cardiovascular diseases including pulmonary embolism , atherosclerosis , coronary heart disease , acute myocardial infarction , and stroke are major causes of morbidity and mortality worldwide ( capstick and henry , 2005 ; prasad et al . , 2006 ; van der worp and van gijn , 2007 ; white and chew , 2008 ; jones et al . , 2010 ; collart et al . , 2012 ; these diseases result from obstruction of blood flow by thrombus , and the most effective method of preventing morbidity and mortality associated with these diseases is to prevent thrombus formation . when thrombus has already formed , the best treatment strategy is to achieve rapid recanalization of the occluded vessel by angioplasty , surgery , or thrombolysis / fibrinolysis to remove the thrombus and prevent further propagation ( absar et al . , 2013 ) . stroke is usually treated by thrombolytic therapy , which works by interacting with the body 's intrinsic fibrinolytic system ( kowalski et al . , 2009 ) . many thrombolytic agents have been found to effectively dissolve thrombus , including streptokinase , urokinase , and tissue - type plasminogen activator . these agents comprise proteolytic components of the blood clotting cascade , and as they are circulated throughout the cardiovascular system they do not selectively target specific organs or tissues . the systemic side effects of these agents such as fibrinogenolysis and bleeding ( kowalski et al . 2013 ) result in unavoidable clinical difficulties ( oyama et al . , 2013 ) . 2006 ) reported that addition of a neuroprotective agent can increase the effectiveness of thrombolytic therapy , increase the therapeutic time window , and reduce cerebral ischemia - reperfusion injury . it is hoped that thrombolytic agents with neuroprotective effects can be developed for clinical use . intravascular thrombus formation is a complex physiological process that involves interactions among many factors . when a blood vessel is injured , local accumulation of platelets and fibrin results in thrombus formation to prevent blood loss . after thrombosis , plasminogen activator cleaves the sensitive arg561-val562 peptide bond of plasminogen ( lijnen and collen , 2000 ; lijnen , 2001 ; oyama et al . , 2013 ) , thereby activating plasminogen to form plasmin ( gabriel et al . , 1992 ; castellino and ploplis , 2005 ; kunamneni et al . , 2007 ; baumer et al . , 2013 ; gomaraschi et al . , 2013 ) , which triggers the body 's mechanisms for dissolving thrombus into soluble fibrin degradation products and restores the blood flow ( guyatt et al . , thrombosis is triggered by prothrombin activator , which causes formation of cross - linked polymer fibrin with the addition of ca . endogenous plasminogen is activated and converted to plasmin in the intravascular space by plasminogen activator , and the cross - linked polymer fibrin is degraded into soluble fibrin degradation products by plasmin and then cleared . in an attempt to increase the efficacy and safety of thrombolytic therapy , recent research has focused on the development of targeted thrombolytic agents . thrombolytic agents have been used with the aim of recanalizing the occluded vessel since streptokinase was first used in patients with acute myocardial infarction ( meyer et al . , 1965 ) . four generations of thrombolytic agents have subsequently been used in clinical trials for the treatment of various clotting disorders ( kirmani et al . , 2012 ) . however , thrombolytic therapy is still associated with many problems . the first - generation fibrinolytic agents are effective for thrombolysis , but are not fibrin specific ( bentley and sharma , 2005 ) and they may induce immunological responses resulting in drug resistance , fever , and allergic reactions ( verstraete , 2000 ) . the second - generation fibrinolytic agents are more fibrin specific , do not induce adverse immunological responses ( balami et al . , 2013 ) , and have a shorter half - life ( collen and lijnen , 1991 ; epplera et al . , 1998 ; kim et al . , 2009 ) . the majority of third- and fourth - generation fibrinolytic agents have advantages over second - generation agents , but are currently only available in clinical trials ( longstaff et al . , 2008 ) . there is a need to improve the specificity , efficacy , and safety of the drugs available for clinical use ( toombs , 2001 ) . current research is focused on enhancing the ability to target the site of the thrombus and reduce adverse effects and complications ( table 1 ) . thrombus is mainly composed of a mesh of fibrin