Prophylactic thrombolysis by thrombin-activated latent prourokinase targeted to PECAM-1 in the pulmonary vasculature

Abstract

A recombinant prodrug, single-chain urokinase-type plasminogen activator (scuPA) fused to an anti-PECAM-1 antibody single-chain variable fragment (anti-PECAM scFv/scuPA) targets endothelium and augments thrombolysis in the pulmonary vasculature. 1 To avoid premature activation and inactivation and to limit systemic toxicity, we replaced the native plasmin activation site in scFv/lowmolecular-weight (lmw)-scuPA with a thrombin activation site, generating anti-PECAM scFv/uPA-T that (1) is latent and activated by thrombin instead of plasmin; (2) binds to PECAM-1; (3) does not consume plasma fibrinogen; (4) accumulates in mouse lungs after intravenous injection; and (5) resists PA inhibitor PAI-1 until activated by thrombin. In mouse models of pulmonary thrombosis caused by thromboplastin and ischemiareperfusion (I/R), scFv/uPA-T provided more potent thromboprophylaxis and greater lung protection than plasminsensitive scFv/uPA. Endothelium-targeted thromboprophylaxis triggered by a prothrombotic enzyme illustrates a novel approach to time-and site-specific regulation of proteolytic reactions that can be modulated for therapeutic benefit. IntroductionPlasminogen activators (PAs) used in the treatment of thrombosis convert the zymogen plasminogen into plasmin, which lyses fibrin and restores perfusion. [2][3][4] Unfortunately, the utility of PAs is limited by inadequate delivery (rapid clearance, inactivation, and ineffective penetration of occlusive clots) and side effects, including hemorrhage and extravascular toxicity. [5][6][7] Further, postevent thrombolytic therapy is marred by inevitable delays (time needed for diagnosis, transportation, injection, and lysis) causing ischemia-reperfusion (I/R) injury that perpetuates thrombosis and worsens outcome. 8,9 Attempts to improve the effectiveness of PAs by increasing clot affinity have further diminished permeation into occluding thrombi due to retention on the clot surface 5 and have yet to provide decisively better clinical outcomes. [10][11][12][13][14] Thrombi often recur due to underlying procoagulation states, vascular damage, disturbance in blood flow, inflammation, or/and patients' immobility. 15 Rethromboses often occur within hours to days after acute myocardial infarction, transient ischemic attack, ischemic stroke, and pulmonary embolism. The effectiveness of secondary prevention using existing anticoagulants and antiplatelet agents is limited and dosing is constrained by the risk of bleeding. 16 In theory, prophylactic delivery of a PA into nascent thrombi, which are more susceptible to dissolution than mature occlusive clots, would expedite thrombolysis and minimize ischemiareperfusion injury in patients at high risk of imminent thrombosis. Unfortunately, PAs are not suitable for thromboprophylaxis due to unfavorable pharmacokinetics and toxicity.Conceivably, the failure to exploit PAs for thromboprophylaxis could be overcome by optimization of drug and targeting strategies. Single-chain urokinase-type plasminogen activator (scuPA) seems suitable for t...

Anchoring Fusion Thrombomodulin to the Endothelial Lumen Protects against Injury-induced Lung Thrombosis and Inflammation

“…To avoid any Fc fragment-mediated side effects and unwanted internalization of PECAM-1 that might be induced by multivalent antibody conjugates (25), we have previously generated fusion proteins consisting of a monovalent single-chain variable fragment of a PECAM-1 antibody fused with urokinaseplasminogen activator (scFv/uPA) that accumulates along the pulmonary vasculature and provides local fibrinolysis (26,27). This prototype supports the concept of vascular targeting, but does not provide antiinflammatory activity.…”

“…APC activation was assayed by incubating scFv/TM or sTM with thrombin and protein C and measuring the optical density (OD 405nm ) with Spectrozyme PCa (American Diagnostica, Inc., Stamford, CT). Binding of scFv/TM to PECAM-1 was measured by ELISA using an anti-TM antibody (27).…”

A Humanized Single-Chain Variable Fragment Antibody Against Beta3 Integrin in Escherichia coli

“…Therefore, to avoid the possible side effects, this newly generated scFv Ab should be fused with other anti-thrombotic agents such as plasminogen activators to maximize its therapeutic window of efficacy, while being administered at a relatively lower dose. (11)(12)(13) In the future, this proof of principle needs to be further evaluated in at least one animal thrombotic model by monitoring the platelet counts upon injection of the scFv Ab. Nevertheless, the current data have established the concept of developing a different approach to combat arterial thrombosis by generating an scFv for the lysis of platelet thrombus.…”

“…The scFv form has additional unparalleled advantages for clinical utility, (7)(8)(9) with their recombinant feature allowing for further genetic engineering such as affinity maturation and construction of fusion molecules for high clot-target avidity. (10)(11)(12)(13) Binding activity of A11 to platelet integrin GPIIIa49-66…”

Lung vascular targeting through inhalation delivery: Insight from filamentous viruses and other shapes

“…Muzyknatov et al have been investigating the targeted delivery of drugs to pulmonary vascular endothelium as an important means for controlling oxidative stress, thrombosis, and inflammation associated with pulmonary diseases. Antibodies to platelet-endothelial cell adhesion molecule 1 (PECAM-1) take advantage of the stable expression and high density of PECAM-1 on the luminal surface of the endothelium (54,55). McIntosh et al (56) addressed the inverse problem: they demonstrated targeting to alveolar epithelium in rat lung after intravenous injection of gold nanoparticle (6 nm) conjugated to a monoclonal antibody (TX3.833) specific for lung caveolae which rapidly transcytose to the alveolar air space via epithelial caveolae.…”