Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke
Tissue type plasminogen activator is the only approved thrombolytic agent for the treatment of ischemic stroke. However, it carries the disadvantage of a 10-fold increase in symptomatic and asymptomatic intracranial hemorrhage. A safer thrombolytic agent may improve patient prognosis and increase patient participation in thrombolytic treatment. A novel direct-acting thrombolytic agent, Δ(K2-K5) plasmin, promising an improved safety profile was examined for safety in the snare ligature model of stroke in the rat.
Male spontaneously hypertensive rats were subjected to 6 hours middle cerebral artery occlusion followed by 18 hours reflow. Beginning 1 minute before reflow, they were dosed with saline, vehicle, Δ(K2-K5) plasmin (0.15, 0.5, 1.5, and 5 mg/kg) or recombinant tissue-type plasminogen activator (10 and 30 mg/kg) by local intra-arterial infusion lasting 10 to 60 minutes. The rats were assessed for bleeding score, infarct volume, modified Bederson score and general behavioral score. In a parallel study, temporal progression of infarct volume was determined. In an in vitro study, whole blood clots from humans, canines and rats were exposed to Δ(K2-K5). Clot lysis was monitored by absorbance at 280 nm.
The main focus of this study was intracranial hemorrhage safety. Δ(K2-K5) plasmin treatment at the highest dose caused no more intracranial hemorrhage than the lowest dose of recombinant tissue type plasminogen activator, but showed at least a 5-fold superior safety margin. Secondary results include: temporal infarct volume progression shows that the greatest expansion of infarct volume occurs within 2–3 hours of middle cerebral artery occlusion in the spontaneously hypertensive rat. A spike in infarct volume was observed at 6 hours ischemia with reflow. Δ(K2-K5) plasmin tended to reduce infarct volume and improve behavior compared to controls. In vitro data suggests that Δ(K2-K5) plasmin is equally effective at lysing clots from humans, canines and rats.
The superior intracranial hemorrhage safety profile of the direct-acting thrombolytic Δ(K2-K5) plasmin compared with recombinant tissue type plasminogen activator makes this agent a good candidate for clinical evaluation in the treatment of acute ischemic stroke.
- NINDS (rt-PA Stroke Study Group): Tissue plasminogen activator for acute ischemic stroke N Engl J Med 1995, 333:1581–1587.
- Hacke W, Kaste M, Bluhmki E, Brozman M, Davalos A, Guidetti D, Larrue V, Lees KR, Medeghri Z, Machnig T, et al.: Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med 2008, 359:1317–1329. CrossRef
- Hunt JA, Petteway SR, Scuderi P, Novokhatny V: Simplified recombinant plasmin: production and functional comparison of a novel thrombolytic molecule with plasma-derived plasmin. Thromb Haemost 2008, 100:413–419.
- Marder VJ, Manyak S, Gruber T, Goyal A, Moreno G, Hunt J, Bromirski J, Scuderi P, Petteway SR, Novokhatny V: Haemostatic safety of a unique recombinant plasmin molecule lacking kringles 2–5. Thromb Haemost 2010, 104:780–787. CrossRef
- Marder VJ: Historical perspective and future direction of thrombolysis research: the re-discovery of plasmin. J Thromb Haemost 2011,9(Suppl 1):364–373. CrossRef
- Marder VJ, Landskroner K, Novokhatny V, Zimmerman TP, Kong M, Kanouse JJ, Jesmok G: Plasmin induces local thrombolysis without causing hemorrhage: a comparison with tissue plasminogen activator in the rabbit. Thromb Haemost 2001, 86:739–745.
