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Semin Thromb Hemost 2001; 27(4): 337-348
DOI: 10.1055/s-2001-16887
Copyright © 2001 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel.: +1(212) 584-4662

Production and Characterization of Saratin, an Inhibitor of von Willebrand Factor-Dependent Platelet Adhesion to Collagen

Christopher S. Barnes1 , Bianca Krafft3 , Matthias Frech2 , Uwe R. Hofmann2 , Adam Papendieck3 , Ulrike Dahlems3 , Gerd Gellissen3 , Marc F. Hoylaerts4
  • 1Department of Cardiovascular Research, Merck KGaA, Darmstadt, Germany
  • 2Department of Gene Expression and Biotechnology, Merck KGaA, Darmstadt, Germany
  • 3Rhein Biotech GmbH, Düsseldorf, Germany
  • 4Center for Molecular and Vascular Biology, KULeuven, Leuven, Belgium
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Publication History

Publication Date:
31 August 2001 (online)

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ABSTRACT

Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC50 = 0.23 ± 0.004 and 0.81 ± 0.04 μg mL-1, respectively) and to calf skin collagen (IC50 = 0.44 ± 0.008 μg mL-1). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s-1), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC50 = 0.96 ± 0.25 μg mL-1), but at reduced shear (1300 s-1) a rightward shift in the dose-response curve was noted (IC50 = 5.2 ± 1.4 μg mL-1). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (Kd 5 × 10-8 M and 2 × 10-6 M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.

KEYWORD

Saratin - von Willebrand factor - platelet adhesion - antithrombotic agent - Hansenula polymorpha

REFERENCES

  • 1 Sadler J E. Biochemistry and genetics of von Willebrand factor.  Annu Rev Biochem . 1998;  67 395-424
    CrossrefPubMedSearch in Google Scholar
  • 2 Roth G J, Titani K, Hoyer L W, Hickey M J. Localization of binding sites within human von Willebrand factor for monomeric type III collagen.  Biochemistry . 1986;  25 8357-8361
    CrossrefPubMedSearch in Google Scholar
  • 3 Kalafatis M, Takahashi Y, Girma J P, Meyer D. Localization of a collagen-interactive domain of human von Willebrand factor between amino acid residues Gly 911 and Glu 1,365.  Blood . 1987;  70 1577-1583
    PubMedSearch in Google Scholar
  • 4 Pareti F I, Niiya K, McPherson J M, Ruggeri Z M. Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III.  J Biol Chem . 1987;  262 13835-13841
    PubMedSearch in Google Scholar
  • 5 Stel H V, Sakariassen K S, de Groot G P, van Mourik A J, Sixma J J. Von Willebrand factor in the vessel wall mediates platelet adherence.  Blood . 1985;  65 85-90
    PubMedSearch in Google Scholar
  • 6 Turitto V T, Weiss H J, Zimmerman T S, Sussman I I. Factor VIII/von Willebrand factor in subendothelium mediates platelet adhesion.  Blood . 1985;  65 823-831
    PubMedSearch in Google Scholar
  • 7 Sakariassen K S, Nievelstein P F, Coller B S, Sixma J J. The role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium.  Br J Haematol . 1986;  63 681-691
    CrossrefPubMedSearch in Google Scholar
  • 8 Tschopp T B, Weiss H J, Baumgartner H R. Decreased adhesion of platelets to subendothelium in von Willebrand's disease.  J Lab Clin Med . 1974;  83 296-300
    PubMedSearch in Google Scholar
  • 9 Sixma J J, van Zanten H G, Huizinga E G. Platelet adhesion to collagen: an update.  Thromb Haemost . 1997;  78 434-438
    PubMedSearch in Google Scholar
  • 10 Moroi M, Jung S M. Platelet receptors for collagen.  Thromb Haemost . 1997;  78 439-444
    PubMedSearch in Google Scholar
  • 11 Sugiyama T, Okuma M, Ushikubi F. A novel platelet aggregating factor found in a patient with defective collagen-induced platelet aggregation and autoimmune thrombocytopenia.  Blood . 1987;  69 1712-1720
    PubMedSearch in Google Scholar
  • 12 Heemskerk J W, Siljander P, Vuist W M. Function of glycoprotein VI and integrin alpha2beta1 in the procoagulant response of single, collagen-adherent platelets.  Thromb Haemost . 1999;  81 782-792
    PubMedSearch in Google Scholar
  • 13 Moroi M, Jung S M, Okuma M, Shinmyozu K. A patient with platelets deficient in glycoprotein VI that lack both collagen-induced aggregation and adhesion.  J Clin Invest . 1989;  84 1440-1445
    CrossrefPubMedSearch in Google Scholar
  • 14 Nieuwenhuis H K, Akkerman J W, Houdijk W P, Sixma J J. Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia.  Nature . 1985;  318 470-472
    CrossrefPubMedSearch in Google Scholar
  • 15 Tandon N N, Kralisz U, Jamieson G A. Identification of glycoprotein IV (CD36) as a primary receptor for platelet-collagen adhesion.  J Biol Chem . 1989;  264 7576-7583
    PubMedSearch in Google Scholar
  • 16 Chiang T M, Rinaldy A, Kang A H. Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.  J Clin Invest . 1997;  100 514-521
    PubMedSearch in Google Scholar
  • 17 Kulkarni S, Dopheide S M, Yap C L. A revised model of platelet aggregation.  J Clin Invest . 2000;  105 783-791
    CrossrefPubMedSearch in Google Scholar
  • 18 Madan M, Tcheng J E. Coronary intervention and glycoprotein IIb/IIIa integrin blockade.  Coronary Art Dis . 1999;  10 553-560
    PubMedSearch in Google Scholar
  • 19 Frangos S G, Chen A H, Sumpio B. Vascular drugs in the new millennium.  J Am Coll Surg . 2000;  191 76-92
    CrossrefPubMedSearch in Google Scholar
  • 20 Alevriadou B R, Moake J L, Turner N A. Real-time analysis of shear-dependent thrombus formation and its blockade by inhibitors of von Willebrand factor binding to platelets.  Blood . 1993;  81 1263-1276
    PubMedSearch in Google Scholar
  • 21 Turner N A, Moake J L, Kamat S G. Comparative real-time effects on platelet adhesion and aggregation under flowing conditions of in vivo aspirin, heparin, and monoclonal antibody fragment against glycoprotein IIb-IIIa.  Circulation . 1995;  91 1354-1362
    CrossrefPubMedSearch in Google Scholar
  • 22 Ferns G A, Raines E W, Sprugel K H. Inhibition of neointimal smooth muscle accumulation after angioplasty by an antibody to PDGF.  Science . 1991;  253 1129-1132
    PubMedSearch in Google Scholar
  • 23 Bilder G, Wentz T, Leadley R. Restenosis following angioplasty in the swine coronary artery is inhibited by an orally active PDGF-receptor tyrosine kinase inhibitor, RPR101511A.  Circulation . 1999;  99 3292-3299
    PubMedSearch in Google Scholar
  • 24 Margulis T, David M, Maor N. The von Willebrand factor in myocardial infarction and unstable angina: a kinetic study.  Thromb Haemost . 1986;  55 366-368
    PubMedSearch in Google Scholar
  • 25 Jansson J H, Nilsson T K, Johnson O. Von Willebrand factor in plasma: a novel risk factor for recurrent myocardial infarction and death.  Br Heart J . 1991;  66 351-355
    PubMedSearch in Google Scholar
  • 26 Goto S, Sakai H, Goto M. Enhanced shear-induced platelet aggregation in acute myocardial infarction.  Circulation . 1999;  99 608-613
    CrossrefPubMedSearch in Google Scholar
  • 27 Sakai H, Goto S, Kim J-Y. Plasma concentration of von Willebrand factor in acute myocardial infarction.  Thromb Haemost . 2000;  84 204-209
    PubMedSearch in Google Scholar
  • 28 Montalescot G, Philippe F, Ankri A. Early increase of von Willebrand factor predicts adverse outcome in unstable coronary artery disease: beneficial effects of enoxaparin. French Investigators of the ESSENCE Trial.  Circulation . 1998;  98 294-299
    PubMedSearch in Google Scholar
  • 29 Yamamoto H, Vreys I, Stassen J M. Antagonism of vWF inhibits both injury induced arterial and venous thrombosis in the hamster.  Thromb Haemost . 1998;  79 202-210
    PubMedSearch in Google Scholar
  • 30 Schaefer S, Piontek M, Ahn S J. Recombinant hepatitis B vaccines: Characterization of the viral disease and vaccine production in the methylotrophic yeast, Hansenula polymorpha .  In: Dembowski K, Stadler P, eds. Novel Therapeutic Proteins-Selected Case Studies Weinheim, Germany: Wiley-VCH; 2000: 243-272
    Search in Google Scholar
  • 31 Roggenkamp R, Janowicz Z A, Stanikowski B, Hollenberg C P. Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha Mol Gen Genet .  1984;  194 489-490
    PubMedSearch in Google Scholar
  • 32 Hollenberg C P, Janowicz Z A. DNA molecules coding for FMDH control region. European Patent application. 1987; EPA no. 0299108
  • 33 Amuel C, Gellissen G, Hollenberg C P, Suckow M. Analysis of heatshock promoters in Hansenula polymorpha: The TPS1 promoter, a novel element for heterologous gene expression.  Biotechnol Bioprocess Eng . 2000;  5 247-252
    PubMedSearch in Google Scholar
  • 34 van Dijk R, Faber K N, Kiel J K W A. The methylotrophic yeast Hansenula polymorpha: a versatile cell factory.  Enzyme Microb Tech . 2000;  26 793-800
    CrossrefPubMedSearch in Google Scholar
  • 35 Weydemann U, Keup P, Piontek M. High-level secretion of hirudin by Hansenula polymorpha-authentic processing of three different preprohirudins.  Appl Microbiol Biotechnol . 1995;  44 377-385
    CrossrefPubMedSearch in Google Scholar
  • 36 Zurek C, Kubis E, Keup P. Production of two aprotinin variants in Hansenula polymorpha Process Biochem .  1996;  31 679-689
    PubMedSearch in Google Scholar
  • 37 Dohmen R J, Strasser A WM, Höner C B, Hollenberg C P. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera.  Yeast . 1991;  7 691-692
    CrossrefPubMedSearch in Google Scholar
  • 38 Gellissen G, Hollenberg C P, Janowicz Z A. Gene expression in methylotrophic yeasts. In: Smith A, ed. Gene Expression in Recombinant Microorganisms New York: Marcel Dekker 1995: 195-239
    Search in Google Scholar
  • 39 Roggenkamp R, Hansen H, Eckart M, Janowicz Z A, Hollenberg C P. Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors.  Mol Gen Genet . 1986;  202 302-308
    PubMedSearch in Google Scholar
  • 40 Gellissen G, Janowicz Z A, Weydemann U. High-level expression of foreign genes in Hansenula polymorpha Biotech Adv .  1992;  10 179-189
    PubMedSearch in Google Scholar
  • 41 Gellissen G, Janowicz Z A, Merckelbach A. Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase.  Biotechnology . 1991;  9 291-295
    PubMedSearch in Google Scholar
  • 42 Harsfalvi J, Stassen J M, Hoylaerts M F. Calin from Hirudo medicinalis, an inhibitor of von Willebrand factor binding to collagen under static and flow conditions.  Blood . 1995;  85 705-711
    PubMedSearch in Google Scholar
  • 43 Johnsson B, Lofas S, Lindquist G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors.  Anal Biochem . 1991;  198 268-277
    CrossrefPubMedSearch in Google Scholar
  • 44 Johnsson B, Lindquist G. Advances in Biosensors.  London: JAI Press Ltd; 1992
    Search in Google Scholar
  • 45 Watson S P. Collagen receptor signaling in platelets and megakaryocytes.  Thromb Haemost . 1999;  82 365-376
    PubMedSearch in Google Scholar
  • 46 Baskova I, Zavalova L, Berezhnoy S. Inhibition of induced and spontaneous platelet aggregation by destabilase from medicinal leech.  Platelets . 2000;  11 83-86
    CrossrefPubMedSearch in Google Scholar
  • 47 Connolly T M, Jacobs J W, Condra C. An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. I. Identification, isolation, and characterization.  J Biol Chem . 1992;  267 6893-6898
    PubMedSearch in Google Scholar
  • 48 Keller P M, Schultz L D, Condra C, Karczewski J, Connolly T M. An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression.  J Biol Chem . 1992;  267 6899-6904
    PubMedSearch in Google Scholar
  • 49 Munro R, Jones C P, Sawyer R T. Calin-a platelet adhesion inhibitor from the saliva of the medicinal leech.  Blood Coagul Fibrinolysis . 1991;  2 179-184
    CrossrefPubMedSearch in Google Scholar
  • 50 Depraetere H, Kerekes A, Deckmyn H. The collagen-binding leech products rLAPP and calin prevent both von Willebrand factor and alpha2beta1(GPIa/IIa)-I-domain binding to collagen in a different manner.  Thromb Haemost . 1999;  82 1160-1163
    PubMedSearch in Google Scholar
  • 51 van Zanten H G, Connolly T M, Schiphorst M E. Recombinant leech antiplatelet protein specifically blocks platelet deposition on collagen surfaces under flow conditions.  Arterioscler Thromb Vasc Biol . 1995;  15 1424-1431
    PubMedSearch in Google Scholar
  • 52 Deckmyn H, Stassen J M, Vreys I. Calin from Hirudo medicinalis, an inhibitor of platelet adhesion to collagen, prevents platelet-rich thrombosis in hamsters.  Blood . 1995;  85 712-719
    PubMedSearch in Google Scholar
  • 53 Schaffer L W, Davidson J T, Siegl P K. Recombinant leech antiplatelet protein prevents collagen-mediated platelet aggregation but not collagen graft thrombosis in baboons.  Arterioscler Thromb . 1993;  13 1593-1601
    PubMedSearch in Google Scholar
  • 54 Cruz C P, Eidt J, Drouilhet J. Saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model.  J Vasc Surg 2001 (in press).
    PubMed