Inhibition by Recombinant Hirudins of Experimental Venous Thrombosis and Disseminated Intravascular Coagulation Induced by Tissue Factor in Rats

Summary

Antithrombotic potency of recombinant hirudins rHV2, rHV2–Lys47 and rHV2–Arg47 was studied in a model of experimental thrombosis induced by tissue factor in the rat. Venous thrombosis was induced by i.v. injection of 25 mg/kg tissue factor followed by stasis of the inferior vena cava. In this model natural recombinant hirudins, rHV2 and rHV2–Lys47 injected 5 min before thromboplastin totally inhibited thrombosis in the same μg range as heparin or natural hirudin extracted from leeches. However, the mutant variant rHV2–Arg47 gave a maximal 60% inhibition of thrombosis. Variants rHV2–Lys47 (30 μg/kg) and rHV2–Arg47 (157 μg/kg) injected 5 min before thromboplastin prevented by 90 to 100% the drop in platelet count observed during the disseminated intravascular coagulation induced by thromboplastin injection. Recombinant hirudins were less anticoagulant than heparin as measured by an APTT on rat plasma. After rat tail transection, rHV2–Lys47 caused a 2–fold smaller prolongation of the bleeding time than an equivalent antithrombotic dose of heparin. Plasmatic elimination of rHV2–Lys47 from rat plasma after i.v. injection had a fast distribution phase with a half-life of 3 min during which 90% of injected rHV2–Lys47 was lost and was followed by a slower elimination phase. Thus recombinant hirudin rHV2–Lys47 appears as a promising potent antithrombotic agent for the prevention of thrombin-dependent venous thrombosis and disseminated intravascular coagulation

