Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia^[*]

Summary

Venous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1^-/- and TLR4^-/- mice underwent treatment with saline or LPS (10 mg/kg i.p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4^-/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1^-/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated VT.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

TLR4 - cellular adhesion molecules - ICAM-1 - sepsis - LPS - venous thrombosis - venous thromboembolism

^* Presented in part at the 2014 Vascular Research Initiatives Conference and Arteriosclerosis, Vascular Biology and Thrombosis 2014 Annual Scientific Sessions.

• References

• 1 Heit JA, Silverstein MD, Mohr DN. et al. The epidemiology of venous thromboembolism in the community.. Thromb Haemost 2001; 86: 452-463.
• 2 Lagu T, Rothberg MB, Shieh MS. et al. Hospitalisations, costs, and outcomes of severe sepsis in the United States 2003 to 2007.. Crit Care Med 2012; 40: 754-761.
• 3 Bellini G, Teng A, Kotecha N. et al. The identification of risk factors for venous thromboembolism in gastrointestinal oncologic surgery.. J Surg Res 2016; 205: 279-285.
• 4 Bahl V, Hu HM, Henke PK. et al. A validation study of a retrospective venous thromboembolism risk scoring method.. Ann Surg 2010; 251: 344-350.
• 5 Schmidt M, Horvath-Puho E, Thomsen RW. et al. Acute infections and venous thromboembolism.. J Intern Med 2012; 271: 608-618.
• 6 Kaplan D, Casper TC, Elliott CG. et al. VTE Incidence and Risk Factors in Patients With Severe Sepsis and Septic Shock.. Chest 2015; 148: 1224-1230.
• 7 Bloemenkamp KW, Rosendaal FR, Helmerhorst FM, Buller HR, Vandenbroucke JP. Enhancement by factor V Leiden mutation of risk of deep-vein thrombosis associated with oral contraceptives containing a third-generation progestagen. Lancet 1995; 346: 1593-1596.
• 8 Dellinger RP, Levy MM, Rhodes A. et al. Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012.. Crit Care Med 2013; 41: 580-637.
• 9 Shojania KG, Duncan BW, McDonald KM. et al. Making health care safer: a critical analysis of patient safety practices.. Evid Rep Technol Assess 2001; I x 1-668.
• 10 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.
• 11 Massberg S, Grahl L, von Bruehl ML. et al. Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.. Nat Med 2010; 16: 887-896.
• 12 Zarbock A, Ley K, McEver RP, Hidalgo A. Leukocyte ligands for endothelial selectins: specialised glycoconjugates that mediate rolling and signaling under flow. Blood 2011; 118: 6743-6751.
• 13 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.
• 14 Wakefield TW, Myers DD, Henke PK. Mechanisms of venous thrombosis and resolution. Arterioscler Thromb Vasc Biol 2008; 28: 387-391.
• 15 Zonneveld R, Martinelli R, Shapiro NI. et al. Soluble adhesion molecules as markers for sepsis and the potential pathophysiological discrepancy in neonates, children and adults.. Crit Care 2014; 18: 204.
• 16 Fijen JW, Kobold AC, de Boer P. et al. Leukocyte activation and cytokine production during experimental human endotoxaemia.. Eur J Intern Med 2000; 11: 89-95.
• 17 Rossignol DP, Lynn M. TLR4 antagonists for endotoxaemia and beyond. Curr Opin Investig Drugs 2005; 06: 496-502.
• 18 Cox LA, van Eijk LT, Ramakers BP. et al. Inflammation-induced increases in plasma endocan levels are associated with endothelial dysfunction in humans in vivo.. Shock 2015; 43: 322-326.
• 19 Ao L, Song Y, Fullerton DA. et al. The interaction between myocardial depressant factors in endotoxaemic cardiac dysfunction: role of TNF-alpha in TLR4-mediated ICAM-1 expression.. Cytokine 2007; 38: 124-129.
• 20 Darbousset R, Thomas GM, Mezouar S. et al. Tissue factor-positive neutrophils bind to injured endothelial wall and initiate thrombus formation.. Blood 2012; 120: 2133-2143.
• 21 Raeburn CD, Calkins CM, Zimmerman MA. et al. ICAM-1 and VCAM-1 mediate endotoxaemic myocardial dysfunction independent of neutrophil accumulation.. Am J Physiol Regul Integr Comp Physiol 2002; 283: R477-486.
