Download PDF
Thromb Haemost 2016; 115(04): 835-843
DOI: 10.1160/TH15-08-0631
Stroke, Systemic or Venous Thromboembolism
Schattauer GmbH

Pretreatment with rivaroxaban attenuates stroke severity in rats by a dual antithrombotic and anti-inflammatory mechanism

Melanie Dittmeier
1   Department of Neurology, University Hospital Würzburg, Würzburg, Germany
,
Peter Kraft
1   Department of Neurology, University Hospital Würzburg, Würzburg, Germany
,
Michael K. Schuhmann
1   Department of Neurology, University Hospital Würzburg, Würzburg, Germany
,
Felix Fluri   *
1   Department of Neurology, University Hospital Würzburg, Würzburg, Germany
,
Christoph Kleinschnitz   *
1   Department of Neurology, University Hospital Würzburg, Würzburg, Germany
› Author Affiliations Financial support: This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 688, the Interdisziplinäres Zentrum fÜr Klinische Forschung (IZKF), WÜrzburg and Bayer Vital GmbH, Leverkusen, Germany (unrestricted research grant to CK).
Further Information

Publication History

Received: 07 August 2015

Accepted after major revision: 23 September 2015

Publication Date:
29 November 2017 (online)

Also available at
    Permissions and Reprints

Summary

Stroke outcome is more favourable in patients receiving oral anticoagulants compared with non-anticoagulated patients. The reasons for this “stroke-attenuating” property of oral anticoagulants are largely unknown. This study examined whether prestroke anticoagulation with rivaroxaban, a novel direct factor Xa inhibitor, influences stroke severity, thrombin-mediated intracerebral thrombus formation and pro-inflammatory processes in a rat model of brain ischaemia/reperfusion injury. Male Wistar rats were anticoagulated with rivaroxaban and subjected to 90 minutes of transient middle cerebral artery occlusion. Infarct size, functional outcome and the occurrence of intracranial haemorrhage (ICH) were assessed until day 7. Thrombin generation was determined by measuring the amount of thrombin/antithrombin complex. Intracerebral thrombus formation was evaluated by histology and Western blot. CD68-immunoreactivity and the expression of cytokines and adhesion molecules were investigated to assess postischaemic inflammation. The integrity of the blood–brain barrier was analysed using fluorescein isothiocyanate-dextran. Rats pretreated with rivaroxaban developed significantly smaller strokes and less severe functional deficits compared with controls. Although rivaroxaban strongly reduced thrombin-mediated thrombus formation, this was not accompanied by an increased risk of ICH. In addition, rivaroxaban dampened the inflammatory response in the ischaemic brain by downregulating ICAM-1 expression and the activation of CD68+-immune cells. In contrast, rivaroxaban had no effect on the integrity of the blood–brain barrier after stroke. Here, we identified reduced thrombo-inflammation as a major determinant of the stroke-protective property of rivaroxaban in rats. Further studies are needed to assess the therapeutic potential of novel oral anticoagulants in the acute phase after a stroke.

Keywords

tMCAO - experimental stroke - rivaroxaban - thrombin - cerebral inflammation

* Equal contribution/joint senior authors.


 