• References

• 1 Markwardt F. The comeback of hirudin, an old-established anticoagulant agent. Folia Haematol Leipzig 1988; 115: 10-23
• 2 Markwardt F. Hirudin as an inhibitor of thrombin. In: Methods in Enzymology, Vol. 29 Academic Press; New York: 1970. pp 924-932
• 3 Stone SR, Hofsteenge J. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 1986; 25: 4622-4628
• 4 Markwardt F, Hauptmann J, Nowak G, Kleßen C, Walsmann P. Pharmacological studies on the antithrombotic action of hirudin in experimental animals. Thromb Haemostas 1982; 47: 226-229
• 5 Markwardt F, Nowak G, StÜrzebecher J, Grießbaeh U, Walsmann P, Vogel G. Pharmacokinetics and anticoagulant effect of hirudin in man. Thromb Haemostas 1984; 52: 160-163
• 6 Harvey RP, Degryse E, Stefani L, Schamber F, Cazenave J-P, Courtney M, Tolstoshev P, Lecocq J-P. Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech, Hirudo medicinalis. Proc Natl Acad Sci USA 1986; 83: 1084-1088
• 7 Loison G, Findeli A, Bernard S, Nguyen-Juilleret M, Marquet M, Riehl-Bellon N, Carvallo D, Guerra-Santos L, Brown SW, Courtney M, Roitsch C, Lemoine Y. Expression and secretion in S. cerevisiae of biologically active leech hirudin. Biotechnology 1988; 6: 72-7
• 8 Dodt J, Schmitz T, Schafer T, Bergmann C. Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence. FEBS Lett 1986; 202: 373-377
• 9 Bergmann C, Dodt J, Köhler S, Fink E, Gassen HG. Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis . Biol Chem Hoppe-Seyler 1986; 367: 731-740
• 10 Fortkamp E, Rieger M, Heistberg-Moutses G, Schweitzer S, Sommer R. Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech. DNA 1986; 5: 511-517
• 11 Meyhack B, Heim J, Rink H, Zimmermann W, Maerki W. Desulfatohirudin, a specific thrombin inhibitor: expression and secretion in yeast. Thromb Res 1987; Suppl VII 33
• 12 Krstenansky J, Mao SJ T. Antithrombin properties of C-terminus of hirudin using synthetic unsulfated N^α-acetyl-hirudin_45–65 . FEBS Lett 1987; 211: 10-16
• 13 Konno S, Fenton JW II, Villanueva GB. Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin. Arch Biochem Biophys 1988; 267: 158-166
• 14 Braun PJ, Dennis S, Hofsteenge J, Stone SR. Use of site-directed mutagenesis to investigate the basis for the specificity of hirudin. Biochemistry 1988; 27: 6517-6522
• 15 Chang J-Y. The structures and proteolytic specificities of autolysed human thrombin. Biochem J 1986; 240: 797-802
• 16 Chang J-Y. The hirudin binding site of human α-thrombin. Identification of lysyl residues which participate in the combining site of hirudin-thrombin complex. J Biol Chem 1989 264. 7141-7146
• 17 Chang J-Y. The functional domain of hirudin, a thrombin specific inhibitor. FEBS Lett 1983; 164: 307-313
• 18 Dodt J, Köhler S, Baici A. Interaction of site specific hirudin variants with α-thrombin. FEBS Lett 1988; 229: 87-90
• 19 Dejana E, Villa S, de Gaetano G. Bleeding time in rats: a comparison of different experimental conditions. Thromb Haemostas 1982; 48: 108-111
• 20 Riehl-Bellon N, Carvallo D, Acker M, Van Dorsselaer A, Marquet M, Loison G, Lemoine Y, Brown SW, Courtney M, Roitsch C. Purification and biochemical characterization of recombinant hirudin produced by Saccharomyces cerevisiae . Biochemistry 1989; 28: 2941-2949
• 21 Van Dorsselaer A, Lepage P, Bitsch F, Whitechurch O, Riehl-Bellon N, Fraisse D, Green B, Roitsch C. Mass spectrometry analyses of recombinant hirudins (7 kDa). Biochemistry 1989; 28: 2949-2956
• 22 Degryse E, Acker M, Defreyn G, Bernat A, Maffrand J-P, Roitsch C, Courtney M. Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin. Prolein Eng 1989; 2: 459-465
• 23 Freyssinet J-M, Gauchy J, Cazenave J-P. The effect of phospholipids on the activation of protein C by the human thrombin-thrombomodulin complex. Biochem J 1986; 238: 151-157
• 24 Regoeczi E. Iodine-labeled Plasma Proteins. CRC Press; Boca Raton, FL: 1984. pp 49-56
• 25 Singlas E, Tabaret A-M. In:. Abrégé de pharmacocinétique; Hoechst Biologie: 1987. pp 47-50
• 26 Ringler DH, Dabich L. Hematology and clinical biochemistry. In: The Laboratory Rat, Biology and Diseases Vol. 1 Baker HJ, Lindsey JR, Weisbroth SH. eds. Academic Press; New York: 1979. p 108
• 27 Griessbach U, StÜrzebecher J, Markwardt F. Assay of hirudin in plasma using a chromogenic thrombin substrate. Thromb Res 1985; 37: 347-350
• 28 Wessler S, Reimer SM, Sheps MC. Biologic assay of a thrombosis-inducing activity in human serum. J Appl Physiol 1959; 14: 943-946
• 29 Reyers I, Mussoni L, Donati MB, de Gaetano G. Failure of aspirin at different doses to modify experimental thrombosis in rats. Thromb Res 1980; 18: 669-674
• 30 Fareed J, Wallenga JM, Kumar A, Rock A. A modified stasis model to study the antithrombotic actions of heparin and its fractions. Semin Thromb Haemostas 1985; 11: 155-175
• 31 Markwardt F, Fink G, Kaiser B, Klöcking HP, Nowak G, Richter M, StÜrzebecher T. Pharmalogical survey of recombinant hirudin. Pharmazie 1988; 43: 202-207
• 32 Talbot MD, Ambler J, Butler KD, Findlay VS, Mitchell KA, Peters RF, Tweed MF, Wallis RB. Recombinant desulphatohirudin (CGP 39393) anticoagulant and antithrombotic properties in vivo. Thromb Haemostas 1989; 61: 77-80
• 33 Markwardt F, Kaiser B, Nowak G. Studies of antithrombotic effects of recombinant hirudin. Thromb Res 1989; 54: 377-388
• 34 Doutremepuich C, Deharo E, Guyot M, Lalanne MC, Walenga J, Fareed J. Antithrombotic activity of recombinant hirudin in the rat: a comparative study with heparin. Thromb Res 1989; 54: 435-445
• 35 Kaiser B, Markwardt F. Antithrombotic and haemorrhagic effects of synthetic and naturally occurring thrombin inhibitors. Thromb Res 1986; 43: 613-620
• 36 Markwardt F, Nowak G, StÜrzebecher J, Vogel G. Clinico-pharmalogical studies with recombinant hirudin. Thromb Res 1988; 52: 393-400