• 22 Yipp BG, Petri B, Salina D. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.. Nat Med 2012; 18: 1386-1393.
• 23 Moir E, Greaves M, Adey GD, Bennett B. Polymorphonuclear leukocytes from patients with severe sepsis have lost the ability to degrade fibrin via u-PA. J Leukoc Biol 2004; 76: 571-576.
• 24 Engelmann B, Massberg S. Thrombosis as an intravascular effector of innate immunity. Nat Rev Immunol 2013; 13: 34-45.
• 25 Cicala C, Santacroce C, Itoh H. et al. A study on rat platelet responsiveness following intravenous endotoxin administration.. Life Sci 1997; 60: PL31-38.
• 26 Rumbaut RE, Bellera RV, Randhawa JK. et al. Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism.. Am J Physiol Heart Circ Physiol 2006; 290: H1671-1679.
• 27 Kanaan Z, Gardner S, Carruba C. et al. Macrophage Genetic Reprogramming During Chronic Peritonitis is Augmented by LPS Pretreatment.. J Surg Res 2012; 175: 289-297.
• 28 Liu Z, Miner JJ, Yago T. et al. Differential regulation of human and murine P-selectin expression and function in vivo.. J Exp Med 2010; 207: 2975-2987.
• 29 Parant MA, Pouillart P, Le Contel C. et al. Selective modulation of lipopolysaccharide-induced death and cytokine production by various muramyl peptides.. Infect Immun 1995; 63: 110-115.
• 30 Runnels JM, Zamiri P, Spencer JA. et al. Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy.. Mol Imaging 2006; 05: 31-40.
• 31 Halin C, Mora JR, Sumen C, von Andrian UH. In vivo imaging of lymphocyte trafficking. Ann Rev Cell Develop Biol 2005; 21: 581-603.
• 32 Wojcik BM, Wrobleski SK, Hawley AE. et al. Interleukin-6: a potential target for post-thrombotic syndrome.. Ann Vasc Surg 2011; 25: 229-239.
• 33 Henke PK, Varma MR, Moaveni DK. et al. Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis.. Thromb Haemost 2007; 98: 1045-1055.
• 34 Aleman MM, Byrnes JR, Wang JG. et al. Factor XIII activity mediates red blood cell retention in venous thrombi.. J Clin Invest 2014; 124: 3590-3600.
• 35 Magnani JL. The discovery, biology, and drug development of sialyl Lea and sialyl Lex. Arch Biochem Biophys 2004; 426: 122-131.
• 36 McAvoy EF, McDonald B, Parsons SA. et al. The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxaemia.. J Immunol 2011; 186: 2592-2601.
• 37 Pannucci CJ, Laird S, Dimick JB. et al. A Validated Risk Model to Predict 90-Day VTE Events in Postsurgical Patients.. Chest 2014; 145: 567-573.
• 38 Pannucci CJ, Obi A, Alvarez R. et al. Inadequate Venous Thromboembolism Risk Stratification Predicts Venous Thromboembolic Events in Surgical Intensive Care Unit Patients.. J Am Coll Surg 2014; 218: 898-904.
• 39 Myers D, Wrobleski S, Londy F. et al. New and effective treatment of experimentally induced venous thrombosis with anti-inflammatory rPSGL-Ig.. Thromb Haemost 2002; 87: 374-382.
• 40 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.
• 41 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.
• 42 Wakefield TW, Strieter RM, Wilke CA. et al. Venous thrombosis-associated inflammation and attenuation with neutralising antibodies to cytokines and adhesion molecules.. Arterioscler Thromb Vasc Biol 1995; 15: 258-268.
• 43 Eriksson EE, Karlof E, Lundmark K. et al. Powerful inflammatory properties of large vein endothelium in vivo.. Arterioscler Thromb Vasc Biol 2005; 25: 723-728.
• 44 Patel KN, Soubra SH, Bellera RV. et al. Differential role of von Willebrand factor and P-selectin on microvascular thrombosis in endotoxaemia.. Arterioscler Thromb Vasc Biol 2008; 28: 2225-2230.
• 45 Slotta JE, Braun OO, Menger MD, Thorlacius H. Capture of platelets to the endothelium of the femoral vein is mediated by CD62P and CD162. Platelets 2009; 20: 505-512.
• 46 Medzhitov R, Preston-Hurlburt P, Janeway Jr. CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997; 388: 394-397.
• 47 Hoshino K, Takeuchi O, Kawai T. et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.. J Immunol 1999; 162: 3749-3752.
• 48 Qureshi ST, Lariviere L, Leveque G. et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4).. J Exp Med 1999; 189: 615-625.