  • References

  • 1 Hart RG, Pearce LA, Aguilar MI. Meta-analysis: antithrombotic therapy to prevent stroke in patients who have nonvalvular atrial fibrillation. Ann Intern Med 2007; 146: 857-867.
    CrossrefPubMedSearch in Google Scholar
  • 2 Fang MC, Go AS, Chang Y. et al. Warfarin Discontinuation After Starting Warfarin for Atrial Fibrillation. Circ Cardiovasc Qual Outcomes 2010; 03: 624-631.
    CrossrefPubMedSearch in Google Scholar
  • 3 Hylek EM, Evans-Molina C, Shea C. et al. Major Hemorrhage and Tolerability of Warfarin in the First Year of Therapy Among Elderly Patients With Atrial Fibrillation. Circulation 2007; 115: 2689-2696.
    CrossrefPubMedSearch in Google Scholar
  • 4 Patel MR, Mahaffey KW, Garg J. et al. Rivaroxaban versus Warfarin in Nonvalvular Atrial Fibrillation. N Engl J Med 2011; 365: 883-891.
    CrossrefPubMedSearch in Google Scholar
  • 5 Hylek EM, Go AS, Chang Y. et al. Effect of intensity of oral anticoagulation on stroke severity and mortality in atrial fibrillation. N Engl J Med 2003; 349: 1019-1026.
    CrossrefPubMedSearch in Google Scholar
  • 6 O’Donnell M, Oczkowski W, Fang J. et al. Preadmission antithrombotic treatment and stroke severity in patients with atrial fibrillation and acute ischaemic stroke: an observational study. Lancet Neurol 2006; 05: 749-754.
    CrossrefPubMedSearch in Google Scholar
  • 7 Tomita H, Hagii J, Metoki N. et al. Severity and Functional Outcome of Patients with Cardioembolic Stroke Occurring during Non-vitamin K Antagonist Oral Anticoagulant Treatment. J Stroke Cerebrovasc Dis 2015; 24: 1430-1437.
    CrossrefPubMedSearch in Google Scholar
  • 8 Suo Z, Wu M, Citron BA. et al. Persistent Protease-activated Receptor 4 Signaling Mediates Thrombin-induced Microglial Activation. J Biol Chem 2003; 278: 31177-31183.
    CrossrefPubMedSearch in Google Scholar
  • 9 Fan Y, Zhang W, Mulholland M. Thrombin and PAR-1-AP Increase Proinflammatory Cytokine Expression in C6 Cells1. J Surg Res 2005; 129: 196-201.
    CrossrefPubMedSearch in Google Scholar
  • 10 Rahman A, Anwar KN, True AL. et al. Thrombin-Induced p65 Homodimer Binding to Downstream NF-κB Site of the Promoter Mediates Endothelial ICAM-1 Expression and Neutrophil Adhesion. J Immunol 1999; 162: 5466-5476.
    CrossrefPubMedSearch in Google Scholar
  • 11 Sugawara T, Jadhav V, Ayer R. et al. Thrombin Inhibition by Argatroban Ameliorates Early Brain Injury and Improves Neurological Outcomes After Experimental Subarachnoid Hemorrhage in Rats. Stroke 2009; 40: 1530-1532.
    CrossrefPubMedSearch in Google Scholar
  • 12 Dirnagl U. Bench to bedside: the quest for quality in experimental stroke research. J Cereb Blood Flow Metab 2006; 26: 1465-1478.
    CrossrefPubMedSearch in Google Scholar
  • 13 Kubitza D, Becka M, Wensing G. et al. Safety, pharmacodynamics, and pharma-cokinetics of BAY 59-7939-an oral, direct Factor Xa inhibitor-after multiple dosing in healthy male subjects. Eur J Clin Pharmacol 2005; 61: 873-880.
    CrossrefPubMedSearch in Google Scholar
  • 14 Kleinschnitz C, Pozgajova M, Pham M. et al. Targeting Platelets in Acute Experimental Stroke Impact of Glycoprotein Ib, VI, and IIb/IIIa Blockade on Infarct Size, Functional Outcome, and Intracranial Bleeding. Circulation 2007; 115: 2323-2330.
    CrossrefPubMedSearch in Google Scholar
  • 15 Bederson JB, Pitts LH, Tsuji M. et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986; 17: 472-476.
    CrossrefPubMedSearch in Google Scholar
  • 16 Moran PM, Higgins LS, Cordell B. et al. Age-related learning deficits in trans-genic mice expressing the 751-amino acid isoform of human beta-amyloid precursor protein. Proc Natl Acad Sci USA 1995; 92: 5341-5345.
    CrossrefPubMedSearch in Google Scholar
  • 17 Junge CE, Sugawara T, Mannaioni G. et al. The contribution of protease-acti-vated receptor 1 to neuronal damage caused by transient focal cerebral ischemia. Proc Natl Acad Sci USA 2003; 100: 13019-13024.
    CrossrefPubMedSearch in Google Scholar
  • 18 Sun L, Zhou W, Mueller C. et al. Oxygen therapy reduces secondary hemorrhage after thrombolysis in thromboembolic cerebral ischemia. J Cereb Blood Flow Metab 2010; 30: 1651-1660.
    CrossrefPubMedSearch in Google Scholar
  • 19 Kraft P, Göb E, Schuhmann MK. et al. FTY720 Ameliorates Acute Ischemic Stroke in Mice by Reducing Thrombo-Inflammation but Not by Direct Neu-roprotection. Stroke 2013; 44: 3202-3210.
    CrossrefPubMedSearch in Google Scholar
