Inducing heat shock protein 70 expression provides a robust anti-thrombotic effect with minimal bleeding risk

Mikel Allende; Eva Molina; Ramón Lecumberri; Juan Antonio Sánchez-Arias; Ana Ugarte; Elizabeth Guruceaga; Julen Oyarzabal; José Hermida

doi:10.1160/TH17-02-0108

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2017; 117(09): 1722-1729
DOI: 10.1160/TH17-02-0108

Coagulation and Fibrinolysis

Schattauer GmbH

Inducing heat shock protein 70 expression provides a robust anti-thrombotic effect with minimal bleeding risk

Mikel Allende

¹Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Center for Applied Medical Research (CIMA), IdiSNA, Navarra’s Health Research Institute, University of Navarra, Pamplona, Spain

,

Eva Molina

¹Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Center for Applied Medical Research (CIMA), IdiSNA, Navarra’s Health Research Institute, University of Navarra, Pamplona, Spain

,

Ramón Lecumberri

²Hematology Service, Hospital of the University of Navarra, IdiSNA, Navarra’s Health Research Institute, University of Navarra, Pamplona, Spain Spain

,

Juan Antonio Sánchez-Arias

⁴Small Molecule Discovery Platform, Molecular Therapeutics Program. Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

,

Ana Ugarte

⁴Small Molecule Discovery Platform, Molecular Therapeutics Program. Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

,

Elizabeth Guruceaga

⁵Proteomics, Genomics & Bioinformatics Unit, Center for Applied Medical Research (CIMA), IdiSNA, Navarra’s Health Research Institute, University of Navarra, Pamplona, Spain

,

Julen Oyarzabal

⁴Small Molecule Discovery Platform, Molecular Therapeutics Program. Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

,

José Hermida

¹Division of Cardiovascular Sciences, Laboratory of Thrombosis and Haemostasis, Center for Applied Medical Research (CIMA), IdiSNA, Navarra’s Health Research Institute, University of Navarra, Pamplona, Spain

› Author Affiliations

Abstract

Summary

Antithrombotic medications target coagulation factors. Their use is associated with an increased bleeding risk. Safer drugs are needed. The heat shock protein 70 (Hsp70) exhibits antithrombotic properties that do not influence bleeding. By using murine models, we aimed to test the hypothesis that overexpressing Hsp70 with CM-695, a first in class dual inhibitor of HDAC6 and phosphodiesterase 9, protects against thrombosis while leaves bleeding tendency unaltered. CM-695 was used to induce Hsp70 overexpression. Hsp70 overexpressing mice were submitted to three thrombosis-triggering procedures. The ferric chloride carotid artery model was used to compare the antithrombotic role of CM-695 and rivaroxaban, a direct oral anticoagulant. The mouse tail transection model was used to compare the bleeding tendency upon CM-695 or rivaroxaban administration. Intraperitoneal (i. p.) 20 mg/kg CM-695 increased Hsp70 expression markedly in the murine aortic tissue. This treatment delayed thrombosis in the collagen/epinephrine [p=0.04 (Log-Rank test), n=10], Rose Bengal/laser [median vessel occlusion time (OT): 58.6 vs 39.0 minutes (min) in the control group (CG), p=0.008, n≥10] and ferric chloride (OT: 14.7 vs 9.2 min in the CG, p=0.032, n≥10) models. I.p. 80 mg/kg CM-695 (n≥9) and intravenous 3 mg/kg rivaroxaban (n≥8) significantly delayed thrombosis. CM-695 did not induce bleeding [median bleeding time (BT): 8.5 vs 7.5 min in the CG, n≥10]. However, BT was dramatically increased by rivaroxaban (30.0 vs 13.7 min in the CG, p=0.001, n=10). In conclusion, CM-695 is a new antithrombotic small molecule devoid of bleeding risk that may be envisioned as a useful clinical tool.

