials data. Int J Cardiol 2016; 214: 292-298
  • 17 Hoffman M. Remodeling the blood coagulation cascade. J Thromb Thrombolysis 2003; 16 (1-2): 17-20
  • 18 Mann KG, Brummel-Ziedins K, Orfeo T, Butenas S. Models of blood coagulation. Blood Cells Mol Dis 2006; 36 (02) 108-117
  • 19 Mackman N. Triggers, targets and treatments for thrombosis. Nature 2008; 451 (7181): 914-918
  • 20 Mackman N. Tissue-specific hemostasis in mice. Arterioscler Thromb Vasc Biol 2005; 25 (11) 2273-2281
  • 21 Gailani D, Renné T. Intrinsic pathway of coagulation and arterial thrombosis. Arterioscler Thromb Vasc Biol 2007; 27 (12) 2507-2513
  • 22 Machlus KR, Lin FC, Wolberg AS. Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice. Blood 2011; 118 (14) 3960-3968
  • 23 Nicolaes GA, Kulharia M, Voorberg J. , et al. Rational design of small molecules targeting the C2 domain of coagulation factor VIII. Blood 2014; 123 (01) 113-120
  • 24 Schwarb H, Tsakiris DA. New direct oral anticoagulants (DOAC) and their use today. Dent J (Basel) 2016; 4 (01) 5
  • 25 Venkateswarlu D. Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: a computational protein-protein docking and molecular dynamics refinement study. Biochem Biophys Res Commun 2014; 452 (03) 408-414
  • 26 Chow FS, Benincosa LJ, Sheth SB. , et al. Pharmacokinetic and pharmacodynamic modeling of humanized anti-factor IX antibody (SB 249417) in humans. Clin Pharmacol Ther 2002; 71 (04) 235-245
  • 27 Toomey JR, Valocik RE, Koster PF. , et al. Inhibition of factor IX(a) is protective in a rat model of thromboembolic stroke. Stroke 2002; 33 (02) 578-585
  • 28 Howard EL, Becker KCD, Rusconi CP, Becker RC. Factor IXa inhibitors as novel anticoagulants. Arterioscler Thromb Vasc Biol 2007; 27 (04) 722-727
  • 29 Vadivel K, Schmidt AE, Marder VJ, Krishnaswamy S, Bajaj S. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2012
  • 30 Eriksson BI, Dahl OE, Lassen MR. , et al; Fixit Study Group. Partial factor IXa inhibition with TTP889 for prevention of venous thromboembolism: an exploratory study. J Thromb Haemost 2008; 6 (03) 457-463
  • 31 Lincoff AM, Mehran R, Povsic TJ. , et al; REGULATE-PCI Investigators. Effect of the REG1 anticoagulation system versus bivalirudin on outcomes after percutaneous coronary intervention (REGULATE-PCI): a randomised clinical trial. Lancet 2016; 387 (10016): 349-356
  • 32 De Caterina R, Husted S, Wallentin L. , et al; European Society of Cardiology Working Group on Thrombosis Task Force on Anticoagulants in Heart Disease. General mechanisms of coagulation and targets of anticoagulants (Section I). Thromb Haemost 2013; 109 (04) 569-579
  • 33 Gómez-Outes A, Suárez-Gea ML, Lecumberri R, Rocha E, Pozo-Hernández C, Vargas-Castrillón E. New parenteral anticoagulants in development. Ther Adv Cardiovasc Dis 2011; 5 (01) 33-59
  • 34 Anastasopoulos C, Sarigiannis Y, Stavropoulos G. A novel approach in potential anticoagulants from peptides epitope 558-565 of A2 subunit of factor VIII. Amino Acids 2013; 44 (04) 1159-1165
  • 35 Slagboom J, Kool J, Harrison RA, Casewell NR. Haemotoxic snake venoms: their functional activity, impact on snakebite victims and pharmaceutical promise. Br J Haematol 2017; 177 (06) 947-959
  • 36 McCleary RJR, Kini RM. Non-enzymatic proteins from snake venoms: a gold mine of pharmacological tools and drug leads. Toxicon 2013; 62: 56-74
  • 37 Mukherjee AK, Mackessy SP. Pharmacological properties and pathophysiological significance of a Kunitz-type protease inhibitor (Rusvikunin-II) and its protein complex (Rusvikunin complex) purified from Daboia russelii russelii venom. Toxicon 2014; 89: 55-66
  • 38 Sajevic T, Leonardi A, Križaj I. An overview of hemostatically active components of Vipera ammodytes ammodytes venom. Toxin Rev 2014; 33 (1–2): 33-36
  • 39 Latinović Z, Leonardi A, Šribar J. , et al. Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera ammodytes ammodytes envenomation: therapeutic implications. J Proteomics 2016; 146: 34-47
  • 40 Leonardi A, Sajevic T, Latinović Z. , et al. Structural and biochemical characterisation of VaF1, a P-IIIa fibrinogenolytic metalloproteinase from Vipera ammodytes ammodytes venom. Biochimie 2015; 109: 78-87
