PDF herunterladen
Thromb Haemost 1994; 72(03): 421-425
DOI: 10.1055/s-0038-1648882
Original Article
Schattauer GmbH Stuttgart

Antithrombotic Effects of Recombinant Human Soluble Thrombomodulin in a Rat Model of Vascular Shunt Thrombosis

Mayumi Ono
The Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan
,
Katsuhiko Nawa
The Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan
,
Yasumasa Marumoto
The Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan
› Institutsangaben
Weitere Informationen

Publikationsverlauf

Received 21. Dezember 1993

Accepted after revision 29. April 1994

Publikationsdatum:
25. Juli 2018 (online)

Auch verfügbar auf
  als PDF herunterladen  Lizenzen und Reprints

Summary

Thrombomodulin on endothelial cells is a cofactor for thrombin-catalyzed activation of protein C. We have investigated the anticoagulant function of recombinant human soluble thombomodulin (rsTM) in a rat model of arterio-venous (AV)-shunt thrombosis. A bolus injection of rsTM 30 s before the induction of AV-shunt thrombosis inhibited the thrombus formation in a dose-dependent manner. The dose of anticoagulant that inhibited thrombus formation by 50% was 0.4 mg/kg rsTMα, 0.15 mg/kg rsTMβ, and 13 U/kg heparin. Recently, we characterized three monoclonal antibodies (moAbs) against human TM whose epitopes are located in the TM epidermal growth factor-like domain (Nawa et al., 1994). moAb 2A2 inhibited thrombin binding to rsTM, and abolished both TM functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation. moAb 1F2 preserved the latter activities as an anticoagulant, but inhibited cofactor activity. moAb 10A3 had no inhibitory effect on either activity. Analysis of the in vivo anticoagulant mechanism of rsTM was facilitated by the availability of these moAbs. After incubation at rsTM/moAb molar ratios of 1:1.25, the effect of the mixtures were examined in the AV-shunt thrombosis model. An injection of 0.8 mg/kg rsTMα or 0.4 mg/kg rsTMβ resulted in a significant reduction on thrombus formation, as expected. moAb 10A3 had no effect on rsTM activity. However, co-injection of rsTM with moAb 1F2 resulted in a significant decrease of the inhibitory activity on thrombus formation. moAb 2A2 essentially abolished the inhibitory effect of rsTM. These moAb effects were observed for both rsTMα and β. These results suggest that the protein C activation accelerated by the injection of rsTM plays an important role for inhibiting thrombus formation in the AV-shunt thrombosis model.

 

