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Thromb Haemost 2017; 117(06): 1171-1181
DOI: 10.1160/TH16-04-0323
Stroke, Systemic or Venous Thromboembolism
Schattauer GmbH

E-selectin inhibition with GMI-1271 decreases venous thrombosis without profoundly affecting tail vein bleeding in a mouse model

Dorian L. Culmer
1   Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA
,
Misha L. Dunbar
2   Research Animal Resources, University of Minnesota, Minneapolis, Minnesota, USA
,
Angela E. Hawley
3   Department of Surgery, Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Suman Sood
4   Division of Hematology/Oncology, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Robert E. Sigler
5   Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Peter K. Henke
3   Department of Surgery, Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Thomas W. Wakefield
3   Department of Surgery, Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
John L. Magnani
6   GlycoMimetics Inc., Rockville, Maryland, USA
,
Daniel D. Myers Jr.
3   Department of Surgery, Vascular Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA
5   Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA
› Author Affiliations
Further Information

Publication History

Received: 23 April 2016

Accepted after major revision: 21 February 2017

Publication Date:
09 November 2017 (online)

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Summary

Selectins, such as E-selectin (CD62E), function in venous thrombosis by binding and activating immune cells to initiate the coagulation cascade. GMI-1271 is a small molecule antagonist that inhibits E-selectin activity. Here we determine whether inhibition of E-selectin is sufficient to decrease acute venous thrombosis and associated inflammatory events in both prophylactic and treatment protocols without significantly affecting haemostasis. Male C57BL/6 mice underwent surgery for experimental thrombosis induction and were harvested at peak thrombus formation in our animal model, two days post induction. Groups included non-thrombosed true controls, shams, controls, and prophylactic or treatment groups of GMI-1271 (10 mg/kg intraperitoneal BID (twice a day) and low-molecular-weight heparin (LMWH, Lovenox 6 mg/kg subcutaneously (SC), once a day (SID). Compared with control animals, prophylaxis or treatment with LMWH and GMI-1271 in a dose-dependent manner significantly decreased thrombosis. GMI-1271 significantly lowered tail bleeding times when compared to LMWH. GMI-1271 and LMWH prophylactically administered significantly decreased vein wall neutrophil cell extravasation. However, all treatment and prophylactic therapies significantly decreased vein wall monocyte extravasation versus controls. GMI-1271 prophylactic therapy significantly decreased intra-thrombus cell counts versus control animals and other treatment groups. Immunohistochemistry confirmed that both treatments with GMI-1271 and LMWH significantly decreased activated leukocyte migration. GMI-1271 therapy significantly decreased thrombus weight and resulted in significantly lower bleeding times than LMWH. GMI-1271 treated mice showed decreased local and systemic inflammatory effects while modulating neutrophil activation, suggesting that GMI-1271 is a viable therapeutic candidate for venous thrombosis prophylaxis and treatment.

Keywords

E-selectin inhibition - glycomimetic - venous thrombosis - animal models - inflammation
 