- Marder VJ, Novokhatny V: Direct fibrinolytic agents: biochemical attributes, preclinical foundation and clinical potential. J Thromb Haemost 2010, 8:433–444. CrossRef
- Stewart D, Kong M, Novokhatny V, Jesmok G, Marder VJ: Distinct dose-dependent effects of plasmin and TPA on coagulation and hemorrhage. Blood 2003, 101:3002–3007. CrossRef
- Marder VJ, Jahan R, Gruber T, Goyal A, Arora V: Thrombolysis with plasmin: implications for stroke treatment. Stroke 2010, 41:S45–49. CrossRef
- Crumrine RC, Marder VJ, Taylor GM, LaManna JC, Tsipis CP, Novokhatny V, Scuderi P, Petteway SR, Arora V: (k2-k5)Plasmin (TAL6003) is an effective and safe direct acting thrombolytic as compared to rtPA: implications for stroke therapy. In CIRSE 2011; Munich, Germany. 2011 (Abstract)
- Crumrine RC, Marder VJ, Taylor GM, Lamanna JC, Tsipis CP, Scuderi P, Petteway SR, Arora V: Intra-arterial administration of recombinant tissue-type plasminogen activator (rt-PA) causes more intracranial bleeding than does intravenous rt-PA in a transient rat middle cerebral artery occlusion model. Exp Transl Stroke Med 2011, 3:10. CrossRef
- Lisboa RC, Jovanovic BD, Alberts MJ: Analysis of the safety and efficacy of intra-arterial thrombolytic therapy in ischemic stroke. Stroke 2002, 33:2866–2871. CrossRef
- Molina CA: Reperfusion therapies for acute ischemic stroke: current pharmacological and mechanical approaches. Stroke 2011, 42:S16–19. CrossRef
- IST-3 Study Protocol: Third international stroke trial: thrombolysis for acute ischaemic stroke. [http://www.ist3.com]
- Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen HJ: A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry. Stroke 1998, 29:2162–2170. CrossRef
- Zhao Q, Memezawa H, Smith ML, Siesjo BK: Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament. Brain Res 1994, 649:253–259. CrossRef
- Selman WR, Crumrine RC, Ricci AJ, LaManna JC, Ratcheson RA, Lust WD: Impairment of metabolic recovery with increasing periods of middle cerebral artery occlusion in rats. Stroke 1990, 21:467–471. CrossRef
- Selman WR, Crumrine RC, Rosenstein CC, Jenkins C, LaManna JC, Ratcheson RA, Lust WD: Rapid metabolic failure in spontaneously hypertensive rats after middle cerebral artery ligation. Metabolic brain disease 1991, 6:57–64. CrossRef
- Selman WR, Ricci AJ, Crumrine RC, LaManna JC, Ratcheson RA, Lust WD: The evolution of focal ischemic damage: a metabolic analysis. Metabolic brain disease 1990, 5:33–44. CrossRef
- Selman WR, Lust WD: Pathophysiology of irreversible brain damage in a rat model of focal ischemia. Neurosurgeons 1991, 10:18–27.
- Crumrine RC, Selman WR, LaManna JC, Lust WD: Protein kinase C activity in permanent focal cerebral ischemia. Molecular and chemical neuropathology/sponsored by the International Society for Neurochemistry and the World Federation of Neurology and research groups on neurochemistry and cerebrospinal fluid 1992, 16:85–93.
- Finelli DA, Hopkins AL, Selman WR, Crumrine RC, Bhatti SU, Lust WD: Evaluation of experimental early acute cerebral ischemia before the development of edema: use of dynamic, contrast-enhanced and diffusion-weighted MR scanning. Magnetic resonance in medicine : official journal of the Society of Magnetic Resonance in Medicine/Society of Magnetic Resonance in Medicine 1992, 27:189–197. CrossRef
- Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W: Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab 1988, 8:474–485. CrossRef
- Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz SI, Lo EH: Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke 2009, 40:2244–2250. CrossRef
- Howells DW, Porritt MJ, Rewell SS, O'Collins V, Sena ES, van der Worp HB, Traystman RJ, Macleod MR: Different strokes for different folks: the rich diversity of animal models of focal cerebral ischemia. J Cereb Blood Flow Metab 2010, 30:1412–1431. CrossRef
- Welsh FA, Sakamoto T, McKee AE, Sims RE: Effect of lactacidosis on pyridine nucleotide stability during ischemia in mouse brain. J Neurochem 1987, 49:846–851. CrossRef
- Seta KA RCC, TS W, WD L, McCandless D: Experimental models of human stroke. In Neuromethods. Volume 22. Edited by Boulton AA BG, Butterworth RF. Totowa, New Jersey: The Humana Press, Inc.; 1992: 1–50: Animal Models of Neurological Disease, II: Metabolic Encephalopathies and the Epilepsies] 1992, 22:1–50.
- Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H: Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 1986, 17:472–476. CrossRef
- Shaw GJ, Sperling M, Meunier JM: Long-term stability of recombinant tissue plasminogen activator at −80 C. BMC Res Notes 2009, 2:117. CrossRef
- Korninger C, Collen D: Studies on the specific fibrinolytic effect of human extrinsic (tissue-type) plasminogen activator in human blood and in various animal species in vitro. Thromb Haemost 1981, 46:561–565.
- Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, Bartkowski HM: Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 1986, 17:1304–1308. CrossRef
- Landskroner K, Olson N, Jesmok G: Cross-species pharmacologic evaluation of plasmin as a direct-acting thrombolytic agent: ex vivo evaluation for large animal model development. Journal of vascular and interventional radiology : JVIR 2005, 16:369–377. CrossRef
- Haelewyn B, Risso JJ, Abraini JH: Human recombinant tissue-plasminogen activator (alteplase): why not use the 'human' dose for stroke studies in rats? J Cereb Blood Flow Metab 2010, 30:900–903. CrossRef
- Wang CX, Ding X, Shuaib A: Treatment with melagatran alone or in combination with thrombolytic therapy reduced ischemic brain injury. Exp Neurol 2008, 213:171–175. CrossRef
- Christou I, Alexandrov AV, Burgin WS, Wojner AW, Felberg RA, Malkoff M, Grotta JC: Timing of recanalization after tissue plasminogen activator therapy determined by transcranial doppler correlates with clinical recovery from ischemic stroke. Stroke 2000, 31:1812–1816. CrossRef
- Hacke W, Donnan G, Fieschi C, Kaste M, von Kummer R, Broderick JP, Brott T, Frankel M, Grotta JC, Haley EC: et al: Association of outcome with early stroke treatment: pooled analysis of ATLANTIS, ECASS, and NINDS rt-PA stroke trials. Lancet 2004, 363:768–774. CrossRef
- del Zoppo GJ, von Kummer R, Hamann GF: Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke. J Neurol Neurosurg Psychiatry 1998, 65:1–9. CrossRef
- Gautier S, Petrault O, Gele P, Laprais M, Bastide M, Bauters A, Deplanque D, Jude B, Caron J, Bordet R: Involvement of thrombolysis in recombinant tissue plasminogen activator-induced cerebral hemorrhages and effect on infarct volume and postischemic endothelial function. Stroke 2003, 34:2975–2979. CrossRef
- Kahles T, Foerch C, Sitzer M, Schroeter M, Steinmetz H, Rami A, Neumann-Haefelin T: Tissue plasminogen activator mediated blood–brain barrier damage in transient focal cerebral ischemia in rats: relevance of interactions between thrombotic material and thrombolytic agent. Vascul Pharmacol 2005, 43:254–259. CrossRef
- Lewandowski CA, Frankel M, Tomsick TA, Broderick J, Frey J, Clark W, Starkman S, Grotta J, Spilker J, Khoury J, Brott T: Combined intravenous and intra-arterial r-TPA versus intra-arterial therapy of acute ischemic stroke: Emergency Management of Stroke (EMS) Bridging Trial. Stroke 1999, 30:2598–2605. CrossRef
- Jiang Q, Zhang ZG, Zhang L, Ding GL, Li L, Ewing JR, Lu M, Whitton P, Hu J, Li QJ, et al.: MRI evaluation of treatment of embolic stroke in rat with intra-arterial and intravenous rt-PA. J Neurol Sci 2004, 224:57–67. CrossRef
- Shaltoni HM, Albright KC, Gonzales NR, Weir RU, Khaja AM, Sugg RM, Campbell MS, Cacayorin ED, Grotta JC, Noser EA: Is intra-arterial thrombolysis safe after full-dose intravenous recombinant tissue plasminogen activator for acute ischemic stroke? Stroke 2007, 38:80–84. CrossRef
- Messe SR, Tanne D, Demchuk AM, Cucchiara BL, Levine SR, Kasner SE: Dosing errors may impact the risk of rt-PA for stroke: the Multicenter rt-PA Acute Stroke Survey. J Stroke Cerebrovasc Dis 2004, 13:35–40. CrossRef
- Aronowski J, Strong R, Grotta JC: Reperfusion injury: demonstration of brain damage produced by reperfusion after transient focal ischemia in rats. J Cereb Blood Flow Metab 1997, 17:1048–1056. CrossRef
- Overgaard K, Sereghy T, Pedersen H, Boysen G: Effect of delayed thrombolysis with rt-PA in a rat embolic stroke model. J Cereb Blood Flow Metab 1994, 14:472–477. CrossRef
- Parsons M, Spratt N, Bivard A, Campbell B, Chung K, Miteff F, O'Brien B, Bladin C, McElduff P, Allen C, et al.: A randomized trial of tenecteplase versus alteplase for acute ischemic stroke. N Engl J Med 2012, 366:1099–1107. CrossRef
- Nagai N, De Mol M, Van Hoef B, Verstreken M, Collen D: Depletion of circulating alpha(2)-antiplasmin by intravenous plasmin or immunoneutralization reduces focal cerebral ischemic injury in the absence of arterial recanalization. Blood 2001, 97:3086–3092. CrossRef
- Lapchak PA, Araujo DM, Pakola S, Song D, Wei J, Zivin JA: Microplasmin: a novel thrombolytic that improves behavioral outcome after embolic strokes in rabbits. Stroke 2002, 33:2279–2284. CrossRef
- Safety evaluation of a recombinant plasmin derivative lacking kringles 2-5 and rt-PA in a rat model of transient ischemic stroke
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
Experimental & Translational Stroke Medicine
- Online Date
- May 2012
- Online ISSN
- BioMed Central
- Additional Links
- Ischemic stroke
- Δ(K2-K5) plasmin
- Intracranial hemorrhage
- Spontaneously hypertensive rat model
- Recombinant tissue-type plasminogen activator (rt-PA)
- Middle cerebral artery occlusion (MCAo)
- Author Affiliations
- 1. Research and Pre-clinical Development, Grifols Therapeutics, Inc, Research Triangle Park, North Carolina, USA
- 2. Division of Hematology/Medical Oncology, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
- 3. Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA