HD1, a thrombin- and prothrombin-binding DNA aptamer, inhibits thrombin generation by attenuating prothrombin activation and thrombin feedback reactions

Colin A. Kretz; Karl R. Cuddy; Alan R. Stafford; James C. Fredenburgh; Robin Roberts; Jeffrey I. Weitz

doi:10.1160/TH09-04-0237

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2010; 103(01): 83-96
DOI: 10.1160/TH09-04-0237

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

HD1, a thrombin- and prothrombin-binding DNA aptamer, inhibits thrombin generation by attenuating prothrombin activation and thrombin feedback reactions

Colin A. Kretz

¹Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Henderson Research Centre, Hamilton, Ontario, Canada

,

Karl R. Cuddy

¹Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Henderson Research Centre, Hamilton, Ontario, Canada

,

Alan R. Stafford

¹Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Henderson Research Centre, Hamilton, Ontario, Canada

,

James C. Fredenburgh

¹Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Henderson Research Centre, Hamilton, Ontario, Canada

,

Robin Roberts

¹Departments of Medicine and Biochemistry and Biomedical Sciences, McMaster University and Henderson Research Centre, Hamilton, Ontario, Canada

,

Jeffrey I. Weitz^*

› Author Affiliations

Abstract

Summary

HD1, a DNA aptamer, binds exosite 1 on thrombin and blocks its clotting activity. Because HD1 also binds prothrombin and inhibits its activation by prothrombinase, we hypothesised that HD1 would be a more potent inhibitor of coagulation than other exosite 1-directed ligands, such as Hir^54–65(SO₃ ^-). Supporting this concept, the effect of HD1 on the prothrombin time and activated partial thromboplastin time was twofold greater than that of Hir^54–65(SO₃ ^-) even though both agents inhibited thrombin-mediated factor (F) V and FVIII activation to a similar extent. In thrombin generation assays, HD1 (a) delayed the lag time, (b) reduced peak thrombin concentration, and (c) decreased endogenous thrombin potential to a greater extent than Hir^54–65(SO₃ ^-). To eliminate thrombin feedback, studies were repeated in FV- and/or FVIII-deficient plasma supplemented with FVa and/or FVIIIa. Only HD1 prolonged the lag time in FV- and FVIII-deficient plasma supplemented with FVa and FVIIIa. In contrast, HD1 and Hir^54–65(SO₃ ^-) inhibited the lag time in FVIII-deficient plasma supplemented with FVIIIa and in normal plasma. The more potent anticoagulant properties of HD1, therefore, reflect its capacity to attenuate FV activation by thrombin and inhibit prothrombinase assembly. These findings identify prothrombin as a potential target for new anticoagulants.

Keywords

Aptamer - thrombin - thrombin generation assay - cofactor activation - prothrombinase