and platelets ( cadroy and hanson , 1990 ; varin et al . , 2013 ; wadajkar et al . , fibrin ( also called factor ia ) is a fibrous , non - globular , insoluble protein that is produced in response to bleeding , and is the main protein component of thrombus ( ghasemi et al . , 2012 ) . there is a high concentration of fibrin in all types of thrombus , including acute and chronic , and arterial and venous thrombus ( sirol et al . , 2005 ) . fibrin is a tough protein substance arranged in long fibrous chains that form from fibrinogen during blood coagulation ( mosesson , 2005 ) . when tissue damage results in bleeding , fibrinogen in the wound is converted to fibrin monomers by the action of thrombin ( lord , 2007 ; ariens , 2013 ) . the fibrin monomers combine to form long fibrin threads that entangle platelets to build a spongy mass that gradually hardens and contracts , resulting in thrombus formation . several proteins can bind to fibrinogen and/or fibrin and can thereby influence thrombus formation , structure , and degradation . carboxypeptidase n ( cpn ) is an enzyme that cleaves c - terminal arginine residues from bradykinin , and belongs to the same family of zinc metallocarboxy - peptidases as thrombin - activatable fibrinolysis inhibitor ( walker et al . , 2008 ) . talens et al . studied and identified cpn as a novel thrombus component with possible antifibrinolytic properties ( talens et al . , 2012a ) , but could not prove the presence of cpn in thrombus ( talens et al . , 2012b ) . cpn should therefore be investigated further to determine whether it binds directly to fibrin or fibrinogen . the above - mentioned studies used surface plasmon resonance to determine that thrombus - bound cpn has the same molecular forms as cpn in the plasma , and that cpn may bind to fibrinogen and fibrin . fibrin has been considered as a molecular target for the selective delivery of thrombolytic agents to the thrombus . recently , ultrasound - assisted drug delivery has been investigated as a method of targeting a specific area ( klibanov et al . , 2010 ) , and intrinsically echogenic liposomes have been used as a vehicle to achieve ultrasound - triggered controlled drug release ( huang , 2008 ; greineder et al . , 2013 ) . ultrasound was found to improve the effectiveness of tissue plasminogen activator ( tpa ) , but was associated with hemorrhagic side effects ( datta et al . use of tpa - loaded intrinsically echogenic liposomes was found to be similarly effective to other treatment methods , while offering the advantages of ultrasound monitoring and enhanced thrombolysis with site - specific delivery ( shaw et al . , 2009 ; laing et al . , 2012 ) . the fibrin - specificity of thrombolytic agents may be improved by conjugating them with fibrin - specific monoclonal antibodies ( vaidya et al . , 2012 ) . a hybrid molecule conjugated with tpa and the fibrin - specific monoclonal antibody 59d8 by a disulfide bond ( runge et al . , 1987 ) has a 10-fold higher affinity for fibrin than urokinase and a 100-fold higher affinity for fibrin than tpa . furthermore , experimental use of a hybrid recombinant plasminogen activator , antifibrin antibody 59d8low - molecular - weight single - chain urokinase - type plasminogen activator , for antibody targeting of fibrin increased the thrombolytic and antithrombotic potency with less impairment of hemostasis compared with recombinant tpa and recombinant single - chain urokinase - type plasminogen activator ( runge et al . , 1996 ) . current strategies for site - specific delivery are focused primarily on the local release of therapeutic agents by drug - eluting stents . however , this technique is expensive and can only be used in limited situations ( muni and gross , 2004 ; huang et al . , nanoparticles have recently attracted attention as potential vehicles for targeted drug therapy , and have been shown to increase therapeutic effectiveness ( cyrus et al . , 2008 ; ( 2009 ) used in vitro fibrinolysis assays to show that use of 40-nm polystyrene - latex nanoparticles covalently conjugated to tpa and anti - fibrin antibody could deliver tpa directly to the site of the thrombus , thereby lowering the risk of hemorrhage . since then , a thrombolytic agent that conjugates an antifibrin monoclonal antibody and urokinase to a perfluorocarbon nanoparticle has been developed , and its effectiveness for targeted thrombolysis has been evaluated ( marsh et al . platelets ( also called thrombocytes ) are small , disk - shaped , clear , anuclear cell fragments , 23 m in diameter , which are derived from fragmentation of precursor megakaryocytes . platelets circulate in the blood of mammals and play a vital role in the process of thrombus formation to maintain hemostasis ( langer and gawaz , 2008 ) . under pathophysiological conditions , platelet activation can also play a critical role in various thromboembolic diseases ( harrison , 2000 ) . platelet membrane glycoprotein iib / iiia ( gpiib / iiia ) receptor activation is the final common pathway of platelet aggregation ( kulkarni et al . , 2000 ; ten berg et al . , 2001 ; davi and patrono , 2007 ; badimon and vilahur , 2008 ; gladding et al . , 2008 ) . the gpiib / iiia receptor is the most abundant protein on the platelet membrane ( jennings , 2009 ; vaidya et al . , 2011 ) , and is a potential target for novel thrombolytic agents . l - arginine - glycine - aspartic acid peptide ( rgd ) is a gpiib / iiia antagonist that binds to activated gpiib / iiia receptors specifically on aggregated platelets ( meyer et al . , 2006 ) , and has been used to develop targeted thrombolytic drugs . under normal conditions , the spatial configuration of gpiib / iiia receptors is stable , and the platelet is inactive . when platelet agonists such as thrombin , collagen , adenosine diphosphate , and thromboxane a2 bind with their receptors on the platelet membrane , gpiib / iiia forms a functional dimer complex that exposes the platelet membrane ( huang et al . , 2008 ) , resulting in binding with rgd . conversely , the gpiib / iiia receptors are hidden on the unactivated platelet membrane , and can not bind with rgd . rgd therefore binds only with activated platelets in the thrombus , and has no effect on circulating platelets . the hexapeptide h - pro - ser - nva - gly - asp - trp - oh also binds to the gpiib / iiia receptor on activated platelets , and development of thrombus - targeted microbubbles has been attempted by binding this hexapeptide to microbubbles ( zhou et al . , 2011 ) . binding of the hexapeptide to the gpiib / iiia receptor delivers the microbubbles specifically to the thrombus ( wang et al . , 2006 ) . the microbubbles were found to be good carriers of thrombolytic drugs , and to be beneficial for preventing thrombus recurrence in vivo . gpiib / iiia receptors have been used as a target for the delivery of thrombolytic agents by conjugation with a monoclonal antibody modified with n - succinimidyl-3-(2-pyridyldithio ) propionate at 7e3 fab ( bates et al . , 1991 ) to selectively bind urokinase to gpiib / iiia on the platelet membrane . the conjugated urokinase was found to have higher thrombolytic activity than unconjugated urokinase ( bode et al . , 1991 ) . annexin v has a high affinity for phosphatidylserine , and binding of annexin v to the b chain of urokinase - type plasminogen activator by a disulfide bond was found to increase the thrombolytic activity of the plasminogen activator in in vitro tests ( okabayashi et al . , 1996 ) . in most cases , labile and complex biotherapeutic agents such as enzymes require precise delivery to the target site . the best way to achieve this goal is to use coupling drugs such as synthetic or natural polymers with various geometric configurations , phospholipid liposomes , albumin , antibodies , or other biological molecules as carriers ( simone et al . , 2008 ) . rbcs ( also called erythrocytes ) are anuclear , biconcave , discshaped cells with a diameter of 78 m , thickness of 23 m , and membrane surface area of about 160 m , and are the most common type of blood cell ( pierige et al . , 2008 ) . rbcs have many features that make them ideal carriers for drugs in the bloodstream ( magnani et al . , 2002 ; danchin et al . , 2008 ) , especially when sustained action is needed ( bax et al . , 1999 rbcs can transport many substances , and the rbc membrane is supported by a complex cytoskeleton comprising a hexagonal lattice of actin - spectrin filaments interconnected by anchoring integral plasmalemmal proteins via numerous structural and connector proteins ( muzykantov , 2010 ; luo et al . , this problem can not be solved by increasing the dose because of the associated risk of adverse effects ( ganguly et al . , 2006 ) . however , the use of rbcs as drug carriers may help to solve this problem by enabling the use of plasminogen activators as thromboprophylactic agents ( ganguly et al . rbcs have recently been used as intravascular carriers for targeted drug delivery ( serafini et al . , 2004 ; rossi et al . , 2005 once thrombus is established , it becomes progressively more impermeable to rbcs , and rbc carriers of plasminogen activators can therefore potentially prevent vascular occlusion in patients at imminent risk of thrombosis without lysing hemostatic clots . ( 2003 ) hypothesized that tpa conjugated to rbcs would dissolve nascent clots while having minimal effects on preexisting hemostatic clots or extravascular tissues . this rbc - based drug delivery method alters the fibrinolytic profile of tpa , thereby permitting prophylactic fibrinolytic therapy . conjugating of tpa to rbcs also reduces its central nervous system toxicity by spatially confining the drug to the vascular system . administration of rbc - tpa before or after cerebral hypoxia / ischemia may preserve the responses to cerebral vasodilators and prevent neuronal injury mediated through the extracellular signal - related kinase ( mitogen - activated protein kinase ) pathway , indicating that use of rbc - tpa may increase the benefit / risk ratio of thrombolytic therapy ( armstead et al . , 2009 ) . plasminogen activators are currently not used for thromboprophylaxis because of their rapid clearance , associated risk of bleeding , and extravascular toxicity . ( 2006 ) conjugated tpa to a monoclonal antibody against complement receptor type 1 expressed primarily on human rbcs , and found that tpa bound rapidly to rbcs in the bloodstream and circulated safely for many hours after injection in mice , providing prophylactic thrombolysis without hemorrhagic side effects , similar to use of preformed rbc - tpa . this approach provided rapid and tight binding of tpa to rbcs , which markedly prolonged the circulation of tpa , accelerated lysis of venous and occlusive arterial thrombus that formed subsequent to injection , and reduced bleeding from preexisting hemostatic clots . subsequently , a single - chain antibody fragment - tissue type plasminogen activator fusion targeted to rbc glycophorin - a related antigen was developed that bound safely to circulating rbcs and had anti - thrombotic effects in a mouse model of thrombosis that were qualitatively similar to rbc - tpa and superior to tpa ( zaitzev et al . , 2010 ) . pharmacological agents have generally targeted the transformation of plasminogen and plasmin , thereby facilitating the natural process of fibrinolysis . however , these agents do not discriminate between healthy and at - risk vasculature , and are widely distributed in the circulation . development of newer targeted thrombolytic agents should enable selective delivery to specific organs , tissues , or cells to enhance targeting of the thrombus and reduce adverse effects , thereby achieving superior efficacy and safety compared with existing therapeutic options . several approaches have been proposed for the targeted delivery of plasminogen activators , including conjugation with specific monoclonal antibodies , oligopeptides , and nanoparticles . conjugation with fibrin - specific immunoconjugates seems to be a less promising approach because of the poor specificity of antibody binding to fibrin , and the inability to distinguish between hemostatic clots and occlusive thrombus . the effects of platelet - specific agents are obvious , but their safety and effectiveness need to be further studied . use of a single - chain antibody fragment - tissue type plasminogen activator fusion targeted to rbc glycophorin - a related antigen appears to be at least as promising as use of tpa - loaded nanoparticles for the prevention of both venous and arterial thrombus formation . effective clearance of preexisting thrombus using methods such as ultrasound ( uesugi et al . , 2010 ) and enhancement of the effectiveness of thrombolytic agents may provide a good approach to the treatment of stroke .
stroke is usually treated by systemic thrombolytic therapy if the patient presents within an appropriate time window . 
 there is also widespread interest in the development of thrombolytic agents that can be used in cases of delayed presentation . 
 current agents that can be used in cases of delayed presentation of nerve damage by thrombus . 
 current systemic thrombolytic therapy is associated with adverse effects such as fibrinogenolysis and bleeding . in an attempt to increase the efficacy , safety , and specificity of thrombolytic therapy , a number of targeted thrombolytic agents 
 have been studied in recent years . 
 this review focuses on the concepts underlying targeted thrombolytic therapy and describes recent drug developments in this field .