  • References

  • 1 Heit JA. The Epidemiology of Venous Thromboembolism in the Community. Arterioscl Thromb Vasc Biol 2008; 28: 370-372.
    CrossrefPubMedGoogle Scholar
  • 2 Lopez JA, Kearon C, Lee AY. Deep venous thrombosis. Haematology 2004; 439-456.
    PubMedGoogle Scholar
  • 3 Hidalgo A, Peired AJ, Wild MK. et al. Complete identification of E-selectin ligands on neutrophils reveals distinct functions of PSGL-1, ESL-1, and CD44. Immunity 2007; 26: 477-489.
    CrossrefPubMedGoogle Scholar
  • 4 Eppihimer MJ, Wolitzky B, Anderson DC. et al. Heterogeneity of expression of E- and P-selectins in vivo. Circ Res 1996; 79: 560-569.
    CrossrefPubMedGoogle Scholar
  • 5 Lorant DE, Topham MK, Whatley RE. et al. Inflammatory roles of P-selectin. J Clin Invest 1993; 92: 559-570.
    CrossrefPubMedGoogle Scholar
  • 6 Myers Jr. D, Farris D, Hawley A. et al. Selectins influence thrombosis in a mouse model of experimental deep venous thrombosis. J Surg Res 2002; 108: 212-221.
    CrossrefPubMedGoogle Scholar
  • 7 Garcia DA, Baglin TP, Weitz JI. et al. Parenteral anticoagulants: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141 (Suppl. 02) e24S-43S.
    CrossrefPubMedGoogle Scholar
  • 8 Crowther MA, Warkentin TE. Bleeding risk and the management of bleeding complications in patients undergoing anticoagulant therapy: focus on new anticoagulant agents. Blood 2008; 111: 4871-4879.
    CrossrefPubMedGoogle Scholar
  • 9 Yeh CH, Gross PL, Weitz JI. Evolving use of new oral anticoagulants for treatment of venous thromboembolism. Blood 2014; 124: 1020-1028.
    CrossrefPubMedGoogle Scholar
  • 10 Academies NRCoTN. Guide for the care and use of laboratory animals. Eighth ed. Washington, D. C.: The National Academies Press; 2011
    Google Scholar
  • 11 Group NCRRGW. Animal research: reporting in vivo experiments: the ARRIVE guidelines. J Physiol 2010; 588: 2519-2521.
    CrossrefPubMedGoogle Scholar
  • 12 Thackray H. GlycoMimetics, Inc. Investigator’s Brochure. Maryland, USA: July 31 2015
    Google Scholar
  • 13 Diaz JA, Alvarado CM, Wrobleski SK. et al. The electrolytic inferior vena cava model (EIM) to study thrombogenesis and thrombus resolution with continuous blood flow in the mouse. Thromb Haemost 2013; 109: 1158-1169.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Diaz JA, Hawley AE, Alvarado CM. et al. Thrombogenesis with continuous blood flow in the inferior vena cava. A novel mouse model. Thromb Haemost 2010; 104: 366-375.
    PubMedGoogle Scholar
  • 15 Culmer DL, Diaz JA, Hawley AE. et al. Circulating and vein wall P-selectin promote venous thrombogenesis during aging in a rodent model. Thromb Res 2013; 131: 42-48.
    CrossrefPubMedGoogle Scholar
  • 16 Broze Jr. GJ, Yin ZF, Lasky N. A tail vein bleeding time model and delayed bleeding in hemophiliac mice. Thromb Haemost 2001; 85: 747-748.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Wakefield TW, Strieter RM, Wilke CA. et al. Venous thrombosis-associated inflammation and attenuation with neutralizing antibodies to cytokines and adhesion molecules. Arterioscl Thromb Vasc Biol 1995; 15: 258-268.
    CrossrefPubMedGoogle Scholar
  • 18 Downing LJ, Strieter RM, Kadell AM. et al. IL-10 regulates thrombus-induced vein wall inflammation and thrombosis. J Immunol 1998; 161: 1471-1476.
    PubMedGoogle Scholar
  • 19 VanLangevelde LA, Anchill SE, Wrobleski SK. et al. Gender differences in deep venous thrombosis in a rat model: a preliminary study. Comp Med 2005; 55: 55-60.
    PubMedGoogle Scholar
  • 20 Blann AD, Nadar SK, Lip GY. The adhesion molecule P-selectin and cardiovascular disease. Eur Heart J 2003; 24: 2166-2179.
    CrossrefPubMedGoogle Scholar
  • 21 Bucek RA, Reiter M, Quehenberger P. et al. The role of soluble cell adhesion molecules in patients with suspected deep vein thrombosis. Blood Coagul Fibrinolysis 2003; 14: 653-657.
    CrossrefPubMedGoogle Scholar
  • 22 Rectenwald JE, Myers Jr. DD, Hawley AE. et al. D-dimer, P-selectin, and micro-particles: novel markers to predict deep venous thrombosis. A pilot study. Thromb Haemost 2005; 94: 1312-1317.
    PubMedGoogle Scholar
  • 23 Ramacciotti E, Blackburn S, Hawley AE. et al. Evaluation of soluble P-selectin as a marker for the diagnosis of deep venous thrombosis. Clin Appl Thromb Haemost 2011; 17: 425-431.
    CrossrefPubMedGoogle Scholar
  • 24 Hou H, Ge Z, Ying P. et al. Biomarkers of deep venous thrombosis. J Thromb Thrombol 2012; 34: 335-346.
    CrossrefPubMedGoogle Scholar