  • 20 Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-AACT Method. Methods 2001; 25: 402-408.
    CrossrefPubMedSearch in Google Scholar
  • 21 Langhauser F, Göb E, Kraft P. et al. Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation. Blood 2012; 120: 4082-4092.
    CrossrefPubMedSearch in Google Scholar
  • 22 Karolczak K, Rozalska S, Wieczorek M. et al. Poly(amido)amine dendrimers generation 4.0 (PAMAM G4) reduce blood hyperglycaemia and restore impaired blood-brain barrier permeability in streptozotocin diabetes in rats. Int J Pharm 2012; 436: 508-518.
    CrossrefPubMedSearch in Google Scholar
  • 23 Heydenreich N, Nolte MW, Göb E. et al. C1-Inhibitor Protects From Brain Ischemia-Reperfusion Injury by Combined Antiinflammatory and Antithrombotic Mechanisms. Stroke 2012; 43: 2457-2467.
    CrossrefPubMedSearch in Google Scholar
  • 24 Schuhmann MK, Kraft P, Stoll G. et al. CD28 superagonist-mediated boost of regulatory T cells increases thrombo-inflammation and ischemic neurodegeneration during the acute phase of experimental stroke. J Cereb Blood Flow Metab Off J Int Soc Cereb Blood Flow Metab 2015; 35: 6-10.
    CrossrefPubMedSearch in Google Scholar
  • 25 Flaherty ML, Kissela B, Woo D. et al. The increasing incidence of anticoagulant-associated intracerebral hemorrhage. Neurology 2007; 68: 116-121.
    CrossrefPubMedSearch in Google Scholar
  • 26 Ploen R, Sun L, Zhou W. et al. Rivaroxaban does not increase hemorrhage after thrombolysis in experimental ischemic stroke. J Cereb Blood Flow Metab 2014; 34: 495-501.
    CrossrefPubMedSearch in Google Scholar
  • 27 Kono S, Yamashita T, Deguchi K. et al. Rivaroxaban and Apixaban Reduce Hemorrhagic Transformation After Thrombolysis by Protection of Neurovascular Unit in Rat. Stroke 2014; 45: 2404-2410.
    CrossrefPubMedSearch in Google Scholar
  • 28 Fluri F, Heinen F, Kleinschnitz C. Intravenous thrombolysis in a stroke patient receiving rivaroxaban. Cerebrovasc Dis Extra 2013; 03: 153-155.
    CrossrefPubMedSearch in Google Scholar
  • 29 Landais A, Ginoux C. Intravenous Thrombolysis for Acute Ischemic Stroke in a Patient Receiving Rivaroxaban. J Stroke Cerebrovasc Dis 2015; 24: e73-74.
    CrossrefPubMedSearch in Google Scholar
  • 30 Ishihara H, Torii H, Imoto H. et al. Intravenous Thrombolysis with Recombinant Tissue Plasminogen Activator in a Stroke Patient Treated with Riva-roxaban. J Stroke Cerebrovasc Dis 2014; 23: e457-459.
    CrossrefPubMedSearch in Google Scholar
  • 31 Perzborn E, Gruber A, Tinel H. et al. Reversal of rivaroxaban anticoagulation by haemostatic agents in rats and primates. Thromb Haemost 2013; 110: 162-172.
    Thieme ConnectPubMedSearch in Google Scholar
  • 32 Striggow F, Riek M, Breder J. et al. The protease thrombin is an endogenous mediator of hippocampal neuroprotection against ischemia at low concentrations but causes degeneration at high concentrations. Proc Natl Acad Sci USA 2000; 97: 2264-2269.
    CrossrefPubMedSearch in Google Scholar
  • 33 Tripathy D, Sanchez A, Yin X. et al. Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia. Front Aging Neurosci 2013; 05: 19.
    PubMedSearch in Google Scholar
  • 34 Huang C-F, Li G, Ma R. et al. Thrombin-induced microglial activation contributes to the degeneration of nigral dopaminergic neurons in vivo. Neurosci Bull 2008; 24: 66-72.
    CrossrefPubMedSearch in Google Scholar
  • 35 Choi S-H, Lee DY, Kim SU. et al. Thrombin-Induced Oxidative Stress Contributes to the Death of Hippocampal Neurons In Vivo: Role of Microglial NADPH Oxidase. J Neurosci 2005; 25: 4082-4090.
    CrossrefPubMedSearch in Google Scholar
  • 36 Rohatgi T, Henrich-Noack P, Sedehizade F. et al. Transient focal ischemia in rat brain differentially regulates mRNA expression of protease-activated receptors 1 to 4. J Neurosci Res 2004; 75: 273-279.
    CrossrefPubMedSearch in Google Scholar
  • 37 Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med 2011; 17: 796-808.
    CrossrefPubMedSearch in Google Scholar
  • 38 Kaplanski G, Marin V, Fabrigoule M. et al. Thrombin-Activated Human endothelial Cells Support Monocyte Adhesion In Vitro Following Expression of Intercellular Adhesion Molecule-1 (ICAM-1; CD54) and Vascular Cell Adhesion Molecule-1 (VCAM-1; CD106). Blood 1998; 92: 1259-1267.
    PubMedSearch in Google Scholar
  • 39 Okada M, Suzuki K, Takada K. et al. Detection of up-regulated genes in throm-bin-stimulated human umbilical vein endothelial cells. Thromb Res 2006; 118: 715-721.
    CrossrefPubMedSearch in Google Scholar