  • 41 Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32 (05) 1792-1797
  • 42 Sajevic T, Leonardi A, Kovačič L. , et al. VaH3, one of the principal hemorrhagins in Vipera ammodytes ammodytes venom, is a homodimeric P-IIIc metalloproteinase. Biochimie 2013; 95 (06) 1158-1170
  • 43 Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011; 127 (Suppl. 03) S21-S25
  • 44 Lancé MD. A general review of major global coagulation assays: thrombelastography, thrombin generation test and clot waveform analysis. Thromb J 2015; 13: 1
  • 45 Kim DE, Chivian D, Baker D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res 2004; 32 (Web Server issue): W526-31
  • 46 Zeng F, Shen B, Zhu Z. , et al. Crystal structure and activating effect on RyRs of AhV_TL-I, a glycosylated thrombin-like enzyme from Agkistrodon halys snake venom. Arch Toxicol 2013; 87 (03) 535-545
  • 47 Šali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 1993; 234 (03) 779-815
  • 48 Fiser A, Do RK, Šali A. Modeling of loops in protein structures. Protein Sci 2000; 9 (09) 1753-1773
  • 49 Johnson DJD, Langdown J, Huntington JA. Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-A structure of the ternary complex. Proc Natl Acad Sci U S A 2010; 107 (02) 645-650
  • 50 Johnson DJD, Li W, Adams TE, Huntington JA. Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation. EMBO J 2006; 25 (09) 2029-2037
  • 51 Berling I, Isbister GK. Hematologic effects and complications of snake envenoming. Transfus Med Rev 2015; 29 (02) 82-89
  • 52 Waheed H, Moin SF, Choudhary MI. Snake venom: from deadly toxins to life-saving therapeutics. Curr Med Chem 2017; 24 (17) 1874-1891
  • 53 Barrett AJ, Rawlings ND. Families and clans of serine peptidases. Arch Biochem Biophys 1995; 318 (02) 247-250
  • 54 Vaiyapuri S, Thiyagarajan N, Hutchinson EG, Gibbins JM. Sequence and phylogenetic analysis of viper venom serine proteases. Bioinformation 2012; 8 (16) 763-772
  • 55 Brummel KE, Paradis SG, Butenas S, Mann KG. Thrombin functions during tissue factor-induced blood coagulation. Blood 2002; 100 (01) 148-152
  • 56 Young G, Sørensen B, Dargaud Y, Negrier C, Brummel-Ziedins K, Key NS. Thrombin generation and whole blood viscoelastic assays in the management of hemophilia: current state of art and future perspectives. Blood 2013; 121 (11) 1944-1950
  • 57 Serrano SMT, Maroun RC. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon 2005; 45 (08) 1115-1132
  • 58 Spencer FA, Becker RC. The prothrombinase complex: assembly and function. J Thromb Thrombolysis 1997; 4 (3/4): 357-364
  • 59 Lentz BR. Exposure of platelet membrane phosphatidylserine regulates blood coagulation. Prog Lipid Res 2003; 42 (05) 423-438
  • 60 Zwaal RF. Membrane and lipid involvement in blood coagulation. Biochim Biophys Acta 1978; 515 (02) 163-205
  • 61 Bevers EM, Rosing J, Zwaal RF. Development of procoagulant binding sites on the platelet surface. Adv Exp Med Biol 1985; 192: 359-371
  • 62 Monroe DM, Hoffman M, Roberts HR. Platelets and thrombin generation. Arterioscler Thromb Vasc Biol 2002; 22 (09) 1381-1389
  • 63 Perot E, Enjolras N, Le Quellec S. , et al. Expression and characterization of a novel human recombinant factor IX molecule with enhanced in vitro and in vivo clotting activity. Thromb Res 2015; 135 (05) 1017-1024
  • 64 Jagannathan I, Ichikawa HT, Kruger T, Fay PJ. Identification of residues in the 558-loop of factor VIIIa A2 subunit that interact with factor IXa. J Biol Chem 2009; 284 (47) 32248-32255
  • 65 Anastasopoulos C, Sarigiannis Y, Stavropoulos G. Cyclic peptide analogs of 558-565 epitope of A2 subunit of Factor VIII prolong aPTT. Toward a novel synthesis of anticoagulants. Amino Acids 2014; 46 (04) 1087-1096
  • 66 Verhamme P, Pakola S, Jensen TJ. , et al. Tolerability and pharmacokinetics of TB-402 in healthy male volunteers. Clin Ther 2010; 32 (06) 1205-1220
  • 67 Arruda VR, Doshi BS, Samelson-Jones BJ. Novel approaches to hemophilia therapy: successes and challenges. Blood 2017; 130 (21) 2251-2256