• 49 Stark RJ, Aghakasiri N, Rumbaut RE. Platelet-derived Toll-like receptor 4 (Tlr-4) is sufficient to promote microvascular thrombosis in endotoxaemia. PLoS One 2012; 07: e41254.
• 50 Ren M, Li R, Luo M. et al. Endothelial cells but not platelets are the major source of Toll-like receptor 4 in the arterial thrombosis and tissue factor expression in mice.. Am J Physiol Regul Integr Comp Physiol 2014; 307: R901-907.
• 51 Ma AC, Kubes P. Platelets, neutrophils, and neutrophil extracellular traps (NETs) in sepsis. J Thromb Haemost 2008; 06: 415-420.
• 52 Margraf S, Logters T, Reipen J. et al. Neutrophil-derived circulating free DNA (cf-DNA/NETs): a potential prognostic marker for posttraumatic development of inflammatory second hit and sepsis.. Shock 2008; 30: 352-358.
• 53 Brill A, Fuchs TA, Savchenko AS. et al. Neutrophil extracellular traps promote deep vein thrombosis in mice.. J Thromb Haemost 2012; 10: 136-144.
• 54 Fuchs TA, Brill A, Wagner DD. Neutrophil extracellular trap (NET) impact on deep vein thrombosis. Arterioscler Thromb Vasc Biol 2012; 32: 1777-1783.
• 55 Fuchs TA, Brill A, Duerschmied D. et al. Extracellular DNA traps promote thrombosis.. Proc Natl Acad Sci USA 2010; 107: 15880-15885.
• 56 Brill A, Fuchs TA, Savchenko A. et al. Neutrophil Extracellular Traps Promote Deep Vein Thrombosis in Mice.. J Thromb Haemost 2012; 10: 136-144.
• 57 Varma MR, Varga AJ, Knipp BS. et al. Neutropenia impairs venous thrombosis resolution in the rat.. J Vasc Surg 2003; 38: 1090-1098.
• 58 Demers M, Krause DS, Schatzberg D. et al. Cancers predispose neutrophils to release extracellular DNA traps that contribute to cancer-associated thrombosis.. Proc Natl Acad Sci USA 2012; 109: 13076-13081.
• 59 El-Sayed OM, Dewyer NA, Luke CE. et al. Intact Toll-like receptor 9 signaling in neutrophils modulates normal thrombogenesis in mice.. J Vasc Surg 2016; 64: 1450-1458.
• 60 Wagner JG, Roth RA. Neutrophil migration during endotoxaemia. J Leukoc Biol 1999; 66: 10-24.
• 61 Aird WC. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 2003; 101: 3765-3777.
• 62 Aleman MM, Gardiner C, Harrison P, Wolberg AS. Differential contributions of monocyte- and platelet-derived microparticles towards thrombin generation and fibrin formation and stability. J Thromb Haemost 2011; 09: 2251-2261.
• 63 Henke PK, Varma MR, Deatrick KB. et al. Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularisation.. Thromb Haemost 2006; 95: 272-281.
• 64 Semeraro N, Ammollo CT, Semeraro F, Colucci M. Sepsis, thrombosis and organ dysfunction. Thromb Res 2012; 129: 290-295.
• 65 Wu K, Urano T, Ihara H. et al. The cleavage and inactivation of plasminogen activator inhibitor type 1 by neutrophil elastase: the evaluation of its physiologic relevance in fibrinolysis.. Blood 1995; 86: 1056-1061.
• 66 Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320: 365-376.
• 67 Obi AT, Diaz JA, Ballard-Lipka NL. et al. Low-molecular-weight heparin modulates vein wall fibrotic response in a plasminogen activator inhibitor 1-dependent manner.. J Vasc Surg Ven Lymph Dis 2014; 02: 441-450 e1.
• 68 Obi AT, Diaz JA, Ballard-Lipka NL. et al. Plasminogen activator-1 overexpression decreases experimental postthrombotic vein wall fibrosis by a non-vitronectin-dependent mechanism.. J Thromb Haemost 2014; 12: 1353-1363.
• 69 Zeerleder S, Schroeder V, Lammle B. et al. Factor XIII in severe sepsis and septic shock.. Thromb Res 2007; 119: 311-318.
• 70 Lee SY, Chang SK, Lee IH. et al. Depletion of plasma factor XIII prevents disseminated intravascular coagulation-induced organ damage.. Thromb Haemost 2001; 85: 464-469.
• 71 Diaz JA, Obi AT, Myers Jr. DD. et al. Critical review of mouse models of venous thrombosis.. Arterioscler Thromb Vasc Biol 2012; 32: 556-562.
• 72 Manly DA, Boles J, Mackman N. Role of tissue factor in venous thrombosis. Ann Rev Physiol 2011; 73: 515-525.
• 73 Carraway MS, Welty-Wolf KE, Miller DL. et al. Blockade of tissue factor: treatment for organ injury in established sepsis.. Am J Respir Crit Care Med 2003; 167: 1200-1209.
• 74 Yuki K, Bu W, Xi J. et al. Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1.. FASEB J 2012; 26: 4408-4417.