Full Text

References

References
1 Versteeg HH, Ruf W. Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity. J Biol Chem 2007; 282: 25416-25424.
2 van Dieijen G, Tans G, Rosing J. et al. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433-3442.
3 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine factor V and factor Va to the activity of prothrombinase. J Biol Chem 1979; 254: 10952-10962.
4 van Rijn JLML, Govers-Riemslag JWP, Zwaal RFA. et al. Kinetic studies of prothombin activation: Effect of factor Va and phospholipids on the formation of the enzyme-substrate complex. Biochemistry 1984; 23: 4557-4564.
5 Hemker HC, Al Dieri R, DE Smedt E. et al. Thrombin generation, a function test of the haemostatic-thrombotic system. Thromb Haemost 2006; 96: 553-561.
6 Adams TE, Huntington JA. Thrombin-cofactor interactions: structural insights into regulatory mechanisms. Arterioscler Throm Vasc Biol 2006; 26: 1738-1745.
7 Stubbs MT, Bode W. A player of many parts – The spotlight falls on thrombin’s structure. Thromb Res 1993; 69: 1-58.
8 Nogami K, Zhou Q, Myles T. et al. Exosite-interactive regions in the A1 and A2 do-mains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain. J Biol Chem 2005; 280: 18476-18487.
9 Bukys MA, Orban T, Kim PY. et al. The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII. J Biol Chem 2006; 281: 18569-18580.
10 Griffin LC, Toole JJ, Leung LLK. The discovery and characterization of a novel nucleotide-based thrombin inhibitor. Gene 1993; 137: 25-31.
11 Naski MC, Fenton JWI, Maraganore JM. et al. The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen. J Biol Chem 1990; 265: 13484-13489.
12 DiMaio J, Gibbs B, Munn D. et al. Bifunctional thrombin inhibitors based on the sequence of hirudin45–65. J Biol Chem 1990; 265: 21698-21703.
13 Thurieau C, Simonet S, Paladino J. et al. New Nmα-guanidinobenzoyl derivatives of hirudin-54–65 containing stabilized carboxyl or phosphoryl groups on the side chain of phenylalanine-63. J Med Chem 1994; 37: 625-629.
14 Kretz CA, Stafford AR, Fredenburgh JC. et al. HD1, a thrombin-directed aptamer, binds exosite 1 on prothrombin with high affinity and inhibits its activation by prothrombinase. J Biol Chem 2006; 281: 37477-37485.
15 Bloom JW, Nesheim ME, Mann KG. Phospholipid-binding properties of bovine factor V and factor Va. Biochemistry 1979; 18: 4419-4425.
16 Barenholz Y, Gibbes D, Litman BJ. et al. A simple method for the preparation of homogeneous phospholipid vesicles. Biochemistry 1977; 16: 2806-2810.
17 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685.
18 Hemker HC, Beguin S. Thrombin generation in plasma: Its assessment via the endogenous thrombin potential. Thromb Haemost 1995; 74: 134-138.
19 Becker DL, Fredenburgh JC, Stafford AR. et al. Molecular basis for the resistance of fibrin-bound thrombin to inactivation by heparin/serpin complexes. Adv Exp Med Biol 1997; 425: 55-66.
20 Kelinbaum DG, Kupper LL, Muller KE. et al. Applied Regression Analysis and Other Miltivariable Methods. 2nd Ed. PWS; 1988. Boston, MA, USA:
21 Rezaie AR. Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin antithrombin reaction. Biochemistry 1996; 35: 1918-1924.
22 Pospisil CH, Stafford AR, Fredenburgh JC. et al. Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin. J Biol Chem 2003; 278: 21584-21591.
23 Meh DA, Siebenlist KR, Mosesson MW. Identification and characterization of the thrombin binding sites on fibrin. J Biol Chem 1996; 271: 23121-23125.
24 Hemker HC, Al Dieri R, Beguin S. Thrombin generation assays: accruing clinical relevance. Curr Opin Hemat 2004; 11: 170-175.
25 Al Dieri R, Alban S, Beguin S. et al. Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time. J Thromb Haemost 2004; 02: 1395-1401.
26 Anderson PJ, Nesset A, Dharmawardana KR. et al. Characterization of proexosite I on prothrombin. J Biol Chem 2000; 275: 16428-16434.
27 Brummel KE, Paradis SG, Butenas S. et al. Thrombin functions during tissue factor-induced blood coagulation. Blood 2002; 100: 148-152.
28 Mann KG, Brummel-Ziedins K, Orfeo T. et al. Models of blood coagulation. Blood Cells Mol Dis 2006; 36: 108-117.
29 Gerotziafas GT, Elalamy I, Depasse F. et al. In vitro inhibition of thrombin generation, after tissue factor pathway activation, by the oral, direct factor Xa inhibitor rivaroxaban. J Thromb Haemost 2007; 05: 886-888.
30 Wakabayashi H, Fay PJ. Identification of residues contributing to A2 domain-dependent structural stability in factor VIII and factor VIIIa. J Biol Chem 2008; 283: 11645-11651.
31 Wakabayashi H, Varfaj F, Deangelis J. et al. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface. Blood 2008; 112: 2761-2769.
32 Ovanesov MV, Ananyeva NM, Panteleev MA. et al. Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. J Thromb Haemost 2005; 03: 321-331.
33 Anderson PJ, Nesset A, Dharmawardana KR. et al. Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation. J Biol Chem 2000; 275: 16435-16442.
34 Thorelli E, Kaufman RJ, Dahlback B. Cleavage requirements of factor V in tissue-factor induced thrombin generation. Thromb Haemost 1998; 80: 92-98.
35 Allen GA, Wolberg AS, Oliver JA. et al. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J Thromb Haemost 2004; 02: 402-413.
36 Butenas S, Orfeo T, Brummel-Ziedins KE. et al. Influence of bivalirudin on tissue factor-triggered coagulation. Blood Coag Fibrinolysis 2007; 18: 407-414.
37 Croy CH, Koeppe JR, Bergqvist S. et al. Allosteric changes in solvent accessibility observed in thrombin upon active site occupation. Biochemistry 2004; 43: 5246-5255.
38 Duffy EJ, Angliker H, Le Bonniec BF. et al. Allosteric modulation of the activity of thrombin. Biochem J 1997; 321: 361-365.
39 Fredenburgh JC, Stafford AR, Weitz JI. Evidence for allosteric linkage between exosites 1 and 2 of thrombin. J Biol Chem 1997; 272: 25493-25499.
40 Hemker HC, Giesen P, AlDieri R. et al. The calibrated automated thrombogram (CAT): a universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb 2002; 32: 249-253.
41 Sanchez C, Alessi MC, Saut N. et al. Relation between the antithrombin Cambridge II mutation, the risk of venous thrombosis, and the endogenous thrombin generation. J Thromb Haemost 2008; 06: 1975-1977.