Keywords

Thrombosis - antithrombotics - haemorrhage

Full Text

References

References
1 Wysowski DK, Nourjah P, Swartz L. Bleeding complications with warfarin use: a prevalent adverse effect resulting in regulatory action. Arch Intern Med 2007; 167: 1414-1419.
2 Dentali F, Riva N, Crowther M. et al. Efficacy and safety of the novel oral anticoagulants in atrial fibrillation: a systematic review and meta-analysis of the literature. Circulation 2012; 126: 2381-2391.
3 Flaherty ML, Kissela B, Woo D. et al. The increasing incidence of anticoagulant-associated intracerebral haemorrhage. Neurology 2007; 68: 116-121.
4 Sherwood MW, Douketis JD, Patel MR. et al. ROCKET AF Investigators. Outcomes of temporary interruption of rivaroxaban compared with warfarin in patients with nonvalvular atrial fibrillation: results from the rivaroxaban once daily, oral, direct factor Xa inhibition compared with vitamin K antagonism for prevention of stroke and embolism trial in atrial fibrillation (ROCKET AF). Circulation 2014; 129: 1850-1859.
5 Yeh CH, Hogg K, Weitz JI. Overview of the new oral anticoagulants: opportunities and challenges. Arterioscler Thromb Vasc Biol 2015; 35: 1056-1065.
6 Sharma M, Cornelius VR, Patel JP. et al. Efficacy and Harms of Direct Oral Anticoagulants in the Elderly for Stroke Prevention in Atrial Fibrillation and Secondary Prevention of Venous Thromboembolism: Systematic Review and Meta-Analysis. Circulation 2015; 132: 194-204.
7 Chan KE, Edelman ER, Wenger JB. et al. Dabigatran and rivaroxaban use in atrial fibrillation patients on hemodialysis. Circulation 2015; 131: 972-979.
8 Allende M, Molina E, Guruceaga E. et al. Hsp70 protects from stroke in atrial fibrillation patients by preventing thrombosis without increased bleeding risk. Cardiovasc Res 2016; 110: 309-318.
9 Patent WO2014/131855A1 Inventors: Cuadrado-Tejedor MM, Franco R, García-Osta A, Oyarzabal J, Rabal O. Novel Compounds as dual inhibitors of phosphodiesterases and histone deacetylases. Publication Date: 04-September-2014. Application Number: PCT/EP2014/053877
10 Rabal O, Sánchez-Arias JA, Cuadrado-Tejedor M. et al. Design, synthesis, and biological evaluation of first-in-class dual acting histone deacetylases (HDACs) and phosphodiesterase 5 (PDE5) inhibitors for the treatment of Alzheimer’s disease. J Med Chem 2016; 59: 8967-9004.
11 Sánchez-Arias JA, Rabal O, Cuadrado-Tejedor M. et al. Impact of Scaffold Exploration on Novel Dual-Acting Histone Deacetylases and Phosphodiesterase 5 Inhibitors for the Treatment of Alzheimer’s Disease. ACS Chem Neurosci. 2017 in press doi: 10.1021/acschemneuro.6b00370
12 Hutson PH, Finger EN, Magliaro BC. et al. The selective phosphodiesterase 9 (PDE9) inhibitor PF-04447943 (6-((3S,4S)-4-methyl-1-(pyrimidin-2-ylmethyl)pyrrolidin-3-yl)-1-(tetrahydro-2H–pyran-4-yl)-1,5-dihydro-4H–pyrazolo(3,4-d)pyrimidin-4-one) enhances synaptic plasticity and cognitive function in rodents. Neuropharmacology 2011; 61: 665-676.
13 Irizarry RA, Bolstad BM, Collin F. et al. Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 2003; 31: e15
14 Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004 03. Article 3
15 Gene Ontology Consortium. Gene ontology annotations and resources. Nucleic Acids Res 2013; 41: D530-535.
16 Gentleman R, Carey V, Dudoit S. et al. Bioinformatics and computational biology solutions using R and Bioconductor. 2005. Springer; New York, NY:
17 Patent WO2016/120432A1 Inventors Allende M, Hermida J, Montes R, Oyarzabal J. Compounds and methods for anticoagulation therapy. Publication Date: 04-August-2016. Application Number: PCT/EP2016/051892
