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

ABSTRACT: 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. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.

SUBMITTER: Zaitsev S

PROVIDER: S-EPMC2892950 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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

Zaitsev Sergei S Spitzer Dirk D Murciano Juan-Carlos JC Ding Bi-Sen BS Tliba Samira S Kowalska M Anna MA Marcos-Contreras Oscar A OA Kuo Alice A Stepanova Victoria V Atkinson John P JP Poncz Mortimer M Cines Douglas B DB Muzykantov Vladimir R VR

Blood 20100421 25

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 uro ...[more]

PMID: 20410503

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Project description:AbstractFibrinolytics delivered into the general circulation lack selectivity for nascent thrombi, reducing efficacy and increasing the risk of bleeding. Urokinase-type plasminogen activator (uPA) transgenically expressed within murine platelets provided targeted thromboprophylaxis without causing bleeding but is not clinically feasible. Recent advances in generating megakaryocytes prompted us to develop a potentially clinically relevant means to produce "antithrombotic" platelets from CD34+ hematopoietic stem cell-derived in vitro-grown megakaryocytes. CD34+ megakaryocytes internalize and store in alpha granules (α-granules) single-chain uPA (scuPA) and a plasmin-resistant thrombin-activatable variant (uPAT). Both uPAs colocalized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3, but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+ megakaryocytes was mediated, in part, via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV but not endogenous VWF in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid artery injury model in nonobese diabetic-severe combined immunodeficiency IL2rγnull (NSG) mice homozygous for VWFR1326H (a mutation switching binding VWF specificity from mouse to human glycoprotein Ibα) to test whether platelets derived from scuPA- or uPAT-megakaryocytes would prevent thrombus formation. NSG/VWFR1326H mice exhibited a lower thrombotic burden after carotid artery injury compared with NSG mice unless infused with human platelets or megakaryocytes, whereas intravenous injection of uPA-megakaryocytes generated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies describe the use of in vitro-generated megakaryocytes as a potential platform for delivering uPA or other ectopic proteins within platelet α-granules to sites of vascular injury.

Project description:Thromboelastography (TEG) is used to diagnose perturbations in clot formation and lysis that are characteristic of acute traumatic coagulopathy. With novel functional fibrinogen (FF) TEG, fibrin- and platelet-based contributions to clot formation can be elucidated to tailor resuscitation and thromboprophylaxis. We sought to describe the longitudinal contributions of fibrinogen and platelets to clot strength after injury, hypothesizing that low levels of FF and a low contribution of fibrinogen to clot strength on admission would be associated with coagulopathy, increased transfusion requirements, and worse outcomes.A total of 603 longitudinal plasma samples were prospectively collected from 251 critically injured patients at a single Level 1 trauma center from 0 hour to 120 hours. TEG maximal amplitude (MA), FF MA, FF levels, von Clauss fibrinogen, and standard coagulation measures were performed in parallel. Percentage contributions of FF (%MA(FF)) and platelets (%MA(platelets)) were calculated as each MA divided by overall kaolin TEG MA.Coagulopathic patients (international normalized ratio ? 1.3) had significantly lower admission %MA(FF) than noncoagulopathic patients (24.7% vs. 31.2%, p < 0.05). Patients requiring plasma transfusion had a significantly lower admission %MA(FF) (26.6% vs. 30.6%, p < 0.05). Higher admission %MA(FF) was predictive of reduced mortality (hazard ratio, 0.815, p < 0.001). %MA(platelets) was higher than %MA(FF) at all time points, decreased over time, and stabilized at 72 hours (69.4% at 0 hour, 56.2% at 72 hours). In contrast, %MA(FF) increased over time and stabilized at 72 hours (30.6% at 0 hour, 43.8% at 72 hours).FF TEG affords differentiation of fibrin- versus platelet-based clot dynamics. Coagulopathy and plasma transfusion were associated with a lower %MA(FF). Despite this importance of fibrinogen, platelets had a greater contribution to clot strength at all time points after injury. This suggests that attention to these relative contributions should guide resuscitation and thromboprophylaxis and that antiplatelet therapy may be of underrecognized importance to thromboprophylaxis after trauma.Prognostic study, level III.

Dataset Information

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

Publications

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

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