  • References

  • 1 Esmon CT. Molecular events that control the protein C anticoagulant pathway. Thromb Haemost 1993; 70: 29-35
    PubMedSuche in Google Scholar
  • 2 Dittman WA, Majerus PW. Structure and function of thrombomodulin: A natural anticoagulant. Blood 1990; 75: 329-336
    PubMedSuche in Google Scholar
  • 3 Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982; 257: 7944-7947
    PubMedSuche in Google Scholar
  • 4 Esmon NL, Carroll RC, Esmon CT. Thrombomodulin blocks the ability of thrombin to activate platelets. J Biol Chem 1983; 258: 12238-12242
    PubMedSuche in Google Scholar
  • 5 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactor for thrombin-catalyzed activation of protein C. J Biol Chem 1982; 257: 859-864
    PubMedSuche in Google Scholar
  • 6 Kisiel W, Canfield WM, Ericsson LH, Davie EW. Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 1977; 16: 5824-5831
    CrossrefPubMedSuche in Google Scholar
  • 7 Vehar GA, Davie EW. Preparation and properties of bovine factor VIII. Biochemistry 1980; 19: 401-410
    CrossrefPubMedSuche in Google Scholar
  • 8 Walker FJ. Regulation of activated protein C by a new protein: A possible function for bovine protein S. J Biol Chem 1980; 255: 5521-5524
    PubMedSuche in Google Scholar
  • 9 Bourin MC, Ohlin AK, Lane DA, Stenflo J, Lindahl U. Relationship between anticoagulant activities and polyanionic properties of rabbit thrombomodulin. J Biol Chem 1988; 263: 8044-8052
    PubMedSuche in Google Scholar
  • 10 Preissner KT, Koyama T, Muller D, Tschopp J, Muller-Berghaus G. Domain structure of the endothelial cell receptor thrombomodulin as deduced from modulation of its anticoagulant functions. J Biol Chem 1990; 265: 4915-4922
    CrossrefPubMedSuche in Google Scholar
  • 11 Bourin MC, Lundgren-Akerlund E, Lindahl U. Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem 1990; 265: 15424-15431
    PubMedSuche in Google Scholar
  • 12 Nawa K, Sakano K, Fujiwara H, Sato Y, Sugiyama N, Teruuchi T, Iwa-moto M, Marumoto Y. Presence and function of chondroitin-4-sulfate on recombinant human soluble thrombomodulin. Biochem Biophys Res Commun 1990; 171: 729-737
    CrossrefPubMedSuche in Google Scholar
  • 13 Nawa K, Ono M, Uchiyama T, Sugiyama N, Marumoto Y. Recombinant human thrombomodulin as a proteoglycan. Trends in Glycoscience and Glycotechnology 1994; 6: 111-120
    CrossrefPubMedSuche in Google Scholar
  • 14 Parkinson JF, Grinnell BW, Moore RE, Hoskins J, Vlahos CJ, Bang NU. Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells. J Biol Chem 1990; 265: 12602-12610
    PubMedSuche in Google Scholar
  • 15 Koyama T, Parkinson JF, Aoki N, Bang NU, Muller-Berghaus G, Preissner KT. Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. Brit J Haematol 1991; 78: 515-522
    CrossrefPubMedSuche in Google Scholar
  • 16 Kumada T, Dittman WA, Majerus PW. A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood 1988; 71: 728-723
    PubMedSuche in Google Scholar
  • 17 Gomi K, Zushi M, Honda G, Kawahara S, Matsuzaki O, Kanabayashi T, Yamamoto S, Maruyama I, Suzuki K. Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice. Blood 1990; 75: 1396-1399
    CrossrefPubMedSuche in Google Scholar
  • 18 Nawa K, Itani T, Ono M, Sakano K, Marumoto Y, Iwamoto M. The glycosaminoglycan of recombinant human soluble thrombomodulin affects antithrombotic activity in a rat model of tissue factor-induced disseminated intravascular coagulation. Thromb Haemost 1992; 67: 366-370
    Thieme ConnectPubMedSuche in Google Scholar
  • 19 Vogel GMT, Meuleman DG, Bourgondien FGM, Hobbelen PMJ. Comparison of two experimental thrombosis models in rats: Effects of four glyco-saminoglycans. Thromb Res 1989; 54: 399-410
    CrossrefPubMedSuche in Google Scholar
  • 20 Peters RF, Lees CM, Mitchell KA, Tweed MF, Talbot MD, Wallis RB. The characterization of thrombus development in an improved model of arteriovenous shunt thrombosis in the rat and the effects of recombinant desul-phatohirudin (CGP 39393), heparin, and Iloprost. Thromb Haemost 1991; 65: 268-274
    Thieme ConnectPubMedSuche in Google Scholar
  • 21 Nawa K, Ono M, Fujiwara H, Sugiyama N, Uchiyama T, Marumoto Y. Monoclonal antibodies against human thrombomodulin whose epitope is located in epidermal growth factor-like domains. Biochim Biophys Acta 1994; 1205: 162-170
    CrossrefPubMedSuche in Google Scholar
  • 22 Ogata M, Wakita K, Kimura K, Marumoto Y, Oh-i K, Shimizu S. High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells. Appl Microbiol Biotechnol 1993; 38: 520-525
    PubMedSuche in Google Scholar
  • 23 Owen WG, Esmon CT, Jackson CM. The conversion of prothrombin to thrombin. J Biol Chem 1974 249: 594-605
    PubMed
  • 24 Ishii H, Nakano M, Tsubouchi J, Ishikawa T, Uchiyama H, Hiraishi S, Tahara C, Miyajima Y, Kazama M. Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thromb Haemost 1990; 63: 157-162
    Thieme ConnectPubMedSuche in Google Scholar
  • 25 Nakane PK. Preparation and standardization of enzyme-labeled conjugates. In: Immunoassays in the clinical laboratory Nakamura RM, Dito WR, Tucker ES. (eds) Alan R Liss Inc; New York: 1975. pp 81-87
  • 26 Lowry OH, Rosebrough NJ, Farr A, Rindall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275
    PubMedSuche in Google Scholar
  • 27 Okamoto S, Hijikata A, Kikumoto R, Tonomura S, Hara H, Ninomiya K, Maruyama A, Sugano M, Tamao Y. Potent inhibition of thrombin by the newly synthesized arginine derivative No. 805. The important of stereo-structure of its hydrophobic carboxamide portion. Biochem Biophys Res Commun 1981; 101: 440-446
    CrossrefPubMedSuche in Google Scholar
  • 28 Solis MM, Cook C, Cook J, Glaser C, Light D, Morser J, Yu S, Fink L, Eidt JF. Intravenous recombinant soluble human thrombomodulin prevents venous thrombosis in a rat model. J Vase Surg 1991; 14: 599-604
    CrossrefPubMedSuche in Google Scholar
  • 29 Gonda Y, Hirata S, Saitoh K, Aoki Y, Mohri M, Gomi K, Sugihara T, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Antithrombotic effect of recombinant human soluble thrombomodulin on endotoxin-induced disseminated intravascular coagulation in rats. Thromb Res 1993; 71: 325-335
    CrossrefPubMedSuche in Google Scholar
  • 30 Hanson SR, Griffin JH, Harker LA, Kelly AB, Esmon CT, Gruber A. Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates. J Clin Invest 1993; 92: 2003-2012
    CrossrefPubMedSuche in Google Scholar
  • 31 Ohishi R, Watanabe N, Aritomi M, Gomi K, Kiyota T, Yamamoto S, Ishida T, Maruyama I. Evidence that the protein C activation pathway amplifies the inhibition of thrombin generation by recombinant human thrombomodulin in plasma. Thromb Haemost 1993; 70: 423-426
    Thieme ConnectPubMedSuche in Google Scholar
  • 32 Bauer KA, Kass BL, Beeler DL, Rosenberg RD. Detection of protein C activation in humans. J Clin Invest 1984; 74: 2033-2041
    CrossrefPubMedSuche in Google Scholar
  • 33 Okafuji T, Maekawa K, Nawa K, Marumoto Y. The cDNA cloning and mRNA expression of rat protein C. Biochim Biophys Acta 1992; 1131: 329-332
    CrossrefPubMedSuche in Google Scholar
  • 34 Gruber A, Griffin JH. Direct detection of activated protein C in blood from human subjects. Blood 1992; 79: 2340-2348
    PubMedSuche in Google Scholar
  • 35 Orthner CL, Kolen B, Drohan WN. A sensitive and facile assay for the measurement of activated protein C activity levels in vivo. Thromb Haemost 1993; 69: 441-447
    Thieme ConnectPubMedSuche in Google Scholar
  • 36 Ono M, Fujiwara H, Okafuji T, Enjoh T, Nawa K. Recombinant rat protein C: Comparative studies of structure, function and synthesis with plasma protein C. Thromb Haemost 1994; 71: 54-61
    PubMedSuche in Google Scholar