Background: Since thrombolysis is the only approved intervention for ischemic stroke, improving its efficacy and safety is a therapeutic aim of considerable interest. The activated form of thrombin activatable fibrinolysis inhibitor (TAFI) has antifibrinolytic effects, and inhibition of TAFI might thus favor recanalization. The present study compared efficacy between TAFI inhibition alone and TAFI inhibition in combination with rtPA at a suboptimal dose, in a murine model of thromboembolic stroke. Methods: Focal ischemia was induced in mice by thrombin injection in the middle cerebral artery. Animals were placed within the magnet immediately after surgery for baseline MRI (H0). MRI examination comprised diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and T2-weighted imaging (T2-WI). Animals were randomly assigned to 1 of 5 treatment groups: saline, rtPA 5 mg/kg (tPA5: suboptimal or low dose), rtPA 10 mg/kg (tPA10: standard dose), TAFI-I 100 mg/kg (TAFI-I), and rtPA 5 mg/kg + TAFI-I 100 mg/kg (tPA5 + TAFI-I). Treatments were administered inside the magnet, via a catheter placed in the tail vein, using a power injector, as 10% bolus and 90% infusion over a period of 20 min. MRI examination was repeated at 3 h (H3) and 24 h (H24) after surgery. Therapeutic benefit was evaluated by: (1) improvement of reperfusion and (2) reduction in final lesion size. Microhemorrhages were assessed as black spots on T2-WI at H24. Animals were sacrificed after the last MR examination. The surgeon and all investigators were blinded to treatment allocation. Results: A total of 104 mice were operated on. Forty four of these were excluded from the study and 27 from the analysis, according to a priori defined criteria (no lesion or no mismatch), leading to the following distribution: saline (n = 6), tPA5 (n = 8), tPA10 (n = 7), TAFI-I (n = 7), and TAFI-I + tPA5 (n = 5). Standard-dose rtPA treatment (tPA10) significantly improved lesion regression between H0 and H24 compared to saline (-57 ± 18% vs. -36 ± 21%, p = 0.03), which treatment with rtPA5 or TAFI-I alone did not. On the other hand, combined treatment with tPA5 + TAFI-I showed only a trend toward lesion regression (-49 ± 26%), similarly to treatment with tPA10, but not significantly different from saline (p = 0.46). Nine animals showed microhemorrhage on T2-WI at H24. These animals were evenly distributed between groups. Conclusions: The present study showed that the combination of TAFI-I with a suboptimal dose of rtPA is not as effective as the standard dose of rtPA, while TAFI inhibition alone is not effective at all. The thromboembolic model is of particular interest in assessing rtPA association to improve thrombolysis, especially when coupled with longitudinal MRI assessment.

Keywords:
Embolic stroke, Recombinant tissue plasminogen activator (rtPA), Thrombin activatable fibrinolysis inhibitor (TAFI), Combined therapy, Magnetic resonance imaging (MRI)
1.
National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group: Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1995;333:1581-1587.
2.
Hacke W, Kaste M, Fieschi C, von Kummer R, Davalos A, et al: Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute ischaemic stroke (ECASS II). Second European-Australasian Acute Stroke Study Investigators. Lancet 1998;352:1245-1251.
3.
Hacke W, Kaste M, Bluhmki E, Brozman M, Davalos A, et al: Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med 2008;359:1317-1329.
4.
Derex L, Nighoghossian N: Intracerebral haemorrhage after thrombolysis for acute ischaemic stroke: an update. J Neurol Neurosurg Psychiatry 2008;79:1093-1099.
5.
Toyoda K, Koga M, Naganuma M, Shiokawa Y, Nakagawara J, et al: Routine use of intravenous low-dose recombinant tissue plasminogen activator in Japanese patients: general outcomes and prognostic factors from the SAMURAI register. Stroke 2009;40:3591-3595.
6.
Levy DE, Brott TG, Haley EC Jr, Marler JR, Sheppard GL, et al: Factors related to intracranial hematoma formation in patients receiving tissue-type plasminogen activator for acute ischemic stroke. Stroke 1994;25:291-297.
7.
Seet RC, Rabinstein AA: Symptomatic intracranial hemorrhage following intravenous thrombolysis for acute ischemic stroke: a critical review of case definitions. Cerebrovasc Dis 2012;34:106-114.
8.
Fernandez-Cadenas I, Alvarez-Sabin J, Ribo M, Rubiera M, Mendioroz M, et al: Influence of thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 gene polymorphisms on tissue-type plasminogen activator-induced recanalization in ischemic stroke patients. J Thromb Haemost 2007;5:1862-1868.
9.
Khatri P, Mono ML: Combining antithrombotic and fibrinolytic agents: can it be done? Stroke 2013;44:1489-1491.
10.
Rother J, Ford GA, Thijs VN: Thrombolytics in acute ischaemic stroke: historical perspective and future opportunities. Cerebrovasc Dis 2013;35:313-319.
11.
Vercauteren E, Emmerechts J, Peeters M, Hoylaerts MF, Declerck PJ, et al: Evaluation of the profibrinolytic properties of an anti-TAFI monoclonal antibody in a mouse thromboembolism model. Blood 2011;117:4615-4622.
12.
Orset C, Macrez R, Young AR, Panthou D, Angles-Cano E, et al: Mouse model of in situ thromboembolic stroke and reperfusion. Stroke 2007;38:2771-2778.
13.
Durand A, Chauveau F, Cho TH, Bolbos R, Langlois JB, et al: Spontaneous reperfusion after in situ thromboembolic stroke in mice. PLoS One 2012;7:e50083.
14.
Campos F, Qin T, Castillo J, Seo JH, Arai K, et al: Fingolimod reduces hemorrhagic transformation associated with delayed tissue plasminogen activator treatment in a mouse thromboembolic model. Stroke 2013;44:505-511.
15.
El Amki M, Lerouet D, Coqueran B, Curis E, Orset C, et al: Experimental modeling of recombinant tissue plasminogen activator effects after ischemic stroke. Exp Neurol 2012;238:138-144.
16.
Garcia-Yebenes I, Sobrado M, Zarruk JG, Castellanos M, Perez de la Ossa N, et al: A mouse model of hemorrhagic transformation by delayed tissue plasminogen activator administration after in situ thromboembolic stroke. Stroke 2011;42:196-203.
17.
Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, et al: Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009;40:2244-2250.
18.
Albers GW, Thijs VN, Wechsler L, Kemp S, Schlaug G, et al: Magnetic resonance imaging profiles predict clinical response to early reperfusion: the diffusion and perfusion imaging evaluation for understanding stroke evolution (DEFUSE) study. Ann Neurol 2006;60:508-517.
19.
Dobre MC, Ugurbil K, Marjanska M: Determination of blood longitudinal relaxation time (T1) at high magnetic field strengths. Magn Reson Imaging 2007;25:733-735.
20.
Hermitte L, Cho TH, Ozenne B, Nighoghossian N, Mikkelsen IK, et al: Very low cerebral blood volume predicts parenchymal hematoma in acute ischemic stroke. Stroke 2013;44:2318-2320.
21.
Faul F, Erdfelder E, Buchner A, Lang AG: Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav Res Methods 2009;41:1149-1160.
22.
Morser J, Gabazza EC, Myles T, Leung LL: What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse? J Thromb Haemost 2010;8:868-876.
23.
Kraft P, Schwarz T, Meijers JC, Stoll G, Kleinschnitz C: Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice are susceptible to intracerebral thrombosis and ischemic stroke. PLoS One 2010;5:e11658.
24.
Bird E, Tamura J, Bostwick JS, Steinbacher TE, Stewart A, et al: Is exogenous tissue plasminogen activator necessary for antithrombotic efficacy of an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI) in rats? Thromb Res 2007;120:549-558.
25.
Nagashima M, Werner M, Wang M, Zhao L, Light DR, et al: An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model. Thromb Res 2000;98:333-342.
26.
Wang YX, da Cunha V, Vincelette J, Zhao L, Nagashima M, et al: A novel inhibitor of activated thrombin activatable fibrinolysis inhibitor (TAFIa) - part II: enhancement of both exogenous and endogenous fibrinolysis in animal models of thrombosis. Thromb Haemost 2007;97:54-61.
27.
Carmichael ST: Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx 2005;2:396-409.
28.
Stroke Therapy Academic Industry Roundtable (STAIR): Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke 1999;30:2752-2758.
29.
Liu S, Zhen G, Meloni BP, Campbell K, Winn HR: Rodent stroke model guidelines for preclinical stroke trials (1st edition). J Exp Stroke Transl Med 2009;2:2-27.
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2014
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