18 Oyarzabal J, Pastor J, Howe TJ. Optimizing the performance of in silico ADMET general models according to local requirements: MARS approach Solubility estimations as case study. J Chem Inf Model 2009; 49: 2837-2850.
19 Jagadeeswaran P, Cooley BC, Gross PL, Mackman N. Animal Models of Thrombosis From Zebrafish to Nonhuman Primates: Use in the Elucidation of New Pathologic Pathways and the Development of Antithrombotic Drugs. Circ Res 2016; 118: 1363-1379.
20 Wagner NM, Dressel T, Schäfer K, Konstantinides S. Effect of the factor Xa inhibitor rivaroxaban on arterial thrombosis in wild-type and apolipoprotein E-deficient mice. Thromb Res 2012; 130: 793-798.
21 Muller JE, Stone PH, Turi ZG. et al. and the MILIS Study Group. Circadian variation in the frequency of onset of acute myocardial infarction. N Engl J Med 1985; 313: 1315-1322.
22 Tanaka A, Kawarabayashi T, Fukuda D. et al. Circadian variation of plaque rupture in acute myocardial infarction. Am J Cardiol 2004; 93: 1-5.
23 Kovacs JJ, Murphy PJ, Gaillard S. et al. HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol Cell 2005; 18: 601-607.
24 Vihervaara A, Sistonen L. HSF1 at a glance. J Cell Sci 2014; 127: 261-266.
25 Boyault C, Zhang Y, Fritah S. et al. HDAC6 controls major cell response pathways to cytotoxic accumulation of protein aggregates. Genes Dev 2007; 21: 2172-2181.
26 Shrestha L, Young JC. Function and chemotypes of human Hsp70 chaperones. Curr Top Med Chem 2016; 16: 2812-2828.
27 Kurz KD, Main BW, Sandusky GE. Rat model of arterial thrombosis induced by ferric chloride. Thromb Res 1990; 60: 269-280.
28 Rosen ED, Raymond S, Zollman A. et al. Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi. Am J Pathol 2001; 158: 1613-1622.
29 Furie B, Furie BC. Thrombus formation in vivo. J Clin Invest 2005; 115: 3355-3362.
30 Renne T, Nieswandt B, Gailani D. The intrinsic pathway of coagulation is essential for thrombus stability in mice. Blood Cells Mol Dis 2006; 36: 148-151.
31 Tatsumi K, Mackman N. Tissue Factor and Atherothrombosis. J Atheroscler Thromb 2015; 22: 543-549.
32 Williams JC, Mackman N. Tissue factor in health and disease. Front Biosci (Elite Ed) 2012; 04: 358-372.
33 Varon D, Shai E. Platelets and their microparticles as key players in pathophysiological responses. J Thromb Haemost 2015; 13 (Suppl. 01) S40-46.
34 Renné T, Schmaier AH, Nickel KF. et al. In vivo roles of factor XII. Blood 2012; 120: 4296-4303.
35 Mega JL, Simon T. Pharmacology of antithrombotic drugs: an assessment of oral antiplatelet and anticoagulant treatments. Lancet 2015; 386: 281-291.
36 Turpie AG. Rivaroxaban as an oral anticoagulant for stroke prevention in atrial fibrillation. Ther Clin Risk Manag 2014; 10: 197-205.
37 Reddy P, Giugliano RP. The role of rivaroxaban in atrial fibrillation and acute coronary syndromes. J Cardiovasc Pharmacol Ther 2014; 19: 526-532.
38 Antoniou S. Rivaroxaban for the treatment and prevention of thromboembolic disease. J Pharm Pharmacol 2015; 67: 1119-1132.
39 Pérez-Ruiz A, Montes R, Velasco F. et al. Regulation by nitric oxide of endotox-in-induced tissue factor and plasminogen activator inhibitor1 in endothelial cells. Thromb Haemost 2002; 88: 1060-1065.
40 Upmacis RK, Shen H, Benguigui LE. et al. Inducible nitric oxide synthase provides protection against injury-induced thrombosis in female mice. Am J Physiol Heart Circ Physiol 2011; 301: H617-624.
41 Akerfelt M, Morimoto RI, Sistonen L. Heat shock factors: integrators of cell stress, development and lifespan. Nat Rev Mol Cell Biol 2010; 11: 545-555.

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