Red Blood Cells-Coupled tPA Prevents Impairment of Cerebral Vasodilatory Responses and Tissue Injury in Pediatric Cerebral Hypoxia/Ischemia through Inhibition of ERK MAPK Activation

Armstead, William M.; Ganguly, Kumkum; Kiessling, J. Willis; Chen, Xiaohan; Smith, Douglas H.; Higazi, Abd Al‐Roof; Cines, Douglas B.; Bdeir, Khalil; Зайцев, С. В.; Muzykantov, Vladimir R.

doi:10.1038/jcbfm.2009.61

Cited by 33 publications

(44 citation statements)

References 32 publications

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“…6 Infusion of RBC-bound tPA in mice, rats and pigs has been shown to prevent thrombotic occlusion in the peripheral and cerebral vasculature, without the hemorrhagic and CNS toxicities seen with soluble nontargeted tPA. [6][7][8][21][22][23] Two major obstacles must be overcome to translate this new approach into preclinical and clinical development. First, for use as thromboprophylaxis, the respective agent should enjoy a prolonged circulation as a latent pro-drug and then be activated only when and where thrombi begin to form.…”

Section: Discussionmentioning

confidence: 99%

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Зайцев

Spitzer

Murciano

et al. 2010

Blood

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Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. IntroductionPlasminogen activators (PAs; tissue-type, tPA; urokinase, uPA) can provide urgent thrombolysis within a narrow therapeutic window of time in the setting of life-or limb-threatening thrombosis. 1,2 However, their efficacy is limited by plasma inhibitors (eg, PAI-1) and inadequate delivery into impermeable occlusive clots, a situation that is exacerbated by delays in initiating treatment. Endowing tPA derivatives with higher affinity for fibrin 3,4 further impairs clot permeation, 5 while increased dosing and constitutive lytic activity enhances the risk of bleeding and central nervous system (CNS) toxicity.Coupling tPA to carrier red blood cells (RBCs) followed by re-infusion of the RBC/tPA conjugates in animals provides protracted thromboprophylaxis in arteries and veins, including the vulnerable cerebrovascular circulation. 6-8 RBC carriage prolongs the half-life of tPAs in the circulation from minutes to many hours and prevents drug extravasation and access to hemostatic plugs, thereby reducing the risk of bleeding episodes. 7 In the prophylactic setting where tPAs lacked efficacy but caused bleeding and CNS toxicity, RBCs/tPAs mediated timely reperfusion, reducing morbidity and mortality. 8 Translation of RBC/PA thromboprophylaxis into the clinical domain could improve management of patients known to be at high risk of thrombosis or rethrombosis, including after acute myocardial infarction (AMI), transient ischemic attack, pulmonary embolism, angioplasty, and abdominal or other surgical procedures such as knee replacements, where the efficacy of thromboprophylaxis is low and/or the risk of bleeding is high. The goal of this study was to modify an existing prototypic approach (ex vivo coupling of PAs to RBCs followed by RBC/PA re-infusion) into a more clinically applicable approach to thromboprophylaxis. To anchor the injected PAs to circulating RBCs, we used a scFv fragment of the monoclonal antibody Ter-119 that recognizes mouse glycophorin A (GPA), an abundant RBC-specific surface molecule (ϳ 10 6 copies/ RBC, 9,10 similar to its human homologue 11 ). We fused scFv Ter-119 to a recombinant low molecular weight single chain uPA lacking the kringle and growth factor-like domains (lmw scuPA). Lmw scuPA is a zymogen that is naturally activated by plasmi...

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Section: Discussionmentioning

confidence: 99%

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Зайцев

Spitzer

Murciano

et al. 2010

Blood

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“…Thus, the rA2 plus low-dose tPA combination may improve recanalization and cerebral blood flow recovery. Third, despite clot lysis and reperfusion, experimental studies using advanced 'closed cranial window technique' have clearly shown that exogenous tPA may impair physiological responses of cerebral vasodilatation to hypoxia/ ischemia, and as well cause vasoconstriction after hypoxic/ischemic injury in a dose-dependent manner (Armstead et al, 2005(Armstead et al, , 2009Nassar et al, 2004). So by lowering tPA dose, the combination might eliminate tPA direct vasoactivity effect.…”

Section: Discussionmentioning

confidence: 99%

Annexin A2 Combined with Low-Dose tPA Improves Thrombolytic Therapy in a Rat Model of Focal Embolic Stroke

Zhu

Fan

et al. 2010

J Cereb Blood Flow Metab

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Recent studies showed that soluble annexin A2 dramatically increases tissue plasminogen activator (tPA)-mediated plasmin generation in vitro, and reduces thrombus formation in vivo. Here, we hypothesize that combining annexin A2 with tPA can significantly enhance thrombolysis efficacy, so that lower doses of tPA can be applied in ischemic stroke to avoid neurotoxic and hemorrhagic complications. In vitro activity assays confirmed tPA-specific amplification of plasmin generation by recombinant annexin A2. In a rat focal embolic stroke model, combination therapy with tPA and recombinant annexin A2 protein at 2 h post-ischemia decreased the effective dose required for tPA by four-fold and reduced brain infarction. Combining annexin A2 with tPA also lengthened the time window for thrombolysis. Compared with tPA (10 mg/kg) alone, the combination of annexin A2 (5 mg/kg) plus low-dose tPA (2.5 mg/kg) significantly enhanced fibrinolysis, attenuated mortality, brain infarction, and hemorrhagic transformation, even when administered at 4 h post-ischemia. Combination with recombinant annexin A2, the effective thrombolytic dose of tPA can be decreased. As a result, brain hemorrhage and infarction are reduced, and the time window for stroke reperfusion prolonged. Our present findings provide a promising new approach for enhancing tPA-based thrombolytic stroke therapy.

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“…RBC/tPA circulate for many hours and incorporate into and rapidly dissolve newly formed, potentially occlusive clots from within (Murciano et al, 2003). Infusion of RBC/tPA in mice, rats, and pigs provides an effective short-term option to prevent thrombotic occlusion in diverse vascular systems, including the cerebral vasculature, without the hemorrhagic and CNS toxicity profile typically seen with free tPA (Murciano et al, 2003;Ganguly et al, 2005;Ganguly et al, 2006;Ganguly et al, 2007;Danielyan et al, 2008;Armstead et al, 2009).…”

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Targeting of a Mutant Plasminogen Activator to Circulating Red Blood Cells for Prophylactic Fibrinolysis

Зайцев

Spitzer

Murciano

et al. 2009

J Pharmacol Exp Ther

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Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl 3 models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

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Red Blood Cells-Coupled tPA Prevents Impairment of Cerebral Vasodilatory Responses and Tissue Injury in Pediatric Cerebral Hypoxia/Ischemia through Inhibition of ERK MAPK Activation

Cited by 33 publications

References 32 publications

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation

Annexin A2 Combined with Low-Dose tPA Improves Thrombolytic Therapy in a Rat Model of Focal Embolic Stroke

Targeting of a Mutant Plasminogen Activator to Circulating Red Blood Cells for Prophylactic Fibrinolysis

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