  • 25 Vandy FC, Stabler C, Eliassen AM. et al. Soluble P-selectin for the diagnosis of lower extremity deep venous thrombosis. J Vasc Surg Venous Lymphat Disord 2013; 1: 117-125.
    PubMedGoogle Scholar
  • 26 Myers DD, Hawley AE, Farris DM. et al. P-selectin and leukocyte microparticles are associated with venous thrombogenesis. J Vasc Surg 2003; 38: 1075-1089.
    CrossrefPubMedGoogle Scholar
  • 27 Myers Jr. DD, Henke PK, Bedard PW. et al. Treatment with an oral small molecule inhibitor of P selectin (PSI-697) decreases vein wall injury in a rat stenosis model of venous thrombosis. J Vasc Surg 2006; 44: 625-632.
    CrossrefPubMedGoogle Scholar
  • 28 Myers Jr. DD, Henke PK, Wrobleski SK. et al. P-selectin inhibition enhances thrombus resolution and decreases vein wall fibrosis in a rat model. J Vasc Surg 2002; 36: 928-938.
    CrossrefPubMedGoogle Scholar
  • 29 Magnani JL. The discovery, biology, and drug development of sialyl Lea and sialyl Lex. Arch Biochem Biophys 2004; 426: 122-131.
    CrossrefPubMedGoogle Scholar
  • 30 Musolino C, Alonci A, Allegra A. et al. Increased levels of the soluble adhesion molecule E-selectin in patients with chronic myeloproliferative disorders and thromboembolic complications. Am J Haematol 1998; 57: 109-112.
    CrossrefPubMedGoogle Scholar
  • 31 Aref S, Salama O, Al-Tonbary Y. et al. L and E selectins in acute myeloid leukemia: expression, clinical relevance and relation to patient outcome. Haematology 2002; 7: 83-87.
    PubMedGoogle Scholar
  • 32 Hidalgo A, Chang J, Jang JE. et al. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury. Nature Med 2009; 15: 384-391.
    CrossrefPubMedGoogle Scholar
  • 33 Chase SD, Magnani JL, Simon SI. E-selectin ligands as mechanosensitive receptors on neutrophils in health and disease. Ann Biomed Eng 2012; 40: 849-859.
    CrossrefPubMedGoogle Scholar
  • 34 Winkler IG, Barbier V, Nowlan B. et al. Vascular niche E-selectin regulates haematopoietic stem cell dormancy, self renewal and chemoresistance. Nature Med 2012; 18: 1651-1657.
    CrossrefPubMedGoogle Scholar
  • 35 Dutta P, Hoyer FF, Sun Y. et al. E-Selectin Inhibition Mitigates Splenic HSC Activation and Myelopoiesis in Hypercholesterolemic Mice With Myocardial Infarction. Arterioscl Thromb Vasc Biol 2016 Epub ahead of print.
    PubMedGoogle Scholar
  • 36 Vestweber D, Blanks JE. Mechanisms that regulate the function of the selectins and their ligands. Physiol Rev 1999; 79: 181-213.
    CrossrefPubMedGoogle Scholar
  • 37 Andre P, Hartwell D, Hrachovinova I. et al. Pro-coagulant state resulting from high levels of soluble P-selectin in blood. Proc Natl Acad Sci USA 2000; 97: 13835-13840.
    CrossrefPubMedGoogle Scholar
  • 38 McEver RP. Selectins: initiators of leucocyte adhesion and signalling at the vascular wall. Cardiovasc Res 2015; 107: 331-339.
    CrossrefPubMedGoogle Scholar
  • 39 Burnand KG, Gaffney PJ, McGuinness CL. et al. The role of the monocyte in the generation and dissolution of arterial and venous thrombi. Cardiovasc Surg 1998; 6: 119-125.
    CrossrefPubMedGoogle Scholar
  • 40 Henke PK, Varga A, De S. et al. Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model. Arterioscl Thromb Vasc Biol 2004; 24: 1130-1137.
    CrossrefPubMedGoogle Scholar
  • 41 Sullivan VV, Hawley AE, Farris DM. et al. Decrease in fibrin content of venous thrombi in selectin-deficient mice. J Surg Res 2003; 109: 1-7.
    CrossrefPubMedGoogle Scholar
  • 42 Vandendries ER, Furie BC, Furie B. Role of P-selectin and PSGL-1 in coagulation and thrombosis. Thromb Haemost 2004; 92: 459-466.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 43 von Bruhl ML, Stark K, Steinhart A. et al. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo. J Exp Med 2012; 209: 819-835.
    CrossrefPubMedGoogle Scholar
  • 44 Hidalgo A, Chang J, Jang J-E. et al. Heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury. Nature Med 2009; 15: 384-391.
    CrossrefPubMedGoogle Scholar
  • 45 van Dongen CJ, van den Belt AG, Prins MH. et al. Fixed dose subcutaneous low molecular weight heparins versus adjusted dose unfractionated heparin for venous thromboembolism. Cochrane Database Syst Rev 2004; 4: CD001100.
    PubMedGoogle Scholar
  • 46 Simon SI, Hu Y, Vestweber D. et al. Neutrophil tethering on E-selectin activates beta 2 integrin binding to ICAM-1 through a mitogen-activated protein kinase signal transduction pathway. J Immunol 2000; 164: 4348-4358.
    CrossrefPubMedGoogle Scholar