The purpose of this study was to design and evaluate hirudin (HIR) derivatives with low bleeding risk. In these derivatives, the factor (F) XIa, FXa, and thrombin recognition peptides (EPR, GVYAR, and LGPR, respectively) were linked to the N-terminus of HIR. The intact derivatives have no anticoagulant activity because of the extension of the N-terminus of HIR. After cleavage by the corresponding coagulation factor that occurs on the activation of the coagulation system and in the presence of the thrombus, its activity is released. This limited the anticoagulant activity of these derivatives to the vicinity of the thrombus, and as a result, systemic bleeding complications were avoided. The definite antithrombotic effect and low bleeding parameters of these derivatives were investigated in rat carotid artery and inferior vena cava thrombosis models. In both models, the three derivatives showed significant antithrombotic effects, indicating that anticoagulant activity could be successfully released in vivo. Moreover, the bleeding parameters of these derivatives were lower than that of HIR as indicated by the values of activated partial thromboplastin time (APTT) and thrombin time (TT). To further assess the safety of these derivatives, bleeding time was measured in a mouse tail-cut model. Although the derivatives had obvious effects on bleeding at a dose of 6 mg/kg, the effect of these derivatives on bleeding was significantly weaker than that of HIR at a dose of 1.5 mg/kg. Thus, the benefit-to-risk profiles of the derivatives were superior to that of HIR.
2
Meyer BJ,
Fernandez-Ortiz A,
Mailhac A.
et al. Local delivery of r-hirudin by a double-balloon perfusion catheter prevents mural thrombosis and minimizes platelet deposition after angioplasty. Circulation 1994; 90: 2474-2480.
6
OASIS-2 Investigators.
Effects of recombinant hirudin (lepirudin) compared with heparin on death, myocardial infarction, refractory angina, and revascularization procedures in patients with acute myocardial ischemia without ST elevation: a randomised trial. Organization to Assess Strategies for Ischemic Syndromes (OASIS)-2 Investigators. Lancet 1999; 353: 429-438.
8
Wallace A,
Dennis S,
Hofsteenge J.
et al. Contribution of the N-terminal region of hirudin to its interaction with thrombin. Biochemistry 1989; 28: 10079-10084.
9
Fortkamp E,
Rieger M,
Heisterberg-Moutses G.
et al. Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech. DNA 1986; 5: 511-517.
10
Castillo MJ,
Kurachi K,
Nishino N,
Ohkubo I,
Powers JC.
Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X. Biochemistry 1983; 22: 1021-1029.
11
Scott CF,
Mentzer RL,
Budzynski AZ,
Colman RW.
Human factor XIa cleaves fibrinogen: effects on structure and function. Arch Biochem Biophys 1986; 249: 480-488.
12
Shi BX,
Li JC,
Yu AP.
et al. Two-step ion-exchange chromatographic purification of recombinant hirudin-II and its C-terminal-truncated derivatives expressed in Pichia pastoris
. Proc Biochemistry 2006; 41: 2446-2451.
15
Towbin H,
Staehelin T,
Gordon J.
Electrophoretic transfer of proteins from polyacryamide gels to nitrocelluslose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354.
16
Forstner M,
Peters-Libeu C,
Contreras-Forrest E.
et al. Carboxyl-terminal domain of human apolipoprotein E: Expression, purification, and crystallization. Protein Expr Purif 1999; 17: 267-272.
21
Visconte C,
Sainte-Marie M,
Lorrain J.
et al. SSR182289A enhances thrombolysis induced by fibrinolytic agents in rabbit models of venous and arterial thrombosis. J Thromb Haemost 2004; 2: 629-636.
22
Kawasaki T,
Sato K,
Hirayama F.
et al. Effect of a synthetic factor Xa inhibitor, YM-60828, on blood vessel patency in combination with a thrombolytic agent and on blood loss from the operation site in a rat model of arterial thrombosis. Thromb Haemost 1998; 79: 859-864.
23
Szemraj J,
Stankiewicz A,
Rozmyslowicz-Szerminska W.
et al. A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity-a staphylokinase variant. Thromb Haemost 2007; 97: 1037-1045.
24
Millet J,
Theveniaux J,
Pascal M.
A new experimental model of venous thrombosis in rats involving partial stasis and slight endothelium alterations. Thromb Res 1987; 45: 123-133.
25
Feuerstein GZ,
Toomey JR,
Valocik R.
et al. An inhibitory anti-factor IX antibody effectively reduces thrombus formation in a rat model of venous thrombosis. Thromb Haemost 1988; 60: 236-239.
26
Toomey JR,
Abboud AA,
Valocik RE.
et al. A comparison of the β–D-xyloside, odiparcil, to warfarin in a rat model of venous thrombosis. J Thromb Haemost 2006; 4: 1989-1996.
28
Berry CN,
Lunven C,
Lechaire I.
et al. Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis. Br J Pharmacol 1998; 125: 29-34.
29
Eisenberg PR,
Siegel JE,
Abendschein DR.
et al. Importance of factor Xa in determining the procoagulant activity of whole-blood clots. J Clin Invest 1993; 91: 1877-1883.
32
Jenny RJ,
Mann KG,
Lundblad RL.
A critical review of the methods for cleavage of fusion proteins with thrombin and factor Xa. Protein Expr Purif 2003; 31: 1-11.
